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Highlights
Modified atmosphere packaging (MAP) was used to preserve blackberry fruits.
Vapors of oregano essential oil (Eo) were used with MAP.
Fungal development was inhibited with MAP and vapors of essential oil (MAP-Eo).
Shelf life of blackberry fruits was extended up to seven days at 23 °C.
MAP-Eo is a useful alternative to extend shelf life of blackberry fruits.
Introduction
Fungal development causes deterioration of horticultural crops in postharvest and limits shelf life (Legrand et al., 2021; Moncayo-Pérez et al., 2020). Fungi of genera including Penicillium (Kharchoufi et al., 2018), Colletotrichum (Shi et al., 2021), Fusarium (Medina-Romero, Roque-Flores, & Macías-Rubalcava, 2017), Rhizopus (Kong et al., 2019), and Alternaria (Li et al., 2021), are among main postharvest deterioration agents. Due to similar factors and phenomena such as dehydration, wrinkling, and aging, blackberry fruits (Rubus spp.) have shelf life of only three days at 25 °C and 7-8 days at 4 °C (Bersaneti, Prudencio, Mali, & Pedrine, 2021; Toscano-Ávila et al., 2020; Vilaplana, Guerrero, Guevara, & Valencia-Chamorro, 2020). On the other hand, many regions lack refrigeration or cooling systems (Frias-Moreno et al., 2019), so postharvest handling must focus on controlling deterioration factors, even without this infrastructure.
Essential oils (Eo) from different plant species have antimicrobial activity and have attracted attention to be part of disease control strategies in postharvest handling (Aguilar‐Veloz, Calderón‐Santoyo, Vázquez González, & Ragazzo‐Sánchez, 2020). Eo are complex, natural, and volatile products, characterized by having a strong odor and being formed by aromatic plants as terpenes (monoterpenes, diterpenes, and sesquiterpenes) and other components such as alcohols, acids, esters, epoxides, aldehydes, ketones, amines, aromatic phenols, and sulfides (Rehman, Hanif, Mushtaq, & Al-Sadi, 2016; Pandey, Kumar, Singh, Tripathi, & Bajpai, 2017).
The use of Eo has been evaluated in the preservation of blackberry fruits through their incorporation in biopolymeric coatings (Potma da Silva, de Carvalho, Ayub, & Celano-Menezes, 2020; Shi et al., 2022). This form of use of Eo through biopolymers is one of the most common practices and currently seeks to improve release through strategies based on nanoemulsions, encapsulation, multilayer systems, and the production of nanofibers through electrospinning (Zhang, Jiang, Rhim, Cao, & Jiang, 2022). However, the controlled release of vapors of Eo in the headspace of products can induce a greater inhibitory effect (López-Gómez et al., 2021; Peretto et al., 2014; Reyes-Jurado, Cervantes-Rincón, Bach, López-Malo, & Palou, 2019), but this implies retaining the active compounds to ensure a certain concentration. In this sense, the use of Eo vapors can be used in conjunction with modified atmospheres (MAP) to promote the retention of these compounds in the head space (López-Gómez et al., 2021).
The fungal development that causes deterioration of blackberry fruits can include species as Botrytis spp., Colletotrichum spp., and Aspergillus spp. (Cosseboom, Schnabel, & Hu, 2020; Liu et al., 2019; Uribe-Gutiérrez, Moreno-Velandia, & Villamizar, 2022), so the antimicrobial agent must control different type of microorganisms. Shi et al. (2022) demonstrated that the use of Eo of oregano applied with polylactic acid/polycaprolactone electro spin nanofibers caused an antimicrobial effect that delayed postharvest decay, deterioration, and storage quality loss in blackberry fruits.
Oregano (Origanum vulgare) is a culinary herb that produces an Eo where compounds with high antimicrobial potential as p-cymene, terpinene-4-ol, carvacrol, and thymol have been identified (Tapiero, Salamanca, & Marín, 2019). Based on this, the objective of the work was to evaluate the use of modified atmosphere packaging incorporated with vapors of Eo of oregano to prolong shelf life of blackberry fruits.
Materials and methods
General organization
Two stages were conducted. First, fungal species that caused deterioration of blackberry fruits were identified and the minimum inhibitory concentration of oregano Eo to prevent fungi rot was determined. Second, the storage of blackberry fruits under modified atmosphere with vapors of Eo of oregano was evaluated.
Identification of essential oil components by GC-MS
Oregano (Origanum vulgare) Eo was provided by Química Laitz S.A de C.V. (México). Eo was analyzed with a gas chromatograph (GC) (Agilent 7890B, Agilent Technologies, USA), coupled to a mass selective detector (MS) (Agilent 5977A, Agilent Technologies, USA), which used helium as carrier gas with flow of 1 mL·min-1. The compounds were separated on a capillary column (DB-WAX Ultra Inert, Agilent Technologies, USA; 60 m × 250 µm × 0.25 µm), with oven at 40 °C during 1 min and heating up to 220 °C during 2 min at 9 °C·min-1. The MS used electron ionization energy of 70 eV, scanning range of 30-550 uma, scan rate of 13.8 spectra·s-1, ionization chamber temperature of 200 °C, and transfer line temperature of 250 °C. Sample handling consisted of filtering through a 45 µm PTFE microfilter, dissolving 200 µL of Eo in 800 µL of HPLC-grade hexane, and injecting 1 µL into the GC inlet at 220 °C in split mode (10:1). Data were processed with the MassHunter Workstation software (Agilent Technologies, USA). Compounds were identified based on their mass spectra fragmentation patterns with the spectral database of the National Institute of Standards and Technology (NIST) and by comparing their Kovats indexes relative to the retention times of a series of alkanes (C7-C40) using the calculator described by Lucero, Estell, Tellez, and Fredrickson (2009). The relative quantities of each compound were expressed as percentage of area.
Inhibitory effect of essential oil
Blackberry fruits (Rubus fructicosus L.) variety Cheyenne, collected at commercial maturity in San Juan Tezontla, Texcoco de Mora, Mexico (19° 32’ 04’’ N and 98° 47’ 17’’ W, at 2476 m a. s. l.) were used. The region experiments average maximum temperatures of 22.62 ± 1.97 °C, average minimum temperatures of 10.25 ± 2.70 °C, and average precipitation of 34.42 ± 30.74 mm. In addition, the soil presents characteristics of clayey sediments (Moreno-Sánchez, 2007).
Fruits were stored at 30 °C until a fungal development was observed. Fruits were submerged in sterile distilled water for 5 min with gentle agitation to form a culture broth. Fungi were isolated in potato-dextrose-agar (PDA) culture media and observed with an optical microscope to identify the species present. The methodology described by Aguilar-González, Palou, and López-Malo (2015) was used to determine a minimum inhibitory concentration (MIC) in vitro and in vivo. PDA culture media were prepared in petri dishes and oregano Eo was incorporated in concentrations of 0.1, 0.2, 0.3, and 0.4 µL·mL-1. Plates were inoculated with the culture broth, they were incubated at 35 °C, and radial mycelial growth was measured with a digital vernier scale for 5 d. Thereafter, blackberry fruits were inoculated at three points on the external surface with a bacteriological loop and placed in 400 mL airtight jars. Sterile cottons containing Eo in quantity to produce concentrations in the range from 0.2 to 0.8 μL·mL-1 were placed inside. Units were placed at 30 °C for 5 d and the percentage of damage was quantified.
Handling of fruits in modified atmosphere
Experimental units were formed with polyethylene terephthalate cylindrical recipients with volume of 400 mL and contained batches of 180 ± 2 g of fruits. Units were stored in an isolated room at 23 ± 0.5 °C and 62 % relative humidity for 7 d under two conditions. One consisted of handling in natural air (control), with containers without lids. The other was formed with closed recipients having three perforations of 100 μm in their lids and containing a cotton impregnated with Eo at 0.8 µL·mL-1 relative the container volume (treatment; MAP-Eo). Three units from each treatment were withdrawn daily to evaluate accumulated weight loss, color, firmness, appearance, pH, antioxidant capacity, and contents of total soluble solids (TSS), titratable acidity (TA), total soluble phenols (TSP), and anthocyanins.
Parameters evaluated
Experimental units were weighted with a digital scale (Ohaus®, USA) at the beginning and at the time of removal from storage. Based on the difference, the cumulative weight loss was calculated. Color was measured on the surface of fruits with a colorimeter (MiniScan XE® Plus, Hunter Associates Laboratory, USA) and was expressed as lightness (L*), hue angle (H*, degrees), and chroma (C*) (Sant’Anna, Gurak, Ferreira, & Tessaro, 2013). Firmness was determined with a texture analyzer equipment (TA-TX2i, Stable Micro Systems, UK), using a 5 mm spherical probe that compressed fruits until a deformation of 2 mm at a speed of 2 mm·s-1. Results were expressed in Newton (N) as the average of five determinations. The fruit appearance was evaluated by three untrained judges, through a categorical scale of five points, defined as “very good” (5), “good” (4), “regular” (3), “bad” (2), and “very bad” (1). TSS content was evaluated in °Brix with a manual refractometer (Alla France®, USA) in juice obtained through the maceration of five fruits (AOAC, 1990). TA (% citric acid) and pH were evaluated through titration with NaOH 0.03 N and a potentiometer (Conductronic, USA), respectively, in juice obtained from 20 g of fruits ground with 50 mL of distilled water.
Contents of TSP and anthocyanins, and antioxidant capacity were evaluated in extracts obtained with the method described by Hernández-Rodríguez et al. (2016). Samples of 1 g of fruits were mixed with methanol 80 % (v/v) at 1:20 (w:v) ratio and the pH was adjusted to 3.0 with HCl (10 %). Agitation in Vortemp® 56 equipment (ThermoFisher Scientific, USA) was applied at 1,000 rpm for 3 min, sonication for 15 min, and agitation at 150 rpm and 30 °C during 30 min. The mixture was centrifuged in equipment Hettich Zentrifugen (Germany) at 2,200 g for 15 min. The supernatant was recovered and calibrated to a final volume of 10 mL with methanol 80 %. The extracts of each treatment were processed in triplicate.
TSP were quantified using the Folin-Ciocalteu (FC) reagent method (Singleton & Rossi, 1965) adapted to a spectrophotometer with a microplate reader (Synergy™ HTX, BioTek Instruments, USA). Twenty-five microliters of the diluted extract (1.5:1, v/v) were mixed with methanol 80 % and 125 µL of distilled water, 20 µL of FC reagent diluted with distilled water (1:10, v/v), and 30 µL of 20 % Na2CO3. The mixture was shaken and left to stand for 30 min in dark. Absorbance at 760 nm was measured and TSP were quantified with the aid of a gallic acid standard curve in the range of 2.5-29.0 μg·mL-1. Results were expressed as milligrams equivalent of gallic acid per 100 g of sample on a fresh basis (mg·100 g-1). All samples were analyzed in triplicate.
Total anthocyanin content was evaluated with the pH differential spectrophotometric protocol described by Lee, Robert, and Wrolstad (2005). An aliquot of 100 μL of the extract was mixed with 900 μL of buffer pH 1 (0.025 M KCl). The absorbance of the mixture was measured at 510 and 700 nm in a spectrophotometer (Synergy™ HTX, USA). Another aliquot of the extract (100 µL) was mixed with 900 µL of 4.5-pH buffer and absorbance was measured at the same wavelengths. The anthocyanin content was determined as: Ant = (A M / 0.38 ε), where M is molecular weight of cyanidin-3-O-glucoside (449.2 g mol-1), ε is the molar extinction coefficient of this compound (26,900 L·mol-1·cm-1), the constant 0.38 is the path length of the light beam, and A was obtained as [A = (A 520nm - A 700nm ) pH 1.0 - (A 520nm - A 700nm ) pH 4.5 ]. Results were expressed as mg equivalent of cyanidin-3-O-glucoside per 100 g of fresh sample (mg·100 g-1).
The antioxidant capacity was determined with the ABTS (2,2’-azino-bis [3-ethylbenzothiazolin-6-sulfonic] acid) (Re et al., 1999) assay and the FRAP (ferric reducing antioxidant power) (Benzie & Strain, 1996) assay. The ABTS+ free radical was generated through the reaction between solutions of ABTS (7.4 mM) and sodium persulfate (2.6 mM), which were mixed at a 1:1 (v/v) ratio and protected from light for 16 h. An aliquot of 600 µL of the mixture was made up to 10 mL with methanol. An aliquot of 20 μL of the standard or the extract under study was mixed with 180 μL of the ABTS+ solution. A spectrophotometer (Synergy™ HTX, USA) was used to evaluate the decrease of absorbance at 734 nm. A Trolox calibration curve was prepared in the range of 4.99 to 59.93 µM. Results were expressed as micromoles equivalent of Trolox per gram of sample on a fresh basis (µmol·g-1). In the second assay, the FRAP reagent was prepared by mixing acetate buffer (300 mM, pH 3.6), 10 mM TPTZ solution in 40mM HCl, and 20mM FeCl3•6H2O solution, at a ratio of 10:1:1, respectively. An aliquot of 20 μL of the extract was mixed with 180 μL of FRAP reagent and 60 μL of distilled water. Absorbance was measured at 595 nm. Antioxidant activity was quantified with the aid of a Trolox calibration curve in the range of 3.8 to 46 µM. Results were expressed as micromoles equivalent of Trolox per gram of fresh sample (µmol·g-1 fw). All evaluations were done in triplicate.
Experimental design and data analysis
The MIC determination was conducted as a completely randomized design, where the oil concentration was the variation factor. The fruit postharvest evaluation was conducted based on a factorial arrangement, were treatments (control and MAP-Eo) and time were considered as variation factors. Analyzes of variance were performed together with treatment mean comparison routines applied with the Tukey’s test (P ≤ 0.05).
Results and discussion
Oregano essential oil analysis
Twenty-two compounds were identified in the Eo, mainly monoterpene hydrocarbons and oxygenated monoterpenes. The most abundant were linalool (24.27 %), o-cymene (14.08 %), and thymol (15.14 %) (Table 1). In this regard, Tapiero et al. (2019) found p-cymene and terpinene-4-ol among the main components of oregano Eo, in addition to carvacrol and thymol.
Table 1 Chemical composition of oregano essential oil.
| Peak | Name | MF | MW | RIexp | RIlit | Area (%) |
|---|---|---|---|---|---|---|
| 1 | α-Thujene | C10H16 | 136.1 | 1012.3 | 1017 | 0.99 |
| 2 | Camphene | C10H16 | 136.1 | 1061.6 | 1068.5 | 0.70 |
| 3 | β-Myrcene | C10H16 | 136.1 | 1153.9 | 1160.9 | 1.74 |
| 4 | α-Phellandrene | C10H16 | 136.1 | 1162.5 | 1167.7 | 0.19 |
| 5 | α-Terpinene | C10H16 | 136.1 | 1178.1 | 1177.8 | 2.08 |
| 6 | Limonene | C10H16 | 136.1 | 1196.2 | 1198.2 | 1.25 |
| 7 | Eucalyptol | C10H18O | 154.1 | 1214.8 | 1211.1 | 0.50 |
| 8 | g-Terpinene | C10H16 | 136.1 | 1247.0 | 1245 | 5.06 |
| 9 | o-Cymene | C10H14 | 134.1 | 1275.2 | 1268 | 14.08 |
| 10 | Isoterpinolene | C10H16 | 136.1 | 1283.6 | 1286 | 0.77 |
| 11 | 3-octanol | C8H18O | 130.1 | 1381.2 | 1383 | 0.20 |
| 12 | Linalool | C10H18O | 154.1 | 1543.4 | 1543.3 | 24.27 |
| 13 | o-Methylthymol | C11H16O | 164.1 | 1602.6 | 1597 | 0.12 |
| 14 | Terpinen-4-ol | C10H18O | 154.1 | 1611.8 | 1616 | 1.44 |
| 15 | Caryophyllene | C15H24 | 204.2 | 1618.9 | 1617 | 2.77 |
| 16 | α-Terpineol | C10H18O | 154.1 | 1709.3 | 1706 | 4.46 |
| 17 | Unknown 1 | 220.2 | 1727.2 | 1.51 | ||
| 18 | Geranyl acetate | C12H20O2 | 196.1 | 1765.6 | 1763 | 0.38 |
| 19 | Thymol acetate | C12H16O2 | 192.1 | 1855.1 | 1845 | 4.60 |
| 20 | Unknown 2 | 207.1 | 1938.6 | 4.74 | ||
| 21 | 2,3,5,6-Tetramethylphenol | C10H14O | 150.2 | 1953.1 | 10.44 | |
| 22 | Thymol | C10H14O | 150.1 | 2146.0 | 2151 | 15.14 |
MF = molecular formula; MW = molecular weight; RIexp = Kovats index calculated from retention time data on a DB-WAX capillary column; RIlit = Kovats index from literature (NIST). Minor peaks were omitted since the similarity with database was below of 70 %. (-) The Kovats index was not reported on a DB-WAX column.
Minimum inhibitory concentration
Blackberry fruits placed at 30 °C showed growth of three species of fungi: Alternaria alternata, Aspergillus carbonarius, and Penicillium digitatum. This coincided in part with information published before, since it has been reported that the postharvest damage of blackberries can occur through the development of fungi such as Botrytis spp., Colletotrichum spp., and Aspergillus spp. (Cosseboom et al., 2020; Junqueira-Gonçalves, Alarcón, & Niranjan, 2016; Liu et al., 2019; Uribe-Gutiérrez et al., 2022).
The incubation of PDA culture media incorporated with Eo and inoculated with the fungal species showed that the growths of A. alternata and P. digitatum were completely inhibited at Eo concentration of 0.2 µL·mL-1 during in vitro evaluations, while A. carbonarius required the use of 0.4 µL·mL-1 (Table 2). On the other hand, the exposure of inoculated blackberry fruits to volatilized Eo at 0.2, 0.4, 0.6, and 0.8 μL·mL-1 showed that the higher the concentration the higher the inhibition. However, such in vivo evaluations showed that a complete absence of fungal development was observed only with the highest value (Table 2), thus the concentration of 0.8 μL·mL-1 was considered as the MIC. These facts indicated that the use of Eo vapors constituted a feasible strategy that did not require a vehicle for the Eo, such as a biopolymeric coating.
Potma da Silva et al. (2020) used biopolymeric coatings to preserve blackberry fruits, by incorporating Eo from lemongrass in the form of nanoparticles in microfibrillated cellulose. A similar strategy was used by Shi et al. (2022), with Eo of oregano applied on blackberry fruits through polylactic acid/polycaprolactone electro spin nanofibers. However, although in these works a lengthening of the shelf life was achieved, the procedure required greater processing of Eo, which can difficult a practical implementation. On the other hand, Reyes-Jurado et al. (2019) demonstrated the antifungal capacity of oregano Eo applied in volatilized form in the headspace of culture media and determined MIC values between 0.25 and 1.00 μg·mL-1 for Aspergillus spp. and Penicillium spp. In this sense, the presence of terpenes in the Eo can affect the membrane permeability and functionality, in addition to their lipophilic property, that gives them the ability to penetrate cell walls and affect enzymes involved in cell-wall synthesis, thus altering the morphological characteristics of fungi (Pandey, et al., 2017).
Table 2 In vitro and in vivo inhibitory effect of essential oil of oregano after five days of inoculation.
| Treatments (µL·mL‑1) | P. digitatum | A. alternata | A. carbonarius |
|---|---|---|---|
| In vitro evaluation (fungal development in Petri dish, cm) | |||
| Control (0) | 1.17 ± 0.04 Caz | 2.10 ± 0.15 Ba | 8.50 ± 0.28 Aa |
| 0.1 | 0.20 ± 0.07 Cb | 0.98 ± 0.06 Bb | 4.07 ± 0.40 Ab |
| 0.2 | 0.0 Bc | 0.0 Bc | 3.00 ± 0.63 Ab |
| 0.3 | 0.0 Bc | 0.0 Bc | 0.56 ± 0.03 Ac |
| 0.4 | 0.0 Ac | 0.0 Ac | 0.0 Ac |
| In vivo evaluations (percentage of fruits damaged) | |||
| 0.4 | 16.67 ± 8.33 Cbc | 33.33 ± 8.33 Bc | 66.67 ± 8.33 Aab |
| 0.6 | 8.33 ± 8.33 Bc | 8.33 ± 8.33 Bc | 41.67± 8.33 Abc |
| 0.8 | 0.00 Ac | 0.00 Ad | 16.67± 8.33 Ac |
zEqual capital letters indicate non-significant difference within a row. Equal lowercase letters indicate non-significant difference within a column (Tukey, P ≤ 0.05). Values in parentheses correspond to standard deviation.
Handling in modified atmosphere
Weight loss
The cumulative weight loss increased in fruits during the storage, at a rate of 3.34 and 0.31 %·d-1 in control (handling in air) and treatment (handling in MAP-Eo), respectively, with significant contrast between them (Figure 1A). At the end of the storage, the cumulative weight loss was 24.85±2.07 % in the control and 2.15±0.47 % in MAP-Eo. The handling in MAP-Eo was efficient to reduce weight loss, because the relative humidity increased in the semi-closed space, which caused the vapor pressure deficit to be reduced, thus reducing the transpiration of the fruits (Hübert & Lang, 2012). A similar phenomenon was reported by Pérez, Gómez, and Castellanos (2021) when a modified atmosphere with micro-perforation was used for the preservation of blackberry fruits.
Figure 1 Variation of weight loss (A), firmness (B), color attributes expressed as lightness (C), hue angle (D) and chroma (E), and fruit appearance (F) of blackberry fruits handled in natural air (control) and modified atmosphere (MAP-Eo). Equal capital letters in legends indicate non-significant difference between treatments. Equal lowercase letters indicate non-significant difference within treatments (Tukey, P ≤ 0.05). Error bars correspond to standard deviation.
Firmness
Fruits presented initial firmness of 1.49±0.08 N and a loss of consistency was recorded in the control during storage (Figure 1B). However, firmness was maintained without significant difference relative to the initial value for 5 d with MAP-Eo, which indicated a positive effect of such strategy. The loss of firmness involves modifications at cell wall level caused by several enzymes such as polygalacturonase, pectinesterase, pectate lyase, among others (Tucker et al., 2017).
In the case of blackberry fruits, Zhang, Xiong, Yang, and Wu (2019) studied the softening phenomenon of two varieties and found noticeable increments in polygalacturonase and cellulase activities in the late softening stages, coinciding with a disassembling of the cell wall, but authors found that other enzymes can be present as a function of the variety. Authors also found that cellulose and hemicellulose declined during ripening of fruits. On the other hand, Horvitz, Chanaguano, and Arozarena (2017) indicated that the microbial growth significantly limited the shelf life of Andean blackberry fruits handled in polyethylene terephthalate containers at 18 °C and observed a reduction in firmness from 4.0 to 2.5 N in 3 d. In the present work, handling was also carried out in modified atmospheres, but with higher temperature (23 °C) and with exposure to volatilized Eo, which indicated that this allowed reducing the loss of consistency of fruits through inhibition of the fungal development.
Color
Fruits presented lightness, hue angle, and chroma of 16.61±0.30, 286.09±5.93°, and 1.62±0.10, respectively. These values were similar to those reported by Mikulic-Petkovsek, Koron, Zorenc, and Veberic (2017), who evaluated different blackberry varieties and determined, on average, lightness of 14.08, hue angle of 266.37°, and chroma of 2.09, for fruits harvested at consumption maturity. The hue angle suggests that blackberry fruits exhibit hue close to blue (Sant’Anna et al., 2013); however, the low lightness and chroma values cause the dark-purple appearance of fruits at edible maturity.
During the storage, lightness presented non-significant variations in both handlings, with no significant difference between them (Figure 1C). In the case of chroma, a downward trend was identified, to an average value of 1.37±0.04 (Figure 1E), which was equivalent to a change of 15.4 % and occurred without contrast between treatments. According to Mikulic-Petkovsek et al. (2017), blackberry fruits show descending changes in lightness and chroma of about 2.20 and 21.05 %, respectively, during the transition from optimal consumption maturity to over-ripeness, so the behavior that was found was considered normal.
On the other hand, the fruits of the control showed an increase in hue angle up to 318.33±8.85° on the third day and remained in that condition until day seven (Figure 1D). However, the MAP-Eo fruits showed an increase in hue angle up to 334.71±2.56° on the third day of storage and a subsequent decrease to 301.47±12.30°, although MAP-Eo values were higher than those of the control. However, this behavior was not appreciated visually, due to the low values of lightness and chroma. Color reversion is a factor that deteriorate the quality of blackberries in postharvest and consists of the occurrence of drupelets in a fruit where hue turns from dark purple or black to red, which generates an appearance of irregular coloration (Armour, Worthington, Clark, Threlfall, & Howard, 2021).
Lawrence and Melgar (2018) defined that a blackberry fruit presented color reversion when five or more drupelets showed a clearly red hue. This situation was not observed in the present work, so the increment in the hue angle in the control was attributed to an increase in concentration of anthocyanins (Figure 2D), due to the weight loss of fruits (Figure 1A), but a continuous increase in pH (Figure 2B) prevented a continuous elevation in hue after day three. However, in the case of MAP-Eo, where weight loss did not significantly vary and neither did the concentration of anthocyanins (Figures 1A and 2D), it is believed that the fruits tended to show a phenomenon of color reversion, but the increase of pH prevented a clear manifestation of this disorder and induced the reduction of hue from day three.
Figure 2 Variation of total soluble solids (A), acidity (B), content of total soluble phenols (C), content of total anthocyanins (D), and antioxidant capacity evaluated with the ABTS (E) and FRAP tests (F) in blackberry fruits. Equal capital letters in legends indicate non-significant difference between treatments. Equal lowercase letters indicate non-significant difference within treatments (Tukey, P ≤ 0.05). Error bars correspond to standard deviation.
Appearance
Fruits of the control showed continuous deterioration of the appearance from the first day of storage and such attribute was classified as regular on day three, due to wilting and the beginning of fungal development, which increased in the following days (Figure 1F). Toscano-Ávila et al. (2020) indicated that the shelf life of blackberry fruits is limited by dehydration and an appearance of wilting and aging, which they qualified as a physiological disorder and typified it as the cause of a shelf life of only 3 d at 25 °C. On the other hand, fruits stored in MAP-Eo maintained their appearance without significant changes until day five of storage. Therefore, the use of MAP-Eo caused a beneficial effect on fruits, since they had a shelf life between 6 and 7 d at 23 °C.
The results obtained are similar to the shelf life of 6 d at 22 °C obtained for blackberry fruits by Heras-Mozos, Gavara, and Hernández-Muñoz (2021), through the antifungal capacity of biopolymeric coatings based on the covalent union of trans-2-hexenal, and aldehyde with antifungal properties, with primary amino groups of chitosan with a Schiff base or imine formation. In that work, the mechanism of action was based on the hydrolysis of imine bond and the release of the active agent. On the other hand, different works have suggested the use of refrigeration at 4 °C to achieve shelf life of 7-8 d (Bersaneti et al., 2021; Vilaplana et al., 2020) and, with the use of biopolymeric coatings at the same thermal condition, shelf life between 9 and 12 d has been achieved (Heras-Mozos et al., 2021; Toscano-Ávila et al., 2020). However, refrigeration infrastructure is lacking in many production areas, which justifies the use of a simpler strategy, such as the one shown in this work, through the release of Eo vapors in the head space of fruits.
Total soluble solids, titratable acidity and pH
Fruits presented average content of TSS of 8.66±0.33 °Brix at the beginning and, throughout the storage, values remained in the range of 8.76 to 10.66 °Brix, without significant difference between control and MAP-Eo, except on day seven, when an increase in SST was observed in the control and a reduction in MAP-Eo (Figure 2A). Cortés-Rodríguez, Villegas-Yépez, Gil-González, and Ortega-Toro (2020) reported TSS content of 8.17 °Brix in Andean blackberry fruits and the reduction of this value in fruits handled without any treatment, which was attributed to the consumption of sugars and organic acids in the respiratory process. In the same work, an increase in TSS was observed in fruits handled at 4 °C with a biopolymeric coating, which was attributed to a concentration process derived from the weight loss of fruits during storage.
The TA decreased throughout the storage, from 0.76±0.01 to 0.16±0.01 %, which occurred similarly in both treatments (data not shown). The initial values were similar to those reported by Kim, Perkins-Veazie, Ma, and Fernandez (2015), between 0.92 and 0.94 %. The reduction of TA has been reported in other works and may be due to the consumption of organic acids in the respiration process (Cortés-Rodríguez et al., 2020; Kim et al., 2015), to obtain energy (Martínez-Damián, Cruz-Arvizu, & Cruz-Álvarez, 2020). Consistent with the behavior of acidity, a significant increase in the pH of fruits was observed, from an initial value of 3.37±0.07 to 5.09±0.11 (control) and 4.82±0.13 (MAP-Eo) (Figure 2B), with no general contrast between both conditions. The initial pH value was similar to that reported in other works. In this regard, Schulz et al. (2019) reported a value of 3.48, Cortés-Rodríguez et al. (2020) found values between 2.97 and 3.00, while Kim et al. (2015) reported a value of 3.73. Likewise, the increase in pH during the postharvest period has been coherent with the decrease of acidity (Cortés-Rodríguez et al., 2020).
Total soluble phenols and total anthocyanins
The content of TSP was 270.97±15.56 mg·100 g-1 at the storage beginning. Schulz et al. (2019) identified 26 phenolic compounds in blackberry (R. ulmifolius) fruits, highlighting the presence of quercetin, isoquercetin, kaempferol, (+)-epicatechin, (+)-catechin, gallic acid, sinapic acid, 3,4-dihydroxybenzoic acid, caffeic acid, and p-coumaric acid. During the storage, the concentration increased in fruits of the control, up to 366.39±14.54 mg·100 g-1 on the day seven, but remained without significant changes, in the range of 268 to 282 mg·100 g-1, in fruits of MAP-Eo, which caused that the TSP average content was significantly higher in control than in MAP-Eo (Figure 2C). Similarly to the control, Cortés-Rodríguez et al. (2020) found that the total phenolic content presented an increasing trend to a maximum value on day six in blackberry fruits harvested at full maturity and stored in natural air at 4 °C. Also, Horvitz et al. (2017) reported the increment of the total phenolic content in fruits of blackberry harvested at full maturity and stored at 18 °C in natural air. Mikulic-Petkovsek et al. (2017) evaluated separately the variation of anthocyanins, flavanols ellagitannins, and flavonol derivatives in five cultivars of blackberry fruits, and found an increment in all the compounds between optimal ripe and over-ripe maturity states.
The behavior observed in the control may be indicative of senescence, since the reactive oxygen species (ROS) may increase with such condition, causing an increase in phenolic compounds in defense of oxidative stress (Neves, Tosin, Benedette, & Cisneros-Zevallos, 2015). In this sense, the absence of an increment of TSP in the treatment MAP-Eo (Figure 2C) may indicate that fruits did not activate a defense mechanism against ROS, which constituted a positive effect on delaying senescence.
Total anthocyanin content was 221.18±23.61 mg·100 g-1 at the storage beginning, which was higher than values of 112.81 and 153.40 mg·100 g-1 reported by Mikulic-Petkovsek et al. (2017) for fruits at optimum consumption and over-ripe maturities, respectively. These authors indicated that cyanidin-3-O-glucoside was the principal anthocyanin found in blackberry fruits. During the storage, the content increased in the control up to 321.45±23.50 mg·100 g-1, parallel to the TSP behavior (Figure 2D), which represented a change of 45.3 % and was consistent with the 40 % of variation found by Mikulic-Petkovsek et al. (2017) between optimal maturity and over-ripeness. However, the anthocyanin content remained between 179 and 221 mg·100 g-1 in MAP-Eo fruits, without significant changes throughout the storage. The color of blackberry fruits is directly associated with the content of anthocyanins and with the maturity stage (Li et al., 2022), which suggested that the handling in MAP-Eo caused a delay in the metabolic activity that leads to senescence.
Antioxidant capacity
The ABTS and FRAP assays indicated antioxidant capacity of 9.13±0.48 and 13.44±1.126 μmol·g-1 at the storage beginning. The ABTS assay is based on an electron transfer principle, while the FRAP assay is based on a hydrogen atom transfer phenomenon (Apak, Özyürek, Güçlü, & Çapanoğlu, 2016), so it is common that results are different. The antioxidant capacity increased throughout storage in the control, up to 10.35±3.80 μmol·g-1, according to the ABTS test and up to 18.53±1.02 μmol·g-1 according to the FRAP one, while remained between 8.6-9.5 and 11.7-13.7 μmol·g-1, respectively, without significant changes, in MAP-Eo fruits (Figures 2E and 2F).
The antioxidant potential of blackberry fruits is commonly associated with the presence of bioactive substances such as phenolic compounds. In this regard, Mikulic-Petkovsek et al. (2017) reported that phenolic compounds increased in blackberry fruits of different varieties, which caused that in over-ripe fruits the antioxidant capacity (FRAP values) was in the range of 25.9-43.2 μmol·g-1. Likewise, Li et al. (2022) determined in fruits of two blackberry varieties that the non-anthocyanin flavonoid fraction diminished from early to late maturity stages and also its antioxidant activity, while the anthocyanin fraction increased, along with its antioxidant potential. These facts pointed out that the advance of an over-ripe state is accompanied by an increment of the antioxidant capacity and suggested that the less value that was found in fruits of MAP-Eo corresponded to a delaying the senescence of them. Besides, although the increase in antioxidant capacity during storage may be a beneficial factor, it may actually be indicative of the increase in oxidative stress in fruits, derived from a process of over-ripening or senescence and therefore the reduction in the marketing potential of fruits.
Conclusions
The use of oregano essential oil in the head space of blackberry fruits reduced significantly the fungal development, principally for Aspergillus spp., followed by Alternaria spp., and Penicillium spp. The shelf life of blackberry fruits was extended up to 6-7 d at 23 °C relative to fruits of the control that exhibited postharvest life of 3 d.
The use of modified atmosphere systems with microperforation and with vapors of essential oil (MAP-Eo) in the head space allowed reducing the rate of weight loss, the loss of consistency, and that of alteration of color attributes in fruits, allowing a better appearance along the storage at room temperature conditions. Likewise, the use of MAP-Eo allowed maintaining the content of phenolic compounds and anthocyanins, as well as the antioxidant capacity, without significant changes. The use of MAP-Eo systems is a useful alternative for postharvest handling of blackberry fruits at room conditions.
