See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/327971201 A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive Coatings Article in International Journal of Green Energy · January 2018 CITATIONS READS 2 320 5 authors, including: Diana Elizabeth Nieto-Oropeza Jorge Aguirre Autonomous University of Coahuila Autonomous University of Coahuila 3 PUBLICATIONS 121 CITATIONS 22 PUBLICATIONS 276 CITATIONS SEE PROFILE SEE PROFILE Raul Rodriguez Leonardo Sepúlveda universidad autonoma de caohuila Autonomous University of Coahuila 410 PUBLICATIONS 6,957 CITATIONS 91 PUBLICATIONS 1,077 CITATIONS SEE PROFILE All content following this page was uploaded by Leonardo Sepúlveda on 30 September 2018. The user has requested enhancement of the downloaded file. SEE PROFILE International Journal of Green Technology, 2018, 4, 24-28 24 A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive Coatings Diana Nieto-Oropeza, Jorge Aguirre-Joya, Raúl Rodríguez Herrera, Leonardo SepúlvedaTorre and Cristóbal Noé Aguilar* Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280 Coahuila, México Abstract: Bioactive films and coatings have been used as carriers for food additives such as antioxidant or antimicrobial agents. Larrea tridentata polyphenols and Aloe vera gel have demonstrated to act as efficient antimicrobial agents on food systems and can be incorporated into a coating matrix. In this research, bioactive coatings were prepared on a candelilla/pectin-based formulation. Bioactive coating antifungal activity against necrotic pathogenic fungi (Botritis cinerea and Fusarium oxysporum) was investigated by determining fungal radial growth, and the best in vitro fungicide concentration (MIC50) of Larrea tridentata polyphenols was determined. The highest fungal growth control was observed on B. cinerea (88%). Bioactive coatings presented MIC50 of 558 and 612 mg/L for F. oxysporum and B. cinerea, respectively. Candelilla wax-pectin-Aloe vera bioactive coatings incorporated with L. tridentata polyphenols inhibited the growth of necrotic pathogenic fungi and can be useful for preservation of avocado and increase of its shelf life. After all, avocado (Persea americana Mill.) is a susceptible fruit because of extreme environment conditions and invasion of microorganisms which may induce a negative reaction on quality. Technology of bioactive coatings is an alternative for extend fruit shelf-life and keeping organoleptic characteristics of Avocado cv. Hass. Keywords: Avocado, bioactive coating, shelf-life quality, necrotic fungi. 1. INTRODUCTION Persea Americana Mill. (Avocado) is a food with origin in Central America and South of Mexico [1]. This fruit has high nutritional value being its principal compounds: ascorbic acid, carotenoids, phenols, palmitic, oleic, and linoleic acids, A, B, C, and E vitamins, and fiber [2, 3]. The most common and commercial cultivar is Hass, a cross between Guatemalan and Mexican races [4]. The principal quality parameter for avocado commercialization is maturity, because fruit has large harvest periods and only maturates when it is cut from the tree [2]. Worldwide, the principal producer of avocado cv. Hass is Mexico [5] with 30% of global production, which is mainly sold to USA, France, Japan and Canada. In 2017, Mexico produced 1,997,629 tons of avocado [6]. Nevertheless, its commercialization and global distribution stills facing some problems such as: fruit short shelf life, mediated mainly by two factors; a natural accelerated respiration task and phytopathogenic fungi spoilage [7]. Average time of avocado shelf life at room temperature is between 5 to 7 days and 2 to 4 weeks under optimum conditions of temperature and relative humidity [5]. Some methods for disease control are: fungicides, cold treatment and careful handling to avoid fruit * Address correspondence to this author at the Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280 Coahuila, México; Tel: +52844 4155752, 45161238; Fax: *52 844 4159534; E-mail: [email protected] damage [8]. However, Botrytis cinerea and Fusarium oxysporum are common necrotic pathogens found under field conditions and can damage to agricultural commodities, decreasing crop quality and production [9]. As it was described previously [10], the main factor responsible for crop losses around the world is microbial deterioration. A green alternative to control avocado spoilage is using natural based coatings added with antimicrobial compounds [11]. Natural coatings are an easy and environmental friendly technology, in this case, coating cover the food with fine films (0.3 mm or less) which can regulating vapor and carbon dioxide permeability, reducing food quality losses and extending shelf life [12, 13]. Natural bioactive coatings represent a primary packaging that helps to maintain moisture and firmness [14] and are designed to intentionally interact with food and/or the surrounding environment [15]. Some reports [16] indicated that most used biopolymers to manufacture bioactive coatings and films are proteins, lipids and saccharides. Coatings are applied over the food in a liquid form, then it is dried on the food surface to make a solid and continuous barrier [17]. With the aim to improve, final characteristics of packaging, such as moisture retention and gas migration control, most authors have focused on composite or multicomponent coatings [18]. One of the most green promising technologies for traditional packaging are the bioactive coatings, where active compounds are added to improve characteristics of E-ISSN-2414-2077 © 2018 International Journal of Green Technology A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive Coatings packaging such as polyphenols to reduce oxidation in food and/or to control spoilage as antimicrobial agents [19]. In the present study, candelilla (Euphorbia antisiphilityca Zucc.) wax (CAS N° 8006-44-8) was used as lipid source, since it has been reported as an efficient ingredient for edible and bioactive coatings, specially to prolong quality and shelf life of fruits and vegetables [20-22]. Candelilla wax is an edible nonrestrictive ingredient approved by FDA, widely used in the food and candy industries. Pectin was employed as biopolymer for coating since it was recognized as GRAS (CAS N° 9000-69-5), and has capability of stabilizing all ingredients and is used by food industries, without limits other than current good manufacturing practices [5], with the intention to make a handle film glycerol was added as plasticizer, due to its compatibility, stability and food grade (CAS N° 5682-5). Aloe mucilage (Aloe vera) was added according to previous results of our research group, to improve characteristics of coating, also Larrea tridentata purified polyphenols where added as antioxidant/antifungal component [7]. Based on these antecedents, the main objective of present work was to evaluate the control of necrotic fungi of avocado, by applying a green technology of bioactive coatings made with pectin, glycerol, Aloe mucilage and candelilla wax functionalized with Larrea tridentata purified polyphenols. 2. MATERIALS AND METHODS 2.1. Reagents and Biological Material Isolates of Fusarium oxysporum and Botrytis cinerea where kindly provided by the Food Research Department at Universidad Autónoma de Coahuila, Saltillo, Coahuila, Mexico. Avocados (Persea americana Mill.) cv. Hass, and leaves of Aloe vera were acquired in a super market at Saltillo, Coahuila, Mexico. National Forest Commission (CONAFOR) from Mexico provided leaves of creosote bush (Larrea tridentata L.) and candelilla wax (Euphorbia antisyphilitica Zucc.) from communities with legal permission to exploit non-timber natural resources. Pectin from citric (at least 65 % galacturonic acids) and glycerin were obtained from Faga Lab (Favela Pro, SA de CV, Cerro Agudo, Sinaloa, Mexico). PDA was purchased from Sigma-Aldrich (Sigma-Aldrich Inc., Germany). All other chemicals where from analytical grade. International Journal of Green Technology, 2018, Vol. 4, 25 2.2. Extraction of Polyphenols from Creosote Bush The leaves of creosote bush were dehydrated using an oven at 60°C by 24 hours and pulverized in a mill; the obtained powder (12.5 g) of creosote bush leaves was placed in distilled water (500 ml) at 70°C during 30 min with constant agitation. The obtained extract was decanted and filtered using a filter paper. The filtered ® solution was passed through Amberlite-XAD16 with the conditions described for polyphenols recovery [23] to obtain a powder rich in total polyphenols from L. tridentata (PTP). 2.3. Determination in vitro of Fungicide Activity The poisoned medium method was used for determinate the fungicide activity. The PDA medium was sterilized. After that, PTP was added before agar solidified. The tested PTP concentrations were 250, 500, 1000 and 1250 ppm, and control was a Petri dish with PDA without polyphenols and each fungal strain. Botrytis cinerea and Fusarium oxysporum were activated on Petri plates with PDA. An explant of 5 mm diameter with necrotic fungal was placed on poisoned medium with polyphenols. Radial growth was measured with a Vernier in a kinetical way until the control plate was completely invaded by the fungal strain. 2.4. Bioactive Coating Manufacturing Pectin (1.1% w/v) was dispersed in distilled water at 80°C with constant agitation. Candelilla wax (0.16% w/v) and glycerol (0.3% v/v) were added when pectin was completely dispersed at room temperature. Thereafter, Aloe vera mucilage (5 % w/v) was incorporated. TPT were added in different concentrations to obtain treatments: C1 (320 ppm), C2 (620 ppm) and C3 (920 ppm), CS represents control avocado without coating and CC represents a control coating without bioactive from L. tridentata. Final dispersion was homogenate at 1000 rpm/15 min [7]. 2.5. Application of the Bioactive Coating Agar PDA medium was used to reactive fungal strains, then, spores were recovered with Tween 80. The number of spores was counted using a 7 Neubauer´s chamber and was adjusted to 1 x 10 spores/mL. Avocados were washed with hypochlorite solution at 5 % and dried at room temperature, after that were inoculated with 20 µL droplet of each fungus 7 (Botrytis cinerea and Fusarium oxysporum) at 1 x 10 spores/mL for each one and were let at room 26 International Journal of Green Technology, 2018, Vol. 4 Diana et al. Table 1: In Vitro Growth Rate for Minimum Inhibitory Concentration Microorganism Growth Rate Control (mm/h) Growth Rate 1000 ppm (mm/h) Percent Delay (%) MIC 50 (ppm) Botrytis cinerea 0.88 0.24 88 612 Fusarium oxysporum 0.4 0.2 50 558 Concentrations of L. tridentata polyphenols used during the in vivo assay and added to the coatings were selected according the results obtained from in vitro assay, in this case, 320, 620 and 920 ppm were used (C1, C2 and C3, respectively). Results showed that fungal (B. cinerea and F. oxysoprum) invasion into avocado mesocarp diminished in comparison to control 100 80 60 40 C 3 C 2 C 1 C 0 C 20 S With the methodology used for polyphenols extraction from Larrea tridentata leaves was possible to achieve 2.98 % of recuperation yield (dried weight), while some studies reported to being achieved 6.8 % of recuperation yield [24], differences may be due to plant physiological state and genotype, soil type, geographical region and season of tissue recollection. Determination of fungicide activity in vitro (MIC50) was evaluated by measuring B. cinerea and F. oxysoprum growth on poisoned medium with polyphenols of L. tridentata (Table 1). In the present study, MIC50 concentrations are lower than those reported by [25], where those authors used 1000-4000 ppm of Larrea tridentata leaf resin to inhibit 96-99 % of B. cinerea and 42-63 % of F. oxysporum. However, using polyphenols powder it was not possible to obtain 100% of F. oxysoprum inhibition. It should be mentioned that, there are reports where aqueous extracts of L. tridentata were used and an 40 % of Botrytis sp. inhibition was observed [26] while 60-90 % of F. oxysoprum inhibition was determined [27]. Extracts of L. tridentata also has been used to treat food borne pathogens, in concentrations from 1000 to 5000 ppm, the best control for Staphylococcus aureus was observed using 5000 ppm [28]. C 3. RESULTS AND DISCUSSION (CC) (Figure 1). Treatment C3 (920 ppm) displayed the lowest fungal invasion, for B. cinerea was observed 15.5 % ± 3.31 and for F. oxysoprum 21.9 % ± 9.99, comparing with control without and with coating. Other authors [29] evaluated the efficacy of L. tridentata extracts against gram negative bacteria with promising results against Staphylococcus aureus. The present study demonstrated that it is possible prevent microorganism infection (B. cinerea and F. oxysoprum) in avocado by using bioactive coating. Similar results has been mentioned [30], who consigned that fungal invasion (1.2 %) by C. gloesporioides with coatings of candelilla wax and ellagic acid (reagent). Fungal invasion (%) temperature (25 °C) for 12 hours. After this time, avocados were two times immersed completely for 3 s in the emulsion for each treatment, C1 (320 ppm), C2 (620 ppm), C3 (920 ppm), CC represents a control coating without bioactive from L. tridentate and CS represents control avocado without coating. Avocados were storage at 25°C and 60 % RH [7]. Treatments Figure 1: Fungal invasion of Botrytis cinerea and Fusarium oxysporum. Treatments C1 (320 ppm), C2 (620 ppm) and C3 (920 ppm), CS represents control avocado without coating and CC represents a control coating without bioactive from L. tridentate, the line of each bar represents its standard deviation. CONCLUSION Bioactive coating presented antifungal activity against the two fungal strain. The best treatment to control fungi invasion was C3 (920 ppm of polyphenols) with 15.5 and 21.9 % of necrotic invasion in comparison to controls. These results showed that bioactive coating functionalized with Larrea tridentata purified polyphenols had an important antifungal potential, which might be utilized in the food industry. A Green Technology for Control of Avocado Necrotic Fungi Using Bioactive Coatings The bioactive coatings could be an excellent alternative to preserve avocado for attack of B. cinerea and F. oxysoprum and extend fruit shelf life. CONFLICT OF INTEREST Hernández-Téllez CN, Plascencia-Jatomea M and CortezRocha MO. Chitosan-Based bionanocomposites: Development and perspectives in food and agricultural applications. In: Romanazzi G, Jiménez-Aparicio A, editors. Chitosan in the Preservation of Agricultural Commodities. 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