Physiological properties of novel melon cultivars (cv. Meloni and cv. Tacapa Green Black) during storage

Melon ( Cucumis melo L.) has polymorphic varieties that affect genetic diversity. Melon cv. Meloni and cv. Tacapa Green Black are new cultivars produced by Universitas Gadjah Mada. The information about respiration rate, volatile compounds, and firmness, including the impact on storage time of those novel cultivars, is not available yet. This work aimed to investigate the respiration rate, volatile compounds, and firmness of melon cv. Meloni and Tacapa Green Black during 20 days of storage at a temperature of 21°C. Based on respiration rate measurement, melon cv. Meloni and cv. Tacapa Green Black are classified into climacteric and non - climacteric fruit, respectively. The volatile compounds were extracted using solvent extraction followed by gas chromatography - mass spectrometry (GC - MS). It identified more than seventy volatile compounds in melon cv. Meloni, whereas only 20 volatile compounds were found in cv. Tacapa Green Black at harvest time and altered during storage. Melon cv. Meloni has a highly aromatic volatile compound but short shelf life according to firmness. Whereas cv. Tacapa Green Black could be stored until 20 days in form shape but lacking volatile compounds. Melon cv. Meloni and cv. Tacapa Green Black has good potential and could be a promising commercial horticultural product.


Introduction
Melon fruits have unique ripening behaviour. It could belong to climacteric and non-climacteric fruit (Obando-ulloa et al., 2008). The respiration rate is one of the parameters to determine the ripening behaviour of fruit. The measurement of respiration rate could be one of the ways to predict the fruit shelf life. This is due to respiration being strongly related to fruit quality parameters such as volatile compounds to compose aroma and firmness. During storage, the rapid respiration rate can lead to shorter shelf life and vice versa (Workneh and Osthoff, 2010). The characteristic of climacteric melons is usually more aromatic but loses firmness rapidly rather than non-climacteric melons (Shalit et al., 2001;Saladie et al., 2015). The aroma and firmness are the crucial attributes of melon fruit quality perceived by consumers (Lester, 2006). The volatile compounds in charge of the key contributors to the unique aroma of melon are various and cultivar dependence (Nuñez-Palenius et al., 2008). Melon cv. Meloni and Tacapa Green Black are new cultivars produced by Universitas Gadjah Mada. Both cultivars have prominent characteristics. Melon cv. Meloni has an oval shape of the fruit, orange-fleshed colour, sweet (7-16% Brix), pleasant aroma, and smooth yellow skin (Amalia, 2016). Moreover, melon cv. Tacapa Green Black has a big round shape of the fruit, green yellowish fleshed colour, sweet (7-11% Brix), resistant to powdery mildew, and long storage time (Aristya and Daryono, 2012). Those novel cultivars have different characteristics compared with the other melon cultivars. Since the melons are new cultivars, the respiration rate, volatile compounds, and firmness, including the impact of storage time of those characteristics in melon cv. Meloni and Tacapa Green Black are not available yet. Therefore, this study aimed to investigate the respiration rate, volatile compounds, and firmness of those novel cultivars and their alteration during storage.

Materials
Melon cv. Meloni and Tacapa Green Black were grown in a greenhouse at Universitas Gadjah Mada experimental farm from September until November 2020. The fruits were harvested at the full ripe stage at 39 days after pollination (DAP) and transferred gently to the Laboratory of Food Engineering in Faculty of Agricultural Technology Universitas Gadjah Mada using partitioned corrugated boxes among melon fruit to avoid mechanical injury during transportation. All chemicals used in the experiment were analytical grades such as Npentane, dichloromethane, Ethanol, Sodium hypochlorite, 2.5% glutaraldehyde, sodium phosphate buffer pH 7.2 (Merck), ethyl decanoate as internal standard (Sigma), and CO 2 standard (Samator).

The storage condition of melon fruits
The selected melon fruits without decay were washed thoroughly in cold running tap water. Then immersed in 50 ppm sodium hypochlorite (Merck) 5.25% for 1 min for reducing the microbial in the skin of melon fruits. The melons were rinsed in a cold running tap again to remove sodium hypochlorite residue and allowed to drain. Samples were kept in a storage room at 21⁰C that simulated commercial temperature and 74% RH during 20 days of storage in a partitioned corrugated box.

Respiration rate measurement
The respiration rate of melon was examined by the following method of Oh et al. (2011) with minor modification. Three fruits from each cultivar were retained individually in a 16.3 L air-tight jar for 3 hrs at 20°C. After incubation, 1 mL of headspace gas was withdrawn by tight syringe and injected into gas chromatography (GC-2010 Plus, Shimadzu, Oregon, USA) equipped with Rt-Q-BOND (30m, 0.32 mm ID, 10 µm df) (Restek, Bellefonte, PA, USA), and thermal conductivity detector (TCD). The carrier gas was nitrogen at the flow rate of 0.8 mL/s. The temperatures in the injector, oven, and detector were 150, 70, and 200°C, respectively. The quantification of carbon dioxide used was the carbon dioxide standard. The CO 2 production was expressed mg kg -1 fruit h -1 .

Volatile compounds extraction
Approximately 400 g of fruit flesh were extracted using a maceration method following the Nussbaumer and Hostettler (1996) method with some modifications. The fruit fleshes were homogenized and macerated with 540 mL mixture of n-pentane and dichloromethane (Merck, Darmstadt, Germany) (2:1, v/v), followed by an addition 10 µL ethyl decanoate (Sigma-Aldrich, Saint Louis, MO, USA) (2.15 µg/mL) as internal standard. The macerated sample was incubated at -20⁰C for 24 hrs. After the maceration, the extract was decanted and dried over anhydrous sodium sulphate (Merck, Darmstadt, Germany). The free water extract was concentrated by rotary evaporator to approximately 5 mL and finally flushed with nitrogen gas to 1 mL and stored at -20⁰C before analysis

Volatile compounds identification
The identification of volatile compounds was based on the reference method (Lignou et al., 2014), with minor modifications. The volatile compounds of the extracted sample were identified by GC-MS Agilent 7890A with CTC PAL auto sample with MS Agilent 5975C detector (Agilent, Santa Clara, CA). The instrument was equipped with a capillary column DB-WAX Agilent JW (30m × 250 µm × 0.25 µm) and a split -less injector. This study used helium was used as a carrier gas, and the flow rate was 0.8 mL/min. The injector temperature was 250°C, and the volume of injection was 0.5 µL. The oven temperature program was 40°C for 2 mins and raised to 240°C at 4°C/min. The mass spectrometer was recorded in the electron ionization mode at 70 eV, a source temperature of 200°C and a scan range from m/z 29 to 550. Each component was identified using a mass spectra database (NIST 14 Library), Linear Retention Indices (LRI), and literature data. The retention time of the n-alkanes series (C 11 -C 28 ) was used to calculate linear retention indices (LRI) according to the Van den Dool and Kratz equation (Van den Dool and Kratz, 1963).

Flesh firmness measurement
The flesh firmness was measured at 0, 10, and 20 days of storage. The fruits were peeled and sliced longitudinally. Seeds were removed, and the seed cavity was cleaned by removing 1 to 2 mm tissue. Melon was cut into three parts as follows: (1) near the fruit stalk, (2) in the middle, and (3) at the end of the fruit. Cut into 2×2×2 cm cubes. Every cube was run in duplicate. The texture parameter was analysed by CT3 Texture Analyzer (Brookfield, Middleborough, MA, USA) equipped with a TA41 probe. The probe diameter was 6 mm. The penetration depth was 5 mm, with a probing rate was 1 mm/s (Munira et al., 2013)

Microstructure cell wall
The microstructure of the cell wall was determined by performing the reference method (Samuel et al., 1993). The flesh was cut into 1 × 1 × 0.5 cm, then immersed in 2.5% glutaraldehyde, buffered with 0.1 M sodium phosphate buffer pH 7.2 for 24 hrs at room https://doi.org/10.26656/fr.2017.7(4).841 © 2023 The Authors. Published by Rynnye Lyan Resources FULL PAPER temperature. The fixed pieces were dried with a vacuum drier and observed by scanning electron microscopy (SEM) JSM-6510LA (JEOL, Tokyo, Japan).

Statistical analysis
The data obtained were statistically analysed using Univariate analysis of variance (ANOVA) followed by Duncan's Multiple Range Test with a 95% confidence level to determine the degree of difference in values between treatments. Principal Component Analysis (PCA) was conducted to investigate the interaction among volatile compounds using Minitab 19.

Respiration rate of melon cv. Meloni and Tacapa Green Black during storage
The respiration rate during storage has a strong influence on fruit quality, such as aroma and texture properties. As new cultivars, melon cv. Meloni and cv. Tacapa Green Black respiration rate should be measured and classified into climacteric or non-climacteric. The respiration rate of cv. Meloni and cv. Tacapa Green Black during 20 days of storage was shown in Figure 1. The respiration rate of cv. Meloni increased transients until the sixth day (89 mg/kg. Hour) and declined considerably until the 20 th day. The melon cv. Meloni showed climacteric behaviour with respiration having a well-defined peak at the sixth day of storage. According to Mishra and Gamage (2007), the respiration rate of melon cv. Meloni was included in the high respiration group. A high respiration rate led to a high breakdown of carbon compounds to produce volatile compounds. Unfortunately, the high respiration rate could lead to loss of flesh firmness and shorter shelf life of melon fruit (Saladie et al., 2015).
Vice versa, the respiration rate of melon cv. Tacapa Green Black tended to decrease from 50 to 30 mg/kg. An hour at the end of storage and no respiratory burst. The pattern of cv. Tacapa Green Black respiration shows non -climacteric behaviour, corresponding with Obandoulloa et al. (2008) and Miccolis and Saltveit (1995) reported that carbon dioxide production of several melon cultivars declined during storage. The low respiration rate could delay flesh softening during postharvest storage and prolong the shelf life of melon fruit (Pech et al., 2008) but led to a lack of aromatic volatile compounds (Obando-ulloa et al., 2008).

Volatile compounds profile of melon cv. Meloni and Tacapa Green Black during storage
The volatile compounds of melon cv. Meloni and Tacapa Green Black were identified by Gas Chromatography-Mass Spectrometry (GC-MS Agilent 7890A with CTC PAL auto sample with MS Agilent 5975C detector). The results of GC-MS are summarized in Table 1. At the time of harvest (0 days) and after 20 days of storage, solvent extraction revealed 74 and 58 volatile compounds in melon cv. Meloni, respectively. Meanwhile, only twenty volatile compounds were found in melon cv. Tacapa Green Black during harvest (0 days) and storage (20 days). During 20 days of storage, the total volatile compound concentration in cv. Meloni decreased by 64%, while it increased by 41% in cv. Tacapa Green Black.
From Table 1, it is shown that melon cv. Meloni has much more volatile compounds compared to cv. Tacapa Green Black. This result aligns with the respiration rate measurement of this study where melon cv. Meloni has been indicated as a climacteric fruit that shows climacteric behaviour with high levels of respiration. Aromatic volatile compounds are abundant in climacteric melon varieties such as the reticulatus and cantalupensis groups (e.g., cantaloupes). Non-climacteric melon varieties, such as the indoors variety, do not experience a respiratory burst, resulting in a lack of aroma (Saladie et al., 2015). Table 1 shows the highest number of volatile compounds in cv. Meloni at 0 days is benzyl alcohol. After storage, the concentration declined from 1917 µg/ kg dry weight (DW) to 313 µg/kg DW. The odour description of benzyl alcohol is floral, fruity, and balsamic (Verzera et al., 2011). This compound could be used as a substrate to form benzyl acetate through benzyl -alcohol acyltransferase enzyme activity (Shalit et al., 2000). This result was proven by the increasing concentration of benzyl acetate from 43 µg/kg DW to 569 µg/kg DW at the end of storage. In contrast, Beaulieu (2005) reported that the benzyl acetate concentration in melon cv. Athena and cv. Sol Real declined during 11 days of storage. Benzyl acetate is a significant compound in several melon cultivars (Aubert    (Pino et al., 2005), mangaba (Hancornia speciosa Gomes) fruit (Sampaio and Nogueira, 2006), and raspberry (Aprea et al., 2015). Benzyl acetate has an odour threshold of 15µg/kg (Buttery et al., 1982). The odour description of benzyl acetate is fruity, fresh, floral, sweet, and pine (Amaro et al., 2012). It could be an important key compound contributing to the aroma of melon cv.
Meloni. The identification of volatile compounds after 20 days of storage revealed that 3-ethyl-2-pentanol and benzyl acetate are the most volatile compounds in cv. Meloni.
Furaneol and trans-β-ionone were found exclusively in cv. Meloni in this study. Furaneol content was 18 g/kg FULL PAPER DW at harvest and increased to 59 g/kg DW after 20 days of storage. Furaneol is scarcely found in melon fruits and only in trace amounts (Hayata et al., 2003;Lignou et al., 2013). Furaneol has an odour described as strawberry-like, caramel-like, and sweet (Hayata et al., 2003). Furaneol, which has a low odour threshold in the water of 5 g/kg (Du et al., 2010), could contribute to the aroma of melon cv. According to Hayata et al. (2003), furaneol had high flavour dilution and emerged to be a potent odorant in Miyabi melon. Moreover, trans-βionone with flower and raspberry odour description can only be detected at harvest time in cv. Meloni (36 µg/kg DW). The melon cv. Meloni has orange flesh. Trans-βionone which is mostly found in orange-fleshed melon, is a carotenoid-derivate volatile compound produced from β-carotene cleaved by carotenoid cleavage dioxygenase (El Hadi et al., 2013). The low odour threshold of trans-β-ionone (0.1 µg/kg in water (Du et al., 2010) also could have contributed to the aroma of cv.
Meloni. Table 1 shows that alcohols were found to be the dominant volatile compounds in melon cv. Tacapa Green Black. During 20 days of storage, the dominant volatile compound in melon cv. Tacapa Green Black is 3-ethyl-2 -pentanol. The concentration increased from 388 to 587 g/kg DW after 20 days of storage. Shalit et al. (2001) reported that aldehyde and alcohol were the most abundant volatile compounds in the cultivar Rochet fruit, a non-climacteric melon. For the first time, 2(3H)-Furanone, dihydro-4-hydroxy-was discovered in melon fruit in this study. Furthermore, Pereira et al. (2011) also reported this compound was found for the first time in the green flesh kiwi cv. Hayward and the yellow flesh Chickasaw plum cv.Marsh. The odour description of this compound was fruity . Parthasarathy et al. (2015) identified 2(3H)-Furanone, dihydro-4hydroxy as the second highest (18.61%) antifungal metabolite in unripe healthy mango peel cv. Neelum against Colletotrichum gleosporiodes and Lasiodiplodia theobromae.
The odour activity value (OAV) was calculated as the ratio of the concentration of a volatile compound to its odour detection threshold in water (Zhu et al., 2018). The OAV was used to assess the volatile compounds that contribute to the overall melon aroma. The threshold values using data from the literature. According to Hasbullah et al. (2021), compounds that have the odour active value (OAV) of more than one are considered to contribute to the aroma of melon. Calculating the OAV is required to determine the volatile compounds that contribute to the melon cv. Meloni and cv. Tacapa Green Black aroma. However, searching through several papers does not reveal that all volatile compounds found in melon cv. Meloni and cv. Tacapa Green Black have a threshold. Only volatile compounds with known thresholds will be calculated for the OAV in this study. Table 2 shows the OAV of volatile compounds in melon cv. Meloni and cv. Tacapa Green Black during storage has a high presence and an important role in the aroma. Generally, odorants with high OAVs are more likely to be important, although aroma synergy and suppression exist (Du et al., 2010). Table 2 indicates that there are nine different compounds (OAV > 1) in cv. Meloni is both at 0 and 20 days of storage. Meanwhile, during 0 and 20 days of storage, the volatile compounds with an OAV greater than one in melon cv. Tacapa Green Black are three and four distinct compounds, respectively ( Table 2). During the storage period, Trans-β-Ionone, furaneol, Benzaldehyde, Benzyl alcohol, (6Z)-Nonen-1ol, 1-Butanol, 2-methyl-, Benzyl acetate, Hexyl acetate, and Butyl acetate were the compounds with the highest odour-activity values (OAV > 10) in cv. Meloni. The compounds with OAVs > 10 in cv. Tacapa Green Black was benzaldehyde, benzyl acetate, and hexyl acetate ( Table 2). Benzaldehyde has an aroma aromatic, sweet, and almond (Verzera et al. 2011;Zhu et al., 2018). Benzaldehyde had an OAV of 1328 in melon cv. Meloni, which was 8 times higher than in cv. Tacapa Green Black at 0 days of storage. Hexyl acetate and benzyl acetate could be important odorants in both cultivars. The OAV of hexyl acetate and benzyl acetate in cv Meloni was 3 and 2 times higher, respectively than in cv. Tacapa Green Black. Both compounds contributed to the fruity aroma. Furthermore, in cv. Meloni also had 19 times higher OAV of benzyl alcohol compared to cv. Tacapa Green Black, which also contributes to the higher fresh fruit aroma in cv. Meloni.

Change of melon cv. Meloni and Tacapa Green Black flesh firmness during storage
The flesh firmness of melon cv. Meloni and cv. Tacapa Green Black during 20 days of storage was shown in Figure 3. Jackman et al. (1990) described that firmness values reflect the integrity of pericarp tissue, where fruit-softening enzymes are primarily localized. According to Figure 3, the flesh firmness of melon cv. Meloni declined remarkably from 2711 g force at 0 days to 680 g force at 20 days conforming with the mostly climacteric melons during storage (Fallik et al., 2001;Supapvanich et al., 2011;Munira et al., 2013;Supapvanich and Tucker, 2013) reported that the firmness of Galia melon declined during market storage simulation. The loss of firmness or softening could occur due to the disruption of the primary cell wall (Crookes and Grierson, 1983). This presumption will be proven by the result of scanning electron microscopy analysis to observe the cell walls during storage. Simandjuntak et al. (1996) suggested that one of the problems with melon fruits was softening during storage. Based on consumer acceptance, quality fruit is rejected if the loss of initial firmness is more than 30% (Harker et al., 2008;Goncalves et al., 2017). Melon cv. Meloni had an approximate loss of initial firmness of 45.67% on the 10 th day. It assumed that cv. Meloni at 39 DAP or full ripe could not be stored for more ten than days to perceive the excellent quality of melon cv. Meloni according to texture properties. This result was aligned with Wyllie et al. (1996) reported that cv. Makdimon, as a highly aromatic cultivar that was picked in full maturity, tended to have a short shelf life.
In contrast, the flesh firmness of cv. Tacapa Green Black remained stable during 20 days of storage. The storage period did not alter the flesh firmness of the cv. Tacapa Green Black significantly (p<0.05). That can be correlated with non-climacteric behaviour. That contrasts with the previous studies that reported firmness reduction during storage in some inodourus melons such as cv Honey World (Supapvanich et al., 2011) and cv.
Amarillo (Di Venere et al., 2000). Miccolis and Saltveit (1995) reported firmness of flesh decreased during three weeks in all six cultivars of var. inodourus (Amarelo, Golden Casaba, Honeydew, Honey Loupe, Juan Canary, and Paceco). The shelf life of cv. Tacapa Green Black was longer than cv. Meloni. It could be stored until 20 days in a firm shape. This excellent trait of cv. Tacapa Green Black could be beneficial during supply chain distribution until the consumer's hand.

Microstructure cell wall identification of melon cv. Meloni and Tacapa Green Black flesh during storage
Scanning electron microscopy was conducted to explain how cell walls of parenchyma cells (Lecha, 2000) form or condition from the mesocarp part of flesh during storage and is related to firmness. Melon cv. Meloni lost firmness after storage, although melon cv. Tacapa Green Black did not. Figure 4 shows scanning electron microscopy of the flesh of cv. Meloni and Tacapa Green Black at a magnification of 500x via SEM. The cell wall microscopy of flesh cv. Meloni is shown in Figures 4A-B-C. At day 0 ( Figure 4A), the cell walls of cv. Meloni was still firm and the form was evident.
On the tenth day of storage, the firmness began to deteriorate ( Figure 4B). A section of the cell walls was damaged and lost its rigidity. The cell walls had virtually all been damaged after 20 days of storage ( Figure 4C), and the shape was no longer visible. The microstructure of cell walls changes during storage possibly due to the loss of firmness. It might be because of cell disruption during storage (Crookes and Grierson, 1983). Brummell (2006) reported that the enhancement of electrolyte leakage indicates a membrane integrity degradation in the fruit cell walls. Ergun et al. (2005) also reported that the loss of firmness in the whole Galia melon during 20 FULL PAPER days at 20⁰C was consistent with increasing electrolyte leakage. Furthermore, in melon cv. Veldrantais and Dulce, cell wall degradation and softening of the fruit are expected due to increased activity of polygalacturonases, glucan endo-1,3-β-glucosidases and β-d-xylosidases enzymes (Saladie et al., 2015). Meanwhile, Rojas-Graü et al. (2006) found that the loss of firmness in Honeydew melon correlated with the loss of turgor pressure.
In contrast, the cell walls of cv. Tacapa Green Black ( Figures 4D, 4E and 4F) did not change remarkably during 20 days of storage. It was relatively the same at 0, 10, and 20 days. It aligned with the firmness result of cv. Tacapa Green Black reminded stable during storage. Saladie et al. (2015) reported that the firmness of nonclimacteric melon such as Piel de Sapo varieties which is commonly green-fleshed remained firm during storage.

Conclusion
Melon cv. Meloni and cv. Tacapa Green Black are new cultivars from Indonesia. Based on the respiration rate, cv. Meloni and Tacapa Green Black are classified as climacteric and non-climacteric melon, respectively. The volatile compounds in fruit are the key important factors related to consumer preferences. The volatile compounds were extracted from two different melon cultivars during storage using maceration extraction and then analyzed using GC-MS. Principal component analysis showed that Melon cv. Meloni and Tacapa Green Black contain different volatile compound profiles, of which cv. Meloni is more aromatic than cv. Tacapa Green Black due to the higher number of volatile compounds that have high odour activity values. During 20 days of storage, the firmness of cv. Meloni declined, whereas melon cv. Tacapa Green Black remained firm. The firmness result is assisted by a scanning electron microscopy (SEM) result that showed the microstructure of cell walls during storage. These physiological properties are essential as potential markers for a different cultivar of melon fruit.

Conflict of interest
The authors declare no conflict of interest. A is meloni at 0 day, B is meloni at 10 days, C is meloni at 20 days. D is Tacapa Green Black at 0 day, E is Tacapa Green Black at 10 days, F is Tacapa Green Black at 20 days. Magnification 500×.