Combined Effects of Drying Methods, Extract Concentration, and Film Thickness on Efficacy of Antimicrobial Chitosan Films Wasina Thakhiew, Sakamon Devahastin, and Somchart Soponronnarit

An idea of using a suitable drying method to minimize the loss of added antimicrobial agent and, at the same time, to modify the structure, and hence the release characteristics of chitosan films was proposed. Chitosan film-forming solution was incorporated with galangal extract (0% and 1.5% w/w) and formed into films with the thickness of 15 and 30 μm via hot air drying (HD) (40 °C) and low-pressure superheated steam drying (LPSSD) (70 °C, 10 kPa). The extract retention, release characteristics, and antimicrobial efficacy of the films were then assessed; fresh-cut cantaloupe was used as the test food material, while Staphylococcus aureus was the test pathogenic microorganism. The retention and release of 1,8-cineole, a major bioactive compound in the galangal extract, was monitored during 5-d storage at 25 °C. The film swelling was also evaluated and their results used to interpret the release characteristics of 1,8-cineole from the films to the cantaloupe. At the same thickness, the films prepared by HD had lower extract retention and higher degree of swelling, thus exhibiting faster extract release and lower antimicrobial efficacy than the films prepared by LPSSD. Within the same drying method, the increased film thickness led to higher extract retention and antimicrobial efficacy. The concentration of the extract in the cantaloupe matched well with the extract retention and release characteristics as well as the antimicrobial efficacy of the films.

Abstract:

E: Food Engineering & Physical Properties

Keywords: 1,8-cineole, extract retention, galangal extract, release characteristics, swelling

The results of this work illustrate the feasibility of using a suitable drying technology to improve the quality of antimicrobial films from edible biopolymer and form the basis for future development of an improved antimicrobial film production process.

Practical Application:

Introduction Antimicrobial edible films are considered promising food packagings as they have potential to inactivate foodborne pathogen and represent a green alternative to plastic packagings. Several substances have been proposed for the preparation of antimicrobial films. Use of naturally derived antimicrobial agents is nevertheless an attractive alternative (Seydim and Sarikus 2006; Tajkarimi and others 2010). Antimicrobial films incorporated with different plant essential oils such as clove, cinnamon, basil, coriander, anise, garlic, galangal, mint/pomegranate peel, oregano, rosemary, and thyme have been reported to be effective against Salmonella Enteritidis, Bacillus cereus, Escherichia coli O157:H7, Pseudomonas aeroginosa, Listeria monocytogenes, or Staphylococcus aureus (Pranoto and others 2005; Zivanovic and others 2005; Seydim and Sarikus 2006; Sangsuwan and others 2008; Hosseini and others 2009; Mayachiew and others 2010; Kanatt and others 2012). MS 20140123 Submitted 1/23/2014, Accepted 4/7/2014. Author Thakhiew is with Energy Technology Div., School of Energy, Environment and Materials, King Mongkut’s Univ. of Technology Thonburi, 126 Pracha u-tid Rd., Tungkru, Bangkok 10140, Thailand; and with Dept. of Nutrition, Faculty of Public Health, Mahidol Univ., 420/1 Ratchathewi Rd., Ratchathewi, Bangkok 10400, Thailand. Author Soponronnarit is with Energy Technology Div., School of Energy, Environment and Materials, King Mongkut’s Univ. of Technology Thonburi, 126 Pracha u-tid Rd., Tungkru, Bangkok 10140, Thailand. Author Devahastin is with Dept. of Food Engineering, Faculty of Engineering, King Mongkut’s Univ. of Technology Thonburi, 126 Pracha u-tid Rd., Tungkru, Bangkok 10140, Thailand. Direct inquiries to author Devahastin (E-mail: [email protected]).

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For any antimicrobial film to be effective, retention of added antimicrobial agent, both during and after the film forming process, is of importance. Major loss of antimicrobial agent typically occurs during drying, which is an important film forming step (Mastromatteo and others 2009; Mayachiew and Devahastin 2010). Drying method (and hence time) is an important factor influencing such a loss; the loss generally increases with an increase in the drying time (Monedero and others 2010; Kurek and others 2012). Using a suitable drying method that results in shorter required drying time, especially at a lower temperature, is a solution to such a problem (Figiel and others 2010). Increasing of the film thickness is another simple alternative that could help retain an extract within the polymer matrix (S´anchez-Gonz´alez and others 2011). In addition to reducing the loss, the rate of release from the film and hence the amount of added antimicrobial agent that would reach the food surface must be well regulated. The release characteristics of an antimicrobial agent from the film matrix depends on several factors, in particular the structure of the polymeric network (Zhang and Kosaraju 2007; Mastromatteo and others 2009; S´anchez-Gonz´alez and others 2011; Kuorwel and others 2013; Pinheiro and others 2013). The desired film structure is the one that allows gradual transport of antimicrobial agent from the film to food surface, resulting in the maintenance of the needed concentration of the agent throughout the storage period. Previous studies (Mayachiew and Devahastin 2010; Mayachiew and others 2010; Thakhiew and others 2013) have revealed that the drying methods as well as the concentration of an added antimicrobial R  C 2014 Institute of Food Technologists

doi: 10.1111/1750-3841.12488 Further reproduction without permission is prohibited

Antimicrobial chitosan films . . .

Materials and Methods Materials Chitosan powder (degree of deacetylation = 90.2%; molecular weight = 900 kDa) was supplied by S.K. Profishery Co., Ltd. (Samut Sakhon, Thailand). Sodium chloride, ethanol (95% v/v), and glacial acetic acid (98% v/v) were purchased from Merck (Darmstadt, Germany). Analytical-grade glycerol was provided by Carlo Erba (Val de Reuil, France). For antimicrobial tests, buffer peptone water and tryptic soy broth (TSB) were supplied from BactoTM (Becton, Dickinson and Co., Sparks, Md., U.S.A.). Plate count agar (PCA), MullerHinton agar (MHA), and Muller-Hinton broth (MHB) were supplied from DifcoTM (Difco Laboratories, Detroit, Mich., U.S.A.). S. aureus (TISTR 118) was supplied from the TISTR culture collection of Thailand Inst. of Scientific and Technological Research (TISTR, Pathumthani, Thailand).

For gas chromatographic analysis, 1,8-cineole (eucalyptol) was obtained from Fluka-Analytical (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland).

Galangal extract preparation Preparation of the galangal extract was as recommended by Mayachiew and Devahastin (2008a) with some adjustments. Galangal rhizome was dried by hot air at 40 °C and ground. Ten gram of the galangal powder was introduced to an Erlenmeyer flask along with 100 mL of 95% (v/v) ethanol. The flask was placed in an incubator shaker (New Brunswick Scientific, model G24, Edison, N.J., U.S.A.) and shaken at 200 rpm at ambient temperature for 48 h; this procedure is different from that of Mayachiew and Devahastin (2008a) who suggested to leave the extract overnight without shaking. The extract was filtered and then evaporated by a rotary evaporator (Buchi Labortechnik AG, model R-215, Flawil, Switzerland) at 40 °C, 175 mbar until 10% of the initial volume had been reached. The extract was contained in an amber glass bottle and stored at 4 °C. Determination of chemical compositions of galangal extract Determination of the chemical compositions of the extract by gas chromatography-mass spectrometry (GC-MS) was as suggested by Mayachiew and Devahastin (2008a). The extract was diluted 100 folds with 10% (v/v) ethanol. One microliter of the diluted extract was injected into the GC system (Agilent Technologies, model 7890A, Santa Clara, Calif., U.S.A.) and the MS system (Agilent Technologies, model 5975C) via an auto sampler. An HP-5 capillary column (Agilent Technologies, model 19091J-413) was used; temperature was held at 40 °C for 2 min and then raised at a rate of 10 °C/min to 250 °C and held for 5 min. The carrier gas was helium at a flow rate of 0.9 mL/min. The retention time of the chromatogram peaks and their mass spectra were used to recognize the chemical compositions of the extract (against NIST08 libraries). Film-forming solution preparation Preparation of the solution was as recommended by Mayachiew and Devahastin (2008b). Chitosan powder was dissolved in aqueous solution of glacial acetic acid (1% v/v) to reach a final chitosan solution concentration of 1.5% (w/v); stirring was then carried out at 300 rpm at ambient temperature for 24 h. After complete dissolution of chitosan, glycerol at 25% (w/w chitosan) was added; stirring was continued for another hour. A centrifuge (Hitachi, model Himac CR21, Ibaraki, Japan) was used to remove foreign matters at 12400 rpm for 15 min. Galangal extract was incorporated to the solution to a final concentration of either 0% (control) or 1.5% (w/w solution). R A rotor–stator homogenizer (IKA Werke GmbH & Co. KG, R , Staufen, Germany) model T 25 digital ULTRA-TURRAX was used to homogenize the mixture at ambient temperature at 9600 rpm for 4 min. In order to relieve bubbles, the solution was left overnight. Antimicrobial chitosan films with the final thickness of 15 and 30 μm were cast by pouring 16 and 32 g of the film-forming solution onto an 13×10 cm acrylic casting plate, respectively. From our preliminary experiments, the films could not inhibit the growth of S. aureus on cantaloupe when the galangal extract concentration was below 1.5% (w/w solution). Therefore, in this study, the extract concentration of 1.5% (w/w solution) was chosen. Vol. 79, Nr. 6, 2014 r Journal of Food Science E1151

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agent have significant effects on the film structure, especially in terms of swelling, which, in turn, affects the release characteristics of the added agent. The goals of this study were to examine the combined effects of the drying methods, added antimicrobial agent concentration and film thickness on the retention and release of the antimicrobial agent as well as the efficacy of antimicrobial chitosan films as applied on the surfaces of cantaloupe during storage at 25 °C for 5 d. This storage condition was chosen to accelerate and amplify the changes that may take place at a typical lower storage temperature. Galangal extract was chosen as antimicrobial agent as it has proved to inhibit S. aureus, which was selected as the test pathogenic microorganism; galangal is also readily and inexpensively available in Thailand (Mayachiew and Devahastin 2008a). Low-pressure superheated steam drying (LPSSD) at 70 °C, 10 kPa, and hot air drying at 40 °C were used to prepare the films; the film thickness was fixed at 15 and 30 μm. Concentration of 1,8-cineole, which is a major and active component of the galangal extract, was monitored. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the galangal extract on S. aureus were evaluated. The degree of film swelling was also measured and the results used to interpret the release characteristics of 1,8-cineole from the films to cantaloupe. The hypothesis of the present study was that an increase in the film thickness and the use of a suitable drying method could minimize the loss of added antimicrobial agent and at the same time modify the structure and hence the release characteristics of chitosan films. The present work is quite different from our previous study (Mayachiew and others 2010), for in this study the test material was a real fruit, while in the case of Mayachiew and others (2010) distilled water was used as a food simulant. Since the antimicrobial chitosan films behave quite differently when in contact with different test materials, their release characteristics and hence antimicrobial efficacy are quite different. In addition, the concentration evolution of 1,8-cineole in distilled water was not reported and used to explain the antimicrobial efficacy of the films in the work of Mayachiew and others (2010). Since the drying methods are expectedly affect the properties and the release characteristics of the films, without such a concentration evolution, no clear conclusion on the influence of the drying methods on the release characteristics and antimicrobial efficacy of the films could be established.

Antimicrobial chitosan films . . . Antimicrobial films preparation Hot air drying (HD). A hot air tray dryer was used to dry the film-forming solution at 40 °C (Mayachiew and Devahastin 2008b). LPSSD. Low-pressure superheated steam dryer proposed by Devahastin and others (2004) was used to dry the film-forming solution. Drying at 70 °C and 10 kPa was selected based on the suggestion of Mayachiew and Devahastin (2008b) and Mayachiew and others (2010) as the condition that produced the films with the highest inhibition efficacy against S. aureus and with the best mechanical properties when compared with the LPSSD-dried films prepared at 80 and 90 °C at the same extract concentrations.

E: Food Engineering & Physical Properties

Film thickness determination A micrometer with an accuracy of ±2 μm (Mitutoyo, model 102-309, Tokyo, Japan) was used to measure the film thickness at 5 fixed positions. Antimicrobial efficacy evaluation A film sample (moisture content = 14% d.b.) was equilibrated at 75% RH and ambient temperature for 48 h prior to further characterization (Mayachiew and Devahastin 2008b). The sample was kept in a desiccator containing saturated solution of sodium chloride, which mimicked the typical environment of Thailand. Microorganism and culture method. Stock culture of S. aureus was maintained on MHA slopes at 4 °C and subcultured every month to refresh the organism. Before bacterial inoculation, 1 loopful of the stock culture was transferred to 100 mL of TSB in a sterile Erlenmeyer flask, which was then incubated in the incubator shaker with continuous shaking at 200 rpm for 18 h at 37 °C in order to obtain the bacteria cells in their stationary phase (cell concentration of 109 CFU/mL). Antimicrobial activity of galangal extract on S. aureus. Initial evaluation of the antimicrobial activity of the galangal extract on S. aureus was conducted using an agar diffusion method (Mayachiew and Devahastin 2008a). One hundred microliter of the inoculum that was diluted to contain S. aureus of approximately 106 CFU/mL was spread on the surface of MHA in a petri dish. A sterile paper disc was cut into 5.5 mm in diameter and placed on the MHA that had previously been spread. Twenty microliter of the extract was then impregnated on the paper disc; the control plates were impregnated with 5% (v/v) and 95% (v/v) ethanol. The dishes were incubated at 37 °C for 24 h. Microbial inhibition was determined by measuring the total diameter of an inhibition zone (clear zone), which included the paper disc. The antimicrobial activity of the extract was evaluated in triplicate. A broth macrodilution method as suggested by Andrews (2001) and Stagos and others (2012) was employed to determine the MIC and MBC of the galangal extract on S. aureus. Serial dilutions of the extract to the concentrations of 87.5, 175, 350, 700, 1400, 2800, 5600, 11200, and 22400 ppm were prepared with 5% (v/v) ethanol. The S. aureus inoculum was diluted in MHB to obtain the bacterial culture at a concentration of approximately 105 CFU/mL. One milliliter of the broth medium was transferred into each tube; 1 mL of each serial dilution of the extract was then added. The tubes containing only 5% (v/v) ethanol and the broth medium without the extract were used as controls. The tubes were incubated at 37 °C for 24 h. A spectrophotometer (Thermo Fisher Scientific, model GENESYSTM 20, Waltham, Mass., U.S.A.) was used to evaluate the bacterial growth by measuring the absorbance of a sample at 630 nm (Stagos and others 2012). E1152 Journal of Food Science r Vol. 79, Nr. 6, 2014

MIC was determined as the lowest extract concentration, which did not permit any noticeable growth (turbidity of the solution of less than 0.05 optical density at 630 nm; Stagos and others 2012). MBC was, on the other hand, evaluated by spreading 100 μL of the culture medium from each tube that had no visible growth on MHA. The plate was incubated at 37 °C for 24 h. MBC was noted as the lowest concentration of the extract that resulted in no growth on the MHA. MIC and MBC of the galangal extract were evaluated in triplicate.

Determination of antimicrobial efficacy of films as applied on fresh-cut cantaloupe Preparation of antimicrobial films. An antimicrobial chitosan film was cut into the size of 3 × 3 cm and steriled by UV radiation in a biohazard safety cabinet class II (Microtech, Bangkok, Thailand) for 15 min according to the method of Thangvaravut and others (2012). Preparation of cantaloupe. Cantaloupe cv. Sunlady (Cucumis melo L. cv. Sunlady) with the mass between 1300 and 1600 g was purchased from a local supermarket. The total soluble solids of the cantaloupe were measured to ensure the same maturity; total soluble solids were in the range of 8.0 to 9.0 o Brix. A whole cantaloupe fruit was washed with tap water. The rind and seed were removed. The flesh was cut into the size of 2.5 × 2.5 × 0.5 cm. In order to remove background microorganisms that might naturally contaminate the flesh, a sample was immersed in 70% (v/v) ethanol at the ratio of 1 : 10 for 30 s and then twice rinsed with sterile-distilled water. The cantaloupe specimen was ambient dried in the biohazard safety cabinet for 20 min prior to bacterial inoculation (Thangvaravut and others 2012). S. aureus inoculation S. aureus inoculation on the cantaloupe surface was performed as suggested by Thangvaravut and others (2012). S. aureus inoculum was diluted to obtain the bacterial culture at a concentration of approximately 106 CFU/mL. Twenty microliter of the diluted inoculum was spread onto the cantaloupe surface and ambient dried in the biohazard safety cabinet for 20 min. A sterile antimicrobial film was placed onto the cantaloupe surface, while an aluminum foil was placed on the edges of the sample. The cantaloupe sample with the antimicrobial film was stored in a sterile petri dish, which was then wrapped with a parafilm to avoid rapid evaporation of the antimicrobial agent to the environment. Commercial plastic stretch film (M wrapTM , MMP Corp. Ltd., Bangkok, Thailand) was used as a control. The samples were stored at 25 °C for 5 d. Enumeration of S. aureus survivors and total aerobic bacteria was performed on a daily basis. The initial S. aureus load was selected based on the guidelines for microbiological quality of ready-to-eat foods sampled at the point of sale that was performed by a working group of the PHLS Advisory Committee for Food and Dairy Products in the United Kingdom (Gilbert and others 2000). In the case of S. aureus, a ready-to-eat food sample having the bacterial count of less than 20 CFU/g would be considered as satisfactory quality, 20 to 100 CFU/g as acceptable quality, 100 to 104 CFU/g as unsatisfactory quality, and ࣙ104 CFU/g as unacceptable/potentially hazardous quality. In this study, the initial bacteria number of ࣙ104 CFU/g was used to represent the worst case scenario that may probably take place in a market. Enumeration of S. aureus and total aerobic bacteria survivors. The numbers of S. aureus and total aerobic bacteria survivors were enumerated according to the method of Thangvaravut

and others (2012). A cantaloupe sample was homogenized with 8 mL of 0.1% peptone water in a stomacher (Interscience, Model 400VW, St. Nom, France) for 4 min at the highest speed. One hundred microliter aliquot was spread on MHA for S. aureus and on PCA for total aerobic bacteria counts. The number of S. aureus and total aerobic bacteria survivors were determined by the colony count after an incubation period of 24 h at 37 °C.

Results and Discussion

Determination of 1,8-cineole concentration in films and fresh-cut cantaloupe The 1,8-cineole concentration was evaluated following the method of Sun and others (2011) and Thangvaravut and others (2012). A film sample was homogenized with 10 mL of 10% (v/v) ethanol in the stomacher for 4 min at the highest speed and held at ambient temperature for 2 h. One mL of aliquot, 4 mL of distilled water, and 1.5 g of sodium chloride was submitted to a 20-mL glass vial. A magnetic bar was put into the vial before sealing with a screw cap; the content was stirred at 40 °C for 15 min. In order to adsorb the headspace 1,8-cineole, solid-phase microextraction (SPME) fiber coated with divinylbenzene/carboxen/polydimethylsiloxane (Supelco, Bellefonte, Pa., U.S.A.) was applied to the headspace for 20 min at 40 °C. The fiber was afterward desorbed for 15 min in the GC (Hewlett Packard, model 5890 Series II Plus, Palo Alto, Calif., U.S.A) with an Inert Cap Wax polyethylene glycol capillary column (GL Science, Tokyo). The initial oven temperature was held at 60 °C for 8 min, raised at a rate of 3 °C/min to 100 °C, and held at the final temperature for 5 min. Helium was used as the carrier gas. The temperatures of the detector and injector were 250 and 240 °C, respectively (Thakhiew and others 2013). The 1,8-cineole concentration in a cantaloupe sample was evaluated by the same method as that in a film sample.

Antimicrobial efficacy and release characteristics of films Changes in the number of S. aureus survivors on the cantaloupe surfaces applied with antimicrobial chitosan films during storage at 25 °C for 5 d are shown in Figure 2. The initial number of S. aureus in all cases was approximately 104 CFU/mL. In the case of commercial stretch film (control film), the number of S. aureus survivors continuously increased and reached the highest values after 3 d. This is expected because the film is passive and contained no antimicrobial agent. In the case of chitosan films without the extract, the number of S. aureus survivors was lower than that in the case of the control film; even chitosan films without the galangal extract exhibited some antimicrobial activity against S. aureus. At the same film thickness, the drying methods did not significantly influence the antimicrobial efficacy of the films. On the contrary, the thickness of both the films prepared by HD and LPSSD had a significant effect on the antimicrobial efficacy; the number of S. aureus survivors decreased with an increase in the film thickness. Since chitosan is known to have antimicrobial characteristics, the film with a larger amount of chitosan expectedly led to better microbial inactivation (Fernandez-Saiz and others 2008). The surface positive charges on the C-2 (amino group) are believed to interact with the negative charges on bacterial cell membranes, resulting in the leakage of proteinaceous and other intracellular constituents of the cells and hence their death (No and others 2007; Sangsuwan and others 2009; Wang and others 2011; Pinheiro and others 2013). The evolution of the 1,8-cineole concentration in the films incorporated with 1.5% (w/w) extract is shown in Figure 3(a). At the same thickness, the films prepared by HD had lower concentration of 1,8-cineole than the films prepared by LPSSD all along the storage period. This is because larger extent of evaporation of the antimicrobial agent might have occurred during the longer period of hot air drying (Figiel and others 2010; Kurek and others 2012). The concentrations of 1,8-cineole in both the films prepared by HD and LPSSD increased with an increase in the film thickness. This is probably because the larger amount of chitosan

Identification of chemical compositions of galangal extract GC-MS data of major compounds in the galangal extract are shown in Figure 1. 1,8-Cineole (22.58%) and β-bisabolene (23.96%) were the predominant compounds and accounted for 46.54% of the total composition of the galangal extract. Farnesene (14.22%), β-selinene (13.60%), pentadecane (12.35), β−caryophyllene (8.76%), and α-bisabolene (4.53%) were the Film swelling determination minor compounds of the extract. This result agreed well with the Determination of film swelling was as recommended by result of Mayachiew and Devahastin (2008a) who reported that Mayachiew and others (2010). A vacuum oven (Sanyo, model the major component of galangal extract is 1,8-cineole. Gallenkamp/OM-09980, Loughborough, U.K.) was used to dry a 2 × 2 cm film sample until its mass was constant. The dehyAntimicrobial activity of galangal extract against S. aureus drated film was weighed and soaked in distilled water for 24 h. Agar diffusion method was used for rapid assessment of the anAfter equilibrium had been reached, the water on the saturated timicrobial activity of the galangal extract against S. aureus. Inhifilm surface was immediately removed by blotting with tissue pabition zones of 5% (v/v) ethanol, 95% (v/v) ethanol, and galangal per. The film was again weighed. The degree of swelling was then extract are shown in Table 1. Galangal extract (35.7±2.1 mm) and calculated as: 95% (v/v) ethanol (23.7±1.5 mm) were noted to exhibit strong activity against S. aureus as their inhibition zones were larger than Ms − Md Degree of swelling = × 100 (1) 20 mm (Jord´an and others 2013). Md MIC and MBC of the extract against S. aureus were 700 and where Ms is the mass of the film at equilibrium (g) and Md is the 2800 ppm, respectively. The MBC/MIC ratio was equal to 4; S. aureus is therefore susceptible to the galangal extract (Canillac mass of the dehydrated film (g). and Mourey 2001).

Statistical analysis Individual main effect among the film thickness or extract concentration or drying methods as well as the combined effects of all parameters on the numbers of S. aureus and total aerobic bacteria survivors, the 1,8-cineole concentrations in the films and in cantaloupe as well as the degree of film swelling were assessed by 3-way multivariate analysis of variance (MANOVA) (Thakhiew and others 2013). All experiments were conducted in triplicate. R (version 17; SPSS Inc., Chicago, Ill., U.S.A.) was applied SPSS to analyze the data at 95% confidence level.

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Antimicrobial chitosan films . . .

Antimicrobial chitosan films . . . Table 1–Antimicrobial activity (in terms of diameter of inhibition zone) of different substances.

E: Food Engineering & Physical Properties helped entrap more extract within its matrix; thicker layer of the films might also help reduce the loss of the volatile compound during drying (S´anchez-Gonz´alez and others 2011). In the case of chitosan films with the thickness of 15 μm and 1.5% (w/w) galangal extract, it is seen from Figure 2 that the dry-

ing methods significantly affected the antimicrobial efficacy of the films. The MIC and MBC of the galangal extract against S. aureus were 700 and 2800 ppm (as mentioned earlier in “Antimicrobial activity of galangal extract against S. aureus” section), which were, respectively, equivalent to the concentrations of 1,8-cineole

Figure 1–Chemical compositions of galangal extract: 1,8-cineole (RT = 7.46; 22.58%) (1); β-caryophyllene (RT = 12.85; 8.76%) (2); farnesene (RT = 13.09; 14.22%) (3); α-bisabolene (RT = 13.42; 4.53%) (4); β-selinene (RT = 13.84; 13.60%) (5); pentadecane (RT = 13.98; 12.35%) (6); β-bisabolene (RT = 14.10; 23.96%) (7).

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of 118.55 and 492.2 ppm as assessed by the GC. The MIC of 1,8-cineole for a dried film was then obtained by dividing the mass of 1,8-cineole in the film-forming solution by the mass of the dried film. The MIC of 1,8-cineole for the cantaloupe was obtained by dividing the mass of 1,8-cineole in the dried film by the mass of the cantaloupe. Noted that the MIC of 1,8-cineole against S. aureus was 47.4 ppm (per g cantaloupe) or 6322.7 ppm (per g dried film) and the MBC of 1,8-cineole against S. aureus was 196.9 ppm (per g cantaloupe) or 26250.7 ppm (per g dried film). The MIC and MBC results will be later used to describe the relationship between the 1,8-cineole concentration in the cantaloupe and the numbers of S. aureus and total aerobic bacteria survivors on the cantaloupe surfaces. Based on MIC consideration, the films prepared by LPSSD led expectedly to the lower number of S. aureus survivors than the films prepared by HD; the air-dried films could not reduce the number of S. aureus throughout the storage period since the 1,8-cineole concentration in the cantaloupe was lower than the MIC for all over the whole storage period (see Figure 3b). The films prepared by LPSSD could, on the other hand, reduce the number of S. aureus by 1.3 to 2.3 log cycles since

the 1,8-cineole concentration in the cantaloupe was higher than the MIC even for a rather short period of time. Monoterpenes such as 1,8-cineole are highly hydrophobic substances; this allows them to interact well with the lipids and penetrate through the lipid structure of the bacteria cell walls, leading to protein denaturation and coagulation and aggregation of the cell membranes. These, in turn, lead to changes of the cell membrane functions. The fluidity and permeability of the cell membrane would increase, resulting in the leakage of ions and other intracellular components as well as infestation of the conformation of membrane-embedded proteins, leading to cell death (Oonmetta-aree and others 2006; Bajpai and others 2012; Hamoud and others 2012). When the film thickness increased to 30 μm, the films prepared by LPSSD led to dramatic decrease in the bacterial cell survivors when compared with the films prepared by HD. Only 0.2 to 0.7 log cycle could be reduced by the films prepared by HD. The higher degree of swelling of the films prepared by HD probably led to more rapid release and loss of the antimicrobial agent from the films to the environment; the films could not maintain adequate concentration of the antimicrobial agent to inactivate

10

Figure 2–Number of S. aureus survivors on cantaloupe wrapped with films enriched with 0% and 1.5% (w/w) galangal extract during storage at 25 °C for 5 d. Same letters mean that the values are not significantly different at 95% confidence level among various groups of combined effects.

3.2c 3.2c

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Number of cells (log CFU/mL)

9 8

day 0 day 1 day 2 day 3 day 4 day 5

3 2 1 0 plastic

HD 0%

LPSSD 0%

HD 1.5%

LPSSD 1.5%

Film type (thickness 15 µm) 10

8 7

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4.5ghijjk 4.8 5.2lm 5.3mn 5.6mn 5.5mn 4.4ghi 4.9kl 5.7n 5.3mn 5.6m 5.4lmn 4.5ghij d 3.8 4.3fgh 3.9defde 3.9 3.7d 4.6ghij

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Number of cells (log CFU/mL)

9

0 HD 0%

LPSSD 0%

HD 1.5%

LPSSD 1.5%

Film type (thickness 30 µm) Vol. 79, Nr. 6, 2014 r Journal of Food Science E1155

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Antimicrobial chitosan films . . .

Antimicrobial chitosan films . . .

1,8-Cineole concentration (ppm, per g dried film)

1,8-Cineole concentration (ppm, per g cantaloupe)

E: Food Engineering & Physical Properties

microorganism. On the other hand, the films prepared by LPSSD could almost completely eradicate S. aureus by reducing its number by 4.6 log cycles throughout the storage period. This superior performance is due to the fact that the films prepared by LPSSD had larger amount of the extract in their matrix (see Figure 3a). Moreover, thermal crosslinkage that occurred in LPSSD-dried films (Thakhiew and others 2013) might have allowed the films to slowly and continuously release the antimicrobial agent to the cantaloupe surfaces. The synergistic effect of the larger amount of the extract and appropriate release rate of the LPSSD-dried films thus led to the higher microbial cell reduction. The antimicrobial efficacy matched well with the 1,8-cineole concentration in both the films and cantaloupe. As can be seen in Figure 3(a) and 3(b), the higher 1,8-cineole concentration in the dried films led to more release and more accumulation of 1,8-cineole in the cantaloupe. The film prepared by LPSSD with the thickness of 30 μm had the highest concentration of 1,8-cineole and led to the maintenance of 1,8-cineole

70000

concentration in the cantaloupe at above the MIC throughout the storage period. Although the maximum concentration of 1,8cineole in the cantaloupe applied with the film prepared by LPSSD of 30 μm thickness was lower than the MBC, this film could almost completely eradicate S. aureus. This is probably because 1,8cineole is not the only active component in the galangal extract; the antimicrobial activity of the extract might be ascribed to the effect of 1,8-cineole along with other minor compounds such as β-bisabolene and farnesene, which are sesquiterpenes that could probably inactivate S. aureus by the mechanisms similar to those of other terpenes (Oonmetta-aree and others 2006). The number of total aerobic bacteria survivors on the cantaloupe surfaces wrapped with antimicrobial chitosan films enriched with 0% and 1.5% (w/w) galangal extract is presented in Figure 4. The number of total aerobic bacteria survivors exhibited a similar trend (and values) to that of S. aureus survivors. Therefore, the total aerobic bacteria survivors were most probably only the inoculated S. aureus.

a

HD 15 µm HD 30 µm LPSSD 15 µm LPSSD 30 µm

60000 50000 40000 30000 20000 10000

MIC

0 0

180

20

40

60 Time (h)

80

b

160

100

120

HD 15 µm HD 30 µm LPSSD 15 µm LPSSD 30 µm

140 120 100 80 60

MIC

40 20 0 0

20

40

60

80

100

120

Time (h) Figure 3–1,8-cineole concentration in (a) films enriched with 1.5% (w/w) galangal extract and (b) cantaloupe during storage at 25 °C for 5 d.

E1156 Journal of Food Science r Vol. 79, Nr. 6, 2014

day 0 day 1 day 2 day 3 day 4 day 5

4 3

Figure 4–Number of total aerobic bacteria survivors on cantaloupe wrapped with films enriched with 0% and 1.5% (w/w) galangal extract during storage at 25 °C for 5 d. Same letters mean that the values are not significantly different at 95% confidence level among various groups of combined effects.

4.5ghi 2.2b 2.3b b 2.5 3.3c 3.5cd

4.5fghi 4.4fg ijk 4.9 5.2kl 5.0jk 4.9hijk

5

4.5fghi

6

4.4fghi

7

6.5n

7.3pq

8

4.8ghijk

Number of cells (log CFU/mL)

9

8.4rs 8.0r 7.4q 7.3pq

8.6s 8.6s 8.3rs 8.2rs

10

with an increase in the film thickness. This is probably because the larger amount of chitosan could entrap more water within its body. When incorporating 1.5% (w/w) extract in the films, the number of chemical crosslinkage interactions between the extract and chitosan matrix increased, resulting in reduced degree of swelling in all cases (Thakhiew and others 2013). The swelling of the films prepared by HD and LPSSD increased and decreased with an increase in the film thickness, respectively. This might be because the films prepared by LPSSD had higher number of crosslinkage interactions from the synergistic effect of thermal and chemical crosslinkage. At the same extract concentration, the degrees of swelling of the films prepared by HD were higher than those of the films prepared by LPSSD. The increased degree of film swelling resulted in the increased release of the extract. The higher degree of swelling of the films prepared by HD led to more rapid but short-term release of the extract during the storage period, contributing to the lower inhibitory effect against microbial growth in comparison with the films prepared by LPSSD.

6.6no 6.7no 7.1pq 7.2pq 6.9pq

Swelling of films Previous studies (Mayachiew and Devahastin 2010; Mayachiew and others 2010; Thakhiew and others 2013) have revealed that the drying methods as well as extract concentration significantly affect the film structure, especially the film swelling. Different degrees of swelling, in turn, lead to different release characteristics and hence different antimicrobial efficacy of the films (Mayachiew and Devahastin 2010; Mayachiew and others 2010). It is therefore interesting to evaluate swelling in conjunction with the antimicrobial efficacy of the films. Based on MANOVA the combined effects of the drying methods, extract concentration and film thickness had an effect on the film swelling. The degrees of swelling are listed in Table 2. In the case of chitosan films without the extract, at the same film thickness, the degrees of swelling of the films prepared by LPSSD were lower than those of the films prepared by HD. This is due to the use of a higher temperature during LPSSD, which might probably induce more extensive thermal crosslinkage interactions in the film structure (Thakhiew and others 2010). The degrees of swelling of both the films prepared by HD and LPSSD increased

2 1 0 plastic

HD 0%

LPSSD 0%

HD 1.5%

LPSSD 1.5%

Film type (thickness 15 µm)

10

day 0 day 1 day 2 day 3 day 4 day 5

7

6 5 4

4.0ef 3.6cd 3.8de 4.0ef 3.8de 3.3c 4.5fghi

8

4.6ghij 4.9kl 5.7m 5.9m 5.8lm 5.4lm 4.4fgh 5.1k 5.9m 5.6lm 5.2klkl 5.2

3 2 1

0.0a 0.0a 0.0a 0.0a 0.0a

Number of cells (log CFU/mL)

9

0 HD 0%

LPSSD 0%

HD 1.5%

LPSSD 1.5%

Film type (thickness 30 µm) Vol. 79, Nr. 6, 2014 r Journal of Food Science E1157

E: Food Engineering & Physical Properties

Antimicrobial chitosan films . . .

Antimicrobial chitosan films . . . Table 2–Degree of swelling of films (thickness of 15 and 30 μm) enriched with 0% and 1.5% (w/w) galangal extract. Degree of Swelling (%) Drying Method

Galangal extract concentration (% w/w)

15 μm

30 μm

0% 1.5% 0% 1.5%

117.4 ± 9.4E 88.1 ± 4.8C 106.7 ± 10.5D 57.5 ± 4.7B

221.9 ± 20.7H 128.0 ± 8.2F 141.7 ± 8.7G 37.9 ± 3.3A

Hot air drying at 40 °C LPSSDat 70 °C, 10 kPa

Same letters mean that the values are not significantly different at 95% confidence level among various groups of combined effects.

Conclusions

E: Food Engineering & Physical Properties

The combined effects of the drying methods, galangal extract concentration, and film thickness on the retention and release characteristics of the extract as well as the efficacy of antimicrobial chitosan films as applied on the surfaces of cantaloupe during storage at 25 °C for 5 d were investigated. It was found that the rate of release of the extract increased with an increase in the film swelling. The lower swelling of the films prepared by LPSSD led to slower and more continuous release, resulting in the maintenance of adequate concentration of the extract on the cantaloupe surfaces and contributing to the higher inhibitory effect against microbial growth than the films prepared by HD. At the same film thickness, the films prepared by HD had lower extract retention. If considered the films produced by the same drying method, the extract retention of the films increased with an increase in the film thickness, resulting in increased antimicrobial efficacy. Although the films with the thickness of 30 μm exhibited strong potential to inactivate S. aureus, these films are not too appropriate to be used in practice. This is because the films may excessively adsorb water from food, swell, and become bulky. A better alternative to retain and release an antimicrobial agent such as the use of a double-layer composite film structure should be investigated.

Acknowledgments The authors are thankful for the financial support provided by the Natl. Science and Technology Development Agency (NSTDA). Author Thakhiew thanks the Commission on Higher Education for a grant fund under the Strategic Scholarships for Frontier Research Network Program that was used to support her doctoral study.

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Combined effects of drying methods, extract concentration, and film thickness on efficacy of antimicrobial chitosan films.

An idea of using a suitable drying method to minimize the loss of added antimicrobial agent and, at the same time, to modify the structure, and hence ...
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