As shown in Figure 1
), 102 compounds were detected, including 34 aldehydes (ALD), 15 alcohols (ALC), 1 ester (EST), 8 ketones (KET), 2 acids (ACD), 7 hydrocarbons (AHA), 25 terpenes and derivatives (TER), and 10 other compounds (OTH). After 30 days of aging (Figure 1
), 88 compounds were identified, including 27 aldehydes, 14 alcohols, 2 esters, 10 ketones, 7 acids, 3 hydrocarbons, 16 terpenes and derivatives, and 9 other compounds. Finally, in the samples stored for 24 months (Figure 1
), only 45 analytes were identified: 9 aldehydes, 7 alcohols, 2 esters, 2 ketones, 7 acids, 1 hydrocarbon, 10 terpenes and derivatives, and 6 other compounds. Figure 2
shows the trend of the eight molecular classes for each sample and provides a better representation of the data.
The identified analytes are listed in the tables below and divided into their chemical classes.
The number of aldehydes was the highest in the WDF_t0
sample, with 34 out of the 102 molecules detected being aldehydes, and was the second class in terms of total absolute abundance. During storage, the number of identified aldehydes decreased (27 and 9 for WDF_t1
, respectively). The total abundance in terms of the TIC area decreased by 45% after 30 days of storage, from 1109 × 106
to 608.5 × 106
, and subsequently increased to a value of 940.4 × 106
. This increase is related to the degradation of more complex molecules, such as amino acids, fatty acids, and carotenoids, which release volatile aldehydes as byproducts. In contrast, the TIC area of more unstable aldehydes decreased over time, some of which eventually disappeared. They characterize the WDF_t0
aroma and are typical flavor compounds of the fresh fruit, i.e., Gavina®
watermelon. They probably underwent degradation due to oxidative, biological, and photochemical processes, thus giving rise to other species, and leading to a change in the aroma profile of the sample. Table 1
collects the results relating to this molecular class.
C9 aldehydes characterize the flavor and aroma of different species of Cucurbitaceae [3
] and have been previously reported in several studies on similar samples [5
]. We identified 4-nonenal, nonanal, (Z,Z)-3,6-nonadienal, (Z)-2-nonenal, (E,E)-2,6-nonadiena€(E)-2-nonenal, and 2,4-nonadienal. They are among the species that mainly define the characteristic aroma of Gavina®
watermelon, since they were particularly abundant in WDF_t0
. The abundance of these species decreased sharply during storage, and only nonanal was detected after 24 months (WDF_t2
). The most unstable and rapidly degrading species (4-nonenal, (Z)-2-nonadienal, 3,6-nonadienal, and (E,E)-2,6-nonadienal) were not detected after 30 days (WDF_t1
). At the biological level, C9 aliphatic aldehydes are produced by the enzymatic oxidation of polyunsaturated fatty acids (PUFA), particularly linolenic and linoleic acids, by lipoxygenase [6
]. Figures S1 and S2
show the degradation mechanisms of these fatty acids and the VOCs formation. Some enzymes such as alcohol dehydrogenase and cis/trans isomerase convert these compounds into their corresponding alcohols and cis/trans isomers, respectively [30
(Z,Z)-3,6-nonadienal is often cited in the literature, but is not easily identified because it rapidly isomerizes to 2,6-nonadienal and 2-nonenal [31
]. Some authors have defined it “watermelon aldehyde” [30
], since it has a low odor threshold and a strong aroma of “freshly-cut watermelon”. Despite its instability, (Z,Z)-3,6-nonadienal was detected in WDF_t0
. It is likely that the fresh fruit of this cultivar had a particularly strong aroma that persisted in WDF_t0
. 2,6-nonadienal and 2-nonenal are also particularly unstable. They have a “green” and “waxy” aroma and are characteristic of cucumber flavor. A progressive decrease in their abundance occurred over time until complete disappearance after 24 months. Various mechanisms have been proposed to explain this decrease during storage, including hydration followed by condensation [36
], oxidation to the corresponding acids, or enzymatic reduction [34
In contrast, other aldehydes are considered to contribute to a lesser extent [26
], but they enrich the aroma of WDF samples with pleasant fragrances. Hexanal, 2-hexenal, 4-heptenal, 2,4-hexadienal, 2-heptenal, 2,4-heptadienal, and 2,4-decadienal can be generated by the oxidation of polyunsaturated fatty acids and, to a lesser extent, by autoxidation [1
]. These molecules, already detected in some watermelon cultivars, are particularly abundant in foods rich in fatty acids such as avocado, fish oil, and olive oil [5
The C6 aldehydes hexanal and 2-hexenal play very important roles in the food and perfume industries, since they impart “green” and fresh fragrances, which are particularly appreciated and sought after [1
]. Some authors [25
] have suggested the possible formation of hexanal in small quantities due to lycopene degradation, a carotenoid abundant in watermelon fruit. This aldehyde reached an amount 2.5 times higher after 24 months (WDF_t2
Similarly, 2-methyl-propanal, pentanal, 2-methyl-butanal, and 3-methyl-butanal levels increased over time. The origin of these last two compounds has been associated with the catabolism of the amino acid leucine [38
] in a complex degradation process that involves some sugars, which are certainly present in the analyzed samples. The oxidation of 2-methyl-butanal and 3-methyl-butanal gave rise to volatile compounds present only in aged samples, such as 2-methyl butanoic and 3-methyl butanoic acids, species absent in WDF_t0
, and present in increasing quantities after 30 days (WDF_t1
) and 24 months (WDF_t2
) of storage (Table 1
). 2-methyl-propanal was also assumed to have the same origin [38
], which could explain the significant increase in its concentration over time. These analytes impart a “green”, “fermented”, and “fresh” aroma.
The results of HS-SPME-GC-MS analysis showed a strong loss of the typical aroma of the fresh fruit, watermelon, during storage. The abundance of C9 aldehydes decreased significantly over time and was practically absent after 24 months. After 30 days, the “green” and “herbaceous” notes prevail with some “fruity” nuances, which disappear completely in WDF_t2, where the pleasant fragrance perceived is given only by the first two aromas.
3.7. Terpenes and Derivatives
Terpenes play a fundamental role in defining the aroma and fragrance of fruits, flowers, and spices [1
]. They probably constitute one of the largest groups of compounds known in nature, with approximately 30,000 molecules identified to date, isolated from plants, microorganisms, and animals. Lighter species are important constituents of essential oils and plant resins, whereas heavier species include carotenoids. Similar to monoterpenes, carotenoids are biosynthesized starting from geranyl diphosphate (Figure S6
), which, through further coupling of isoprene units, each catalyzed by specific enzymes, progressively forms derivatives with a lipophilic chain gradually longer, up to the tetraterpenes (C40). They typically have a 40-carbon atom chain backbone, and are among the most important pigments in fruits and flowers [4
]. They show different colors, from the yellow of lutein, to the orange of β-carotene, to the red of lycopene, owing to their system of conjugated double bonds, on which their chemical, physical, and biological properties depend. The high delocalization of π electrons allows them to stabilize reactive intermediates, such as carbocations or radicals, by resonance. Therefore, they have efficient antioxidant properties and protect cells from oxidative stress induced by reactive species, such as free radicals. Foods containing carotenoids have a high antioxidant capacity [45
], and their nutritional intake is strongly recommended, since it is associated with the prevention of several diseases, including cardiovascular diseases and cancers, and with increased immune response [4
]. However, because of their high number of double bonds, carotenoids are particularly sensitive to degradation induced by heat or atmospheric oxygen, biological oxidation processes, and exposure to electromagnetic radiation, leading to the formation of characteristic volatile compounds. For this reason, many carotenoids have considerable commercial value in the food and cosmetic industries, not only as pigments but also as substances capable of imparting pleasant aromas to products [47
]. Given the increase in demand for natural and healthy products and the problems related to the synthesis of these analytes, the flavor and fragrance industry is increasingly searching for new methods to obtain natural species to replace their synthetic counterparts. Among the emerging production methods, the use and transformation of matrices containing carotenoids are becoming increasingly invasive, as a natural alternative capable of supplying precious aroma volatiles [48
The monitoring of carotenoid degradation compounds in commercial products can provide information on storage time, product quality and acceptability, nutritional value, vitamin and aroma content [48
], color, and visual impact. Based on their detection, it is possible to draw conclusions regarding the presence of a given carotenoid. These observations applied to the watermelon WDF can be exploited to evaluate, in an approximate way, the variation of the carotenoid content during storage and to make considerations both on its nutritional value and on its aroma. All the results relating to the terpenes and derivative class are shown in Table 7
Monoterpenes and monoterpenoids were identified in WDF_t0 only. After 30 days, these volatile analytes were not detected probably because they underwent degradation.
HS-SPME-GC-MS analysis highlighted the presence of limonene in an unexpected and positive way. This analyte is one of the characteristic constituents of the citrus family but has also been found in other fruits, including watermelon [6
]. It is a particularly sought-after molecule, appreciated in the food, cosmetic, and soap industries, as it provides a very pleasant citrus and fresh fragrance. It was the most abundant species in WDF_t0
, but it was not detected in any of the aged samples, probably because natural limonene is highly unstable and undergoes isomerization and autoxidation [51
Another monoterpene is linalool (Figure S3
), which is typically present in the essential oils of lavender, citrus, jasmine, and rosewood, to which it provides citrus and floral notes. This species undergoes epoxidation, followed by rearrangement, which leads to the formation of trans-linalool oxide. This explains the decrease of linalool and the increase of its oxidized product in the aged samples.
Β-Terpinene, γ-Terpinene, and α-Terpineol (Figure S4
) are cyclic monoterpenes only present in WDF_t0
, while isoterpinolene was detected only after 24 months of storage. We do not know whether this analyte is formed over time or if it was already present in WDF_t0
and had not been identified because of the high complexity of the chromatogram. Although present in modest quantities, they help enrich and complete the aroma of Gavina®
WDF with floral, citrus, and woody notes.
Cymene was identified only in WDF_t0
. It is an aromatic volatile compound that is very common in thyme and oregano essential oils but has been found in more than 100 plants and 200 fruits [1
]. It performs various biological functions, and it also possesses anxiolytic, anticancer, and antimicrobial properties, as well as antiviral and antifungal activities [1
Borneol (Figure S5
) is a bicyclic molecule with a fresh and woody fragrance, which was detected in WDF_t0
in comparable quantities.
As shown in Figure 3
, lycopene degradation gives rise to several VOCs, following various degradation pathways.
With the term “citral”, or 3,7-Dimethyl-2,6-octadienal, we mean the cis- and trans- mixture of the neral and geranial acyclic monoterpenes, unsaturated aldehydes that impart a “citrus-like fresh and fruity” aroma. In nature, they are found mainly in citrus fruits and are exploited in the food and cosmetic industries because of their characteristic pleasant fragrances. However, these are unstable molecules that are sensitive to oxidation [25
The highest concentrations of (Z)-3,7-Dimethyl-2,6-octadienal and (E)-3,7-Dimethyl-2,6-octadienal were detected in WDF_t0
(85.4 × 106
and 89.8 × 106
on a TIC basis, respectively). These quantities are reduced to approximately a quarter in WDF_t1
(21.6 × 106
and 24.1 × 106
for the (Z) and (E) isomer, respectively) and reduced to zero after 24 months of storage. In addition to watermelon, they are also detected in some cultivars of apples, tomatoes, and paprika [2
], and more generally, in species containing high levels of lycopene or its precursors [4
]. Their formation is probably due to the interaction and subsequent degradation of lycopene with atmospheric [2
]. We also identified 2,3-epoxy-geranial, the epoxidation product of citral, previously detected in similar matrices by other authors [25
]. It is formed by a mechanism that is easily established in contact with atmospheric oxygen. Furthermore, only in WDF_t0
, the citral structural isomer is present, isocitral, in a cis-trans mixture.
The most abundant species associated with lycopene degradation was 6-methyl-5-hepten-2-one, which accounted for approximately 17% of the VOCs fraction of WDF_t0
, with a TIC abundance of 810 × 106
. It imparts a fruity, waxy, green fragrance with citrus notes, and its abundance tends to decrease strongly (approximately −70%) after 30 days and remains almost constant over time up to 24 months. Several authors have correlated its formation with the oxidation or degradation of lycopene, α-farnesene, citral, or conjugated trienols [2
]. The presence of 6-methyl-5-hepten-2-one in non-negligible abundance in the freshly prepared sample highlights the high instability of lycopene, which underwent rapid degradation.
The species shown in Figure 4
are commonly related to β-carotene degradation [29
] by breaking bonds in the different structural positions of the molecule. Among the degradation products, the most representative ones are:
β-ionone, produced following the breaking of the C9-C10 bond, is present in comparable quantities in WDF_t0
(35.8 × 106
and 31.2 × 106
, respectively), while it is much less abundant after 24 months of storage (0.93 × 106
). It is a molecule with a floral, slightly fruity, and pleasant aroma [44
]; is widely used in all sectors of perfumery; and is used industrially for the synthesis of vitamin A [1
]. In our samples, β-ionone was also identified in its epoxidized form (5,6-ionone epoxide), which reached a higher abundance in WDF_t1
(34.4 × 106
). In this aging step, the quantities of β-ionone and 5,6-ionone epoxide were comparable. As previously reported for 2,3-epoxy-geranial, the epoxidation of these terpenes occurs quite easily in the presence of atmospheric oxygen. These two species were the most abundant among those involved in β-carotene degradation. Therefore, we can hypothesize that the C9-C10 bond is the most susceptible to breaking.
Dihydroactinidiolide, whose formation mechanism is not well understood, is hypothesized to form starting from 5,6-ionone epoxide according to a radical oxidation mechanism [44
] or following the oxidation of the C8-C9 bond. Like 5,6-ionone epoxide, dihydroactinidiolide also reaches its maximum quantity after 30 days of storage, and then halves after two years (44.7 × 106
and 22.0 × 106
samples, respectively), giving the aged fiber a fruity aroma with woody notes.
Although β-carotene is not the most abundant carotenoid in watermelon pulp, it contributes significantly to its aroma, as its degradation products have a particularly low odor threshold. Therefore, even if present in small quantities, they are easily perceived by humans sense of smell [4
]. After 24 months of storage, the revealed the presence of some species considered secondary in the degradation process of β-carotene, such as 2,2,6-trimethylcyclohexanone and β-cyclocytral.
ζ-carotene is an acyclic, faint yellow carotenoid [4
], a precursor of lycopene and β-carotene, structurally similar to lycopene, but with a lower number of unsaturations (Figure 5
). Its presence has been hypothesized in several fruits that contain carotenoids, including watermelons and tomatoes.
We assume that ζ-carotene is present in our samples because its main degradation product, geranylacetone, was detected at each aging step, albeit in progressively lower quantities over time (181 × 106
, 104 × 106
, and 2.36 × 106
, and WDF_t2
, respectively). Geranylacetone is a volatile molecule with a floral and fruity aroma and a high odor threshold [39
], lacking a distinctive effect on the overall fragrance of our samples. Furthermore, 6-methyl-5-hepten-2-one is derived from ζ-carotene, a ketone already described above in relation to lycopene, and has a high odor threshold [28
]. As 6-methyl-5-hepten-2-one can result from the simultaneous degradation of two precursor analytes, the significant amount detected in the WDF samples could be justified.
The results of the HS-SPME-GC-MS analysis showed a strong loss of terpenes during storage. They provide the samples with a characteristic and pleasant aroma, and their decrease contributes to the loss of the typical fragrance of the initial fresh matrix. Carotenoids degradation VOCs were mainly identified in WDF_t0, while in the aged samples, their content is minimal. This leads to a significant change in the WDF aroma profile, considering that these VOCs impart pleasant nuances and have a high odor threshold. Moreover, the carotenoid content probably decreases drastically during storage. It follows that the beneficial properties associated with the carotenoid content of Gavina® WDF are also compromised.
3.10. Potential Applications of Gavina® Watermelon WDF
In addition to the environmental benefits of sustainable development, the enhancement of watermelon WDF is extremely interesting because of its numerous beneficial effects on human health. The lycopene and β-carotene contents, confirmed using HS-SPME-GC-MS analysis (Section 3.8
), make the freshly prepared sample (WDF_t0
) a source of antioxidants that protect against several health issues [4
], as outlined in the previous sections. Moreover, the food industry is increasingly looking for dietary fiber sources, as they can physically and chemically modify food preparations and enhance specific sought-after properties [53
]. Dietary fibers also have numerous health benefits, since their consumption is associated with the prevention of several diseases, such as cardiovascular disease, diabetes, and colon cancer [11
]. In recent years, the consumer demand for foods enriched with natural supplements capable of conferring health benefits has increased significantly [53
]. Consumers are more concerned about the consumption of healthy foods with a high content of dietary fibers, polyphenols, vitamins, and minerals, and low-calorie intake. Gavina®
watermelon WDF not only fully satisfies this demand, but is also extremely low-cost, since it can be recovered from pre-waste fruits or from industrial processing that considers it a by-product. Therefore, this represents an opportunity for the food industry.
The products in which dietary fibers are mostly included are bakery goods [54
], beverages, dairy products, frozen dairy products [58
], pasta, meat [59
], and soups. For example, several studies used fruit powder to partially replace traditional wheat flour to produce biscuits [55
]. The final products had significantly higher antioxidant activity, mineral and fiber contents, and reduced starch content.
In several studies, the inclusion of fruit pomace powder led to a significant increase in sensory characteristics, i.e., aroma and flavor [58
]. In fact, they impart fruity notes and improve consumer acceptability of certain food products, such as cakes, ice cream, beverages, and confectionery.
The high potential of watermelon WDF can also been extended to other sectors, such as animal feeds and nutraceuticals [62
]. The use of fruit pomace for feeding can lead to significant changes in animal growth and health.
However, carotenoid degradation, highlighted using HS-SPME-GC-MS analysis, leads to a decrease in the antioxidant content of Gavina® watermelon WDF. Therefore, some of the beneficial effects were reduced over time, but those associated with dietary fiber remained unchanged. To maximize its benefits, it must be consumed within a short time after preparation. A possible solution may concern changing the storage method. In this study, WDF samples were stored at room temperature in the dark. Under these conditions, a significant loss of aromas related to carotenoids occurred after only 30 days of storage. Probably, refrigeration can increase the conservation of watermelon WDF for a longer time.