Non­destructive determination of ripening in melon fruit using time­resolved spectroscopy

: The aim of this work was to explore the feasibility of time­resolved reﬂectance spectroscopy (TRS) in determining the ripening degree and the quality of orange­ﬂeshed melons. Sixty ‘Honey Moon’ melons were measured by TRS in the 540­1064 nm range and classiﬁed as less (LeM), medium (MeM), and more (MoM) mature according to increasing values of μ a 540. MoM fruit showed yellower peel color, slightly more orange pulp, higher juiciness and higher carotenoid contents than LeM ones. MoM fruit also showed higher internal ethylene concentration and lower ﬁrmness than LeM ones, even if the diﬀerences were not signiﬁcant. The μ a 540 was positively related to internal ethylene, carotenoid accumulation, and juiciness, indicating that μ a 540 was linked to diﬀerent ripening processes in melons. However, the relationship between μ a 540 and total carotenoid content was not as high as expected due to the low variability of pulp color and of carotenoid content. Changes in ﬂesh color toward a more orange shade were accompanied by increased juiciness and ethylene production and by carotenoid accumulation, while changes in peel color were associated with changes in ﬂesh ﬁrmness and juiciness. In con­ clusion, the absorption coeﬃcient measured at 540 nm ( µ a 540) by TRS could be used to sort melons in diﬀerent ripening degrees; however, its applicability will need to be evaluated on a larger number of fruits and on other varieties.


Introduction
Melon (Cucumis melo L.) fruit are particularly appreciated by con sumers for their sweetness, flavour, texture, and attractive flesh color, as well as for their nutritional and phytochemical properties.Melons are a good source of carotenoids, ascorbic acid, and phenolic compounds (GómezGarcía et al., 2020;Manchali et al., 2021;Singh et al., 2022).Melon quality depends on the balance between sugars, organic acids, volatiles, and texture characteristics (Kyriacou et al., 2018).These traits are strongly affected by genotype, agroclimate conditions, harvest matu rity, postharvest handling, and storage conditions (Kyriacou et al., 2018).Melons picked at the optimal harvest maturity have premium quality as maturity at harvest has a large impact on sugars, volatiles, and texture.During ripening, rind color changes from green to yellow, orange, or creamy yellow, depend ing on melon genotype; mesocarp color turns from pale green to orange in orangefleshed melons, along with carotenoid accumulation; firmness and acidity decrease, while sucrose as well as soluble solids, vita min C and pH markedly increase (Beaulieu and Lea, 2007;Tristan et al., 2022).Melons harvested mature develop the strongest flavour, whereas fruit harvest ed early develop a less aromatic flavour: esters are predominant in ripe melons, aldehydes are present at higher concentration in immature fruit while a sharp increase of alcohols is typical of overripe mel ons (Senesi et al., 2005;Beaulieu and Lea, 2007;Vallone et al., 2013;Lignou et al., 2014).During ripening, the sensory scores for color intensity, fruity aroma, juiciness, and sweetness increased while the sensory scores for firmness and sourness generally decreased (Vallone et al., 2013).In climacteric mel ons, ethylene was produced at higher levels in fully ripe fruits and was negatively related to firmness and positively related to sensory juiciness and fruity flavour and aroma (Senesi et al., 2005;Vallone et al., 2013).
For more than two decades, several efforts have been made to measure quality characteristics of mel ons in a nondestructive way (Zeb et al., 2021).In industrial fruit sorting, accuracy, cost, and detection speed are important factors.Thus, spectroscopic techniques being fast, simple, and costeffective, have been widely studied.In melons, VisNIR spec troscopy (Sanchez et al., 2014;Lu et al., 2015;Khurnpoon and Sirisomboon, 2018;Zeb et al., 2021), computer vision (Calixto et al., 2022) and hyperspec tral imaging (Sun et al., 2017;Cho et al., 2022) were studied with less or more successful results for pre dicting soluble solids, moisture, pulp color, and firm ness and for classifying fruit according to different sweetness degree or as suitable or not for harvesting.Among spectroscopic techniques, timeresolved reflectance spectroscopy (TRS) is gaining increasing interest in assessing fruit quality.Due to its accuracy in measuring optical properties in deep tissues it allows the evaluation of maturity and internal defects of horticultural products with a relatively thick sur face layer (Lu et al., 2020).TRS allows the complete optical characterization of a diffusive medium through the measurements of the absorption (μ a ) and of the scattering (μ s ) coefficients by probing flesh at a depth of 12 cm with no or limited influence from the skin (Cubeddu et al., 2001;Rizzolo and Vanoli, 2016).While scattering is related to the struc tural properties of fruits and vegetables, absorption depends on the chemical composition of the tissue, mainly on the presence of pigments such as chloro phylls, anthocyanins, and carotenoids (Lu et al., 2020).TRS has been mainly applied in postharvest studies on fruit and vegetables for estimating fruit ripening, for the detection of internal defects, and for discriminating fruit with different texture and senso ry characteristics (Rizzolo and Vanoli, 2016).The absorption coefficient measured at 670 nm (μ a 670) at harvest was shown to be a maturity index for various fruit, such as peaches, nectarines, plums, pears, and apples, as it declines with fruit ripening (Vanoli and Buccheri, 2012).The μ a 540 (carotenoid tail) has been used as a nondestructive maturity index for man goes, as it was able to classify intact mangoes of dif ferent cultivars according to pulp color and to the contents of total and individual carotenoids (Rizzolo and Vanoli, 2016;Vanoli et al., 2018).
TRS has not yet been studied on melons, so the aim of this work was to explore the feasibility of TRS in determining the ripening degree and the quality of orangefleshed melons.

Fruit
The experiment was carried out on 'Honey Moon' (Cucumis melo L. cantalupensis) melons.'Honey Moon' fruits have round shape, medium size, a smooth greygreen skin that turns yellow when fully ripe.Fruits has firm, deep orange flesh and show high sugar content and a pleasant and almost exotic aroma.In order to have high variability in fruit matu rity, 60 melons were picked and selected in a pack inghouse in Sermide (MantovaItaly) on a peel color basis (15 fruit for four color stages: blue, graygreen, yellowgreen and yellow).Then fruit were immedi ately transported to the CREAIT lab in Milan, mea sured by TRS in the 5401064 nm range and classified as less (LeM), medium (MeM) and more (MoM) mature according to increasing μ a 540 values (low μ a 540=less mature fruit; high μ a 540=more mature fruit).All the 60 melons were also individually evalu ated for color (peel and flesh) and for standard matu rity indices (flesh firmness, soluble solids content, acidity).Among the 60 melons, 20 fruits covering the whole range of μ a 540 (i.e., the highest, the lowest and 18 intermediate values of μ a 540), were chosen for internal ethylene concentration, total carotenoid content and juiciness analyses.

Time-resolved reflectance spectroscopy (TRS)
Whole melons were measured by TRS on two opposite sides in the fruit equatorial region.A portable compact setup working at discrete wave lengths developed at Politecnico di Milano (Zhao et al., 2022) was employed.The light source is a super continuum fiber laser (SC4506W, Fianium, UK) pro viding whitelight pulses, with duration of a few picoseconds.Two custommade filter wheels loaded with overall 14 bandpass interference filters (MaxLine series, Semrock, NY, USA, and TECHSPEC series, Edmund Optics, New Jersey, USA) are used for spectral selection in the range 5401064 nm.Light is delivered to the sample by a 200 μm core stepindex fiber and collected by a 1 mm core gradedindex fiber; interfiber distance was set to 1.5 cm.A pair of filter wheels identical to the previous one is used for cutting off the fluorescence signal originated from the sample when it is illuminated in the visible spec tral region.The light then is detected by a cus tomized Silicon PhotoMultiplier module (Martinenghi et al., 2016) and the photon timeofflight distribu tion is measured by a timecorrelated singlephoton counting board (SPC130, Becker&Hickl, Germany).The instrumental response function has a full width at half maximum of about 100 ps and the typical acquisition time is 1 s per wavelength.A semiinfinite model for photon diffusion in a turbid medium was used to analyze TRS data to assess the bulk optical properties of the samples (Martelli et al., 2009) to obtain the estimates of µ a and µ s at each wavelength.

Peel and flesh color
Color was measured on the equatorial region on two opposite side of whole fruit and, after cutting longitudinally the melons, on two opposite portions of the flesh, 1.5 cm from the fruit edge.A CM2600D spectrophotometer (Konica Minolta Sensing, Inc., Osaka, Japan) with the primary illuminant D65 and 2° observer, was used to perform the color measure ments in the L* (lightness) a* (greenred), b* (yellow blue) color space.From a* and b* values, hue (h°) and chroma (C*) were computed according to h° = arctangent (b*/a*) × 360/(2×3.14)and C*= (a* 2 + b* 2 ) 2 .In the flesh also the color spectra (350740 nm) were considered.Color readings were averaged for each fruit.

Standard maturity indices and juiciness Flesh firmness
After having cut the melons into two parts along the longitudinal axis, flesh firmness was measured on two opposite sides in the equatorial part of the fruit (around 1,5 cm from the fruit edge) using an 8 mm diameter plunger mounted on a TAXT plus Texture Analyzer (Stable Micro Systems, Godalming, UK) at a crosshead speed of 200 mm min 1 .The two measure ments were averaged for each fruit.

Soluble solids content (SSC) and titratable acidity
Two longitudinal slices around 4 cm thick, taken from two opposite sides of each fruit (in the same places of the firmness measurements) were frozen for the evaluation of soluble solids content (SSC) and of titratable acidity.After thawing, SSC was deter mined on few juice drops from each slice by using an automatic refractometer (RFM81, BellinghamStanley Ltd., England).Titratable acidity (TA) was measured by titrating 10 g of juice plus 50 mL of distilled water with 0.1 N NaOH to pH 8.

Juiciness
Juiciness was measured on pulp cylinders (diame ter = 15 mm; height = 10 mm) taken from two oppo site sides of the equatorial part of the fruit.Each cylinder was compressed between two plates with a TAXT plus Texture Analyzer (Stable Micro Systems, Godalming, UK) at a deformation rate of 100 mm min 1 by a compression of 50% of the original height of the cylinder (Eccher Zerbini et al., 1999).This method correlated with sensory analysis, and only the juice that could be easily and quickly released by the pulp cylinder was measured.Juiciness was calcu lated as the ratio between the juice weight spilled out after compression and the cylinder's original weight.

Internal ethylene concentration
1 mL sample of internal gas was taken from the seed cavity of each melon using a syringe equipped with a 15 cm long, 15gauge needle.The sample was injected in a DANI GS 86.10 gas chromatograph and analyzed for the ethylene content following the con ditions reported by Rizzolo et al. (2005), using a deac tivated aluminum oxide F1 (80100 mesh) column (1/8 in × 200 cm Alltech Italia, Sedriano, Italy), col umn temperature 100°C, injection temperature 100°C, and a flame ionization detector temperature of 225°C.Ethylene was identified and quantified by relating the peak area of the sample to that of a 10 μL L 1 external standard and was expressed as ppm.

Total carotenoid content
Two slices per fruit were frozen for total carotenoid content analysis.1 g of frozen sample was rapidly homogenized (UltraTurrax, IKAWerk, Germany) in 5 ml of an icecold solution of hexane: acetone:ethylacetate (2:1:1) containing 1% of 1% butylated hydroxitoluene (BHT) in methanol.The homogenate was sonicated for 10 min, centrifuged at 10000 g for 15 min at 4°C and the organic phase was collected.The absorbance of the organic phase, after proper dilution, was measured spectrophotometri cally at 450 nm.Total carotenoid content was esti mated using the molar extinction coefficient for b carotene in hexane (139200 l mol 1 cm 1 ), as reported in Craft and Soares (1992).

Statistical analysis
Data were submitted to analysis of variance (Statgraphics ver.7, Manugistic Inc., Rockville, MD, USA) considering TRS maturity class as a source of variation, and means were compared by Tukey's test at P≤0.05%.The relationships between μ a 540 and quality characteristics and the relationships among quality parameters were studied using regression analysis.For each parameter, the model with the higher performance was considered.Only the models with correlation coefficient r>0.50 were considered.

TRS absorption spectra
TRS absorption spectra of 'Honey Moon' melons (Fig. 1A) showed a maximum at 980 nm (correspond ing to water), high absorption values at 540 nm (cor responding to the tail of carotenoids) and very high variability in the chlorophyll absorption range (610 690 nm).
The variability in the chlorophyll absorption was not linked to the presence of chlorophyll in the pulp but depended on the presence of a green layer (about 3.57 mm) between the rind and the pulp, which interfere with TRS measurements that pass through the pulp, up to 2 cm.The color spectra of the pulp confirmed the TRS data, showing a very low absorption in the chlorophyll range and a very high absorption in the carotenoid range (400500 nm) (Fig. 1B).Then, the absorption coefficient measured at 540 nm (µ a 540) was chosen as a possible ripening index for melons due to its correlation to the carotenoid content in the pulp, as previously found by Rizzolo and Vanoli (2016) and Vanoli et al. (2018) in mangoes.The µ a 540 ranged from 0.194 to 0.663 cm 1 .Melons were ranked according to increasing µ a 540 (increasing maturity) and sorted into three maturity classes: low (µ a 540 = 0.383±0.008cm 1 ), medium (µ a 540 = 0.451±0.004cm 1 ) and more mature (µ a 540 = 0.,537±0.014cm 1 ).

Peel and flesh color
Peel and pulp color significantly changed with TRS maturity classes (Table 1).Peel color turned from green to yellowgreen with advancing TRS maturity class, as b* and C* values increased and h° values decreased from LeM to MoM melons.Pulp color became slightly more orange, as b* and h° were lower in MoM fruit than in LeM ones.There was no

Standard maturity indices, juiciness, internal ethylene and total carotenoids content
Flesh firmness ranged from 7.09 to 27.55 N, SSC from 7.4 to 12.2% and acidity from 1.70 to 5.57 g l 1 citric acid.Flesh firmness, SSC and acidity were not significantly affected by TRS maturity classes (Fig. 2 A,  B, C).However, firmness and acidity were lower and SSC was higher in MoM fruit than in LeM ones.Juiciness ranged from 6.86 to 22.27% and was signifi cantly higher in MoM fruit than in the LeM ones (Fig. 2 D).Internal ethylene concentration ranged from 51.9 to 153.3 ppm.It did not significantly change with TRS maturity class, even if the LeM fruit showed the lowest concentration and MoM ones the highest (Fig. 2E).Total carotenoids content ranged from 16.95 to 28.25 mg Kg 1 fw; it was significantly higher in MoM fruit than in LeM ones (Fig. 2F).

Regression analysis
The results of regression analysis between µ a 540 and quality parameters are summarized in Table 2.The µ a 540 was significantly related to juiciness (r=0.53),internal ethylene concentration (r=0.86) and total carotenoid content (r=0.66).The µ a 540 was also positively related to skin color (except for a*)   with r<0.5.No significant correlation was found between µ a 540 and pulp color, flesh firmness, SSC and acidity.Significant relationships were also found among peel color, firmness and juiciness (Table 3).Firmness was related to b* peel (r=0.62),C* peel (r=0.60) and to h° peel (r=0.58);juiciness was related to b* peel (r=0.57),C* peel (r= 0.55) and to h° peel (r= 0.54).As for pulp color, total carotenoid content was positive ly related to a* pulp (r=0.68) and negatively to h° pulp (r= −0.88); internal ethylene was related to a* pulp (r= −0.61) and juiciness to h° pulp (r= 0.58).Significant relationships were also found between juiciness and total carotenoids (r= 0.64) and between juiciness and firmness (r= 0.73).

Discussion and Conclusions
'Honey Moon' melons used in this experiment showed pulp color similar to that of fruit of the same cultivar picked at commercial maturity, even if firm ness, acidity and SSC had lower values (Cavicchi and Pasotti, 2004).Total carotenoid content showed val ues typical of orangefleshed fruit (Saladie et al., 2015;Singh et al., 2022).Internal ethylene concen tration confirms that 'Honey Moon' is a climacteric genotype, as there was an increase in ethylene pro duction with advancing fruit maturity (Senesi et al., 2005;Vallone et al., 2013;Saladie et al., 2015).
TRS measurements of melons showed some prob lems.In fact, the absorption in the chlorophyll range cannot be used as a maturity index as already done for other fruits, such as apples, pears, peaches and nectarines (Rizzolo and Vanoli, 2016), since chloro phyll was almost absent in the melon pulp while it was present in a layer just under the peel.On the other hand, µ a 540 was able to distinguish MoM mel ons from LeM ones, as MoM fruit showed yellower peel color, slightly more orange pulp, higher juiciness and higher carotenoid contents, in agreement with Kyriacou et al. (2018), Senesi et al. (2005) Beaulieu and Lea (2007), Vallone et al. (2013), Saladie et al. (2015) and Tristan et al. (2022).MoM fruit also showed higher internal ethylene concentration and lower firmness than LeM ones, even if the values showed high variability and the differences were not significant.The µ a 540 was also positively related to internal ethylene, carotenoid accumulation and juici ness, indicating that µ a 540 is linked to different ripen ing processes in melons.However, the relationship between µ a 540 and total carotenoid content was not as high as expected (r=0.66) and as previously found in mangoes when r ranged from 0.78 to 0.91 depend ing on the cultivar (Vanoli et al., 2018).It seems that in 'Honey Moon' melons, the range of µ a 540 (0.194 0.663 cm 1 ) is quite similar to that of mangoes (0.117 0.835 cm 1 ), while the variability in total carotenoids  Moreover no correlation was found in melons between µ a 540 and pulp color while in mangoes µ a 540 was negatively related to h° pulp with r= 0.83 0.98 (Vanoli et al., 2018).In 'Honey Moon' fruit, with advancing TRS ripening degree, the changes in flesh color toward a more orange shade were accompa nied by increased juiciness values and ethylene pro duction and by carotenoid accumulation, while changes in peel color toward a yellow shade were associated with fruit softening.
In conclusion, the absorption coefficient mea sured at 540 nm (µ a 540) by the TRS technique could be used to sort melons in different ripening degrees; however, its applicability will need to be evaluated on a larger number of fruits and on other varieties.

Table 1
Peel and pulp color parameters of 'Honey Moon' melons in relation to TRS maturity classesMeans in the same column followed by different letters are statistically different at P≤0.05 (Tukey's test).

Table 2
Regression models between µ a 540, juiciness, internal ethylene concentration and total carotenoid contents