1. Introduction

Mexico produces approximately 30% of all avocados that are cultivated worldwide (FAO, 2018). The Mexican state of Michoacán is the leader in avocado production. In this state, avocado production involves the participation of 28,000 growers and 62 housekeepers. Avocado exportation to the USA during the 2019–2020 season was 962,000 t (APEAM, 2020). The avocado supply chain is not exempt from suffering food losses. Diseases associated with phytopathogens are the principal cause of avocado postharvest losses. In this context, antifungal active packaging can be used to manage postharvest losses associated with phytopathogens in fruit and vegetables. Active packaging interacts intentionally with food or its environment to increase its quality and then extend its shelf life. The active function can be acquired by adding a chemical substance with a specific function, like antioxidant, antifungal, or antibacterial into the packaging (Colín-Chávez et al., 2013). The methods in which active packaging system functions using active compounds can be classified into two types to perform further. First, sachets or pads consist of active components and can place inside the package. The second, active ingredients incorporated into or on the packaging materials like films and coatings (Robertson, 2012). The following is essential to keep in mind when developing antifungal packaging for fruits: 1) Determine the phytopathogen that produces the fruit decay, 2) Identify a chemical substance with the capacity to control the growth of the phytopathogen, 3) Determine whether the chemical substance will be incorporated into films/coatings or as a sachet/pads in the packaging's headspace, 4) Analyze the mechanism of interaction of antifungal packaging with the fruit, and 5) Validate the technology in controlled and real conditions of storage and distribution of fruits.

Anthracnose is the principal disease of avocado fruit; it is caused by Colletotrichum species. The species registered in Mexico that are associated with anthracnose are Colletotrichum acutatum sensu lato (s.l.), Colletotrichum boninense s.l., Colletotrichum fructicola, Colletotrichum gloeosporioides s.l., Colletotrichum godetiae, Colletotrichum karsti, Colletotrichum siamense, Colletotrichum fioriniae, Colletotrichum cigarro, Colletotrichum chrysophilum, Colletotrichum jiangxiense, Colletotrichum tropicale, and Colletotrichum nymphaeae (Fuentes-Aragón et al., 2020; Hernandez-Lauzardo et al., 2015; Silva-Rojas and Avila-Quezada, 2011; Velázquez-del Valle et al., 2015). Anthracnose in avocados is characterized by the presence of small black spots that become larger and more sunken over time. When the symptoms appear on larger fruit, the quality is reduced, and these fruits are not suitable for export because consumers may reject them.

Mexican oregano oil is an essential oil obtained from Lippia graveolens and Poliomintha longiflora. This oil contains high concentrations of carvacrol and thymol (Cid-Pérez et al., 2016). The antifungal mechanism of carvacrol may be induced reactive oxygen species production, increased membrane permeability, abnormalities of ultrastructure, and loss of intracellular contents (Pei et al., 2020). Thymol can inhibits the activity of pathogens by disrupting the integrity and function of the cell membrane due to its hydrophobicity, causing leakage of the intracellular components, and resulting in cell death (Kong et al., 2021). The antifungal activity of oregano oil in the vapor phase has been tested against diverse Colletotrichum species, such as C. acutatum, C. gloeosporioides, and C. fructicola (Cid-Pérez et al., 2016; Nevárez-Moorillón et al., 2014; Numpaque et al., 2011; Pei et al., 2020). The antifungal properties of oregano oil against Colletotrichum species can be used in the design of active films/coatings or sachets/pads. Little data have been published about the control of avocado anthracnose by means of antifungal active packaging. We found that the microorganism target was C. gloeosporioides, and its growth was controlled by 25% using active packaging (Bill et al., 2018). Thyme oil was the active chemical used in all these packaging designs. This design trait was incorporated into the packaging by the two mechanisms previously mentioned: (1) inside polylactic acid (PLA) and low density polyethylene (LDPE) films and (2) in the headspace of tray packs as PLA sachets filled with LDPE/thyme oil pellets (Bill et al., 2018; Boonruang et al., 2017; Pillai et al., 2016).

Food-antifungal packing interactions describe the mechanism by which the antifungal compound moves from the packaging to the food and exerts its antifungal activity. Frequently, food packaging interactions are associated exclusively with the kinetic release of the active compound to the food. However, when the food is a fruit (living being), the active compound can generate a reaction cascade that enhances or decreases the effect of this compound. Recently, publications have suggested that antifungal compounds can produce or enhance specific defense responses in vegetables and fruit. The induction of biochemical plant defense responses is associated with the enzymatic activities of catalase (CAT), peroxidase (POD), chitinase, β−1,3-glucanase, and phenylalanine ammonia lyase (PAL), which can participate in protecting from microbial infection and inhibiting phytopathogens (Romanazzi et al., 2016). PAL is involved in the biosynthesis of a diverse array of phenylpropanoid metabolites, such as phytoalexins, coumarins, flavonoids, lignin, and stilbenes, all of which have been implicated in plant defense (Vogt, 2010). CAT and POD enzymes are associated with antioxidant enzymatic systems for protecting cells against oxidative damage. β−1,3-glucanase and chitinase are enzymes that degrade fungal cell wall polymers, reducing fruit decay via delaying fungal growth (Theis and Stahl, 2004).

The control of pathogenic diseases of fruit and vegetables with essential oils is due to their chemical compounds acting directly on the pathogen. However, essential oils can trigger defense mechanisms through the induction of defense-related enzymes (Fontana et al., 2021). Sellamuthu et al. (2013) found that the application of thyme essential oil vapors to avocado fruit induced biochemical fruit defense mechanisms by increasing the activity of chitinase, β−1,3-glucanase, and phenylamine ammonia lyase. Similarly, Bill et al. (2014) showed that the application of thyme oil and edible coatings was effective in the activation of defense responses via the accumulation of resistance compounds in avocado fruit against anthracnose during postharvest period.

The objective of this work was to evaluate the effect of active sachets on fungal development, fruit quality, and induction of defense mechanisms of avocado fruit during postharvest storage. The active sachets were developed with Mexican oregano oil microencapsulated by spray drying, using starch and agave fructans as a carrier. Starch is one of the most common polysaccharide-based carriers used as a reference material in the encapsulation of active ingredients due to its physicochemical and functional properties, and it is a low-cost encapsulating agent (Hoyos-Leyva et al., 2018). Agave fructans are polymers of fructose synthesized as reserve carbohydrates in the different agave species. These biopolymers have also been used as wall materials in the spray-drying encapsulation of antioxidants and essential oils (Esquivel-Chávez et al., 2021).

2. Materials and methods

2.1. Solvents and reagents

Potato dextrose agar (PDA, BD Bioxon, Mexico), distilled water (J.T. Baker, Mexico), and ampicillin (96–100.5% anhydrous base, Sigma-Aldrich, USA) were used for antifungal activity. Carvacrol (standard ≥ 98.5%, Sigma-Aldrich, France), acetonitrile (HPLC grade 99.9%, Honeywell, USA), and glacial acetic acid (HPLC grade 99.99%, J.T. Baker, USA) were employed for the quantification of carvacrol. For oregano oil encapsulation, the materials used were native agave fructans (with a weight average molecular weight of 3 kDa and a degree of polymerization around of 18, Tiasa, Mexico), modified corn starch (Ingredion, USA), and Tween 80 (Meyer, Mexico).

2.2. Fruit

Avocado fruit (Persea americana Mill. cv. Hass) at the first stage of maturity, without mechanical injury, 48- and 60-caliber exportation-quality fruit were selected. The fruit was obtained from Frutas Finas de Valles de Michoacan located in Santa Clara del Cobre, Michoacan (Mexico).

2.3. Phytopathogens strains

C. gloeosporioides, C. acutatum, Diaporthe passiflorae, and Neoscytalidium hyalinum were separated from Hass avocado with typical signs of anthracnose and grown on PDA agar plates. The isolation and characterization of the phytopathogens were performed at the Phytopathology Laboratory at CIDAM (Agrofood Innovation and Development Center), where research was conducted.

2.4. Antifungal compound

Oregano oil was acquired from Procesadora de Oregano Silvestre. According to the manufacturer's instructions, oregano oil was extracted from Lippia graveolens. The carvacrol concentration in the oil was 488.88 g kg–1. Carvacrol concentration was determined by high performance liquid chromatography using an HPLC-system (Agilent model 1200, Germany), equipped with a quaternary pump, autosampler, a Supelco Ascentis® Express C18 column (250 × 4.60 mm i.d., 5 µm), and a photodiode array detector (Agilent model G4212B) set at 274 nm. The mobile phase was acetonitrile:water (60:40, v/v) with a flow rate of 0.8 mL min–1. For the carvacrol quantification, a standard curve was developed using standard solutions of carvacrol (assay ≥ 98.5%) at concentrations from 0.005 to 5 g L–1.

2.5. Antifungal active sachets development

2.5.1. Encapsulation of Mexican oregano oil

The formulation for the emulsions was as follows: 75.90% (w/w) of distilled water, 9.48% (w/w) of starch, 9.48% (w/w) of agave fructans, 4.74% (w/w) of Mexican oregano oil, and 0.40% (w/w) of Tween 80. A control emulsion was prepared without the addition of the oil. On an advanced hot plate, the emulsions were kept for overnight magnetic stirring at 400 rpm (VWR, USA). Next, the emulsions were homogenized for 10 min at 10,000 rpm using an L 5M-A homogenizer (Silverson, England), and the oil was added. The temperatures of the lab-scale spray dryer (SD-BASIC Son SDB1113061, Lab. Plant, England) used during processing were 190 °C (input) and 85 °C (output). This process was performed three times to corroborate the results. The capsules were identified as control and active. Control capsules were 0.28 ± 0.003 µm of size and 7.46 ± 0.36% (w/w) of moisture. Active capsules were 0.20 ± 0.02 µm of size and 4.22 ± 0.21% (w/w) of moisture. The moisture content was determined gravimetrically using a thermobalance (Sartorius, Germany) at 110 °C until constant weight, and the results were expressed as percentage (%, w/w). The average particle size of the capsules was measured using a SZ-100 nanoparticle size analyzer (Horiba Ltd, Japan) according to Esquivel-Chávez et al. (2021).

2.5.2. Carvacrol quantification in capsules and encapsulation efficiency

Encapsulation efficiency was determined according to Bae and Lee (2008) with some modifications. To determine the non-encapsulated carvacrol on the surface of capsules, 0.25 g of capsules were added to 2.5 mL of acetonitrile:water (80:20, v/v) and shaken at 40 °C for 2 min. To avoid oxidation of the carvacrol during the extraction process, 0.05 mg mL–1 butylated hydroxytoluene was added to the extraction solution. Then the mixture was filtered (Whatman No. 1) and the capsules were rinsed three times with acetonitrile/water (80:20, v/v). After, the content of carvacrol in the filtrate was determined by HPLC as describe above. The difference between the initial carvacrol amount added in the emulsion and the surface free carvacrol on the capsules gives the encapsulated amount of carvacrol. Encapsulation efficiency was expressed as a percentage and was calculated according to Eq. (1).

2.5.3. Sachet preparation

Nylon fabric (thickness: 9.75 ± 0.60 µm) was purchased from a local store. The porosity of the Nylon according to the manufacturer's specification is close to 60%. Nylon was used to make the sachets and the capsules were incorporated into individual sachets. Sachets were loaded with 0.15, 0.25 and 1 g of active capsules and then sealed with a manual sealed machine (Dilitools, México) to form 4 × 4 cm rectangles. The control sachets were filled with 0.15 g of control capsules without the incorporation of oregano oil. Sachets were prepared before to use.

2.6. Evaluation of in vitro antifungal activity of sachets

The in vitro antifungal activity of active sachets was tested for its inhibition against C. gloeosporioides, C. acutatum, D. passiflorae, and N. hyalinum. Petri dishes containing the PDA medium were individually inoculated with a disk of phytopathogens (6 mm in diameter). The sachet was attached to the inside surface of the upper lids of previously inoculated Petri dishes. The dishes were sealed with parafilm and incubated at 30 °C for 12 d (Esquivel-Chávez et al., 2021). Treatments included sachets filled with control and active capsules. The negative control was a dish without sachet. The percentage of inhibition (%I) of mycelium growth was obtained using the next equation: I% = [(A – B)/B]*100, where A and B are the diameter of mycelial growth in the treatments and control (Iñiguez-Moreno et al., 2020). The treatments were assessed in five repetitions and were performed three times to verify the results.

2.7. In vivo assay of active sachets in sealed humidified chamber

The fruit was sanitized with sodium hypochlorite (3%, w/v), cleaned with distilled water, and dried at 24 °C for 2 h. The peduncle was removed, and in this area, the avocados were inoculated with 30 µL of a conidial suspension containing 1 × 106 conidia mL–1C. gloeosporioides. Rectangular humidified chambers (2.93 × 10–3 m3) with 90% relative humidity were prepared and conditioned according to Esquivel-Chávez et al. (2021). The sachet was fixed to the inside center of the lid of the humiditfied chamber. One inoculated avocado fruit was carefully placed in each humidified chamber, and it was sealed with parafilm to prevent volatiles releases. One avocado fruit in one chamber was considered as one treatment, and each treatment consisted of thirty fruits. Treatments included active sachets loaded with 0.15 g (T-0.15), 0.25 g (T-0.25), and 1 g (T-1) of active capsules. Sachets loaded with 0.15 g of control capsules served as the control. All avocados were stored for 5 d at 20 ± 2 °C with 90 - 95% relative humidity. The experiment was performed on three occasions to validate the findings.

2.7.1. Control of C. gloeosporioides incidence and severity with active sachets

The incidence percentage (I%) of C. gloeosporioides was obtained using the next equation:% I= [IA/TA] *100, where IA is the number of injured avocados, and TA is the total avocados observed. The severity was recorded from the flesh pulp after cutting according to the area of the lesion (cm2). The area of the lesion around the inoculation point was measured by Digimizer version 5.4.4 software. The severity percentage (%) was evaluated by comparing the injured area among the fruits treated with T-0.15, T-0.25, T-1, and the fruits inoculated with the phytopathogen and without sachet (CP).

2.8. Impact of antifungal active sachets on the avocado's quality

The effects of active sachets treatments on quality of avocado fruit were determined using previously reported method with slight modifications (Virgen-Ortíz et al., 2020). Firmness was tested at three points on the fruit with a texturometer (Brookfield CT3, USA). The following conditions were used for the analysis: test speed = 5 mm s–1, trigger force = 10 g, penetration depth = 5 mm, and 5-mm diameter flat probe. The results are expressed in Newtons (N) and are presented as the mean ± SD of thirty avocados.

The color parameters L, a*, b*, Hue, and Chroma of the fruit were measured using a Colorimeter CR-410 (D65 illuminant, Konica Minolta, Japan).

2.9. Effect of active sachets on defense-related enzyme activities in avocado fruit

The enzyme assays for PAL, β−1,3-glucanase and chitinase were performed from avocados inoculated with C. gloeosporioides and treated with active and control sachets as described in Section 2.7.

2.9.1. Phenylalanine ammonia-lyase activity

The PAL activity (EC 4.3.1.5) was determined as previously described by Lafuente et al. (2001) with some modifications. Briefly, the extraction buffer contained sodium borate buffer (pH 8.8), 0.5% (w/v) Triton X-100, 1% (w/v) polyvinylpyrrolidone (PVP), and 5 mM β-mercaptoethanol. The enzyme extracts were prepared by homogenizing 1 g of sample fruit with 5 mL of extraction buffer. Then, the homogenates were centrifuged at 10 000 × g at 4 °C for 15 min, and the supernatants were used for PAL assays. The reaction mixture consisted of 0.45 mL of 100 mM L-phenylalanine, 2.55 mL of 100 mM borate buffer (pH 8.8), and 1.5 mL of the enzyme extract. The mixture was incubated at 37 °C for 60 min under gentle stirring. The reaction was terminated by adding 1.5 mL of 1 M HCl, and the absorbance of the supernatant was measured at 290 nm. One unit (U) of PAL enzyme activity was defined as the amount of enzyme catalyzing the production of 1 µmol t-cinnamic acid min–1. The data for PAL activity was reported in relation to the fresh weight as U kg–1.

2.9.2. Chitinase and β−1,3-glucanase activities

The β−1,3-glucanase and chitinase activities were determined according to a previously described method (Virgen-Ortiz et al., 2020), with some modifications. Briefly, 1.0 g of sample fruit was homogenized at 4 °C in 5 mL of extraction buffer (50 mM sodium acetate buffer, pH 5.0, containing 5 g L–1 Triton X-100 and 1 g L–1 PVP). The homogenate was centrifuged at 10 000 × g at 4 °C for 15 min, and the supernatant was collected for enzyme assays.

Chitinase activity (EC 3.2.1.30) was determined by measuring the amount of p-nitrophenol (pNP) released by enzyme-catalyzed hydrolysis from p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG). The reaction mixture contained 1 mL of 50 mM acetate buffer (pH 5.0), 0.5 mL of 4.8 mM pNP-NAG, and 0.5 mL of crude enzyme extract. Incubation was carried out at 37 °C for 60 min under shaking. The enzyme reaction was terminated by adding 2 mL of 0.25 M sodium carbonate and the absorbance was measured at 405 nm. The amount of p-NP was determined by using a molar extinction coefficient of 18,500 M–1 cm–1. One unit of chitinase activity was defined as the amount of enzyme that produces 1 µmol p-NP min–1. The data for chitinase activity was expressed based on the fresh weight as U kg–1.

The β−1,3-Glucanase activity was assayed by the release of reducing sugar from laminarin. The reaction mixture contained 0.1 mL of enzyme extract, and 1 mL of 5 g L–1 laminarin (substrate was prepared in 100 mM sodium acetate buffer, pH 5.0). Incubation was carried out for 60 min at 40 °C with gentle stirring. After the incubation, the reaction was terminated by placing the reaction tubes in a boiling water bath for 5 min. Then, the amount of reducing sugar produced from laminarin was measured using the dinitrosalicylic acid method. One unit of β−1,3-glucanase activity was defined as the amount of enzyme that catalyzed the formation of 1 mmol glucose min–1 under conditions described above. The data for β−1,3-glucanase activity were expressed based on fresh weight as U kg–1.

2.9.3. Catalase (CAT) and peroxidase (POD) activities

The extraction of CAT and POD enzymes was carried out by previously described methods with modifications (Sellamuthu et al., 2013). Briefly, the extraction buffer contained 100 mM potassium phosphate buffer (pH 7), 10 g L–1 PVP, and 5 g L–1 Triton X-100. For enzyme extraction, 1.0 g of frozen tissue obtained from “thirty fruit per treatment” was homogenized with 5 mL of extraction buffer. After 3 h of extraction at 4 °C, the homogenate was centrifuged at 10 000 × g for 15 min at 4 °C. The supernatant was taken as the crude extract for the measurement of CAT and POD enzyme activities. POD activity (EC 1.11.1.7) was assessed with the method described by Sellamuthu et al. (2013) using guaiacol as the substrate, with slight modifications. The analysis mixture containing 50 µL of crude enzyme extract in 2.1 mL buffered substrate (20 mM guaiacol prepared in 100 mM sodium phosphate buffer, pH 7.0) was incubated for 5 min at 30 °C. Thereafter, the reaction started with the addition of 850 µL of 30 mM H2O2, and the increase in absorbance at 460 nm was measured for 120 s. One unit of POD activity was defined as ΔA460 s–1. The data for the POD activity was expressed based on fresh weight as U kg–1. CAT activity (EC 1.11.1.6) was measured using the method described by Aebi (1984) with slight modifications. Catalase activity was evaluated by adding 50 µL of crude enzyme extract to 3 mL buffered substrate (30 mM H2O2 prepared in 100 mM sodium phosphate buffer, pH 7.0). The absorbance of the mixture reaction was measured at 240 nm every 30 s for 5 min. One unit of POD activity was defined as the amount of enzyme that decomposed 1 mmol H2O2 s–1. The activity of CAT was expressed based on fresh weight as U kg–1.

2.10. Determination of total flavonoids and phenolic content

The analysis of total flavonoids and phenolic content were performed from avocados inoculated with C. gloeosporioides and treated with active and control sachets as described in Section 2.7. Methanolic extracts were obtained as described by Nowicka et al. (2019) with minor modifications. Two grams of sample fruit were homogenized with 8 mL of 80% (v/v) acidified methanol (1% v/v, HCl) at 4 °C. The homogenate was sonicated for 30 min and centrifuged at 10 000 × g for 10 min. The supernatant was collected, and the extraction procedure was repeated one more time, as previously described. Thereafter, the two supernatants were combined and brought to a total volume of 20 mL with 80% (v/v) acidified methanol. Total phenolic content was estimated according to the Folin–Ciocalteu assay (Singleton et al., 1999). The data were reported based on fresh weight basis as grams of gallic acid equivalents (GAE) kg–1. The total flavonoid content was determined by aluminum chloride assay according to Zhishen et al. (1999) with slight modification. Briefly, 1 mL of methanolic extract was mixed with 4 mL of distilled water, and 0.3 mL of 5 g L–1 NaNO2 was added. After stirring, the mixture was left to stand for 5 min. Thereafter, 0.3 mL of 100 g L–1 aluminum chloride was added. After incubation for 6 min in the dark at room temperature, 1 mL of 1 M NaOH was added to the reaction mixture, and the final volume was made up to 10 mL with distilled water. The absorbance was measured at 510 nm against methanol as a blank. The total flavonoid content was expressed based on fresh weight as g quercetin equivalents kg–1 of fruit.

3. Results

3.1. Active sachets characterization

3.1.1. Loading and encapsulation efficiency of carvacrol in capsules

One of the principal properties of antifungal active sachet is that the active compound should be at a suitable concentration to control the target microorganism. The development of active sachet should preserve the antifungal activity of the compound. In this case, it is crucial to preserve the concentration of carvacrol and its functionality. The microcapsules obtained had 95.97 g kg–1 of carvacrol initially added (122.22 g kg–1), and 78.5% (w/w) was retained during the process. This value agrees with those reported by other authors. After spray drying of oregano oil or carvacrol, its retention is in the range of 5.1 to 91.79% (Alvarenga Botrel et al., 2012; Hernández-Nava et al., 2020). The differences in retention of the oregano oil could be attributed to the solid content of the infeed material, molecular weight and vapor pressure of volatile compound concentration or oregano oil, the viscosity of the dryer infeed material, the atomization process, drying air velocity and mixing with atomized infeed, dryer inlet and exit air temperatures, relative humidity of the dryer inlet air, emulsion size, and dryer feed temperature (Reineccius, 2004).

3.2. Evaluation in vitro of active sachets

As shown in Fig. 1, all sachets containing different concentrations (0.15, 0.25, and 1 g) of active capsules exhibited high antifungal activity against all the tested phytopathogens. The microorganisms studied were 100% inhibited in vitro with Mexican oregano oil via the volatiles released from the sachets. The results found are in agreement with previous studies that show the in vitro antifungal effect of oregano oil in the vapor phase against C. gloeosporioides and C. acutatum (Grahovac et al., 2012). Also, C. gloeosporioides has been inhibited with thyme oils via the vapor phase and via active films (Pillai et al., 2016). However, this is the first time that oregano oil in the vapor phase has been tested to inhibit the mycelium growth of D. passiflorae and N. hyalinum. These are important pathogens associated with avocado decay. Furthermore, there are few reports regarding the control of these genus of fungal pathogens with essential oils. For example, citronella oil inhibited (60–85.42%) Neoscytalidium dimidiatum growth, and this phytopathogen is associated with dragon fruit stem canker (Taguiam et al., 2020). Additionally, cinnamon leaf oil repressed (53% and 92.31%) the growth of D. helianthi and D. phaseolorum, which are associated with sunflower stalk and soybean disorders (Jasenka et al., 2010). The essential oils exert their antimicrobial effect by: a) inhibition of cell wall formation, b) cell membrane disruption, c) dysfunction of fungal mitochondria, d) inhibition of cell division, e) inhibition of RNA/DNA synthesis or protein synthesis, and f) inhibition of efflux pumps (Freiesleben and Jäger, 2014). The encapsulation process can lead to a positive or negative effect on the antimicrobial activity of the essential oil in comparison to that of free oil. In this sense, previous studies demonstrated that the encapsulation enhances the antimicrobial activity of essential oils by increasing their ability to disrupt cell membrane integrity (Moghimi et al., 2016). In this study, the microencapsulation of Mexican oregano oil in starch/agave fructans allowed an efficient controlled release of this antifungal compound, as deduced from the total inhibition of fungal growth compared with control sachets (Fig. 1). The results of this study suggest that active sachets can inhibit in vitro the growth of the main phytopathogens associated with avocado decay.

1. Download : Download high-res image (552KB)
2. Download : Download full-size image

Fig. 1

3.3. In vivo assay of active sachets in sealed humidified chamber

3.3.1. Control of C. gloeosporioides incidence and severity with antifungal active sachets

T-0.15 (14.39 mg of carvacrol) reduced a substantial portion of the injured area due to C. gloeosporioides infection (Fig. 2, Fig. 3). T-0.25 (23.99 mg of carvacrol) and T-1 (95.97 mg of carvacrol) had a smaller impact on the reduction of the injury area than that of T-0.15 treatment. There was no statistically significant difference between the T-0.25 and T-1 treatments. Interestingly, the lower carvacrol concentration in active sachets was the most impactful in relation to the reduction of the injured area. Similar behavior was reported for the control of anthracnose in mango with thyme oil active sachets (Esquivel-Chávez et al., 2021). This behavior can be attributed to the humidified chamber becoming saturated or supersaturated with volatile compounds derived from the capsules. There could be a limiting concentration of volatile compounds derived from oregano oil. When the volatiles reach this concentration, their positive effect turns off. It is necessary to verify this behavior under natural fruit storage conditions (carton packaging, temperature, relative humidity, size of refrigerated rooms or trucks). As discussed in the defense-related enzyme activities section, the activity of chitinase was markedly higher in avocados exposed to active sachets with the lower carvacrol concentration, compared with that of the other sachets. In this sense, essential oils have been reported to have an important role in inducing resistance of fruits against fungal decay by enhancing the secretion of defense-related enzymes like chitinases (Cindi et al., 2016). This is the first report that active sachets with Mexican oregano oil are used for controlling C. gloeosporioides in avocado during postharvest storage. Previous studies have shown the efficiency of other essential oils to control avocado anthracnose (Bill et al., 2018; Boonruang et al., 2017). These authors controlled avocado anthracnose with thyme oil (active sachets), thymol (active films), and R-(-)-carvone (active films). In this packaging, the active compounds accounted for 10% (w/w) of the content, the same as that reported in this work. Our findings make the use of oregano oil-starch-agave fructan sachets an attractive antifungal component in the development of active sachets because they control the growth of C. gloeosporioides, and it is expected that when sachets place inside carton boxes the shelf life of avocado will be longer.

1. Download : Download high-res image (608KB)
2. Download : Download full-size image

Fig. 2

1. Download : Download high-res image (226KB)
2. Download : Download full-size image

Fig. 3

3.4. Impact of active sachets on the quality of avocados

In general, none of the applied treatments reduced the avocado firmness (Table 1). Our results agree with those reported for avocado and other fruits packed in essential oil-based packaging. For example, avocados packaged with thyme oil-active sachets did not experience adverse effects on this quality parameter (Bill et al., 2018). In addition, mangoes treated with thyme oil-active sachets and papaya packaged in sealed paper pouches with oregano, cinnamon, or lemongrass oil-active sachets did not significantly affect this characteristic (Espitia et al., 2012; Esquivel-Chávez et al., 2021). This result demonstrated that an active sachet could protect avocado from phytopathogens, maintaining the fruit's firmness.

Table 1. Impact of antifungal active sachets on the avocado (Persea americana Hass) quality after 5 d of inoculation with C. gloeosporioides in sealed humidified chamber.

The results are presented as the mean  ±  SD of thirty avocados.

The treatments evaluated were as follows: T-0.15 loaded with 0.15 g of active capsules into sachets; T-0.25 loaded with 0.25 g of active capsules into sachets; and T-1 loaded with 1 g of active capsules into sachets. The controls were: negative control (fruits not inoculated and without sachets [CN]), and positive control (fruits inoculated and without sachets [CP]). Different superscript letters within the same columns indicate significantly different values (P < 0.05).

Avocado color is preserved utilizing low-weight oregano oil microcapsules in active sachets. Avocados treated with T-0.15 displayed a red-dull-bright color with b* in the yellow direction and a* at the beginning of the red spectrum. The color parameters (L, Hue, and a*) of this fruit did not present significant differences with respect to avocados from CN (fruits not inoculated and without sachets). In addition, no differences were observed between avocados treated with T-0.15 and CP (fruits inoculated and without sachets) in chrome value. In contrast, avocados treated with T-0.25 and T-1 displayed a yellow-dull-bright color, with b* in the yellow direction and a* at the beginning of the red spectrum. The color parameters from avocados packaged with T-0.25 and T-1 were significantly different from those of CN and CP. There is a relationship between ΔE and oregano oil-microcapsule weight. However, it is important to emphasize that ΔE in avocados treated with T-0.15 is smaller than that in avocados from CP. The change in avocado color during ripening is a chemical process resulting from chlorophyll degradation and the production of cyanidin 3-O-glucoside (Lin et al., 2020). Based on our results, we hypothesize that low Mexican oregano oil microcapsule concentrations hold fruit color or cancel out the effect of phytopathogens on color change. Also, it is likely that the effect of active sachets on the color can be attributable to the changes in content of total phenolics. The involvement of phenolic compounds in the color of fruits has been documented by others (Elsayed et al., 2022). As shown in Fig. 3, postharvest treatments with T-0.25 and T-1 exhibited a higher increase in the content of total phenolics compared with the treatment with T- 0.15.

3.5. Effect of active sachets on defense-related enzyme activities in avocado fruit

Table 2 shows the effects of treatment on the PAL, chitinase, β−1,3-glucanase, CAT, and POD activities of avocado fruit after 5 d of storage at 20 ± 2 °C. All avocados treated with active sachets showed significantly (P < 0.05) higher PAL enzyme activity than the control fruit. However, the avocados treated with T-0.25 had the highest PAL activity when compared to other treatments. It has also been documented that postharvest treatments with thyme essential oil vapors elicit PAL gene expression (Bill et al., 2017) and PAL activity in avocado fruit (Sellamuthu et al., 2013). This enzyme is one of the indicators of disease resistance in plants since PAL can convert phenylalanine into phenolic acids and flavonoids that are toxic to plant pathogens (Vogt, 2010). Therefore, active sachets could contribute to disease resistance by activating the PAL enzyme.

Table 2. Effect of active sachets with oregano oil on the activity of defense-related enzymes in ‘Hass’ avocado fruit after 5 d of storage at 20 ± 2 °C in sealed humidified chamber.

Different letters in the same column indicate significant differences (P < 0.05, according to Tukeyʼs test) between the treatments. PAL, phenylalanine ammonia-lyase; POD, peroxidase; CAT, catalase.

As shown in Table 2, chitinase and β−1,3-glucanase activities significantly (P < 0.05) increased when the avocados were exposed to active sachets with oregano oil compared to the untreated control fruit. Interestingly, the activity of chitinase in avocado fruit treated with the lowest doses of active capsules in sachets (0.15 g of active capsules into packaging) was the highest in all treatments. The difference in chitinase activity was not significant between fruit treated with 0.25 g and 1 g of active capsules. Similarly, the avocado fruit treated with active capsules showed significantly higher β−1,3-glucanase activity than untreated control fruit. Avocado fruit exposed to T-0.15 and T-0.25 showed the highest β−1,3-glucanase enzyme activity. β−1,3-glucanase and chitinase enzymes are two important pathogenesis-related proteins within plant tissues. These enzymes can decompose chitin and β−1,3‐glucan in the cell wall of the pathogen and contribute to resistance against pathogen invasion (Romanazzi et al., 2017). The induction of chitinase and β−1,3-glucanase activities in fruits has been associated with plant defensive responses against phytopathogens (Jin et al., 2017). Previous studies have reported that the application of essential thyme oil was able to stimulate the gene expression and enzymatic activities of β−1,3-glucanase and chitinase in avocado fruit (Bill et al., 2017; Sellamuthu et al., 2013). Our results show that active capsules with essential oregano oil may play an important role in stimulating induced resistance in avocado fruit by increasing β−1,3-glucanase and chitinase activities.

The effect of treatments with active sachets on the CAT and POD activities of avocado compared to control fruit is presented in Table 2. The avocados exposed to active sachets exhibited significantly higher CAT and POD activities than the control fruit. For CAT and POD, the activity in the three different treatments showed similar values with no significant differences. The observed increase in the biochemical activities of CAT and POD agrees with a previous study showing a stimulation of CAT activity in avocado fruit exposed to thyme essential oil vapors (Sellamuthu et al., 2013). POD and CAT are antioxidative enzymes that play an important role in plant defense responses. These enzymes induce resistance against pathogen invasion and constitute the primary defense system against reactive oxygen species in fruit (Huang et al., 2021). Therefore, higher activities of these enzymes are beneficial for alleviating oxidative stress during storage. Recent research has demonstrated that diverse postharvest treatments could increase the resistance to pathogens by enhancing the activities of CAT and POD in fruit (Zhao et al., 2020). Our results also show that the active sachets with oregano oil could induce higher levels of PAL, chitinase, β−1,3-glucanase, CAT, and POD, indicating the activation of avocado defense mechanisms.

3.6. Effect of active sachets on total phenolic and flavonoid contents in avocado fruit

The effects of active sachets with essential oregano oil on the total phenolic and flavonoid contents in avocado fruit after 5 d of storage at 20 ± 2 °C are shown in Figs. 3b and 3c. Avocado fruit exposed to the T-0.25 and T-1 treatments had significantly (P < 0.05) higher total phenolic contents than the control fruit (Fig. 3b). However, no significant difference in the total phenolic content was found between avocados treated with the lowest doses of active capsules in sachets (0.15 g of active capsules into sachets) and untreated fruit. Regarding the total flavonoid content (Fig. 3c), all avocado fruit treated with active capsules showed significantly higher total flavonoid contents than untreated control fruit. Avocados treated with 0.25 g of active capsules (T-0.25) had the highest total flavonoid content, which was consistent with a higher PAL enzyme activity (Table 2). Our results agreed with data reported by Sellamuthu et al. (2013), who found that the level of total phenolics in thyme oil-treated avocados was higher than that in untreated (control) fruit. Phenolic compounds are involved in plant defense mechanisms against the invasion of pathogens and are correlated with enhanced disease resistance (Jin et al., 2017). Our results suggest that the active sachets with oregano oil may play an important role in stimulating the synthesis of phenolic compounds and flavonoids by increasing PAL activity in avocado fruit.

4. Conclusions

Sachets with Mexican oregano oil have antifungal properties to control in vitro and in vivo phytopathogens associated with avocado decay. Sachets loaded with 0.15 g of active capsules had a satisfactory effect for postharvest controlling of C. gloeosporioides in avocado fruit stored in humidified chamber. The use of active sachets preserves avocado quality. In addition, our results suggest that treatment with sachets containing Mexican oregano oil improved resistance against decay by inducing defense mechanisms, including activating defense-related enzymes such as chitinase, β‐1,3-glucanase, PAL, CAT, and POD and enhancing the total phenolic and flavonoid contents in postharvest avocado fruit during storage. Some of the avocado-packaging interactions were elucidated. However, it is important to evaluate the effectiveness of active sachets in combination with carton boxes under storage and distribution conditions (temperatures, relative humidity, and time) of Mexican avocados. Given these findings, this study suggests that sachets with Mexican oregano oil might be a promising technique for decreasing decay and controlling avocado fruit quality during distribution and storage.

Funding

This work was supported by the National Council for the Science and Technology (CONACyT) of Mexico through Cátedras-CONACyT project number 770.

Ethical

The authors declare that the research did not involve humans or animals.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

"

"