Localization

This test was carried out in the plant physiology laboratory of the Universidad Pedagógica and Tecnológica de Colombia (Boyaca, Colombia) with avocado fruits that were harvested from a commercial crop on the La Esperanza farm in the Alto el Tigre village in the municipality of Lejanias, with coordinates the 3° 29' 39” N and 73° 59' 47” W, an annual average temperature of 23°C, and a relative humidity that oscillates around 59%.

Plant material

Avocado fruits of the Lorena variety, commonly known as Papelillo, were used, harvested from the middle third of the plant and classified in such a way that were in physiological maturity, free of mechanical damage, with good size (462 g on average), good appearance and marketable. Fruits were taken the same day of the harvest to the laboratory to start the measurements. The fruits were obtained from a 3-year-old crop whose plants were grafted on a so-called 'common' or 'criollo' rootstock, and which are planted in a soil characterized as Inceptisol. The crop receives ten applications per year of “Triple 18” as base fertilization, with an average dose of 1 kg per tree in each fertilizer, it also has a drip irrigation system of five drippers per plant of 4 L h^-1.

The fruits were classified in the laboratory according to the hardness of the epidermis into two degrees of maturity (maturity I: fruits with more hardness to the touch according to the ^{Icontec standard [2003]} NTC 5209; maturity II: fruits with less hardness to the touch), of which, none were in a mature state of consumption.

Experiment design

An experiment design with complete random blocks (BCA) was used, since this is the statistical arrangement that uses the least amount of experimental units (UE). The blocking criterion was the state of maturation of the fruits, with two blocks (maturity I and maturity II), five maturation treatments, three ethylene applications (2, 4 and 6 mL L^-1), fruits wrapped in unprinted newspaper and a control, for a total of 10 EU. Each UE had 24 fruits, for a total of 240.

For the ethylene applications, Ethrel^® was used as a 50% commercial product, from which the concentrations in 4 L buckets were calculated using 16 mL of Ethrel^® to achieve a concentration of 2 mL L^-1 of ethylene, 32 mL of ethrel for the concentration of 4 mL L^-1, and 48 mL of ethrel for the concentration of 6 mL L^-1. In all cases, the avocado fruits were completely immersed in the ethylene solution for 10 min.

Response variables

The following variables were evaluated every 3 d in 3 fruits per EU as follows: the pH was measured in 1 mL of avocado fruit juice graduated to 50 mL with distilled water using a Hanna HI8424 potentiometer (Hanna Instruments, Woonsocket, RI) calibrated with pH 7.0 and 4.0 buffer solutions; the respiratory intensity (IR) was recorded using 2 L SEE BC-2000 hermetic chambers (Vernier Software & Technology, Beaverton, OR, USA); and the CO₂ concentration was determined using the infrared sensor VER CO2-BTA (Vernier Software & Technology, OR, USA) and a Labquest2 interface (Vernier Software & Technology, Beaverton, OR, USA) for 5 min, expressed in mg of CO₂ kg^-1 h^-1 and calculated using Equation (1):

Where, RR was respiratory intensity, Cv chamber volume (2 L), M mass of the fruits (kg) and m slope of the linear regression in Labquest2.

The total soluble solids (SST) were measured with a Hanna HI 96803 digital refractometer (Hanna Instruments, Woonsocket, RI) with a scale from 0 to 85% and precision of 0.1 °Brix. The total titratable acidity (ATT) was quantified by measuring the volume of sodium hydroxide (NaOH) incorporated in 10 mL of fruit juice, brought to 50 mL with distilled water and adding 5 drops of phenolphthalein in potentiometric titration until a pH of 8.2, and was calculated using Equation (2):

Where, A was volume of NaOH spent, B normality of NaOH (0.1 N), C equivalent mass expressed in grams of predominant acid in the fruit (tartatic acid 0.075 g meq^-1) and M mass in grams of the sample used.

The fruit firmness (N) was determined using a PCE-PTR200 digital penetrometer (PCE Iberica, Spain) with a precision of 0.05 N. The accumulated mass loss (PM) was evaluated using an Acculab VIC 612 electronic balance with 0.01 g precision (Sartorius Spain SA, Madrid) during the postharvest phase, taking six fruits for each UE, measured over time and calculated using Equation (3):

Where, ML percentage of accumulated mass loss, Mi initial mass of the fruit and Mf mass of the fruit at each moment.

The epidermis color was measured with a Minolta CR 300 digital colorimeter (Minolta Co., Osaka, Japan), considering the parameters luminosity (L*), chromaticity from green to red (a*) and chromaticity from blue to yellow (b*), always using the same measurement point as a reference.

Analysis of the data

The data were analyzed using a Kolmogorov-Smirnov normality test. Then, an analysis of variance and a Tukey test (P≤0.05) were performed with SAS v 9.2e (Sas Institute Inc., Cary, NC).

Accumulated mass loss

No significant differences were observed between the maturity stages (Fig. 1) or between the accelerating ripening treatments. The maturity II stage presented a greater mass loss (30.21%) than the maturity I stage, which had 25.76% at 16 dah. All treatments, on average, lost mass as the storage time elapsed because of the ripening process of the avocado fruits; however, a greater loss of mass was observed in the fruits treated with 6 mL L^-1 of ethylene (30.94%) than in the fruits without applications (23.55%) at 16 days after harvest (dah), which was higher than the 10% mass loss reported by ^{Ruiz (2018)} in Lorena variety fruits without treatments at 14 dah.

Figure 1. Accumulated mass loss in ‘Lorena’ avocado fruits harvested in two stages of maturity. Maturity I: fruits with more hardness to the touch; Maturity II: fruits with less hardness to the touch; ns: no significant effect between treatments according to the Anova (P≤0.05). Vertical bars indicate the Tukey range test. 

Likewise, ^{Márquez et al. (2014)} found an increasing behavior in the loss of mass during the postharvest stage with final values that ranged between 22.4 and 23% for avocado cv. Hass, similar to ^{Herrera-González et al. (2017)}, who reported mass losses of 21.5% in avocado cv. Hass, and ^{Vallejo-Pérez et al. (2015)}, who obtained values of 26.1 and 23% for fruits of cv. Vigorous and sunspot viroid (ASBVd) infected ‘Hass’, respectively. Water loss is the main cause of deterioration in the postharvest quality of fruits and is associated with a greater loss of mass when fruits mature (^{Vallejo-Pérez et al., 2015}). Cell membranes, being semi-permeable, allow water to exit via transpiration, which depends on the vapor pressure (DPV) of water between the fruit and the environment. So, the longer the storage time is, the greater the loss of water is (^{Díaz-Pérez, 2019}).

Firmness

Figure 2. Firmness in ‘Lorena’ avocado fruits harvested in two stages of maturity. Maturity I: fruits with more hardness to the touch; Maturity II: fruits with less hardness to the touch; ns: no significant, * and ** indicate significant effect between treatments according to the Anova (P≤0.05 and P≤0.01, respectively). Vertical bars indicate the Tukey range test. 

There were significant differences between the maturity stages at 9, 12 and 16 dah. The fruits harvested in maturity stage I presented the highest firmness values throughout storage, which agrees with the initial classification by stages since they were the hardest to touch. After 3 dah, a significant loss of firmness was observed in the avocado fruits, which decreased from 123.1 N to 20.82 N at 6 dah, followed by values of 6.38 N at 16 dah, on average for all treatments and maturity stages (Fig. 2). This agrees with ^{Ruiz (2018)}, who found values of 101.2, 24.5 and 10.6 N in preclimateric, climacteric and postclimacteric fruits, respectively, which confirmed a drastic decrease in firmness during storage similar to that obtained by ^{González-Cuello et al. (2017)} in the first week of postharvest for avocado ‘Hass’. Likewise, ^{Caparrotta et al. (2015)} reported that, in avocado fruits, firmness is a good indicator of the ripening stages, and ^{Benítez (2018}) found a range of harvest maturity between 3 to 9 dah, a consumption maturity that ranged between 9 to15 dah, and an overripeness between 15 and 21 dah.

The treatments only showed significant differences at 12 dah. The fruits without applications of the ripening accelerator maintained higher firmness values (55.34 N) during storage, followed by the treatment wrapped with newsprint paper (52.24 N), as compared to the fruits treated with 6 mL L^-1 of ethylene (47.41 N). These results agree with ^{Vallejo-Pérez et al. (2015)}, where firmness showed a drastic reduction at 7 dah and presented an average value of 47.7 N at the end of postharvest. Likewise, these results are within the range of 37 to 45 N for ripe fruit consumption reported by ^{Icontec (2003)} in NTC 5209.

Once the increase in ethylene synthesis is generated, an increase in respiratory rate occurs, hese processes can be simoultanoeus and promote a decrease in firmness in fruits and a release of available energy through the action of enzymes, such as β-galactosidase, which carry out hydrolysis in the cell wall of terminal galactosyl residues of glycoproteins, galactans and carbohydrates (^{Sañudo-Barajas et al., 2019}). However, ^{Blakey et al. (2012)} stated that the concentrations of sugars, such as D-mannoheptulose, correlate positively with firmness and are increased in slow-ripening avocado varieties.

pH

The pH did not show differences between the treatments; however, the fruits with applications of 2 mL L^-1 of ethylene showed the highest values. When analyzing the repeated measurements over time, there were significant differences, and the pH had a slight increase towards 9 dah, followed by a slight decrease after the second week of postharvest, with values between 6.2 and 6.4 (Tab. 1), as reported by ^{Márquez et al. (2014)}, 6.42 to 6.63 for avocado ‘Hass’ at consumption maturity, and ^{Astudillo-Ordóñez and Rodríguez (2018}), who found pH values between 6.58 and 7.14. There was an increase in pH between weeks zero and three of storage, with maximums in the third week, followed by a slight decrease. Likewise, ^{Buelvas-Salgado et al. (2012)} obtained pH values in three degrees of maturity of 6.26, 6.30 and 6.41 in the green, pintón and mature states, respectively. ^{Vargas-Ortiz et al. (2016)} obtained ranges of 6.8 and 5.5 for the control treatment and 6.3 for avocados heated for 20 min.

The behavior of pH was associated with the content of organic acids present in the fruit since, in the ripening period, they tend to decrease because they are consumed in the different metabolic cycles. In addition, many act as precursors of volatile substances during ripening (^{Márquez et al., 2014}).

Total soluble solids (TSS)

The TSS had significant differences between the maturity stages at three and 16 dah, presenting values for maturity I that ranged from 1.66 to 8.50 °Brix and for maturity II between 1.66 and 8.26 °Brix (Tab. 1). This increase in °Brix was related to the degradation of polysaccharides and organic acids converted into sugars (^{Caparrotta et al., 2015}). According to ^{Buelvas-Salgado et al. (2012)}, the increase in the °Brix content was due to the higher concentration of sugars from respiration and the loss of water in perspiration, making avocados a climacteric fruit with a high increase in concentrations of ethylene at the beginning of ripening.

The TSS had significant differences over time and showed a constant behavior between days one and three of postharvest. Then, there was a strong increase up to 6 dah, going from 1.99 to 8.38 °Brix, after which they remained almost constant with a slight downward trend until 16 dah (7.41 °Brix) when storage ended. These maximum values at 6 dah coincided with the increase in CO₂ concentration in the avocado fruits, which was possibly due to an increase in metabolic activity, which involves the enzymes α-amylase and ß-amylase, which hydrolyze starch to the simpler compounds of disaccharides and monosaccharides during maturation (^{Yahia et al., 2019}).

Likewise, ^{Astudillo-Ordóñez and Rodríguez (2018)} found values of 7.26, 6.22 and 5.90 °Brix, for avocado ‘Hass’ in three harvest indices (early, intermediate and late, respectively), in which the TSS were higher in the early stages of maturity, as observed by ^{Benítez et al. (2021)}, who reported decreases from 7.4 to 5.1% in fruits with consumption ripeness.

Total titratable acidity (TTA)

The TTA did not show differences between the degrees of harvest maturity or between the treatments. The highest values were presented in the fruits that received the control treatment (0.44%), and the lowest values were in those that received 4 mL L^-1 of ethylene (0.33%) at 9 dah (Tab. 1).

The TTA had significant differences over time and presented a decrease at the beginning of postharvest up to 3 dah, with values between 0.31 to 0.24%, followed by an increase up to 9 dah (0.37%). After 12 dah, it began to decrease, indicating senescence, going from 0.37 to 0.22%. These values are similar to ^{Márquez et al. (2014)}, who found decreases in acidity from 0.25 to 0.16% during 21 dah in storage for avocado ‘Hass’ fruits grown in El retiro and El Carmen de Viboral municipalities in Antioquia (Colombia).

This decrease in TTA is attributed to the consumption of organic acids in the metabolism of the avocado fruits (product of the increase in respiratory intensity or in response to senescence), in which tartaric acid predominates, which provides energy for ripening (^{Caparrotta et al., 2015}).

Maturity index (MI)

The MI did not show significant differences between the maturity stages or between the treatments. The fruits that received 2 mL L^-1 of ethylene at 16 dah had a higher MI value, 57.84, than the other treatments (Tab. 1). In this regard, ^{Buelvas-Salgado et al. (2012)} found MI values of 15.38 in the green state, 24.55 in the pintón state and 38.80 in the ripe state in ‘Hass’ avocado fruits.

Over time, the MI had significant differences, with an increase from 3 to 6 dah, from 8.16 to 34.63, and then decreased, meaning the fruits can be consumed in that state. Towards day 16, the MI increased to 37.44 with senescence, and, despite the fact that sugars remained constant, the TTA decreased as a result of the consumption of acids in respiration (^{Yahia et al., 2019}), raising the MI. ^{Márquez et al. (2014)} found MI values that oscillated between 6.4 and 5.2 at harvest and between 15 and 10 at 21 dah for fruits harvested in the municipalities of El Retiro and El Carmen, respectively.

Respiratory rate (RR)

Although the RR did not show significant differences for the maturity stages or for the ripening treatments, the avocado fruits that received the application of 4 mL L^-1 of ethylene showed higher values, 62.79 mg CO₂ kg^-1 h^-1, than the control (38.73 mg CO₂ kg^-1 h^-1) at 16 dah, which suggests the effect of ethylene in increasing respiratory metabolism.

The RR showed significant differences over time throughout postharvest. During the first 6 dah, the RR increased by 170%, which could indicate the climacteric peak. Then, from 6 to 12 dah, it remained constant and, towards the end, had a slight increase, reaching values of 54 mg CO₂ kg^-1 h^-1 (Fig. 3). This is similar to the results of ^{Ruiz (2018)}, who found values of 50.89 mg CO₂ kg^-1 h^-1 in the climacteric peak in ‘Lorena’ avocados at 8 dah. Also, ^{Chen et al. (2017)} found an ethylene peak of 40.5 and 36 mg CO₂ kg^-1 h^-1 after 3 d of storage in avocado fruits treated with exogenous ethylene and from 41 to 38 mg CO₂ kg^-1 h^-1 at 6 and 8 dah in naturally ripened fruits. Furthermore, ^{Herrera-González et al. (2017)} found the climacteric peak in ‘Hass’ and ‘Méndez’ avocado fruits at 7 and 8 d after harvest in refrigerated fruits.

Figure 3. Respiratory rate in ‘Lorena’ avocado fruits. ns: no significant effect between treatments for the same time according to the Anova (P≤0.05) before the comma and different letters indicate significant differences between the measurements over time according to the Tukey test groupings (P<0.05) after the comma. Vertical bars indicate the Tukey range test. 

These values are lower than those of ^{Márquez et al. (2014)}, who observed a maximum value of 150 mg CO₂ kg^-1 h^-1 at 8 d postharvest with an increasing and continuous behavior in ‘Hass’ avocados, similar to that observed in the present study with ‘Lorena’ avocados. The climacteric peaks that occur in respiration are probably due to the stress that fruits undergo when removed from the plant, which generates an increase in mitochondrial activity in combination with the degradation of starch through enzymatic action and formation of carbohydrates (^{Saltveit, 2019}).

Color

The color parameters L*, a* and b* did not present significant differences for any of the treatments in the different periods of measurement; however, the epidermis of the fruits that received applications of ethylene wrinkled towards senescence. The L* value had a downward behavior during postharvest, going from 75.6 to 63.54 towards the end of storage, with an average of 72.5±8.7, higher than that reported by ^{Ruiz (2018)}, of 57.5. This indicates that the fruits lose brightness and luminosity through oxidation of the tissues, perspiration and internal browning as storage increases (^{Cogo et al., 2011}), which indicate a darkening of the avocado.

The b* parameter had a downward behavior and went from 76.1 to 51.8 during postharvest, with an average of 62.8 ± 9.2, which is higher than that obtained for the Lorena variety (47) by ^{Ruiz (2018)} at 12 dah. The a* value went from -65.1 to -39.1, which indicated a slight loss in the intensity of the green color. These values are lower and contrast with ^{Ruiz (2018)}, who reported that the a* value decreased from -20.9 to -22.2 during the postharvest phase and stated that the fruits had a slight gain in the intensity of the green color.