Preservative effectiveness of essential oils in vapor phase combined with modified atmosphere packaging against spoilage bacteria on fresh cabbage

Food Control 51 (2015) 307e313

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Preservative effectiveness of essential oils in vapor phase combined with modified atmosphere packaging against spoilage bacteria on fresh cabbage Jeong-Eun Hyun, Young-Min Bae, Jae-Hyun Yoon, Sun-Young Lee* Department of Food Science and Technology, Chung-Ang University, 72-1 Nae-ri, Daedeok-myeon, Anseong-si, Gyeonggi-do, 456-756, South Korea

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2014 Received in revised form 14 November 2014 Accepted 23 November 2014 Available online 28 November 2014

This study was conducted to investigate the antibacterial effects of various essential oils (EOs) against pathogens using the disc volatilization method. Also, combined effects of EOs in vapor phase and MAP were evaluated for reducing levels of total mesophilic microorganisms on fresh cabbage. The vapor phase activities of EOs (thyme-1, oregano-1, lemongrass-1, and lemongrass-2 oils) observed strong inhibitory effects. The MAP results showed that 100% CO2 gas packaging reduced significantly levels of total mesophilic microorganisms on cabbage and radish sprouts, and their reduction level was 1.55 and 2.26 log10 CFU/g compared to control after 21 days of storage (p
0.05). Based on previous results, combined effects of EOs in vapor phase and MAP (100% CO2) showed that lemongrass-2 oil with 20 discs showed complete inactivation by <1.0 log10 CFU/g after 14 days of storage. These results could provide useful information for developing alternative preservation method to improve the freshness and shelflife of fresh produce using natural antimicrobials. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Antibacterial effect Essential oils Vapor phase Modified atmosphere packaging Combination Fresh produce

1. Introduction Consumers have an increased interest in fresh and health foods such as fruits and vegetables. However, fresh-cut fruits and vegetables deteriorate quickly and have limited shelf-life (Ghidelli, Mateos,
rez-Gago, 2014). Major problems associated with Rojas-Argude, & Pe fresh-cut fruits and vegetables during storage including tissue softening, discoloration, off-flavors, and off-odors are occurred because
pez-Ga
lvez, of microbial growth in the product (Gram et al., 2002; Lo Peiser, Nie, & Cantwell, 1997). Also, foodborne outbreaks associated with consumption of raw vegetables have been increased (Santos et al., 2012). A number of studies have investigated foodborne pathogens isolated from raw vegetable (Beuchat, 1996; Nguyen-the & Carlin, 1994), including Listeria monocytogenes (Schlech III et al., 1983), Salmonella sp. (Doyle, 1990), and Escherichia coli (Nguyenthe & Carlin, 1994). Raw fresh produce could be contaminated with pathogens during harvesting through fecal material (manure, both of human and animal origin), human handling, washing procedure, processing equipment, transportation, and distribution (Beuchat, 1996; Johannessen, Loncarevic, & Kruse, 2002). * Corresponding author. Tel.: þ82 31 670 4587; fax: þ82 31 676 8741. E-mail addresses: [email protected], [email protected] (S.-Y. Lee). http://dx.doi.org/10.1016/j.foodcont.2014.11.030 0956-7135/© 2014 Elsevier Ltd. All rights reserved.

Many studies deal with washing chemical agents such as chlorine to inactivate pathogens on fruits and vegetables. Chemical agents are also used to extend shelf-life for minimally processed foods (Rodgers, Cash, Siddiq, & Ryser, 2004). Chlorine is widely used as a sanitizer for washing fruits, vegetables, and fresh-cut produce. It is commonly used at concentrations of 50e200 mg/L (Beuchat, 2008; Beuchat, Nail, Adler, & Clavero, 1998). However, it has limited effect in reduce level of microorganisms on fruits and vegetables (Beuchat, 1996). Moreover, chlorine may react with organic matter to form carcinogenic products in water (Parish et al., 2003). The inadequacy of chlorine as a sanitizer has stimulated interest in finding safer, more effective sanitizers (Ruiz-Cruz, Luo,
lez-Aguilar, 2006). Gonzalez, Tao, & Gonza Recently, there has been an increasing consumer demands to replace chemically synthesized antimicrobial with natural antimicrobials for food preservation, such as essential oils (EOs) (Xu et al., 2007). EOs are considered generally regarded as safe (GRAS) and aromatic oily liquids extracted from various plant material as flowers, fruits, herbs, leaves, roots, seeds, and stem (Burt, 2004). EOs and some of their major components have shown antibacterial effects against pathogens and spoilage bacteria (Bakkali, Averbeck, Averbeck, & Idaomar, 2008; Burt, 2004; Holley & Patel, 2005). Several studies showed that EOs from spices and plants

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significantly decreased the levels of foodborne pathogens and food spoilage bacteria, especially E. coli O157:H7, Shigella dysenteria, Bacillus cereus, Staphylococcus aureus, L. monocytogenes, Clostridium botulinum, Enterococcus faecalis, Staphylococcus spp., Bacillus spp., enterobacteria, Vibrio parahaemolyticus, and Pseudomonas fluorescens (Burt, 2004; Ceylan & Fung, 2004; Pandit & Shelef, 1994; Ultee, Kets, & Smid, 1999). The antimicrobial activity of EOs in vapor phase has been demonstrated using direct contact methods (Delaquis, Ward, Holley, Cliff, & Mazza, 1999; Fisher & Phillips,
pez, Sa
nchez, 2006; Inouye, Takizawa, & Yamaguchi, 2001; Lo Batlle, & Nerín, 2005; Suhr & Nielsen, 2003; Weissinger, McWatters, & Beuchat, 2001). However, the direct contact methods on food have been limited due to high hydrophobicity and volatility of the EOs (Nedorostova, Kloucek, Kokoska, Stolcova, & Pulkrabek, 2009). Modified atmosphere packaging (MAP) is one of the most important preservation techniques used to maintain the quality and extend the shelf-life of foods (Jayas & Jeyamkondan, 2002;
rez-Muntoz, & Chaparro, 2002; Martínez-Ferrer, Harper, Pe Phillips, 1996). This technique replaces the air that surrounds the food in the package with a specific gas mixture, most commonly CO2, O2, and N2 (Rao & Sachindra, 2002; Smigic et al., 2009). It can extend the shelf-life of perishable products such as meat, poultry, fish, fruits, and vegetables (Sandhya, 2010). The potential of MAP to prolong the shelf-life for many foods has been researched (Brecht, Chau, Fonseca, & Oliveira, 2001; Jacxsens, Devlieghere, & Debevere, 2001; Saltveit, 2003). Low levels of O2 and high levels of CO2 reduce the respiration rate, which in turn delays senescence, thus extending the storage time of the fresh produce (Saltveit, 1993). However, there are very limited studies that investigated the antibacterial efficacy of EOs in fresh produce. The use of EOs as a food preservative may be limited, since the required high concentrations of EOs for food preservation cause changes in the organoleptic properties of food (Devlieghere, Vermeulen, & Debevere, 2004). Accordingly, low concentrations of EOs in vapor phase combined with physical or chemical treatments have been yet proposed. Developing an effective sanitization using natural antimicrobials against food spoilage microorganisms could result in an alternative preservation method to improve the freshness and shelf-life of fresh produce. Therefore, in this study we investigated the antibacterial effects of various EOs against pathogens and fresh produce spoilage bacteria using the disc volatilization method and MAP. We also combined effects of EO in vapor phase and MAP was evaluated for reducing the levels of total mesophilic microorganisms on fresh cabbage. 2. Materials and methods 2.1. Antibacterial effect of the EO treatments 2.1.1. Bacterial strains and growth conditions Five strains of pathogens (E. coli O157:H7 ATCC 43895, Salmonella Typhimurium ATCC 19585, L. monocytogenes ATCC 19115, S. aureus ATCC 4012, and B. cereus ATCC 10876) were obtained from the bacterial culture collection of Chung-Ang University (Anseongsi, Korea) and used in this study. All strains were maintained at 80  C in 20% glycerol and were activated by cultivation in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI, USA, pH 7.3) for 24 h at 37  C before use. 2.1.2. Preparation of EOs EOs used in this study were purchased from Aromasavor (Seoul, Korea) and Whatsoap (Gwangju-si, Korea). In all cases, EOs were of the highest grade available (99e100% pure). The EOs was chosen on

the basis of their potential in preservation of food produce against pathogens. The main components of the tested EOs are presented in Table 1. 2.1.3. Disc volatilization method
pez et al., 2005) was used to The disc volatilization method (Lo determine the diameter of the inhibition zone by various EOs against the 5 pathogens (E. coli O157:H7 ATCC 43895, S. Typhimurium ATCC 19585, L. monocytogenes ATCC 19115, S. aureus ATCC 4012, and B. cereus ATCC 10876). Tryptic soy agar (TSA; Difco Laboratories, Detroit, MI, USA) was autoclaved at 121  C for 15 min. Sterile medium (20 mL) was poured into a petri dish (90 mm diameter), and the plates were dried for 20 min in a safety cabinet. Bacterial cultures (0.1 mL) were inoculated onto the TSA and dried for 10 min in a safety cabinet. A sterile paper disc was laid on the inside surface of the upper lid, and 10 ml each of 100% EOs was pipetted onto the disc. The plates inoculated with pathogens were immediately inverted on top of the lid and sealed with parafilm to prevent leakage of EO vapor. The plates were incubated at 37  C for 24 h. Antibacterial effect was evaluated by measuring diameters of the inhibition zones (mm) against tested pathogens. 2.2. Application of EOs on fresh produce 2.2.1. Modified atmosphere packaging (MAP) Fresh cabbage and radish sprouts were purchased from a local supermarket (Anseong-si, Korea) and stored at 4 ± 2  C before use. Fresh produce was washed with running tap water and dried for 20 min in a laminar flow biosafey hood. Samples (25 g) were placed in UV-sterilized plastic bags (NY bag, thickness 80 mm, polyethylene terephralate/aluminum/linear low-density polyethylene (PET/AL/L-LDPE), and O2 permeability 23 cm2/ m2 day atm at 23  C; Gasung Pak Co., Gwangju-si, South Korea) and packed under 6 different atmosphere conditions: five different passive modified atmosphere (0% CO2 and 100% N2, 25% CO2 and 75% N2, 50% CO2 and 50% N2, 75% CO2 and 25% N2, 100% CO2 and 0% N2) and air packaging. Air packaging consisted of sealing without eliminating air in the bag with a vacuum packaging machine (AVS 200, CSE Any Vac, CSE Co.). The MAP gas mixture was prepared using a gas mixer (MAP Mix 9001 ME gas mixer, PBI Dansensor, Ringsted, Denmark). Bags were heat-sealed using a vacuum sealer (AZ-450E, Airzero, Ansansi, South Korea) connected to the gas mixer. Packed fresh produce were stored at 4 ± 2  C for 0, 7, 14, and 21 days. 2.2.2. Disc volatilization method combined with MAP From results of earlier part of this study, 4 EOs (thyme-1, oregano-1, lemongrass-1, and lemongrass-2) were chosen to combine with MAP on cabbage. Fresh cabbage was purchased from a local supermarket (Anseong-si, Korea) and stored at 4 ± 2  C before use. Fresh cabbage was washed with running tap water and dried for 20 min in a laminar flow biosafey hood. Samples (25 g) were placed in UV-sterilized plastic bags (PET/AL/ L-LDPE) and packed 100% CO2 and 0% N2 gas. And a ventilationsterilized sachet with disc 1 piece, 10 pieces, and 20 pieces (10 ml/ piece, 100% EOs) oil added into each packaging. For this, samples (25 g) were packaged in a sterilized stomacher bag with the combination of MAP and EOs, and then stored for 21 days at 4 ± 2  C. 2.2.3. Gas composition measurement The gas composition inside the packages was analyzed in each package before opening. The concentrations of CO2 and O2 in the

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Table 1 Main composition and type of essential oils (EOs) used in this study. Abbreviation

Common name

Plant species

Main components (%)

Distilled part

Country of origin

Manufacturer

Le1

Lemon-1

Citrus limonum

Fruit feel

Australia

Le2

Lemon-2

Citrus limonum

Fruit feel

Sicily

Sydney Essential Oil Absolute aromas

Cs1

Clary sage-1

Salvia sclarea

Flower, leaf

France, England

Cs2

Clary sage-2

Salvia sclarea

Bud, leaf

Russia

Th1

Thyme-1

Thymus vulgaris

Flower, leaf

France

Th2

Thyme-2

Flower, leaf

Spain

Lg1

Lemongrass-1

Thymus vulgaris ct linalool Cymbopogon citratus

Dry leaf

India

Lg2

Lemongrass-2

Cymbopogon citratus

Leaf

Guatemala

Cy1

Cypress-1

Cupressus sempervirens

Leaf, twig

Australia, France

Cy2

Cypress-2

Cupressus sempervirens

Leaf, twig

Spain

Ro1

Rosemary-1

Rosmarinus officinalis

Flower, leaf, stem

France

Ro2

Rosemary-2

Rosmarinus officinalis

Bud

Tunisia

Tt1

Tea tree-1

Melaleuca alternifolia

Leaf, twig

Australia

Tt2

Tea tree-2

Melaleuca alternifolia

Leaf, stem

Australia

Pe1

Peppermint-1

Mentha arvensis

Flower

England

Pe2

Peppermint-2

Mentha piperita

Flower, leaf

England

Ma1

Marjoram-1

Origanum marjorana

Flower

Australia

Ma2

Marjoram-2

Origanum marjorana

Dry flower

Egypt

Ci1

Cinnamon-1

Cinnamomum zeylanicum

Leaf, twig

Tanzania, India

Ci2

Cinnamon-2

Cinnamomum zeylanicum

Leaf

Sri Lanka

Ju1

Juniper berry-1

Juniperus communis

Fruit

Croatia

Ju2

Juniper berry-2

Juniperus communis

Fruit

Croatia

Fe1

Fennel-1

Fruit

Bulgaria

Fe2

Fennel-2

Fruit

Eastern Europe

Sydney Essential Oil Absolute aromas

Or1

Oregano-1

Foeniculum vulgare var. dulce Foeniculum vulgare var. dulce Origanum vulgare

Flower, leaf

Southern Europe

Garden of green

Or2

Oregano-2

Origanum vulgare

Leaf

USA

Plantlife

Cb

Clove bud

Eugenia caryophyllata

Dry bud

Madagascar

Co

Coriander

Coriandrum sativum

Leaf, seed

Russia

Sydney Essential Oil Absolute aromas

La

Lavandin, super

Lavandula hybrida

Limonene (73.61) b-Pinene (13.39) Limonene (74.91) b-Pinene (8.83) Linalyl acetate (73.60) Linalool (16.00) Linalyl acetate (67.14) Linalool (20.75) Thymol (52.9) p-Cymene (34.00) Linalool (19.05) Terpinen-4-ol (18.29) a-Citral (40.80) b-Citral (32.00) a-citral (46.05) b-citral (37.64) a-Pinene (60.50) Cedrol (8.30) a-Pinene (44.32) d-3-carene (28.15) 1, 8-Cineole (46.60) a-Pinene (11.80) 1, 8-Cineole (38.12) Camphor (14.76) Terpinen-4-ol (39.80) g-Terpinene (17.80) Terpinen-4-ol (43.75) g-Terpinene (20.36) n-Menthol (44.00) n-Menthone (23.68) n-Menthol (52.09) n-Menthone (20.18) Terpinen-4-ol (20.80) g-Terpinene (14.10) 1, 8-Cineole (58.97) Linalool (19.56) Cinnamaldehyde (68.40) Limonene (13.20) Eugenol (80.83) Eugenyl acetate (3.94) a-Pinene (29.17) b-Pinene (17.84) a-Pinene (29.17) b-Pinene (17.84) Trans-anethole (87.85) Methyl cavicol (5.16) Trans-anethole (80.60) Fenchone (5.33) Carvacrol (68.5) Thymoquinone (12.1) Carvacrol (68.5) Thymoquinone (12.1) Eugenol (89.63) b-Caryophyllene (5.40) Linalool (73.60) a-Pinene (7.07) Linalyl acetate (40.97) Linalool (31.49)

Flower

France

Absolute aromas

Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas Sydney Essential Oil Absolute aromas

bags were measured using a needle sensor attached to a gas analyzer (Check point, PBI Dansensor, Ringsted, Denmark).

The plates were incubated at 37  C for 24e48 h, and colonies were counted.

2.2.4. Bacterial enumeration Samples (25 g) treated were transferred aseptically in sterile plastic stomacher bags and homogenized for 90 s using a stomacher (BagMixer 400, Interscience Laboratory Inc., St Nom, France). After homogenization, samples were serially 10fold diluted with 9 mL sterile peptone water, and 0.1 mL of sample or diluent was plated onto plate count agar (PCA; Difco).

2.3. Statistical analyses All experiments were repeated three times with duplicate plates. Before analysis, the mean of duplicate plate counts from three replicates was converted to log10 CFU/g. Analysis of variance (ANOVA) for a completely randomized design was conducted using SAS (version 9.1, SAS Institute, Cary, NC, USA). When the effect was

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significant (p
0.05), means were separated using Duncan's multiple range test. 3. Results 3.1. Determination of antibacterial effect by disc volatilization method Table 2 shows the antibacterial effects of 29 EOs in vapor phase against 5 strains of pathogens tested by the disc volatilization method. Among the EOs, thyme-1, oregano-1, lemongrass-1, and lemongrass-2 oils showed strong inhibitory effects. Oregano-1 oil showed antibacterial effects against all tested pathogens. Whereas, other EOs showed no inhibitory effects (data not shown). 3.2. Antibacterial effect of MAP against microorganisms on fresh produce

3.3. Antibacterial effect of EOs in vapor phase combined with MAP against microorganisms on fresh produce Both the CO2 and O2 composition did not changed inside the packages on cabbage during storage for 21 days (data not shown). The initial bacterial populations for control (no packaging and 100% CO2 packaging) were 3.02 and 2.88 log10 CFU/g, respectively (Table 4). Lemongrass-2 oil with 20 discs showed the strongest antibacterial effects against spoilage bacteria and the next, lemongrass-1 and thyme oils with 20 discs during storage for 21 days. In particular, lemongrass-2 oil with 20 discs showed complete inactivation by <1.0 log10 CFU/g after 14 days. After storage for 21 days, thyme-1, lemongrass-1, and lemongrass-2 oils with 20 discs were effective in maintaining reduced levels of spoilage bacteria on fresh cabbage compared to control. However, cabbage packaged with EOs showed a color change to light yellow after 14 days (data not shown). 4. Discussion

Table 3 shows the changes of atmosphere composition (CO2 and O2) in the packages on cabbage and radish sprouts during storage for 21 days. Both the CO2 and O2 composition did not show any changes inside the packages of fresh produce (p > 0.05). Fig. 1 shows the antibacterial activities against total mesophilic microorganisms on fresh produce were treated with MAP different CO2 concentrations. The initial populations of total mesophilic microorganisms on cabbage and radish sprouts were 2.44e2.97 and 7.75e7.87 log10 CFU/g, respectively. The average bacterial populations of cabbage increased during storage for 21 days. However, packaging with gas composition of 50% CO2 and 50% N2, 75% CO2 and 25% N2, 100% CO2 and 0% N2 effectively delayed the growth of total mesophilic microorganisms on cabbage after 7 days (p
0.05). In particular, the 100% CO2 gas packaging showed significant reduction of total mesophilic microorganisms on cabbage, and their reduction level was 1.55 log10 CFU/g compared to control after 21 days (p
0.05). Similar to cabbage, the average bacterial populations of radish sprouts were increased during storage for 21 days. For radish sprouts, the 100% CO2 gas packaging showed significant reduction of total mesophilic microorganisms, and their reduction levels were 2.12 and 2.26 log10 CFU/g after 14 and 21 days, respectively (p
0.05). After storage for 7 days, CO2 gas packaging showed the highest effectiveness in maintaining reduced levels of total mesophilic microorganisms on fresh cabbage and radish sprouts compared to control. However, occurrence of color changes to light yellow on cabbage when packaged CO2 gas compared to the air (data not shown). After storage for 14 days, radish sprouts observed softening in visual quality when packaged CO2 gas compared to the air. However, we found no substantial differences in the effects of the different gas compositions.

Table 2 Zones of inhibition (mm) around sterile discs loaded with 10 ml essential oils (EOs) in vapor phase against 5 pathogens. Pathogens

EOs Thyme- Oregano- Lemongrass- Lemongrass1 1 1 2

E. coli O157:H7 ATCC 43895 S. Typhimurium ATCC 19585 L. monocytogenes ATCC 19115 S. aureus ATCC 4012 B. cereus ATCC 10876 No inhibition zone.

15 11 e

9 8 15

10 e e

14 e 22

24 12

26 16

8 15

20 20

EOs derived from many different plants has strong antifungal and antibacterial activities. Several studies also reported strong antibacterial activities of EOs against pathogens (Bouhdid et al., 2010; Piskernik, Klancnik, Tandrup, Brondsted, & Smole-Mozina, 2011), spoilage bacteria (Tyagi & Malik, 2011a), yeast, and mold (Tserennadmid et al., 2011). EOs in vapor phase (thyme-1, oregano-1, lemongrass-1, and lemongrass-2 oils) showed strong inhibitory effectiveness against 5 pathogens (E. coli O157:H7 ATCC 43895, S. Typhimurium ATCC 19585, L. monocytogenes ATCC 19115, S. aureus ATCC 4012, and B. cereus ATCC 10876) in this study. Dorman and Deans (2000) also showed antibacterial activities of EOs in vapor phase for oregano (Origanum vulgare), and thyme (Thymus vulgaris) against pathogens (Bacillus subtilis NCIB 3610, E. coli NCIB 8879, S. pullorum NCTC 10704, and S. aureus NCIB 6571). Nedorostova et al. (2009) investigated that the effects of 27 EOs in vapor phase against foodborne pathogens (E. coli, L. monocytogenes, S. enteritidis, S. aureus, and P. aeruginisa) using the disc volatilization method. Mentha piperita in vapor phase are effective against a range of bacteria including E. coli isolates, Pseudomanas sp., B. subtilis, and S. aureus (Tyagi & Malik, 2011b). Antimicrobial effects of EOs in liquid phase against spoilage microorganisms and pathogens have been widely studied while EOs in vapor phase have been limitedly available. MAP is one of the most important preservation techniques for a wide variety of agricultural and food products (Beltran, Selma, Tudela, & Gil, 2005). Effective MAP depends on many factors, such as type of produce, gas concentration, gas diffusion through the film, storage temperature, storage time, and film type (Church & Parsons, 1994; Fonseca, Oliveira, & Brecht, 2002; Sandhya, 2010; Zagory, 1995). Our results showed that high-level CO2 gas packaging was more effective in maintaining reduced levels of total mesophilic microorganisms on fresh cabbage and radish sprouts compared to control. Similar results were also observed in other studies (Bennik, Vorstman, Smid, & Gorris, 1998; Devlieghere, Debevere, & Van Impe, 2000; Hendricks & Hotchkiss, 1997). Furthermore, our studies showed the higher antimicrobial efficacy of lemongrass oil in vapor phase combined with MAP on fresh cabbage. Lemongrass oil demonstrated antibacterial effects against E. coli O157:H7, B. subtilis, S. aureus, S. Typhimurium, and other pathogens (Aiemsaard, Aiumlamai, Aromdee, Taweechaisupapong, & Khunkitti, 2011; Friedman, Henika, Levin, & Mandrell, 2004; Maizura, Fazilah, Norziah, & Karim, 2007). The main composition of lemongrass oil was approximately a or b-citral (73~84%; lemongrass oil-1: 40.80% a-citral & 32.00% b-citral, lemongrass oil2: 46.05% a-citral & 37.64% b-citral) in this study. Antibacterial and

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Table 3 Change of CO2 and O2 composition on fresh cabbage and radish sprouts with modified atmosphere packaging (MAP) for storage 21 days at 4 ± 2  C. Fresh produce CO2 composition (%) CO2 (%)

O2 (%)

0a Cabbage

0 25 50 75 100 Radish sprouts 0 25 50 75 100 a

0.2 24.6 49.0 74.2 96.9 0.7 25.6 50.4 74.5 96.9

1 ± ± ± ± ± ± ± ± ± ±

0.1 0.9 1.5 1.5 0.7 0.2 0.3 0.4 0.3 0.9

3

1.0 24.0 47.8 71.7 95.8 1.0 24.8 48.6 73.7 96.8

± ± ± ± ± ± ± ± ± ±

0.9 2.7 2.4 1.9 0.4 0.2 0.7 0.2 1.2 1.7

1.1 24.0 47.2 73.2 96.8 1.8 25.8 49.7 74.4 96.0

7 ± ± ± ± ± ± ± ± ± ±

0.1 1.0 1.7 1.9 0.7 0.2 0.6 1.0 0.6 1.4

14

2.3 25.9 48.3 73.1 96.0 2.9 25.8 48.9 73.3 95.0

± ± ± ± ± ± ± ± ± ±

0.4 0.7 1.4 1.8 0.9 1.3 1.9 0.7 1.1 0.4

21

2.9 23.7 45.3 71.0 94.6 3.4 24.6 48.1 70.5 96.4

± ± ± ± ± ± ± ± ± ±

0.9 2.8 2.4 3.4 2.2 1.6 3.0 3.5 7.8 3.1

3.3 22.8 44.2 68.1 94.8 3.7 25.5 48.7 72.7 97.2

0 ± ± ± ± ± ± ± ± ± ±

1.2 2.4 2.8 2.7 1.1 0.2 0.3 0.4 0.8 0.3

1

0.2 0.3 0.4 0.4 0.5 0.3 0.1 0.4 0.4 0.4

± ± ± ± ± ± ± ± ± ±

0.2 0.2 0.2 0.4 0.3 0.5 0.2 0.1 0.2 0.2

0.3 0.3 0.3 0.3 0.4 0.1 0.0 0.1 0.2 0.4

3 ± ± ± ± ± ± ± ± ± ±

0.2 0.1 0.0 0.1 0.1 0.1 0.0 0.1 0.0 0.3

7

0.1 0.0 0.1 0.1 0.3 0.0 0.0 0.1 0.2 0.3

± ± ± ± ± ± ± ± ± ±

0.1 0.0 0.2 0.1 0.1 0.1 0.0 0.0 0.1 0.1

0.0 0.0 0.1 0.2 0.6 0.0 0.2 0.2 0.3 0.3

14 ± ± ± ± ± ± ± ± ± ±

0.0 0.1 0.1 0.2 0.3 0.0 0.1 0.2 0.0 0.2

0.1 0.1 0.2 0.1 0.4 0.4 0.3 0.4 0.4 0.4

21 ± ± ± ± ± ± ± ± ± ±

0.2 0.1 0.2 0.1 0.3 0.3 0.3 0.3 0.3 0.3

0.7 0.5 0.5 0.5 0.4 0.3 0.0 0.1 0.2 0.2

± ± ± ± ± ± ± ± ± ±

0.3 0.4 0.2 0.2 0.1 0.5 0.0 0.1 0.1 0.1

Storage time (days).

▵

Fig. 1. Bacterial inhibition effect of MAP on fresh cabbage (A) and radish sprouts (B) for storage 21 days at 4 ± 2  C. (C) Control (air packaging); (B) CO2 0%; (;) CO2 25%; ( ) CO2 50%; (-) CO2 75%; ( ) CO2 100%.

▫

antifungal activity of citral and/or citronellal, aldehydes has been demonstrated (Kim, Marshall, & Wei, 1995). Also, several studies have indicated that lemongrass in vapor phase is more effective than the liquid phase (Tyagi & Malik, 2010a, b). Recently, there have been studies that EOs in vapor phase are more effective than liquid

phase including Eucalyptus globules (Inouye, Abe, Yamaguchi, & Asakura, 2003; Tyagi & Malik, 2011b), Mealaleuca alternifolia (Mondello, Girolamo, Scaturro, & Ricci., 2009), and thyme, fennel, and lavender EOs (Soylu, Soylu, & Kurt, 2006; Tullio et al., 2007). EOs in vapor phase could be highly effective against pathogens and

Table 4 Antibacterial effect of EOs in vapor phase combined with MAP against spoilage bacteria on fresh cabbage for storage 21 days at 4 ± 2  C. Disc (piece)

EOs

Storage time (days)

0

Control-1 (air packaging) Control-2 (CO2 100%) Thyme-1 Oregano-1 Lemongrass-1 Lemongrass-2 Thyme-1 Oregano-1 Lemongrass-1 Lemongrass-2 Thyme-1 Oregano-1 Lemongrass-1 Lemongrass-2

3.02 2.88 2.68 2.34 2.76 2.24 3.12 2.75 2.01 2.89 2.39 2.85 2.46 2.83

0

1

10

20

AD

1 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.11Ab 0.50Ab 0.95Aab 0.37Abc 0.62Aa 0.80Ab 0.45Aab 0.19Abc 1.05Aab 0.49Aab 0.69Aa 0.07Abc 0.53Aa 0.66Aa

2.66 2.56 2.53 3.37 1.96 2.52 2.19 2.83 2.42 3.24 2.17 2.71 2.24 2.94

3 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.30Ab 0.39Ab 0.53Ab 0.39Aabc 1.70Aa 0.91Ab 0.72Ab 0.51Abc 0.82Aab 0.83Aab 0.62Aa 0.36Abc 0.80Aab 0.40Aa

3.93 2.94 2.38 2.16 2.75 2.53 2.25 2.33 2.78 3.23 1.34 2.34 2.69 2.86

7 ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.42Aab 0.95ABCb 0.54BCb 0.37BCc 0.27ABCa 0.33ABCb 0.95BCb 0.75BCc 0.20ABCa 0.25ABab 1.17BCa 0.82BCc 0.60ABCa 0.40ABa

4.83 3.16 3.20 3.82 3.52 2.86 3.81 3.59 3.15 4.14 3.13 3.70 2.72 1.96

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.49Aa 0.34ABb 1.49ABab 1.42ABab 1.18ABa 1.83ABb 0.52ABab 1.07ABb 1.44ABa 0.97ABa 0.80ABa 1.31ABab 0.96ABa 1.72Ba

14

21

4.76 ± 0.61Aa 3.92 ± 0.19ABCab 4.41 ± 0.92ABa 4.37 ± 1.08ABa 3.06 ± 0.86BCDa 3.16 ± 0.13BCDab 4.35 ± 0.83ABa 2.21 ± 0.24Dc 2.15 ± 0.19Dab 2.00 ± 1.33Dbc 2.04 ± 1.77Da 3.27 ± 0.08ABCDbc 2.74 ± 0.85CDa <1.00Eb

5.64 4.74 4.18 2.86 2.69 4.90 1.99 4.94 0.88 1.09 1.20 4.90 0.88 1.20

± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.09Aa 1.24Aa 0.90ABab 0.19BCbc 0.46BCDa 1.48Aa 1.73CDb 0.55Aa 0.78Db 0.95CDc 1.05CDa 0.52Aa 0.78Db 1.05CDab

Means in the same column with different superscript capital letters denote significant difference (p
0.05) between the values for the different oil treatment of storage days according to Duncan test. aec Means in the same row with different lowercase letters denote significant difference (p
0.05) between the values for the different days of storage for each oil treatment according to Duncan test. The limits of detection was 1.00 log10 CFU/g.

312

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spoilage bacteria at lower concentrations than the liquid phase, thereby causing minimum adverse effects on the organoleptic
pez et al., 2005; Serrano properties of food (Laird & Philips, 2011; Lo et al., 2008). Karabagias, Badeka, and Kontominas (2011) observed that the combined effect of essential oils (0.1% thyme oil) and MAP (CO2 80% and N2 20%) on shelf life of lamb meat. Most studies have shown that treatment of fruits and vegetables with 200 ppm of chlorine results in reduction levels of less than 2 log10 CFU/g (Kondo, Murata, & Isshiki, 2006; Takeuchi & Frank, 2000). Natural EO solutions can be used as an alternative to synthetic sanitizers for the disinfection of fresh produce. Although EOs are classified as GRAS, their use in foods as preservatives has been limited, because of the required high concentrations which may cause changes in food that exceed organoleptically acceptable € nül, & Karapınar, 2010). In this study, we evallevels (Gündüz, Go uated the occurrence of discoloration and off-flavor on fresh produce after treatment with thyme, oregano, and lemongrass oils (data not shown). Rojas-Grau, Soliva-Fortuny, and Martín-Belloso (2009) observed that the use of oregano oil as an edible-coated on fresh cut apples resulted in residual oil taste, which decreased the overall acceptance of fruits. Uyttendaele, Neyts, Vanderswalmen, Notebaert, and Debevere (2004) also reported adverse sensory properties (chemical smell, softening of the tissue, and moisture loss) noted for bell peppers treated with thyme oil. These results indicate that high concentrations of these EOs would be needed to reduce total mesophilic microorganisms. Gutierrez, Rodriguez, Barry-Ryan, and Bourke (2008) observed that oregano and marjoram oils did not cause any adverse effect on the sensory quality of lettuce and carrots. It was indicated that unacceptable organoleptic effects can be limited, by careful selection of EOs, according to the type of food (Burt, 2004), and the treatment concentration. Therefore, further studies should investigate the effects of EOs at various concentrations and in combination with other sanitizing methods on more types of fresh produce to identify broadspectrum natural antimicrobials suitable for application in a wide range of fresh produce.

5. Conclusion In conclusion, the results of this study indicate the antibacterial effects of various EOs in vapor phase combined with MAP against total mesophilic microorganisms in fresh produce. Four EOs (thyme, oregano, lemongrass-1, and lemongrass-2) were evaluated using the disc volatilization method. EOs in vapor phase combined with MAP (CO2 100%) showed good inhibitory effects against total mesophilic microorganisms. In particular, lemongrass-2 oil with 20 discs showed strong antibacterial effects during 21 days storage. These results could provide the useful information for developing an alternative preservation method to improve the freshness and shelf-life of fresh produce using natural antimicrobials. The potential of EOs for practical application in combination with other preservation techniques, such as high hydrostatic pressure or lowdose irradiation, need to be investigated.

Acknowledgment This research was a part of the project titled “Development of rapid detection system for foodborne pathogens to strengthen the food safety and to promote the seafoods consumption” funded by the Ministry of Land, Transport and Maritime Affairs, Republic of Korea.

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