Edible coating containing carvacrol for postharvest microbiological
conservation of tomato
Revestimento comestível contendo carvacrol para conservação microbiológica
pós-colheita de tomate
DOI:10.34117/bjdv6n1-344
Recebimento dos originais: 30/11/2019 Aceitação para publicação: 30/01/2020
Cláudia Moreira Santa Catharina
Graduanda em Engenharia de Alimentos pela Universidade Tecnológica Federal do Paraná Universidade Tecnológica Federal do Paraná – Câmpus Francisco Beltrão
Linha Santa Bárbara, s/n, interior, CP 135, Francisco Beltrão-PR, Brasil E-mail: cmsc1996@gmail.com
Felipe Guilerme Brunetto Bretschneider
Graduando em Engenharia de Alimentos pela Universidade Tecnológica Federal do Paraná Universidade Tecnológica Federal do Paraná – Câmpus Francisco Beltrão
Linha Santa Bárbara, s/n, interior, CP 135, Francisco Beltrão-PR, Brasil E-mail: felipebretschneider@gamil.com
Bianca Piva
Graduanda em Engenharia de Alimentos pela Universidade Tecnológica Federal do Paraná Universidade Tecnológica Federal do Paraná – Câmpus Francisco Beltrão
Linha Santa Bárbara, s/n, interior, CP 135, Francisco Beltrão-PR, Brasil E-mail: bianca.piva2011@gmail.com
Cleusa Ines Weber
Doutora em Ciência de Alimentos pela Universidade Estadual de Londrina Universidade Tecnológica Federal do Paraná – Câmpus Francisco Beltrão
Linha Santa Bárbara, s/n, interior, CP 135, Francisco Beltrão-PR, Brasil E-mail: cleusaines@utfpr.edu.br
Franciélly Stadler
Mestre em Tecnologia de Alimentos pela Universidade Tecnológica Federal do Paraná Universidade Tecnológica Federal do Paraná – Câmpus Londrina
Avenida dos Pioneiros, 3131, Jardim Morumbi, Londrina-PR, Brasil E-mail: stadler.alipr@gmail.br
Ana Paula Romio
Doutora em Engenharia Química pela Universidade Federal de Santa Catarina Universidade Tecnológica Federal do Paraná – Câmpus Francisco Beltrão
Linha Santa Bárbara, s/n, interior, CP 135, Francisco Beltrão-PR, Brasil E-mail: anaromio@utfpr.edu.br
Alessandra Machado-Lunkes
Doutora em Química pela Universidade Federal de Santa Maria Universidade Tecnológica Federal do Paraná – Câmpus Francisco Beltrão
Linha Santa Bárbara, s/n, interior, CP 135, Francisco Beltrão-PR, Brasil E-mail: amachado@utfpr.edu.br
ABSTRACT
Tomato is cultivated in several countries and has a significant participation in world food production. This culture suffers a huge loss in the postharvest period due to fungal disease. To improve the microbiological preservation of tomatoes in postharvest, the aim of work was investigated the antifungal activity of cassava starch/gelatin-based edible coating containing carvacrol against A. alternata using in vitro and in vivo (cherry tomato) analysis. Carvacrol showed fungal inhibitory effect (MIC = 563 µg.mL-1) and edible coating incorporated with 0.60 g of carvacrol per gram of coating solution was the most effective in inhibiting the growth of A. alternata in vivo analysis. These results suggest that the application of edible coating containing carvacrol could be used as alternative for the reduction of microbiological deterioration of tomato in the postharvest period.
Keywords: Antimicrobial coating; carvacrol; postharvest; grape tomato.
RESUMO
O tomate é cultivado em vários países e tem uma participação significativa na produção mundial de alimentos. Essa cultura sofre uma enorme perda no período pós-colheita devido a doenças fúngicas. Para melhorar a preservação microbiológica do tomate em pós-colheita, o objetivo do trabalho foi investigar a atividade antifúngica do revestimento comestível à base de amido de mandioca / gelatina contendo carvacrol contra A. alternata usando análises in vitro e in vivo (tomate cereja). O carvacrol apresentou efeito inibitório fúngico (CIM = 563 µg.mL-1) e o revestimento comestível incorporado com 0,60 g de carvacrol por grama de solução de revestimento foi o mais eficaz na inibição do crescimento de A. alternata na análise in vivo. Esses resultados sugerem que a aplicação de revestimento comestível contendo carvacrol poderia ser utilizada como alternativa para a redução da deterioração microbiológica do tomate no período pós-colheita.
Palavras-chave: Revestimento antimicrobiano; carvacrol; pós-colheita; tomate uva.
1 INTRODUCTION
Tomato requires a large amount of nutrients to develop and, its production and the risk of losing tomatoes in postharvest are high (MORETTI et al., 2012). In Brazil there are records of loss around to 40% of the production of tomatoes and fungal contamination is one of the main causes (SEBRAE, 2015).
Tomato loss occurs mainly in the postharvest period, similar to observe for other fruits and vegetables (ALVARENGA, 2013). The Brazilian Agricultural Research Corporation (EMBRAPA) has been studied different preharvest and postharvest treatments to reduce losses of horticultural crops (MORETTI et al., 2006). Tomato is susceptible to postharvest diseases mainly caused by the fungi
Alternaria alternata and Botrytis cinerea (AHMED; SIPES; ALVAREZ, 2017). Specifically, A alternata causes the black spot commonly in the peduncular region of the fruit (TOFOLI,
Postharvest tomato loss has been controlled with the use of edible coating containing carvacrol to increase the microbiological conservation of the fruit (FAKHOURI et al., 2015; MARTÍNEZ-ROMERO et al., 2006; TASTAN et al., 2006).
In fact, carvacrol is a natural antimicrobial extracted from aromatic plants mainly from oregano and thyme that acts as a preservative being qualified as safe by the Food and Drugs Administration (FDA) (SUNTRES; COCCIMIGLIO; ALIPOUR, 2015). Previous research in group of this works showed that cassava starch/gelatin-based edible coating containing carvacrol to inhibit the growth of Colletotrichum sp in strawberries (BRETSCHNEIDER et al., 2019, ROMIO et al., 2017).
In this context, in order to improve the microbiological conservation of tomato during postharvest, the objective of this work was to determine effect of cassava starch/gelatin-based edible coating with antifungal properties on the development of postharvest disease caused by A. alternata.
2 MATERIAL AND METHODS
Grape tomato (Sweet Grape®) fruits from a local market of Francisco Beltrão-PR were selected based on homogeneous size, maturity, color, and absence of injuries.
Coating solution was prepared according to Romio et al. (2017) using cassava starch (Pinduca Alimentos, Brazil), gelatin type A (Gelita, Brazil), glycerol (Synth, Brazil) and carvacrol (Sigma-Aldrich, United States), Miglyol 812 (Sasol, Germany) and lecithin (Alfa Aesar, United States). Aseptic conditions and sterile water were applied to produce the active coating solution (BRETSCHNEIDER et al., 2019). For antifungal analysis, a blank cassava starch/gelatin-based edible coating was prepared and three active edible coating (0.15 mg carvacrol. g-1 coating solution, 0.3 mg carvacrol. g-1 coating solution e 0.6 mg carvacrol. g-1 coating solution).
In vitro antifungal activity was evaluated by liquid growth inhibition assay in 96-well
microplate, as described by Fieira et al. (2013), with some modifications (BRETSCHNEIDER et al., 2019). Carvacrol was tested at concentrations of 94.6 µg. mL-1 to 45.1 mg. mL-1 using sterile ethanol 70% (v/v) as a diluent. Briefly, 10 µL of carvacrol, blank edible coating or active edible coating was added to 80 µL of A. alternata (CCT 2816) (1 x 105 spores. mL-1) in potato dextrose (PD) broth, and diluted in sterile distilled water to a final volume of 100 µL. The positive control was thiabendazole (Sigma-Aldrich, United States) at concentration of 0.1 µg. mL-1 to 32.2 mg. mL-1. For negative
control, carvacrol, edible coating or thiabendazole were replaced by sterile water and ethanol 70% (v/v). Fungal growth was evaluated using 2,3,5-tetrazolium chloride (TTC). The minimal inhibitory concentration (MIC) was determined, following incubation at 25 ºC for 96 h. The MIC is the lowest concentration of sample preventing visible antifungal growth. To determine the minimum fungicidal
concentration (MFC), the total content of the well that did not show fungal growth was removed and inoculated on agar (spread plate method) in the ideal growth condition of A. alternata. After 7 days of incubation the colonies were counted. MFC was determined as the minimum concentration in which there was no growth in plaque.
For the in vivo antifungal assay, a total of 165 grape tomato were superficially disinfected by dipping the fruit in a sodium hypochlorite solution (2.5 % active chlorine) for 15 min, rinsing with sterile distilled water, and drying them at room temperature. After draining, the grape tomatoes were immersed in coating solution for 2 minutes and drained again. Five treatments were prepared to in
vivo assay (Table 1). One wound (2 mm wide by 3 mm deep) was made on each fruit. This wound
was inoculated with A. alternata by placing 10 µL of a spore suspension containing 1 x 105 spores. mL-1. The fruit were placed into polypropylene plastic packages (previously disinfected with 70% ethanol) and incubated at 25 ºC. The effect of edible coatings on disease incidence was evaluated daily for 5 days. Disease incidence (alternaria black spot) data was expressed as the percentage of fruit showing rot symptoms out of the total number of fruits in each treatment. Each treatment included three replicates of 15 fruit each. In vivo antifungal data were subjected analysis of variance (ANOVA) and the Tukey test was used to compare the mean values in different treatment and storage intervals (p < 0.05) using the Statistica version 7.0 program.
Table 01 – Cassava starch/gelatin-based edible coating and control treatment used in vivo assay.
Code Treatment
A 0.6 mg carvacrol. g-1 coating solution
B 0.0 mg carvacrol. g-1 coating solution (blank)
C control (uncoated grape tomato)
D 0.3 mg carvacrol. g-1 coating solution E 0.15 mg carvacrol. g-1 coating solution
3 RESULTS AND DISCUSS
Antifungal activity of carvacrol and thiabendazole are described in Table 2. The results showed that carvacrol exhibited antifungal activity against A. alternata and thiabendazole (commercial fungicide) is the most effective among compounds tested. On the other hand, Abbaszadeh et al. (2014) demonstrated that carvacrol exhibited antifungal activity against A.
alternata, at a concentration of 350 µg. mL-1. This difference can be related to multiple factors that
microbial cells are exposed to, such as microbial concentration, the origin of the antimicrobial substance, the resistance of microorganisms, among other factors (SINIGAGLIA et al., 1998).
Table 2- Minimal inhibitory concentration (MIC) and minimal fungicidal concentraction (MFC) carvacrol e
tiabendazol against Alternaria alternata
Compound MIC (µg. mL-1) MFC (µg. mL-1)
Carvacrol 563 563
Thiabendazole 100.6 >3220
Thus, as the MIC and MFC results of the in vitro antifungal analysis agaisnt A. alternata were 0.3 mg carvacrol. g-1 coating solution and 0.6 mg carvacrol. g-1 coating solution, respectively, it can be said that carvacrol is responsible for the expression of antifungal activity. Similar results were related by Bretschneider et al., (2019) in antifungal analysis using Colletrochium sp. In other works, quinoa protein/chitosan edible film with thymol (carvacrol isomer) nanoparticles was able to inhibit micellar growth and sporulation of one of the decaying tomato fungi (Botrytis cinerea) (ROBLEDO
et al., 2018; MEDINA et al., 2019). Still, carvacrol packaging film based on polyamide 6 exhibit in vitro antifungal efficacy against A. alternata (SHEMESH et al., 2016).
In order to confirm the promising results of in vitro analysis and literature data, an antifungal assay (in vivo analysis) was performed to determine which active coating maintains antifungal properties in tomatoes.
As expected, the fruit deterioration increased over the days for all treatments. The control (uncoated tomato) was fully deteriorated on the third day indicating that the coating has protective action against physical damage to fruits and thus decreasing the incidence of fungal disease (Table 02). Fagundes et al. (2015) described that hydroxypropyl methylcellulose-beeswax edible coatings reduced alternaria black spot incidence on cherry tomatoes.
Table 02 – Disease incidence (% )╪ of control and coated tomatoes
Day Treatment # A B C D E 1 0.00 Ca ± 0.00 0.00 Da ± 0.00 0.00 Da ± 0.00 0.00 Ca ± 0.00 0.00 Da ± 0.00 2 9.09 Cc ± 9.09 27.27 Cb ± 0.00 51.51 Ca ± 5.24 9.09 Cc ± 9.09 39.39 Cab ± 5.25 3 48.48 Bc ± 5.24 78.78 Bb ± 10.49 100.00 Ba ± 0.00 63.63 Bbc ± 15.74 78.78 Bb ± 5.25 4 100.00 Aa ± 0.00 100.00 Aa ± 0.00 100.00 Aa ± 0.00 100.00 Aa ± 0.00 100.00 Aa ± 0.00
╪ Results expressed as mean ± standard deviation. Values with different letters are significantly different (P
< 0.05). Capital letters in the same column = differences to the same treatment. Lowercase letters in the same row = differences between the treatments during the same day (Tukey test).
There was no statistically significant difference between treatment B (0.0 mg carvacrol. g-1 coating solution) and treatment E (0.15 mg carvacrol. g-1 coating solution), so it can be said that the concentration of 0.15 mg carvacrol. g-1 coating solution has no significant effect against A. alternata.
However, the coating is a significant factor of difference, because treatment E was more effective than treatment C (coating without carvacrol).
It can be said that the most effective treatment was treatment D (0.3 mg carvacrol. g-1 coating solution) because when compared to D and A (0.6 mg carvacrol. g-1 coating solution) there was no
statistically significant difference. Interestingly, the lower the concentration of carvacrol in the coating solution, the greater the sensory acceptance of the coated fruits, as it is known that carvacrol alters food aroma and taste. (PASSARINHO et al., 2014).
After three days, treatment B (uncoated tomato) showed 100% disease incidence, whereas the coated tomatoes showed a reduction in the disease manifestation characteristic of A. alternate. The results are consistent with those reported in other studies using edible coating containing carvacrol for postharvest tomato preservation. (BARRETO et al., 2016; FAGUNDES et al., 2015; SHEMESH
et al., 2016). The same active coating was able to control the growth of Colletotricum sp. In
strawberries (0.3 mg carvacrol. g-1 coating solution) (BRETSCHNEIDER et al., 2019).
4 CONCLUSION
The cassava starch/gelatin-based edible coating containing carvacrol is able to inhibit the growth of A. alternata at a 0.3 mg carvacrol. g-1 coating solution for both in vivo and in vitro antifungal assay. Results indicate active edible coating are a viable alternative to extend shelf life of tomato in postharvest. Also, as the concentration of carvacrol is low, can have a minimal impact on the sensory attributes of tomato. The next steps of this work are to evaluate the sensory indicators of tomatoes in the postharvest period with this edible coating.
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