Plant material

Fruit from the most common pepino selection cultivated in Chile were collected between January and March 2015, and between February and April 2016, from a commercial field located in Ovalle (30°33’22.854” S, 71°38’43.004” W), Coquimbo Region, northern Chile. According to International Plant Genetic Resources Institute (IPGRI, Rome, Italy), some important pepino descriptors of this common selection include: traditional cultivar/landrace, mode of reproduction- vegetative (cuttings), dessert fruit, fruit curvature- none, predominant fruit shape- ellipsoid, fruit apex shape- protruded, immature fruit color- white, and fruit predominant color at commercial harvest- pale yellow. Both years, fruit samples from six developmental stages based on fruit size and external color were studied from a very immature fruit (stage 1) to a senescent fruit (stage 6). Sound plants with similar characteristics were selected in the field, and 150 uniform pepino fruits were labeled approximately 10 d after fruit set (stage 1). Then, 25 labeled fruits were hand-picked from the field every 2-3 wk (stages 2-5) during fruit development until ~ 90 d after fruit set (stage 6) (^{Contreras et al., 2017}). The collected fruit were placed in fruit trays to avoid bruising and abrasion during transportation. The fruit was transported to the laboratory facilities by a covered ground transportation during ~ 5 h at 20-25 °C, avoiding vibration damage, reducing water loss and without affecting the progression of ripening as shown in preliminary studies (^{Contreras et al., 2016}), to the Postharvest Laboratory at INIA-La Platina in Santiago (Chile). After arrival to the laboratory, fruit were quickly selected and stored overnight at 20 °C for analyses the next day.

Additionally, a maturity assay was carried out in 2015 season. At harvest time, fruit from the same field were collected with two different maturity stages: M1 (pepinos with green background color), M2 (pepinos with white background color), and M3 (pepinos with a yellow background color) to test quality differences in an over-ripe fruit.

Physiological and quality parameters

Quality parameters were determined for each sampling date, during development, from 20 fruit as biological replicates. The results were expressed as the standard error (SE) of the mean. Ethylene production and the respiration rate were determined using intact fruit with a static system. Briefly, fruit from developmental stage 1 were weighed and placed in 0.5 L jars, from stages 2, 3, 4, and 5 in 0.75 L jars, and from stage 6 in 1.56 L jars, which were sealed and kept at 20° C for 30 min prior to measurements. Fruit from the maturity assay were weighed and placed in 1.56 L jars, stored at 20 °C and measured every 3-4 d. The concentrations of carbon dioxide (μg CO₂ kg^-1 s^-1) and ethylene (ng C₂H₄ kg^-1 s^-1) in the jar headspace were then determined using a gas analyzer (PBI-Dansensor Checkmate 9900, Ringsted, Denmark) and a gas chromatograph (GC-8A; Shimadzu, Tokyo, Japan) equipped with a flame ionization detector (FID). The remaining fruit quality parameters were assessed with same methodology for the developmental study and maturity assay. Fruit firmness was assessed by two measurements performed on opposite sheets of peeled fruit using a penetrometer (Effegi, Milan, Italy) equipped with a 4 mm (at stages 3 and 4) or 8 mm (stages 5 and 6) plunger; the values were expressed in Newton (N). Each fruit was subsequently halved, and one half was immediately frozen in liquid nitrogen and stored at -20 °C for organic acid and sugar analysis. From the other half, a sample of 10 g was homogenized in a mortar, and the juice was analyzed for soluble solid content (SSC) and titratable acidity (TA); SSC was measured using a temperature- compensated refractometer (ATC-1e; Atago, Tokyo, Japan), and the value was expressed as percentage. Titratable acidity measurements were performed by titration with 0.2 N NaOH until pH 8.2 was reached; the values were expressed as a percentage of malic acid equivalents.

Organic acids and sugar determination

Samples of each developmental stage were prepared from frozen homogeneous fruit flesh and three replicates at each sampling time were considered. Organic acids and sugars were extracted, analyzed by HPLC and expressed in fresh weight (FW) according to the methodology described by ^{Manríquez et al. (2014)}. Same methodology was carried out for the developmental study and maturity assay. Below is a brief summary of the procedure. Five grams of tissue were homogenized in an Ultra-Turrax T25 (IKA, Staufen, Germany) with 12.5 mL cold 95% ethanol for 3-5 min. The sample was centrifuged at 8000 rpm for 20 min at 4 °C and vacuum filtered through two layers of Whatman N°1 paper. The solution was brought to a 25 mL with 80% ethanol. Then, an aliquot of 10 mL was dried down under nitrogen gas in a thermal bath at 50 °C. The residue was resuspended in 2 mL 0.2 N H₂SO₄ with 0.05% EDTA.

The sample was loaded onto an activated sorbent cartridge (Sep-Pak plus C18; Waters, Milford, Massachusetts, USA) and the eluate collected. The eluate was filtered through a 0.45 μm filter and analyzed by HPLC. For quantification, calibration curves were designed based on standards for each compound. Calibration curves for D-(-)-fructose (Sigma- Aldrich, St. Louis, Missouri, USA), D-(+)-glucose and sucrose (Supelco Analytical, Bellefonte, Pennsylvania, USA) were used to quantify the sugars. Calibration curves for citric acid (Supelco Analytical), quinic acid (Supelco Analytical), tartaric acid (Fluka, Seelze, Germany), D-(+)-malic acid (Fluka), L-ascorbic acid (Sigma-Aldrich), and succinic acid (Riedel-de Häen, Morristown, New Jersey, USA) were used to quantify the organic acids present in the sample.

Sugars were analyzed in a chromatograph composed of an evaporative light scattering detector (ELSD), Sedex 80 (SEDERE LT-ELSD) and an YL9100 HPLC system (YL Instruments, Anyang, Korea). The separation of sugars was performed using an amino column Kromasil 100-5-NH2 (250 mm × 4.6 mm) (AkzoNobel, Bohus, Sweden) with a mobile phase of 95% acetonitrile and 5% HPLC-grade water, degassed and ultrasonicated. The analysis conditions were held constant at a flow rate of 1.5 mL min^-1 for 35 min at 20 °C. The injection volume was 20 μL. The data were analyzed with software YL-Clarity version 4.0.3. (YL Instruments).

Organic acids were analyzed in a chromatograph with an ultra-violet detector UV-VIS LC-NET II/ADC (JASCO Corporation, Tokyo, Japan) to measure the absorbance at 195 nm. The separation of acids was performed using a Symmetry C-18 (4.6 mm × 250 mm, 5.0 μm) (Waters, Wexford, Ireland). The mobile phase used was 0.0085 N H₂SO₄ degassed and ultrasonicated and the analysis conditions were held constant at a flow rate of 0.4 mL min^-1 for 5 min at 20 °C. The injection volume was 20 μL. The data were analyzed with ChromPass v. 1.7 software (JASCO Corporation).

Volatile extraction

Flesh with peel from pepino samples of 2015 and 2016 were analyzed. Volatiles compounds were determined only for the developmental study. A small-scale liquid/liquid extraction (LLE) method was used for the volatile analysis (^{Aubert et al., 2005}). The extraction method and analysis of volatiles by gas chromatography-mass spectrometry (GC-MS) were performed according to the methodology described in ^{Contreras et al. (2017)}.

RESULTS AND DISCUSSION

Developmental experiment

During the six stages of development spanning nearly 90 d after fruit set, pepino fruit showed low respiration rates, between 20-25 μg CO₂ kg^-1 s^-1 at immature stages down to 5 μg CO₂ kg^-1 s^-1 at riper stages (Figure 1). Also, pepino did not show a climacteric peak after harvest coinciding with the literature that has classified this fruit as having a non- climacteric ripening physiology (^{El-Zeftawi et al., 1988}; ^{Heyes et al., 1994}; ^{Ahumada and Cantwell, 1996}) (Figure 1). Therefore, pepino is a non-climacteric fruit according to our data, however, we do not rule out the possibility that there may be climacteric accessions, such as the case of melon (^{Obando-Ulloa et al., 2008}) or Asian pear (^{Yamane et al., 2007}). The higher respiratory rates in the early developmental stages coincide with the high metabolic activity of the tissue (e.g. cell division) described for immature fruit (^{Wills et al., 2007}). In regards to ethylene, pepino showed very low production values both years, which never exceeded 0.1 ng kg^-1 s^-1, except for the last developmental stage (stage 6), which corresponded to a senescent fruit where ethylene emissions are triggered by senescence rather than ripening. Other authors have classified pepino among fruit with low ethylene production (^{Kader, 2002}).

Other quality parameters, such as SSC and TA, showed a rather stable pattern during fruit development with nonsignificant accumulation (Figure 2). Noteworthy, pepino fruit had low sugar levels around 8%-10%, compared to other fruit that reach 14%-16% (sweet cherry) and up to > 20% (table grape) of total soluble solids (^{Kader, 2002}). Other authors have also reported little changes in SSC content after harvest, confirming that pepinos do not improve flavor after picking (^{Lizana and Levano, 1977}; ^{El-Zeftawi et al., 1988}), and therefore, coinciding with the description of a non- climacteric fruit which needs an optimum harvest time to ensure good quality and flavor for the consumers. Likewise, acidity stayed low, constantly around 0.1% and it was expressed in malic acid. Since pepino has been described as not particularly sweet, many medicinal applications have been reported for the fruit specially to attenuate the progression of diabetes due to its high fiber content and anti-inflammatory and antioxidant activities (^{Maheshwari et al., 2014}).

Values are means ± standard error of 20 biological replicates.

Figure 1 CO₂ and ethylene production in pepino during fruit development in years 2015 and 2016. 

Figure 2 Quality parameters in pepino during fruit development in 2015 season. Physiological stage 5 corresponds to commercial harvest. 

Fruit diameter and weight were reached at early stages of development, stages 3 and 4, respectively (Figure 2). In other words, the equatorial diameter and weight were essentially completed after 40 and 50 d after fruit set. ^{Schaffer et al. (1989)} also studied fruit growth in pepino reporting that it completed the fruit diameter after 60 d post anthesis.

Fruit firmness showed an accelerated loss of flesh firmness during fruit development reaching nearly 60 N at stage 5 (commercial harvest) and < 40 N at stage 6 (senescence) (Figure 2). Other studies have reported a loss of firmness range from 60 to < 20 N during development, however, the developmental stages were based on background color and stripes appearance, rather than days after anthesis or fruit set making it not comparable to our study (^{Heyes et al., 1994}). ^{Galletti et al. (2006)} reported in Chilean pepinos a firmness of 54-51 N at harvest similar to the values obtained in this study.

Color and soluble solids have been reported as harvest indices and maturity measurements (^{El-Zeftawi et al., 1988}). The background color, which has been used as a maturity index for pepinos also changed during maturation. Hue angle decreased during fruit development moving from green color to white during harvest time, and yellow in a senescent fruit (^{Heyes et al., 1994}). Fruit quality parameters were summarized in Table 1 for the two seasons 2015 and 2016.

The organic acids found in pepino were malic, quinic, tartaric, citric and succinic (Figure 3). Different patterns were observed for organic acid accumulation during fruit development. For instance, malic acid was decreasing throughout development whereas the concentration of succinic acid increased especially in riper pepino stages (Figure 3). In fruit from 2015, malic acid was the predominant acid, however, in 2016 fruit, its concentration decreased being the second abundant acid after quinic acid. Other studies have found malic and traces of quinic acid in ‘Suma’ (^{Redgwell and Turner, 1986}). Interestingly, it has been reported in the literature on developed and established cultivars that citric acid is the predominant organic acid increasing 3-fold in riper stages and accounting for nearly 90% of the non-volatile organic acids (^{Redgwell and Turner, 1986}; ^{Kola et al., 2015}). However, we could not confirm this result in our study, in fact, citric acid showed the lowest levels in both years. One possible explanation for this notorious difference between the Chilean pepinos (which correspond to native accessions) and foreign developed cultivars, is that the citric acid found in developed cultivars is the result of breeding programs that targeted for improved flavor, selecting clones with increased sugar and acid concentration. This resulted in cultivars with higher levels of citric acid (^{Rodríguez-Burruezo et al., 2011}). Regardless of this difference, it is important to note that the overall content of organic acids in pepino was very low.

Table 1 Quality parameters of pepino fruit during 91 and 92 d of fruit development for 2015 and 2016, respectively. 

Hue angle: 0° Red; 90° yellow; 180° green; 270° blue.

Values are means ± standard error of three replicates. Organic acids are expressed on a fresh weight basis.

Figure 3 Organic acid concentration in pepino during fruit development in years 2015 and 2016. 

In regard to sugar concentration, fructose, glucose and sucrose were the main soluble sugars as it has been found by other authors (^{Schaffer et al., 1989}) (Figure 4). These sugars showed a similar pattern described by ^{Schaffer et al. (1989)} and ^{Sánchez et al. (2000)}, where sucrose is present in low amounts during immature stages to accumulate at higher levels during ripening. Actually, sucrose has been described to account for nearly 50% of the total sugar content (^{Sánchez et al., 2000}). Fructose and glucose did not show significant changes throughout development, and fructose was slightly higher than glucose. Based on the physiological parameters, 2016 was a colder year since the fruit took a longer period of time to accumulate sugars and organic acids, and the total content was lower compared to 2015. It has been reported that the accumulation of sugars and organic acids are closely linked, and many environmental factors determine their content in the fruit cell, however, temperature is a key factor as it affects the vacuolar storage via several mechanisms (^{Etienne et al., 2013}). The constant low level of sucrose observed during 2016 may be explained by the physiological age of the fruit (Figure 4). Likely, the fruit development was slower during that season due to lower temperatures taking more time for the fruit to fully ripe. It is important to note that soluble sugars do not fully account for the total SSC, since other pigments such as anthocyanins and carotenoids also contribute to the SSC (^{Muñoz-Robredo et al., 2011}). Organic acids, on the other hand, showed decreasing levels during development both years. This behavior has been previously reported in fruit developmental studies, where sugars are the respiratory substrate in green/immature stages, but when ripening occurs there is a shift to organic acids explaining the lower levels at the senescent stage (^{Etienne et al., 2013}).

Values are means ± standard error of three replicates. Soluble sugars are expressed on a fresh weight basis.

Figure 4 Soluble sugar concentration in pepino during fruit development in years 2015 and 2016. 

The volatile profile of Chilean pepinos also coincides with the literature (^{Shiota et al., 1988}; ^{Rodríguez-Burruezo et al., 2004a}) where volatiles derived from the terpenoid pathway are the most predominant aroma constituents, followed by volatiles derived from the lipoxygenase (LOX) pathway (Figure 5). A significant volatile emission in pepino started from phenological stages 5 and 6, which correspond to commercial harvest and senescence, respectively.

The most abundant volatile produced was 3-methyl-3-butenyl acetate, followed by 3-methyl-2-butenyl acetate, which originate from the terpenoid pathway, and trans-2-hexenal from the lipoxygenase pathway (Figure 5, Table 2). The free isoprenols (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol) occurred in minor amounts. ^{Shiota et al. (1988)} also found the isoprenyl esters, which confer a fruity and sweet odor, as the major compounds in ‘El Camino’, ‘Kawi’ and ‘Suma’. The alcohols of the above unsaturated esters (3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol), along with butyl acetate, hexanal, 1-butanol, 3-methyl-butyl acetate and methyl 3-methyl-2-butenoate contribute significantly to the note aroma of ripe pepinos (Figure 5).

trans-2-Hexenal, a 6-C compound and the third major volatile produced, has also been reported to be produced by pepino fruit (^{Rodríguez-Burruezo et al., 2004a}; ^2004b; ²⁰¹¹). Actually, other C-6 compounds from the LOX pathway have also been reported, such as hexanal and hexyl acetate, as important volatiles depending on the clone or cultivar (^{Rodríguez-Burruezo et al., 2004a}; ^2004b; ²⁰¹¹). Five and nine-C compounds from the LOX pathway were also found (Table 2), but these were produced in very low amounts, and in the case of C-9 compounds at immature stages of development (^{Contreras et al., 2017}). The C-9 compounds, which have been described in cucurbit species only, confer the cucumber-like and melon notes to the pepino fruit (^{Shiota et al., 1988}).

Values are means ± standard error of five replicates.

Figure 5 Aroma volatile constituents of pepino during fruit development in years 2015 and 2016. 

Table 2 Identified volatile constituents in ripe pepino fruit (stage 6) during two seasons. 

nd: Not detected at ripe pepino stages; nq: not quantified.

Twenty-two volatile compounds were identified in pepinos by GC-MS (Table 2). Some of them were produced in higher amounts during immature stages (C-9 compounds), and others at ripe stages (C-5, C-6 and terpenes). An open question derived from this study is to identify which are the ‘odor character’ compounds that determine the specific pepino aroma. A preliminary study using GC-olfactometry showed that trans-2-nonenal is one of the key flavor compound in pepino (data not shown), however, further research is needed. Other authors have found that 17 odor-active volatiles (OCVs) contribute significantly to the pepino aroma and classified them in fruity (acetates), green (C-6 and C-9) and exotic (lactones, mesifuran and β-damascenone) (^{Rodríguez-Burruezo et al., 2004}b; ²⁰¹¹).

Maturity experiment

There were nonsignificant differences in quality between both maturities, except in firmness (Figure 6). Respiratory rate, soluble solids and TA showed same patterns for M1 and M2 during ripening at 20 °C. As for firmness, pepino M1 (green background color) had higher firmness values than M2 (white background color) at harvest, showing a drop in firmness within 6 d compared to M2 which already had < 20 N at harvest.

Likewise, organic acids also did not show significant differences between maturities (Figure 7). In both maturities malic acid was the predominant acid and the level stayed constant about 1500-2000 mg kg^-1 fresh weight during ripening. All other organic acids (ascorbic, quinic, tartaric, citric, and succinic) showed low concentration levels < 500 mg g^-1.

Finally, the soluble sugar concentration also evidenced that M2 represents a more advanced physiological stage. It has been described that sucrose content increases sharply in ripe stages and then declines during over-ripe or senescent fruit (^{Schaffer et al., 1989}). It is noticed that in M1, the sucrose content started at low level and increased until day 4 at 20 °C, then remained almost at the same levels as the other two sugars glucose and fructose, to finally increase on day 15 (Figure 8). Whereas in M2, the sucrose content at day 1 was also higher than the glucose and fructose level, increased until 4 d at 20 °C and remained constant for the rest of the storage. Likely, after the 15 d of storage at 20 °C sucrose, fructose and glucose content start to decline as it has been previously reported (^{Schaffer et al., 1989}). As for M3, with the exception of color, did not show any significant differences with M2 in any other physiological or quality parameter (data not shown).

Values are means ± standard error of 20 biological replicates.

Figure 6 Quality parameters of pepino fruit during ripening at 20 °C. M1: Fruit with green background color, and M2: fruit with white background color. 

Values are means ± standard error of three replicates. Organic acids are expressed on a fresh weight basis.

Figure 7 Organic acid concentration in pepino fruit during ripening at 20 °C. M1: fruit with green background color, and M2: fruit with white background color. 

Values are means ± standard error of three replicates. Soluble sugars are expressed on a fresh weight basis.

Figure 8 Soluble sugar concentration in pepino fruit during ripening at 20 °C. M1: Fruit with green background color, and M2: fruit with white background color.