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Thyme oil alginate-based edible coatings inhibit growth of pathogenic microorganisms spoiling fresh-cut cantaloupe
Food Bioscience 32 (2019) 100467
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Thyme oil alginate-based edible coatings inhibit growth of pathogenic microorganisms spoiling fresh-cut cantaloupe
T
Sarengaowaa,b,c, Wenzhong Hub,c,∗, Ke Fengb,c, Zhilong Xiua, Aili Jiangb,c, Ying Laob,c a
School of Bioengineering, Dalian University of Technology, Dalian, 116024, Liaoning, China College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China c Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian, 116600, Liaoning, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fresh-cut cantaloupe Cucumis melo L. Listeria monocytogenes Staphylococcus aureus Salmonella Typhimurium Escherichia coli O157:H7
Fresh-cut cantaloupe (Cucumis melo L.) is a popular food. However, the presence of foodborne pathogen on the cut surfaces leads to both spoilage and food poisoning of fresh-cut cantaloupe. The antibacterial effectiveness of an alginate-based edible coating (EC) containing thyme oil was evaluated against foodborne pathogens on freshcut cantaloupe samples. Cantaloupe samples were treated with alginate-based EC and alginate-based EC containing different concentrations (0, 0.05, 0.35, and 0.65%, v/v) of thyme oil. Uncoated samples served as the control. The viability of naturally occurring microorganisms and artificially inoculated foodborne pathogens on the fresh-cut cantaloupe samples, as well as cantaloupe respiration rate, weight loss, and colour, were determined every 4 days over 16 days at 4 °C. Results showed that alginate-based EC containing thyme oil effectively improved the quality of fresh-cut cantaloupe and prolonged its shelf life. The treatments inhibited the growth of naturally occurring microorganisms and artificially inoculated pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium, and Escherichia coli O157:H7) on fresh-cut cantaloupe samples. Treatment with alginate-based EC containing 0.05% thyme oil did not affect the respiration, prevented weight loss, and maintained the colour and sensory characteristics of fresh-cut cantaloupe samples. Gram-negative bacteria (S. Typhimurium and E. coli O157:H7) were more resistant to alginate-based EC containing thyme oil than Gram-positive bacteria (L. monocytogenes and S. aureus). Therefore, the use of an alginate-based EC containing thyme oil might be a promising approach to increase shelf life and increase safety of fresh-cut cantaloupe.
1. Introduction The popularity and consumption of cantaloupe (Cucumis melo L.) are increasing mainly because of its sweet pulp, pleasant aroma, and recognition as a rich source of vitamins A, C, and folic acid, as well as beta-carotene (Beaulieu & Lea, 2007). Cut or sliced forms of cantaloupe potentially promote bacterial survival and growth because of the nutrient content of the juice and pulp (Ukuku, Mukhopadhyay, & Olanya, 2013). Cantaloupe contamination by foodborne pathogen has been a concern since the associated outbreaks in the United States, Australia, Canada, and other countries (CDC, 2002, 2012a, b). Listeria monocytogenes (LM), Staphylococcus aureus (SA), Salmonella Typhimurium (ST), and Escherichia coli O157:H7 (EC O157:H7) are considered as important foodborne pathogens with a significant impact on public health (Hassanain et al., 2013). LM, SA, ST, and EC O157:H7 cause hemorrhagic colitis, bloody or non-bloody diarrhea, hemolytic uremic
∗
syndrome, self-limited intestinal disease, and death in humans. A cantaloupe-related Listeria outbreak in 2011 was one of the deadliest multistate outbreaks in the United States, resulting in 147 illnesses and 33 deaths (CDC, 2012a). In 2012, a large-scale cantaloupe-related outbreak of salmonellosis caused by ST and Salmonella Newport resulted in 261 reported infections, including 94 hospitalizations and 3 deaths reported to the US Centers for Disease Control and Prevention (CDC, 2012b). In 2019, the outbreak of Salmonella infections linked to pre-cut melons caused 38 illnesses in 137 people infected in the United States (CDC, 2019). Cantaloupes were linked to an outbreak of EC O157:H7 causing 34 people to become sick, but no deaths were reported (Sivapalasingam, Friedman, Cohen, & Tauxe, 2004). SA is a major pathogen due to its enterotoxigenic strains, which are the leading cause of gastroenteritis, resulting from the consumption of enterotoxins-contaminated food (Harris, Foster, & Richards, 2002). The percentage occurrence of SA in melon was 70%, although no cases of
Corresponding author. College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China. E-mail address: [email protected] (W. Hu).
https://doi.org/10.1016/j.fbio.2019.100467 Received 14 April 2019; Received in revised form 16 September 2019; Accepted 16 September 2019 Available online 18 September 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Preparation of alginate-based EC
food poisoning have been associated with SA from cantaloupe (Nwachukwu & Osuocha, 2014). These events indicate the high risk of microbial contamination with fresh-cut cantaloupe. Meanwhile, the severity of cantaloupe-linked outbreaks highlights the role of cantaloupes as a vehicle of foodborne pathogens. There is a need to maintain fruit quality and microbial safety post-cutting. Consumer demands are increasingly focusing on minimally processed and safe foods produced using natural preservatives. Essential oils (EO) are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA, 2018). Many EO have a major role in controlling a wide range of pathogenic and spoilage bacteria associated with food (Cui et al., 2017, 2018; Scollard, McManamon, & Schmalenberger, 2016). However, the odour of EO may negatively affect food quality (Burt, 2004). One method for overcoming this problem might be the use of a combination of an edible coating (EC) and EO. EC acts as a carrier enabling the sustained release of EO to the surface of the fruit or vegetable, reducing the negative effect of EO on the flavour of fresh-cut fruits and vegetables, while maintaining the antibacterial effects (Lin, Zhu, Thangaraj, Abdel-Samie, & Cui, 2018a). EO are being incorporated into EC and films (Lin, Zhu, & Cui, 2018b). Some reports have evaluated the effects of EO, such as lemongrass oil, oregano oil, and vanillin oils, which have been incorporated into different types of EC including sodium alginate, and apple puree-alginate on fresh-cut fruits such as pineapple, and Fuji apples (Rojas-Graü et al., 2007; Azarakhsh, Osman, Ghazali, Tan, & Adzahan, 2014). Previous report showed that thyme oil has the strongest antimicrobial activity against a range of foodborne pathogens (LM, SA, ST, and EC O157:H7) among 15 other types of EO using in vitro experiments (Sarengaowa, Hu, Jiang, Xiu, & Feng, 2018). Therefore, the current study aimed at evaluating the effects of different concentrations of thyme oil incorporated into alginate-based EC on artificially inoculated pathogens, naturally occurring microorganisms, quality, and sensory attributes of fresh-cut cantaloupe during low-temperature storage.
An optimized alginate-based EC was used based on a previous study (Azarakhsh et al., 2014). The EC was prepared by mixing 1.29% (w/v) sodium alginate (food-grade, Henan Qiang Li Chemical Products Co., Ltd., Zhengzhou, Henan, China) with 1.5% (w/v) glycerine (food-grade, Procter & Gamble International Operations, Singapore) in ultrapure water from an ultrapure water machine (PURELAB Classic UF, Elga Ltd., High Wycombe, UK), and stirring at 70 °C until the solution became transparent (Azarakhsh et al., 2014). Different concentrations (0.05, 0.35 and 0.65%, v/v) of thyme oil (Ji'an Zhongxiang Natural Plant Co., Ltd., Jian, Jiangxi, China) were incorporated into the alginate-based EC. The major compounds of the thyme oil were 47.2% thymol, 20.4% p-cresol and 16.3% 2,6-dimethylphenol. The final solutions were homogenized using an Ultra Turrax T25 mixer (IKA® WERKE, Staufen, Baden-Württemberg, Germany) at 1,140 × g at room temperature (~25 °C) for 3 min. For the cross-linking reaction necessary for gel formation, a 2% (w/v) calcium chloride solution (foodgrade, Zhejiang Dacheng Calcium Co., Ltd., Quzhou, Zhejiang, China) containing 1% (w/v) ascorbic acid (food-grade, Henan Qiang Li Chemical Products) and 1% (w/v) citric acid (food-grade, Henan Qiang Li Chemical Products) was prepared. Ascorbic acid and citric acid were added into the calcium chloride solution as the antioxidant and colour fixative, respectively (González-Aguilar, Ruiz-Cruz, Cruz-Valenzuela, Rodríguez-Félix, & Wang, 2004).
2.4. Processing and packaging of fresh-cut cantaloupe Cantaloupe were washed manually with sterile water and sterilized with 75% (v/v) ethyl alcohol (Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China). Samples were air-dried for 10 min at 25 °C in a biosafety cabinet. The cleaned cantaloupe samples were peeled and cut into cubes (1 cm3) using a sterilized knife. Cantaloupe cubes were placed in 100 ml of the coating-forming solutions of sodium alginate and different concentrations of thyme oil for 2 min. They were subsequently immersed in 100 ml of 2% (w/v) calcium chloride solution for 2 min. Uncoated fresh-cut cantaloupe samples were used as the control. Samples (10 cubes) were packaged on polystyrene trays wrapped in PVC films (Lixian Wrap, Jinan ZhenHua Plastic Packaging Co., Ltd., Jinan, Shandong, China) for quality and sensory studies over 16 days at 4 °C. For microbiological evaluation experiments, cantaloupes were cut into 4 × 3 × 0.5 cm3 pieces (~10 g/piece) using a sterile knife. The cantaloupe pieces were placed on petri dishes and then uniformly inoculated as follows. The concentration of bacterial suspension was adjusted to ~108 cfu/ml using the McFarland standard tube (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) to standardize the inoculation amount for LM, SA, ST, EC O157:H7, respectively (Guerreiro, Gago, Faleiro, Miguel, & Antunes, 2015; Pesavento et al., 2015). Subsequently, SA, ST, and EC O157:H7 populations were individually enumerated using trypticase soy agar (TSA, Qingdao Hopebio-Technology). LM populations were enumerated using TSA with 0.6% (w/v) yeast extract (TSA-YE, Qingdao Hopebio-Technology). Suspensions of LM (8.84 log cfu/ml), SA (8.45 log cfu/ml), ST (8.06 log cfu/ml), and EC O157:H7 (8.90 log cfu/ml) were prepared to inoculate the samples. The inoculum of LM, SA, ST, and EC O157:H7 (500 μl each) was spread individually over the entire top surface (4 × 3 cm2) of cantaloupe pieces with a sterile micropipette for the challenge study. Cantaloupe pieces were then air-dried for 1 h at 25 °C in a biosafety cabinet. Then, the samples were EC-coated as described above. Uncoated fresh-cut cantaloupe samples with air drying were used as the control. Each 10 g cantaloupe piece was placed in a Stomacher bag (32 × 20 cm, Qingdao Hopebio-Technology) for storage. The experiments were done in triplicate, with samples measured after 0, 4, 8, 12, and 16 days; overall, 300 bags were analysed.
2. Materials and methods 2.1. Bacterial strains and preparation of bacterial cultures LM (CICC 21633), SA (CICC 21600), ST (CICC 21484), and EC O157:H7 (CICC 21530) were obtained from the China Centre of Industrial Culture Collection (CICC, Beijing, China). All strains except LM were incubated in trypticase soy broth (TSB, Qingdao HopebioTechnology Co., Ltd., Qingdao, Shandong, China) for 24 hat 37 °C. LM was incubated in TSB with 0.6% (w/v) yeast extract (TSB-YE, Qingdao Hopebio-Technology) using the same conditions. The cultures were centrifuged at 2,800 × g for 5 min at 4 °C using a centrifuge (Allegra X30R, Beckman Coulter Inc., Brea, CA, USA), and washed three times in 5 ml of 0.1% (w/v) peptone water (Aobox Biotechnology, Beijing, China) (Joshua, Di, Linda, Donald, & Michelle, 2013). The bacterial suspension was diluted with 10 × serial dilutions in 0.1% (w/v) peptone water to obtain the proper inoculum. Colonies were counted and populations were expressed as log cfu/ml.
2.2. Fruits Fresh cantaloupes were purchased from New-Mart supermarket in Dalian city (Liaoning, China), at commercial maturity stage. Cantaloupes were grown in open fields in Turpan city (Xinjiang, China). They were obtained on the fifth day after harvest directly from the farm. They were chosen for uniformity of maturation stage, size and absence of defects or injuries. The soluble solids content of cantaloupes was determined using a pocket refractometer (PAL-1, Atago Co., Ltd., Tokyo, Japan) and ranged between 8.7 and 9.7 °Brix. Samples were maintained for ~30 min (transportation time) at 4 °C before processing. 2
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specified time point (in g). Colour parameters of cantaloupe cubes, including L* (lightness), a* (+a* = redness, -a* = greenness), and b* (+b* = yellowness, -b* = blueness), were evaluated using a CR400 colorimeter (Konica Minolta Inc., Tokyo, Japan). The experiments were done in triplicate. Overall, 75 trays (10 cubes/tray) were measured. Colour parameters values of each cube were obtained by averaging 6 determinations in the central region of 6 surfaces. Numerical values of a* and b* parameters were used to calculate the whiteness index (WI) using Eq (6).
2.5. Sample analysis 2.5.1. Microbiological analysis Each sample was used for microbiology. The experiments were done in triplicate and 75 bags were measured in total. For the analysis, 90 ml of 0.1% peptone water was added to a Stomacher bag, and cantaloupe pieces were disrupted in a Stomacher (Interscience, Saint Nom la Breteche, France) at the high-speed setting for 1 min. Cantaloupe suspensions (0.1 ml) were plated and cultured on plate count agar (PCA) for 48 h at 37 °C for total plate counts, on potato dextrose agar (PDA) for 48–96 h at 28 °C for yeast and mould counts, on violet red bile dextrose agar (VRBDA) for 24 h at 37 °C for total coliform counts, and on Lactobacilli MRS agar for 48 h at 37 °C for Lactobacillus counts (Gómez & Salvatori, 2012; Siroli et al., 2015). All culturing media were purchased from Qingdao Hopebio-Technology Co., Ltd. The growth of naturally occurring microorganisms on fresh-cut cantaloupe samples was fit to the Gompertz model using MATLAB software (version 7.0, Mathworks Inc., Natick, MA, USA). The Gompertz model is described using Eq (1). Nt = A + C·exp{-exp[-B(t-M)]}
(1)
U = BC/e
(2)
LPD = M – 1/B
(3)
MPD = A + C
(4)
WI = 100 – [(100 – L*)2 + (a*)2 + (b*)2]1/2
2.5.4. Sensory analysis Sensory characteristics of cantaloupe cubes (1 cm3) in control, alginate-based EC, and alginate-based EC containing different concentrations (0.05, 0.35, and 0.65%, v/v) of thyme oil at 4 °C for 8 days were evaluated by regular cantaloupe consumers. Twenty individuals (10 female, 10 male, aged between 22 and 45 years) were recruited from among the students and staff of the Faculty of Food Science and Technology, Dalian Minzu University (Dalian, Liaoning, China). They were trained at the beginning of the experiment to become familiar with the characteristics of fresh-cut cantaloupe. Sensory evaluation was carried out in individual booths in a sensory laboratory. Sample presentation order was randomized and balanced among assessors. The assessors evaluated the colour, appearance, odour, taste, texture, and the overall acceptability of fresh-cut cantaloupe at room temperature (~25 °C) according to a 9-point hedonic scale test, accounting for varying degrees of appreciation, from 1 (dislike very much) to 9 (like very much). The hedonic evaluation scale was as follows: 9, like very much; 8, like a lot; 7, like moderately; 6, like slightly; 5, indifferent; 4, dislike slightly; 3, dislike moderately; 2, dislike a lot; 1, dislike very much (Harich, Maherani, Salmieri, & Lacroix, 2018; Peryam & Pilgrim, 1957). The average response was calculated for each attribute (Guerreiro, Gago, Miguel, Faleiro, & Antunes, 2016). Samples with scores below 6 were deemed unacceptable (Abdollahzadeh, Rezaei, & Hosseini, 2014).
where Nt is the natural logarithm of the microbial population at time t (log cfu/g), A is the initial cell concentration (log cfu/g), C is the difference between the maximum and initial cell concentration (log cfu/ g), B is the maximum growth rate (day−1), M is the time to reach the maximum growth rate (days), U is the maximum specific growth rate (log cfu/g/day), LPD is the lag phase duration (days), and MPD is the maximum population density (log cfu/g) and e = 2.7182. Numerical values for A, C, B, and M were used to calculate U, LPD, and MPD using Eqs (2)–(4). ST, LM, EC O157:H7, and SA, counts on fresh-cut cantaloupes were analysed on Salmonella spp. chromogenic media and Oxford agar base (both from Qingdao Hopebio-Technology), EC O157:H7 chromogenic media, and SA chromogenic media (both from Shanghai Central BioEngineering Ltd., Co., Shanghai, China). All plates were incubated at 37 °C for 24 h, and the colonies were counted using an aCOLyte colony counter (Acolyte Technologies Corp., London, UK). Microbial counts were expressed as log cfu/g.
2.5.5. Statistical analysis All experiments were done in triplicate, as independent experiments. Data were measured using the Statistical Package for the Social Sciences (SPSS, Version 14.0, IBM Corp., Armonk, NY, USA). Significance of the differences between variables was tested using oneway ANOVA (between groups) and repeated measures of ANOVA (within group). The means were compared using Duncan's multiple range test. Statistical significance was determined at p < 0.05.
2.5.2. Respiration rate determination The respiration rate was determined during storage using gas chromatography (GC-2010 Shimadzu, Kyoto, Japan) equipped with an CTR1 column (1.8 m × 3.18 mm inner diameter, 6.35 mm column thickness, stainless steel column filled with porous polymer mixture, No. A8700, Alltech Associates Inc., Deerfield, IL, USA). The gas carrier was helium at 30 ml/min and the temperature of the injector, column and the detector was set at 120, 35, 120 °C, respectively. Coated cantaloupe pieces or control samples (200 g) were placed in 400 ml tightsealed glass containers and incubated at 25 °C for 1 h. Then, 1 ml of headspace gas was withdrawn through a rubber septum of an airtight screw-cap. The respiration rate was calculated based on the carbon dioxide produced [ml CO2/(kg/h)] (Qi, Hu, Jiang, Tian, & Li, 2011).
3. Results and discussion 3.1. Analysis of naturally occurring microorganisms on fresh-cut cantaloupe The population of naturally occurring microorganisms including total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe samples treated with EC with or without thyme oil were fitted with Gompertz model (R2 > 0.98) (Fig. 1). The population considerably increased on fresh-cut cantaloupe samples among treatment groups over 16 days (p < 0.05). The population of naturally occurring microorganisms on samples treated with alginate-based EC were much lower than those on untreated control during storage. The total plate counts, total coliform counts, and yeast and mould counts on samples treated with alginate-based EC containing thyme oil were much lower than that on the alginate-based EC-treated samples (Fig. 1a, b, c). The reduction in naturally occurring microorganisms was primarily associated with the antimicrobial effect of thyme oil. In addition, lactic acid bacterial (LAB) counts on untreated controls over 16 days storage at 4 °C were below 3.00 log cfu/g (data not shown). When fresh-
2.5.3. Determination of cantaloupe weight loss and colour Samples (200 g) were placed on uncovered polystyrene trays. Samples were weighed using a digital balance (PL-2002, Mettler Toledo, Greifensee, Switzerland) during storage. The weight loss rate equation [Eq (5)] was evaluated (Chien, Sheu, & Yang, 2007): Weight loss rate (%) = [(m1 – m2)/m1] × 100
(6)
(5)
Where m1 is the initial weight (in g) and m2 is the weight at the 3
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Fig. 1. Gompertz model of naturally occurring microorganisms on fresh-cut cantaloupe coated with alginate-based EC containing thyme oil at 4 °C. (a) total plate counts; (b) total coliform counts; (c) yeast and mould counts. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil.
was obtained on uncoated fresh-cut cantaloupe. It might be from the pulp and surface of fresh-cut cantaloupe. Other studies showed initial total plate counts, total coliform counts, and yeast and mould counts was ~3.00, 4.80, and 2.00 log cfu/g on fresh-cut cantaloupe, respectively (Graça, Esteves, Nunes, Abadias, & Quintas, 2017; Oms-Oliu, Soliva-Fortuny, & Martín-Belloso, 2007). The MPD of total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe samples coated with EC containing 0.65% thyme oil were significantly reduced (p < 0.05) compared with those of coated samples, and the reduction were 2.13, 1.56, and 0.56 log cfu/g, respectively. Therefore, thyme oil had an important role in the antimicrobial activity against naturally occurring microorganisms on fresh-cut cantaloupe. The LPD of naturally occurring microorganisms on uncoated fresh-cut cantaloupe was shorter than that with other treatments. Compare with samples treated with 0.65% thyme oil, the LPD value of total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe samples coated with EC containing 0.05% thyme oil was the longest among each treatment. It might be that the cell structure was more easily damaged with 0.65% thyme oil compared with 0.05% thyme oil. The juice of cells provide a nutrient rich content for the microorganisms’ growth. Therefore, the microorganisms can grow easily on fresh-cut cantaloupe with 0.65% thyme oil at the early storage times.
cut cantaloupe samples were treated with EC or EC containing thyme oil, no LAB were detected (data not shown). The microbial limit of acceptance for fresh-cut fruits and vegetables is 6.00 log cfu/g according to the UK Institute of Food Science and Technology (IFST, 1999). The total plate count exceeded the limit of 6.00 log cfu/g for the control and EC samples at 8–9 days (Fig. 1a). On the other hand, the same standard suggests that yeasts and moulds might produce toxic metabolites if their counts exceed 6.00 log cfu/g in foods. This value was also the limit for the acceptable shelf life of fruit products (IFST, 1999). The yeast and mould population exceeded 6.00 log cfu/g on uncoated fresh-cut cantaloupe on day 10 according to the growth curve fitted with the Gompertz predictive model at 4 °C (Fig. 1c). However, the total plate counts and yeast and mould counts on samples treated with EC containing thyme oil did not exceed 6.00 log cfu/g during storage. Therefore, the incorporation of thyme oil into EC extended the shelf life of fresh-cut cantaloupe at least 16 days. Another study showed a multi-layered antimicrobial EC extended the shelf life of fresh-cut cantaloupe to only 9 days (Martiñon, Moreira, Castell-Perez, & Gomes, 2014). The extension of the shelf life might be of commercial value to the cantaloupe industry, and the use of edible antimicrobial coatings would provide additional benefits (Martiñon et al., 2014). Parameters derived from Gompertz modeling of naturally occurring microorganisms on fresh-cut cantaloupe are shown in Table 1. The A for total plate counts, total coliform counts, and yeast and mould counts 4
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Table 1 Gompertz modeling parameters of changes in total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe coated with alginate-based EC containing thyme oil over 16 days at 4 °C. Naturally Occurring Microorganisms
Total Plate Counts
Total Coliform Counts
Yeast and Mould Counts
R2
Treatment
Control EC EC + Thy EC + Thy EC + Thy Control EC EC + Thy EC + Thy EC + Thy Control EC EC + Thy EC + Thy EC + Thy
(0.05%) (0.35%) (0.65%)
(0.05%) (0.35%) (0.65%)
(0.05%) (0.35%) (0.65%)
0.9951 0.9960 0.9998 0.9997 0.9996 0.9981 0.9995 0.9996 0.9992 0.9977 0.9880 0.9952 0.9963 0.9947 0.9937
RMSE
0.1827 0.1736 0.0301 0.0338 0.0337 0.1210 0.0540 0.0393 0.0466 0.0756 0.2798 0.1019 0.0813 0.0865 0.0921
SSE
0.1001 0.0904 0.0027 0.0034 0.0034 0.0439 0.0088 0.0046 0.0065 0.0017 0.2349 0.0312 0.0198 0.0224 0.0254
Gompertz Parametersα A
C
U
LPD
MPD
1.95 1.90 1.90 1.85 1.85 1.88 1.80 1.78 1.70 1.70 1.75 1.67 1.60 1.48 1.48
5.24 5.07 3.59 3.35 2.99 5.23 4.44 3.55 3.00 2.98 4.99 2.70 2.48 2.39 2.33
0.65 0.77 0.94 0.88 0.79 0.84 0.86 0.94 0.73 0.46 0.62 0.51 0.38 0.28 0.27
1.41 3.66 6.32 6.19 5.97 5.09 6.61 6.78 6.60 5.84 2.33 3.77 3.85 2.84 2.99
7.20 6.97 5.49 5.20 4.84 7.11 6.24 5.33 4.69 4.68 6.75 4.37 4.08 3.87 3.81
αData obtained from the Gompertz model; Control, uncoated; EC, coated without thyme oil; Thy, thyme oil; RMSE, root mean squared error; SSE, the sum of squares due to error; The parameters are as described in Section 2.5.1.
Fig. 2. Reduction in foodborne pathogen on fresh-cut cantaloupe coated with alginate-based EC containing thyme oil at 4 °C. (a) LM; (b) SA; (c) ST; (d) EC O157:H7. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil. Means designated with the same letters (lowercase, among different treatments at the same time point; uppercase, for the same treatment after different storage times) are not significantly different (p ≥ 0.05).
5
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Fig. 3. Changes in quality parameters of fresh-cut cantaloupe coated with alginate-based EC containing thyme oil at 4 °C. (a) respiration rate; (b) weight loss; (c) lightness; (d) WI. The data are the means ± SD. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil. Means designated by the same letters (lowercase, among different treatments at the same time point; uppercase, for the same treatment after different storage times) are not significantly different (p ≥ 0.05).
fresh-cut melon treated with thyme EO (0.15 mg/ml) alone was 1.00 log cfu/g during day 2 at 8 °C and this effect was no longer observed by day 7 of storage compared to the control (Scollard et al., 2016). It showed further that thyme oil incorporated into EC maintained the antibacterial effect and enhanced safety of fresh-cut cantaloupe during shelf-life compared with thyme oil alone treatment. The reduction in ST, EC O157:H7, LM, SA on fresh-cut cantaloupe treated with alginate-based EC containing 0.65% thyme oil is 2.35, 1.68, 3.29 and 3.76 log cfu/g, respectively, at day 16 compared with the control. It was obviously that Gram-negative bacteria (ST and EC O157:H7) are more resistant to EC containing thyme oil than Grampositive bacteria (LM and SA). It might be that the cell wall of Grampositive bacteria which consists mainly of peptidoglycan allows hydrophobic molecules to easily penetrate the cells and act on both the cell wall and within the cytoplasm (Nazzaro, Fratianni, Martino, Coppola, & Feo, 2013). The hydrophobic nature of thymol and carvacrol may be important for their antibacterial effects (Liolios, Gortzi, Lalas, Tsaknis, & Chinou, 2009). However, the cell walls of Gram-negative bacteria are more complex. The cell wall of Gram-negative bacteria is covered by an outer membrane that contains various proteins as well as lipopolysaccharides. Their characteristics may be more resistant to EO and other hydrophobic natural extracts with antimicrobial activity (Nazzaro et al., 2013; D'Amato, Serio, López, & Paparella, 2018).
3.2. Inhibition of foodborne pathogen on fresh-cut cantaloupe Fig. 2 shows the effect of alginate-based EC with or without thyme oil on LM, SA, ST, and EC O157:H7 inoculated on fresh-cut cantaloupe samples. The alginate-based EC containing thyme oil showed the best antibacterial efficacy against LM, SA, ST, and EC O157:H7 at each time point. Moreover, a significant reduction in the LM, SA, ST, and EC O157:H7 count was noted with increasing concentration of thyme oil during storage. Other studies have also shown thyme oil inhibited the growth of foodborne pathogen on food matrixes including fresh-cut lettuce, cantaloupe melon, pineapple, beef, chicken and cheese (Govaris, Botsoglou, Sergelidis, & Chatzopoulou, 2011; Lin et al., 2018b; Scollard et al., 2016; Solomakos, Govaris, Koidis, & Botsoglou, 2008). Carvacrol and thymol as the major components of thyme oil have strong antibacterial effect against LM, SA, ST, and EC O157:H7 (Sakkas & Papadopoulou, 2017). The combination of thyme oil and alginate-based EC maintained the antibacterial effect against LM, SA, ST, and EC O157:H7 on fresh-cut cantaloupe during storage of 16 days. LM, SA, ST, and EC O157:H7 showed a significant reduction of 0.72, 3.03, 2.36 and 1.50 log cfu/g on fresh-cut cantaloupe treated with EC containing 0.65% thyme oil from day 0 to day 16 (Fig. 2a, b, 2c, 2d). It might be the combination of an EC and EO avoided the depletion of antimicrobials in food and achieved sustained release of EO on the surface of fruits and vegetables (Chong, Lai, & Yang, 2015). However, in another study, the reduction of LM on 6
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EC containing thyme oil did not significantly affect L* (p > 0.05). WI can reflect the extent of translucency due to physiological disorders of fresh-cut cantaloupe (Aguayo, Escalona, & Rtés, 2004; Oms-Oliu et al., 2007). The WI value of fresh-cut cantaloupe treated with EC containing 0.05% thyme oil was higher than that on other treatment groups. It showed that the combination of 0.05% thyme oil and EC maintained the colour of fresh-cut cantaloupe during storage time. 3.4. Sensory analysis of fresh-cut cantaloupe The strong odours and flavours of EO may affect the organoleptic properties of foods (Lee, Kim, Kim, Beuchat, & Ryu, 2018). Sensory evaluation was done after 8 days of storage because the uncoated samples reached the limit of acceptance (6.00 log cfu/g) after that time point (Azarakhsh et al., 2014). Sensory evaluation of differently treated fresh-cut cantaloupe samples after 8 days of storage is shown in Fig. 4. According to the overall acceptability score, samples coated with alginate-based EC and EC containing 0.05% thyme oil scored between “like a little” and “like moderately” (above 6); control samples and ones treated with alginate-based EC containing 0.35 or 0.65% thyme oil scored between “dislike moderately” and “indifferent” (below 6). In colour, appearance, odour, taste, texture attributes, only samples treated with alginate-based EC and EC containing 0.05% thyme oil were acceptable (above 6). Therefore, the use of EC and EO might be acceptable for further commercialization. On the other hand, incorporation of 0.35 and 0.65% thyme oil into EC affected the taste attribute scores of samples (below 4). These observations corresponded with other studies which showed that the high concentration of EO negatively affected the overall acceptability of foods. For examples, when the effects of alginate-based EC as a carrier of lemongrass on the sensory characteristics of fresh-cut melon were evaluated, the firmness score was significantly (p < 0.05) reduced by the incorporation of 0.7% lemongrass in the coating formulation (Raybaudi-Massilia, Mosqueda-Melgar, & Martin-Belloso, 2008). In another study, 0.5% lemongrass incorporated into alginate EC negatively affected the texture score and acceptability of samples (Azarakhsh et al., 2014).
Fig. 4. Effect of alginate-based EC containing thyme oil on the sensory characteristics of fresh-cut cantaloupe after 8 days of storage at 4 °C. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil. The hedonic evaluation scale was used a 9 point as follows: using the 9 point hedonic scale with 9, like very much to 1, dislike very much. For each sample, the means designated by different letters are significantly different (p < 0.05).
3.3. Quality parameters of fresh-cut cantaloupe Fig. 3a shows the changes in the respiration rate. The respiration rate drastically increased after all treatments during storage. The peeling and cutting of fresh-cut cantaloupe resulted in physiological stress of plant tissues, triggering respiratory metabolism (SalviaTrujillo, Rojas-Graü, Soliva-Fortuny, & Martní-Belloso, 2015). The respiration rate of coated samples was significantly lower than that of the uncoated samples (p < 0.05). This was probably a result of the coating acting as a barrier to gas exchange and affecting the internal atmosphere of the fruit, with less oxygen available for plant tissue respiration, leading to a reduced release of carbon dioxide from the product (Chiumarelli & Hubinger, 2012). Meanwhile, the respiration rate increased less rapidly in samples treated with alginate-based EC containing thyme oil than with samples treated with EC without thyme oil. This may be attributed to the prevention of gas diffusion by the coating and microbial inactivation by thyme oil (Salvia-Trujillo et al., 2015). The effect of the incorporation of thyme oil into alginate-based EC on the weight loss of fresh-cut cantaloupe samples was monitored during storage (Fig. 3b). The weight loss in all groups increased significantly (p < 0.05). Since fresh-cut fruits lose their integrity after peeling, cutting, slicing, and shredding, they appear to be more vulnerable to water loss than uncut fruits. The increment was lower in samples coated with alginate-based EC containing 0.05% thyme oil than that of other treatments. This was probably because EC prevented the direct exposure of the fruit to air and simultaneously inhibited the metabolic rate of fresh-cut fruits (Sarengaowa et al., 2018). On the other hand, weight loss of samples treated with alginate-based EC containing 0.35 and 0.65% thyme oil was greater than that of the control, possibly because high concentrations of thyme oil accelerated decay of fruit (Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer, 2011). These findings matched those of an earlier study involving fresh-cut apples (Sarengaowa et al., 2018). Colour is one of the critical factors determining consumer acceptance of fruit quality. All treatments reduced the L* and WI of fresh-cut cantaloupe during storage (Fig. 3c and d). Among these, L* in the control group was reduced significantly, while the coating treatments maintained their L* over 16 days. On the other hand, treatments with
4. Conclusions Alginate-based EC containing thyme oil showed increasing antibacterial activity against naturally occurring microorganisms on freshcut cantaloupe and foodborne pathogen with increasing concentrations of thyme oil. Gram-positive bacteria (LM and SA) were highly sensitive to alginate-based EC containing thyme oil. Gram-negative bacteria (ST and EC O157:H7) were moderately inhibited on fresh-cut cantaloupe. Alginate-based EC containing 0.05% thyme oil effectively assisted in maintaining cantaloupe respiration, reduced weight loss, and preserved the colour of fresh-cut cantaloupe. Therefore, alginate-based EC containing thyme oil might be used as a safe preservative for fresh-cut cantaloupe. Conflicts of interest The authors do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This study was supported by the ‘Thirteenth Five-Year Plan’ for the National Key Research and Development Program, China (Grant No. 2016YFD0400903), the National Natural Science Foundation of China, China (Grant No. 31471923), and the ‘Twelfth Five-Year Plan’ for National Science and Technology Support Program, China (Grant No. 2012BAD38B05). 7
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Food Bioscience journal homepage: www.elsevier.com/locate/fbio
Thyme oil alginate-based edible coatings inhibit growth of pathogenic microorganisms spoiling fresh-cut cantaloupe
T
Sarengaowaa,b,c, Wenzhong Hub,c,∗, Ke Fengb,c, Zhilong Xiua, Aili Jiangb,c, Ying Laob,c a
School of Bioengineering, Dalian University of Technology, Dalian, 116024, Liaoning, China College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China c Key Laboratory of Biotechnology and Bioresources Utilization, Ministry of Education, Dalian, 116600, Liaoning, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fresh-cut cantaloupe Cucumis melo L. Listeria monocytogenes Staphylococcus aureus Salmonella Typhimurium Escherichia coli O157:H7
Fresh-cut cantaloupe (Cucumis melo L.) is a popular food. However, the presence of foodborne pathogen on the cut surfaces leads to both spoilage and food poisoning of fresh-cut cantaloupe. The antibacterial effectiveness of an alginate-based edible coating (EC) containing thyme oil was evaluated against foodborne pathogens on freshcut cantaloupe samples. Cantaloupe samples were treated with alginate-based EC and alginate-based EC containing different concentrations (0, 0.05, 0.35, and 0.65%, v/v) of thyme oil. Uncoated samples served as the control. The viability of naturally occurring microorganisms and artificially inoculated foodborne pathogens on the fresh-cut cantaloupe samples, as well as cantaloupe respiration rate, weight loss, and colour, were determined every 4 days over 16 days at 4 °C. Results showed that alginate-based EC containing thyme oil effectively improved the quality of fresh-cut cantaloupe and prolonged its shelf life. The treatments inhibited the growth of naturally occurring microorganisms and artificially inoculated pathogens (Listeria monocytogenes, Staphylococcus aureus, Salmonella Typhimurium, and Escherichia coli O157:H7) on fresh-cut cantaloupe samples. Treatment with alginate-based EC containing 0.05% thyme oil did not affect the respiration, prevented weight loss, and maintained the colour and sensory characteristics of fresh-cut cantaloupe samples. Gram-negative bacteria (S. Typhimurium and E. coli O157:H7) were more resistant to alginate-based EC containing thyme oil than Gram-positive bacteria (L. monocytogenes and S. aureus). Therefore, the use of an alginate-based EC containing thyme oil might be a promising approach to increase shelf life and increase safety of fresh-cut cantaloupe.
1. Introduction The popularity and consumption of cantaloupe (Cucumis melo L.) are increasing mainly because of its sweet pulp, pleasant aroma, and recognition as a rich source of vitamins A, C, and folic acid, as well as beta-carotene (Beaulieu & Lea, 2007). Cut or sliced forms of cantaloupe potentially promote bacterial survival and growth because of the nutrient content of the juice and pulp (Ukuku, Mukhopadhyay, & Olanya, 2013). Cantaloupe contamination by foodborne pathogen has been a concern since the associated outbreaks in the United States, Australia, Canada, and other countries (CDC, 2002, 2012a, b). Listeria monocytogenes (LM), Staphylococcus aureus (SA), Salmonella Typhimurium (ST), and Escherichia coli O157:H7 (EC O157:H7) are considered as important foodborne pathogens with a significant impact on public health (Hassanain et al., 2013). LM, SA, ST, and EC O157:H7 cause hemorrhagic colitis, bloody or non-bloody diarrhea, hemolytic uremic
∗
syndrome, self-limited intestinal disease, and death in humans. A cantaloupe-related Listeria outbreak in 2011 was one of the deadliest multistate outbreaks in the United States, resulting in 147 illnesses and 33 deaths (CDC, 2012a). In 2012, a large-scale cantaloupe-related outbreak of salmonellosis caused by ST and Salmonella Newport resulted in 261 reported infections, including 94 hospitalizations and 3 deaths reported to the US Centers for Disease Control and Prevention (CDC, 2012b). In 2019, the outbreak of Salmonella infections linked to pre-cut melons caused 38 illnesses in 137 people infected in the United States (CDC, 2019). Cantaloupes were linked to an outbreak of EC O157:H7 causing 34 people to become sick, but no deaths were reported (Sivapalasingam, Friedman, Cohen, & Tauxe, 2004). SA is a major pathogen due to its enterotoxigenic strains, which are the leading cause of gastroenteritis, resulting from the consumption of enterotoxins-contaminated food (Harris, Foster, & Richards, 2002). The percentage occurrence of SA in melon was 70%, although no cases of
Corresponding author. College of Life Science, Dalian Minzu University, Dalian, 116600, Liaoning, China. E-mail address: [email protected] (W. Hu).
https://doi.org/10.1016/j.fbio.2019.100467 Received 14 April 2019; Received in revised form 16 September 2019; Accepted 16 September 2019 Available online 18 September 2019 2212-4292/ © 2019 Elsevier Ltd. All rights reserved.
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2.3. Preparation of alginate-based EC
food poisoning have been associated with SA from cantaloupe (Nwachukwu & Osuocha, 2014). These events indicate the high risk of microbial contamination with fresh-cut cantaloupe. Meanwhile, the severity of cantaloupe-linked outbreaks highlights the role of cantaloupes as a vehicle of foodborne pathogens. There is a need to maintain fruit quality and microbial safety post-cutting. Consumer demands are increasingly focusing on minimally processed and safe foods produced using natural preservatives. Essential oils (EO) are generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA, 2018). Many EO have a major role in controlling a wide range of pathogenic and spoilage bacteria associated with food (Cui et al., 2017, 2018; Scollard, McManamon, & Schmalenberger, 2016). However, the odour of EO may negatively affect food quality (Burt, 2004). One method for overcoming this problem might be the use of a combination of an edible coating (EC) and EO. EC acts as a carrier enabling the sustained release of EO to the surface of the fruit or vegetable, reducing the negative effect of EO on the flavour of fresh-cut fruits and vegetables, while maintaining the antibacterial effects (Lin, Zhu, Thangaraj, Abdel-Samie, & Cui, 2018a). EO are being incorporated into EC and films (Lin, Zhu, & Cui, 2018b). Some reports have evaluated the effects of EO, such as lemongrass oil, oregano oil, and vanillin oils, which have been incorporated into different types of EC including sodium alginate, and apple puree-alginate on fresh-cut fruits such as pineapple, and Fuji apples (Rojas-Graü et al., 2007; Azarakhsh, Osman, Ghazali, Tan, & Adzahan, 2014). Previous report showed that thyme oil has the strongest antimicrobial activity against a range of foodborne pathogens (LM, SA, ST, and EC O157:H7) among 15 other types of EO using in vitro experiments (Sarengaowa, Hu, Jiang, Xiu, & Feng, 2018). Therefore, the current study aimed at evaluating the effects of different concentrations of thyme oil incorporated into alginate-based EC on artificially inoculated pathogens, naturally occurring microorganisms, quality, and sensory attributes of fresh-cut cantaloupe during low-temperature storage.
An optimized alginate-based EC was used based on a previous study (Azarakhsh et al., 2014). The EC was prepared by mixing 1.29% (w/v) sodium alginate (food-grade, Henan Qiang Li Chemical Products Co., Ltd., Zhengzhou, Henan, China) with 1.5% (w/v) glycerine (food-grade, Procter & Gamble International Operations, Singapore) in ultrapure water from an ultrapure water machine (PURELAB Classic UF, Elga Ltd., High Wycombe, UK), and stirring at 70 °C until the solution became transparent (Azarakhsh et al., 2014). Different concentrations (0.05, 0.35 and 0.65%, v/v) of thyme oil (Ji'an Zhongxiang Natural Plant Co., Ltd., Jian, Jiangxi, China) were incorporated into the alginate-based EC. The major compounds of the thyme oil were 47.2% thymol, 20.4% p-cresol and 16.3% 2,6-dimethylphenol. The final solutions were homogenized using an Ultra Turrax T25 mixer (IKA® WERKE, Staufen, Baden-Württemberg, Germany) at 1,140 × g at room temperature (~25 °C) for 3 min. For the cross-linking reaction necessary for gel formation, a 2% (w/v) calcium chloride solution (foodgrade, Zhejiang Dacheng Calcium Co., Ltd., Quzhou, Zhejiang, China) containing 1% (w/v) ascorbic acid (food-grade, Henan Qiang Li Chemical Products) and 1% (w/v) citric acid (food-grade, Henan Qiang Li Chemical Products) was prepared. Ascorbic acid and citric acid were added into the calcium chloride solution as the antioxidant and colour fixative, respectively (González-Aguilar, Ruiz-Cruz, Cruz-Valenzuela, Rodríguez-Félix, & Wang, 2004).
2.4. Processing and packaging of fresh-cut cantaloupe Cantaloupe were washed manually with sterile water and sterilized with 75% (v/v) ethyl alcohol (Tianjin Kemiou Chemical Reagent Co., Ltd., Tianjin, China). Samples were air-dried for 10 min at 25 °C in a biosafety cabinet. The cleaned cantaloupe samples were peeled and cut into cubes (1 cm3) using a sterilized knife. Cantaloupe cubes were placed in 100 ml of the coating-forming solutions of sodium alginate and different concentrations of thyme oil for 2 min. They were subsequently immersed in 100 ml of 2% (w/v) calcium chloride solution for 2 min. Uncoated fresh-cut cantaloupe samples were used as the control. Samples (10 cubes) were packaged on polystyrene trays wrapped in PVC films (Lixian Wrap, Jinan ZhenHua Plastic Packaging Co., Ltd., Jinan, Shandong, China) for quality and sensory studies over 16 days at 4 °C. For microbiological evaluation experiments, cantaloupes were cut into 4 × 3 × 0.5 cm3 pieces (~10 g/piece) using a sterile knife. The cantaloupe pieces were placed on petri dishes and then uniformly inoculated as follows. The concentration of bacterial suspension was adjusted to ~108 cfu/ml using the McFarland standard tube (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) to standardize the inoculation amount for LM, SA, ST, EC O157:H7, respectively (Guerreiro, Gago, Faleiro, Miguel, & Antunes, 2015; Pesavento et al., 2015). Subsequently, SA, ST, and EC O157:H7 populations were individually enumerated using trypticase soy agar (TSA, Qingdao Hopebio-Technology). LM populations were enumerated using TSA with 0.6% (w/v) yeast extract (TSA-YE, Qingdao Hopebio-Technology). Suspensions of LM (8.84 log cfu/ml), SA (8.45 log cfu/ml), ST (8.06 log cfu/ml), and EC O157:H7 (8.90 log cfu/ml) were prepared to inoculate the samples. The inoculum of LM, SA, ST, and EC O157:H7 (500 μl each) was spread individually over the entire top surface (4 × 3 cm2) of cantaloupe pieces with a sterile micropipette for the challenge study. Cantaloupe pieces were then air-dried for 1 h at 25 °C in a biosafety cabinet. Then, the samples were EC-coated as described above. Uncoated fresh-cut cantaloupe samples with air drying were used as the control. Each 10 g cantaloupe piece was placed in a Stomacher bag (32 × 20 cm, Qingdao Hopebio-Technology) for storage. The experiments were done in triplicate, with samples measured after 0, 4, 8, 12, and 16 days; overall, 300 bags were analysed.
2. Materials and methods 2.1. Bacterial strains and preparation of bacterial cultures LM (CICC 21633), SA (CICC 21600), ST (CICC 21484), and EC O157:H7 (CICC 21530) were obtained from the China Centre of Industrial Culture Collection (CICC, Beijing, China). All strains except LM were incubated in trypticase soy broth (TSB, Qingdao HopebioTechnology Co., Ltd., Qingdao, Shandong, China) for 24 hat 37 °C. LM was incubated in TSB with 0.6% (w/v) yeast extract (TSB-YE, Qingdao Hopebio-Technology) using the same conditions. The cultures were centrifuged at 2,800 × g for 5 min at 4 °C using a centrifuge (Allegra X30R, Beckman Coulter Inc., Brea, CA, USA), and washed three times in 5 ml of 0.1% (w/v) peptone water (Aobox Biotechnology, Beijing, China) (Joshua, Di, Linda, Donald, & Michelle, 2013). The bacterial suspension was diluted with 10 × serial dilutions in 0.1% (w/v) peptone water to obtain the proper inoculum. Colonies were counted and populations were expressed as log cfu/ml.
2.2. Fruits Fresh cantaloupes were purchased from New-Mart supermarket in Dalian city (Liaoning, China), at commercial maturity stage. Cantaloupes were grown in open fields in Turpan city (Xinjiang, China). They were obtained on the fifth day after harvest directly from the farm. They were chosen for uniformity of maturation stage, size and absence of defects or injuries. The soluble solids content of cantaloupes was determined using a pocket refractometer (PAL-1, Atago Co., Ltd., Tokyo, Japan) and ranged between 8.7 and 9.7 °Brix. Samples were maintained for ~30 min (transportation time) at 4 °C before processing. 2
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specified time point (in g). Colour parameters of cantaloupe cubes, including L* (lightness), a* (+a* = redness, -a* = greenness), and b* (+b* = yellowness, -b* = blueness), were evaluated using a CR400 colorimeter (Konica Minolta Inc., Tokyo, Japan). The experiments were done in triplicate. Overall, 75 trays (10 cubes/tray) were measured. Colour parameters values of each cube were obtained by averaging 6 determinations in the central region of 6 surfaces. Numerical values of a* and b* parameters were used to calculate the whiteness index (WI) using Eq (6).
2.5. Sample analysis 2.5.1. Microbiological analysis Each sample was used for microbiology. The experiments were done in triplicate and 75 bags were measured in total. For the analysis, 90 ml of 0.1% peptone water was added to a Stomacher bag, and cantaloupe pieces were disrupted in a Stomacher (Interscience, Saint Nom la Breteche, France) at the high-speed setting for 1 min. Cantaloupe suspensions (0.1 ml) were plated and cultured on plate count agar (PCA) for 48 h at 37 °C for total plate counts, on potato dextrose agar (PDA) for 48–96 h at 28 °C for yeast and mould counts, on violet red bile dextrose agar (VRBDA) for 24 h at 37 °C for total coliform counts, and on Lactobacilli MRS agar for 48 h at 37 °C for Lactobacillus counts (Gómez & Salvatori, 2012; Siroli et al., 2015). All culturing media were purchased from Qingdao Hopebio-Technology Co., Ltd. The growth of naturally occurring microorganisms on fresh-cut cantaloupe samples was fit to the Gompertz model using MATLAB software (version 7.0, Mathworks Inc., Natick, MA, USA). The Gompertz model is described using Eq (1). Nt = A + C·exp{-exp[-B(t-M)]}
(1)
U = BC/e
(2)
LPD = M – 1/B
(3)
MPD = A + C
(4)
WI = 100 – [(100 – L*)2 + (a*)2 + (b*)2]1/2
2.5.4. Sensory analysis Sensory characteristics of cantaloupe cubes (1 cm3) in control, alginate-based EC, and alginate-based EC containing different concentrations (0.05, 0.35, and 0.65%, v/v) of thyme oil at 4 °C for 8 days were evaluated by regular cantaloupe consumers. Twenty individuals (10 female, 10 male, aged between 22 and 45 years) were recruited from among the students and staff of the Faculty of Food Science and Technology, Dalian Minzu University (Dalian, Liaoning, China). They were trained at the beginning of the experiment to become familiar with the characteristics of fresh-cut cantaloupe. Sensory evaluation was carried out in individual booths in a sensory laboratory. Sample presentation order was randomized and balanced among assessors. The assessors evaluated the colour, appearance, odour, taste, texture, and the overall acceptability of fresh-cut cantaloupe at room temperature (~25 °C) according to a 9-point hedonic scale test, accounting for varying degrees of appreciation, from 1 (dislike very much) to 9 (like very much). The hedonic evaluation scale was as follows: 9, like very much; 8, like a lot; 7, like moderately; 6, like slightly; 5, indifferent; 4, dislike slightly; 3, dislike moderately; 2, dislike a lot; 1, dislike very much (Harich, Maherani, Salmieri, & Lacroix, 2018; Peryam & Pilgrim, 1957). The average response was calculated for each attribute (Guerreiro, Gago, Miguel, Faleiro, & Antunes, 2016). Samples with scores below 6 were deemed unacceptable (Abdollahzadeh, Rezaei, & Hosseini, 2014).
where Nt is the natural logarithm of the microbial population at time t (log cfu/g), A is the initial cell concentration (log cfu/g), C is the difference between the maximum and initial cell concentration (log cfu/ g), B is the maximum growth rate (day−1), M is the time to reach the maximum growth rate (days), U is the maximum specific growth rate (log cfu/g/day), LPD is the lag phase duration (days), and MPD is the maximum population density (log cfu/g) and e = 2.7182. Numerical values for A, C, B, and M were used to calculate U, LPD, and MPD using Eqs (2)–(4). ST, LM, EC O157:H7, and SA, counts on fresh-cut cantaloupes were analysed on Salmonella spp. chromogenic media and Oxford agar base (both from Qingdao Hopebio-Technology), EC O157:H7 chromogenic media, and SA chromogenic media (both from Shanghai Central BioEngineering Ltd., Co., Shanghai, China). All plates were incubated at 37 °C for 24 h, and the colonies were counted using an aCOLyte colony counter (Acolyte Technologies Corp., London, UK). Microbial counts were expressed as log cfu/g.
2.5.5. Statistical analysis All experiments were done in triplicate, as independent experiments. Data were measured using the Statistical Package for the Social Sciences (SPSS, Version 14.0, IBM Corp., Armonk, NY, USA). Significance of the differences between variables was tested using oneway ANOVA (between groups) and repeated measures of ANOVA (within group). The means were compared using Duncan's multiple range test. Statistical significance was determined at p < 0.05.
2.5.2. Respiration rate determination The respiration rate was determined during storage using gas chromatography (GC-2010 Shimadzu, Kyoto, Japan) equipped with an CTR1 column (1.8 m × 3.18 mm inner diameter, 6.35 mm column thickness, stainless steel column filled with porous polymer mixture, No. A8700, Alltech Associates Inc., Deerfield, IL, USA). The gas carrier was helium at 30 ml/min and the temperature of the injector, column and the detector was set at 120, 35, 120 °C, respectively. Coated cantaloupe pieces or control samples (200 g) were placed in 400 ml tightsealed glass containers and incubated at 25 °C for 1 h. Then, 1 ml of headspace gas was withdrawn through a rubber septum of an airtight screw-cap. The respiration rate was calculated based on the carbon dioxide produced [ml CO2/(kg/h)] (Qi, Hu, Jiang, Tian, & Li, 2011).
3. Results and discussion 3.1. Analysis of naturally occurring microorganisms on fresh-cut cantaloupe The population of naturally occurring microorganisms including total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe samples treated with EC with or without thyme oil were fitted with Gompertz model (R2 > 0.98) (Fig. 1). The population considerably increased on fresh-cut cantaloupe samples among treatment groups over 16 days (p < 0.05). The population of naturally occurring microorganisms on samples treated with alginate-based EC were much lower than those on untreated control during storage. The total plate counts, total coliform counts, and yeast and mould counts on samples treated with alginate-based EC containing thyme oil were much lower than that on the alginate-based EC-treated samples (Fig. 1a, b, c). The reduction in naturally occurring microorganisms was primarily associated with the antimicrobial effect of thyme oil. In addition, lactic acid bacterial (LAB) counts on untreated controls over 16 days storage at 4 °C were below 3.00 log cfu/g (data not shown). When fresh-
2.5.3. Determination of cantaloupe weight loss and colour Samples (200 g) were placed on uncovered polystyrene trays. Samples were weighed using a digital balance (PL-2002, Mettler Toledo, Greifensee, Switzerland) during storage. The weight loss rate equation [Eq (5)] was evaluated (Chien, Sheu, & Yang, 2007): Weight loss rate (%) = [(m1 – m2)/m1] × 100
(6)
(5)
Where m1 is the initial weight (in g) and m2 is the weight at the 3
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Fig. 1. Gompertz model of naturally occurring microorganisms on fresh-cut cantaloupe coated with alginate-based EC containing thyme oil at 4 °C. (a) total plate counts; (b) total coliform counts; (c) yeast and mould counts. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil.
was obtained on uncoated fresh-cut cantaloupe. It might be from the pulp and surface of fresh-cut cantaloupe. Other studies showed initial total plate counts, total coliform counts, and yeast and mould counts was ~3.00, 4.80, and 2.00 log cfu/g on fresh-cut cantaloupe, respectively (Graça, Esteves, Nunes, Abadias, & Quintas, 2017; Oms-Oliu, Soliva-Fortuny, & Martín-Belloso, 2007). The MPD of total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe samples coated with EC containing 0.65% thyme oil were significantly reduced (p < 0.05) compared with those of coated samples, and the reduction were 2.13, 1.56, and 0.56 log cfu/g, respectively. Therefore, thyme oil had an important role in the antimicrobial activity against naturally occurring microorganisms on fresh-cut cantaloupe. The LPD of naturally occurring microorganisms on uncoated fresh-cut cantaloupe was shorter than that with other treatments. Compare with samples treated with 0.65% thyme oil, the LPD value of total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe samples coated with EC containing 0.05% thyme oil was the longest among each treatment. It might be that the cell structure was more easily damaged with 0.65% thyme oil compared with 0.05% thyme oil. The juice of cells provide a nutrient rich content for the microorganisms’ growth. Therefore, the microorganisms can grow easily on fresh-cut cantaloupe with 0.65% thyme oil at the early storage times.
cut cantaloupe samples were treated with EC or EC containing thyme oil, no LAB were detected (data not shown). The microbial limit of acceptance for fresh-cut fruits and vegetables is 6.00 log cfu/g according to the UK Institute of Food Science and Technology (IFST, 1999). The total plate count exceeded the limit of 6.00 log cfu/g for the control and EC samples at 8–9 days (Fig. 1a). On the other hand, the same standard suggests that yeasts and moulds might produce toxic metabolites if their counts exceed 6.00 log cfu/g in foods. This value was also the limit for the acceptable shelf life of fruit products (IFST, 1999). The yeast and mould population exceeded 6.00 log cfu/g on uncoated fresh-cut cantaloupe on day 10 according to the growth curve fitted with the Gompertz predictive model at 4 °C (Fig. 1c). However, the total plate counts and yeast and mould counts on samples treated with EC containing thyme oil did not exceed 6.00 log cfu/g during storage. Therefore, the incorporation of thyme oil into EC extended the shelf life of fresh-cut cantaloupe at least 16 days. Another study showed a multi-layered antimicrobial EC extended the shelf life of fresh-cut cantaloupe to only 9 days (Martiñon, Moreira, Castell-Perez, & Gomes, 2014). The extension of the shelf life might be of commercial value to the cantaloupe industry, and the use of edible antimicrobial coatings would provide additional benefits (Martiñon et al., 2014). Parameters derived from Gompertz modeling of naturally occurring microorganisms on fresh-cut cantaloupe are shown in Table 1. The A for total plate counts, total coliform counts, and yeast and mould counts 4
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Table 1 Gompertz modeling parameters of changes in total plate counts, total coliform counts, and yeast and mould counts on fresh-cut cantaloupe coated with alginate-based EC containing thyme oil over 16 days at 4 °C. Naturally Occurring Microorganisms
Total Plate Counts
Total Coliform Counts
Yeast and Mould Counts
R2
Treatment
Control EC EC + Thy EC + Thy EC + Thy Control EC EC + Thy EC + Thy EC + Thy Control EC EC + Thy EC + Thy EC + Thy
(0.05%) (0.35%) (0.65%)
(0.05%) (0.35%) (0.65%)
(0.05%) (0.35%) (0.65%)
0.9951 0.9960 0.9998 0.9997 0.9996 0.9981 0.9995 0.9996 0.9992 0.9977 0.9880 0.9952 0.9963 0.9947 0.9937
RMSE
0.1827 0.1736 0.0301 0.0338 0.0337 0.1210 0.0540 0.0393 0.0466 0.0756 0.2798 0.1019 0.0813 0.0865 0.0921
SSE
0.1001 0.0904 0.0027 0.0034 0.0034 0.0439 0.0088 0.0046 0.0065 0.0017 0.2349 0.0312 0.0198 0.0224 0.0254
Gompertz Parametersα A
C
U
LPD
MPD
1.95 1.90 1.90 1.85 1.85 1.88 1.80 1.78 1.70 1.70 1.75 1.67 1.60 1.48 1.48
5.24 5.07 3.59 3.35 2.99 5.23 4.44 3.55 3.00 2.98 4.99 2.70 2.48 2.39 2.33
0.65 0.77 0.94 0.88 0.79 0.84 0.86 0.94 0.73 0.46 0.62 0.51 0.38 0.28 0.27
1.41 3.66 6.32 6.19 5.97 5.09 6.61 6.78 6.60 5.84 2.33 3.77 3.85 2.84 2.99
7.20 6.97 5.49 5.20 4.84 7.11 6.24 5.33 4.69 4.68 6.75 4.37 4.08 3.87 3.81
αData obtained from the Gompertz model; Control, uncoated; EC, coated without thyme oil; Thy, thyme oil; RMSE, root mean squared error; SSE, the sum of squares due to error; The parameters are as described in Section 2.5.1.
Fig. 2. Reduction in foodborne pathogen on fresh-cut cantaloupe coated with alginate-based EC containing thyme oil at 4 °C. (a) LM; (b) SA; (c) ST; (d) EC O157:H7. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil. Means designated with the same letters (lowercase, among different treatments at the same time point; uppercase, for the same treatment after different storage times) are not significantly different (p ≥ 0.05).
5
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Fig. 3. Changes in quality parameters of fresh-cut cantaloupe coated with alginate-based EC containing thyme oil at 4 °C. (a) respiration rate; (b) weight loss; (c) lightness; (d) WI. The data are the means ± SD. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil. Means designated by the same letters (lowercase, among different treatments at the same time point; uppercase, for the same treatment after different storage times) are not significantly different (p ≥ 0.05).
fresh-cut melon treated with thyme EO (0.15 mg/ml) alone was 1.00 log cfu/g during day 2 at 8 °C and this effect was no longer observed by day 7 of storage compared to the control (Scollard et al., 2016). It showed further that thyme oil incorporated into EC maintained the antibacterial effect and enhanced safety of fresh-cut cantaloupe during shelf-life compared with thyme oil alone treatment. The reduction in ST, EC O157:H7, LM, SA on fresh-cut cantaloupe treated with alginate-based EC containing 0.65% thyme oil is 2.35, 1.68, 3.29 and 3.76 log cfu/g, respectively, at day 16 compared with the control. It was obviously that Gram-negative bacteria (ST and EC O157:H7) are more resistant to EC containing thyme oil than Grampositive bacteria (LM and SA). It might be that the cell wall of Grampositive bacteria which consists mainly of peptidoglycan allows hydrophobic molecules to easily penetrate the cells and act on both the cell wall and within the cytoplasm (Nazzaro, Fratianni, Martino, Coppola, & Feo, 2013). The hydrophobic nature of thymol and carvacrol may be important for their antibacterial effects (Liolios, Gortzi, Lalas, Tsaknis, & Chinou, 2009). However, the cell walls of Gram-negative bacteria are more complex. The cell wall of Gram-negative bacteria is covered by an outer membrane that contains various proteins as well as lipopolysaccharides. Their characteristics may be more resistant to EO and other hydrophobic natural extracts with antimicrobial activity (Nazzaro et al., 2013; D'Amato, Serio, López, & Paparella, 2018).
3.2. Inhibition of foodborne pathogen on fresh-cut cantaloupe Fig. 2 shows the effect of alginate-based EC with or without thyme oil on LM, SA, ST, and EC O157:H7 inoculated on fresh-cut cantaloupe samples. The alginate-based EC containing thyme oil showed the best antibacterial efficacy against LM, SA, ST, and EC O157:H7 at each time point. Moreover, a significant reduction in the LM, SA, ST, and EC O157:H7 count was noted with increasing concentration of thyme oil during storage. Other studies have also shown thyme oil inhibited the growth of foodborne pathogen on food matrixes including fresh-cut lettuce, cantaloupe melon, pineapple, beef, chicken and cheese (Govaris, Botsoglou, Sergelidis, & Chatzopoulou, 2011; Lin et al., 2018b; Scollard et al., 2016; Solomakos, Govaris, Koidis, & Botsoglou, 2008). Carvacrol and thymol as the major components of thyme oil have strong antibacterial effect against LM, SA, ST, and EC O157:H7 (Sakkas & Papadopoulou, 2017). The combination of thyme oil and alginate-based EC maintained the antibacterial effect against LM, SA, ST, and EC O157:H7 on fresh-cut cantaloupe during storage of 16 days. LM, SA, ST, and EC O157:H7 showed a significant reduction of 0.72, 3.03, 2.36 and 1.50 log cfu/g on fresh-cut cantaloupe treated with EC containing 0.65% thyme oil from day 0 to day 16 (Fig. 2a, b, 2c, 2d). It might be the combination of an EC and EO avoided the depletion of antimicrobials in food and achieved sustained release of EO on the surface of fruits and vegetables (Chong, Lai, & Yang, 2015). However, in another study, the reduction of LM on 6
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EC containing thyme oil did not significantly affect L* (p > 0.05). WI can reflect the extent of translucency due to physiological disorders of fresh-cut cantaloupe (Aguayo, Escalona, & Rtés, 2004; Oms-Oliu et al., 2007). The WI value of fresh-cut cantaloupe treated with EC containing 0.05% thyme oil was higher than that on other treatment groups. It showed that the combination of 0.05% thyme oil and EC maintained the colour of fresh-cut cantaloupe during storage time. 3.4. Sensory analysis of fresh-cut cantaloupe The strong odours and flavours of EO may affect the organoleptic properties of foods (Lee, Kim, Kim, Beuchat, & Ryu, 2018). Sensory evaluation was done after 8 days of storage because the uncoated samples reached the limit of acceptance (6.00 log cfu/g) after that time point (Azarakhsh et al., 2014). Sensory evaluation of differently treated fresh-cut cantaloupe samples after 8 days of storage is shown in Fig. 4. According to the overall acceptability score, samples coated with alginate-based EC and EC containing 0.05% thyme oil scored between “like a little” and “like moderately” (above 6); control samples and ones treated with alginate-based EC containing 0.35 or 0.65% thyme oil scored between “dislike moderately” and “indifferent” (below 6). In colour, appearance, odour, taste, texture attributes, only samples treated with alginate-based EC and EC containing 0.05% thyme oil were acceptable (above 6). Therefore, the use of EC and EO might be acceptable for further commercialization. On the other hand, incorporation of 0.35 and 0.65% thyme oil into EC affected the taste attribute scores of samples (below 4). These observations corresponded with other studies which showed that the high concentration of EO negatively affected the overall acceptability of foods. For examples, when the effects of alginate-based EC as a carrier of lemongrass on the sensory characteristics of fresh-cut melon were evaluated, the firmness score was significantly (p < 0.05) reduced by the incorporation of 0.7% lemongrass in the coating formulation (Raybaudi-Massilia, Mosqueda-Melgar, & Martin-Belloso, 2008). In another study, 0.5% lemongrass incorporated into alginate EC negatively affected the texture score and acceptability of samples (Azarakhsh et al., 2014).
Fig. 4. Effect of alginate-based EC containing thyme oil on the sensory characteristics of fresh-cut cantaloupe after 8 days of storage at 4 °C. Control: uncoated; EC: coated without thyme oil; Thy: thyme oil. The hedonic evaluation scale was used a 9 point as follows: using the 9 point hedonic scale with 9, like very much to 1, dislike very much. For each sample, the means designated by different letters are significantly different (p < 0.05).
3.3. Quality parameters of fresh-cut cantaloupe Fig. 3a shows the changes in the respiration rate. The respiration rate drastically increased after all treatments during storage. The peeling and cutting of fresh-cut cantaloupe resulted in physiological stress of plant tissues, triggering respiratory metabolism (SalviaTrujillo, Rojas-Graü, Soliva-Fortuny, & Martní-Belloso, 2015). The respiration rate of coated samples was significantly lower than that of the uncoated samples (p < 0.05). This was probably a result of the coating acting as a barrier to gas exchange and affecting the internal atmosphere of the fruit, with less oxygen available for plant tissue respiration, leading to a reduced release of carbon dioxide from the product (Chiumarelli & Hubinger, 2012). Meanwhile, the respiration rate increased less rapidly in samples treated with alginate-based EC containing thyme oil than with samples treated with EC without thyme oil. This may be attributed to the prevention of gas diffusion by the coating and microbial inactivation by thyme oil (Salvia-Trujillo et al., 2015). The effect of the incorporation of thyme oil into alginate-based EC on the weight loss of fresh-cut cantaloupe samples was monitored during storage (Fig. 3b). The weight loss in all groups increased significantly (p < 0.05). Since fresh-cut fruits lose their integrity after peeling, cutting, slicing, and shredding, they appear to be more vulnerable to water loss than uncut fruits. The increment was lower in samples coated with alginate-based EC containing 0.05% thyme oil than that of other treatments. This was probably because EC prevented the direct exposure of the fruit to air and simultaneously inhibited the metabolic rate of fresh-cut fruits (Sarengaowa et al., 2018). On the other hand, weight loss of samples treated with alginate-based EC containing 0.35 and 0.65% thyme oil was greater than that of the control, possibly because high concentrations of thyme oil accelerated decay of fruit (Sánchez-González, Vargas, González-Martínez, Chiralt, & Cháfer, 2011). These findings matched those of an earlier study involving fresh-cut apples (Sarengaowa et al., 2018). Colour is one of the critical factors determining consumer acceptance of fruit quality. All treatments reduced the L* and WI of fresh-cut cantaloupe during storage (Fig. 3c and d). Among these, L* in the control group was reduced significantly, while the coating treatments maintained their L* over 16 days. On the other hand, treatments with
4. Conclusions Alginate-based EC containing thyme oil showed increasing antibacterial activity against naturally occurring microorganisms on freshcut cantaloupe and foodborne pathogen with increasing concentrations of thyme oil. Gram-positive bacteria (LM and SA) were highly sensitive to alginate-based EC containing thyme oil. Gram-negative bacteria (ST and EC O157:H7) were moderately inhibited on fresh-cut cantaloupe. Alginate-based EC containing 0.05% thyme oil effectively assisted in maintaining cantaloupe respiration, reduced weight loss, and preserved the colour of fresh-cut cantaloupe. Therefore, alginate-based EC containing thyme oil might be used as a safe preservative for fresh-cut cantaloupe. Conflicts of interest The authors do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This study was supported by the ‘Thirteenth Five-Year Plan’ for the National Key Research and Development Program, China (Grant No. 2016YFD0400903), the National Natural Science Foundation of China, China (Grant No. 31471923), and the ‘Twelfth Five-Year Plan’ for National Science and Technology Support Program, China (Grant No. 2012BAD38B05). 7
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