Use of chitosan-based edible coatings in combination with other natural compounds, to control Rhizopus stolonifer and Escherichia coli DH5a in fresh tomatoes Margarita Ramos-García a , Elsa Bosquez-Molina b , Jesús Hernández-Romano c , Guadalupe Zavala- Padilla d , Eduardo Terrés-Rojas e , Irán Alia-Tejacal f , Laura Barrera-Necha a , Mónica Hernández-López a , Silvia Bautista-Baños a, * a Instituto Politécnico Nacional, Centro de Desarrollo de Productos Bióticos, Carr. Yautepec-Jojutla km. 6, Col. San Isidro, CEPROBI 8, Morelos 62531, Mexico b Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco 186, Michoacán y la Purísima, Col. Vicentina, Mexico D.F. 09340, Mexico c Universidad Politécnica del Estado de Morelos, Boulevard Cuauhnahuac 556, Lomas del Texcal, Jiutepec, Morelos 62550, Mexico d Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico, Av. Universidad 1001, Chamilpa Cuernavaca, Morelos 62209, Mexico e Instituto Mexicano del Petróleo, Eje Central Lázaro Cárdenas 152, Col. San Bartolo Atehuacán, Gustavo A. Madero, Mexico D.F. 07730, Mexico f Facultad de Ciencias Agropecuarias, Universidad Autónoma del Estado de Morelos, Av. Universidad Núm.1001, Chamilpa, Cuernavaca, Morelos 62209, Mexico article info Article history: Received 5 October 2011 Received in revised form 8 February 2012 Accepted 19 February 2012 Keywords: Lime essential oil Beeswax Oleic acid Lycopersicon esculentum Food-borne pathogens abstract During storage of tomatoes, Rhizopus stolonifer rapidly spreads towards adjacent fruits causing severe economic losses while Escherichia coli may cause serious even life threatening diseases. Chitosan-based materials can be used as edible films or coatings to avoid water loss and microbial spoilage. Waxes and essential oils may also be considered for use as antimicrobial agents in chitosan coating. In this study, various chitosan-based formulations (1%) mixed with beeswax (0.1%), oleic acid (1.0%), and lime or thyme essential oil (0.1%) were tested on tomato at three different maturity stages to control R. stolonifer and E. coli DH5a at storage temperatures of 12  C and 25  C. Control fruit were only dipped in water. Overall, fruit were wounded, coated and inoculated. Once the coatings were applied, 20 ml of R. stolonifer spore suspension at 10 5 spores ml 1 concentration and 35 ml of bacterial solution of E. coli DH5a, at 10 5 cfu ml 1 , concentration, were dispensed over the wounded surface. Experiments were carried out in vitro, at a small scale and at semi commercial level. Overall, the protection effect of coating applications was better against E. coli DH5a than R. stolonifer . For in vitro experiments the best coatings was that of chi- tosan (1%) þ beeswax (0.1%) þ lime essential oil (0.1%) since no growth of R. stolonifer and E. coli DH5a took place. Other coating that stopped R. stolonifer growth was that of chitosan (1%) þ oleic acid (1%) þ lime essential oil (0.1%) while for E. coli DH5a were chitosan (1%) þ beeswax (0.1%) þ thyme essential oil (1%) and chitosan (1%) þ beeswax (0.1%). Observation with an electronic scanning micro- scope showed distorted mycelia and no development of R. stolonifer sporangiospores, and no growth of E. coli DH5a when both microorganisms were grown on the formulation of chitosan (1%) þ beeswax (0.1%) þ lime essential oil (0.1%). For E. coli DH5a, this same formulation applied on tomatoes at a small scale and in the semi commercial level completely controlled E. coli DH5a at both storage temperatures. The application of chitosan-based edible coating containing beeswax and lime essential oil is promising to follow since it is an environmentally-friendly alternative to control this important pathogenic microorganism. Export tomato producers might benefit from this nonchemical alternative. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction High quantities of tomato fruit are produced in Mexico, being one of the country’s most important horticultural products for export purposes. Mexico’s tomato exports to the United States and Canada make up 19.7% of world tomato exports. According to the United Nations International Trade Centre, for example, Mexican tomato is the world’s 10th fresh vegetable export (Anonymous, 2010). During field production, tomatoes may be affected by microbial contamination (Serna et al., 2004; Delgado et al., 2010). Among others, the use of irrigation water, soil, field tools and containers are major sources of fungi and food-borne pathogens. In * Corresponding author. E-mail address: sbautis@ipn.mx (S. Bautista-Baños). Contents lists available at SciVerse ScienceDirect Crop Protection journal homepage: www.elsevier.com/locate/cropro 0261-2194/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.cropro.2012.02.016 Crop Protection 38 (2012) 1e6