HS-SPME-GC/MS Analysis for Revealing Carob’s Ripening

Carob’s recognized nutritional and medicinal value next to its unique agriculture importance is associated with an array of social, economic, and cultural activities. The carob fruit is popular for its intense aroma due to the emitted volatile organic compounds (VOCs). The composition of VOCs released from carob fruits changes during ripening, rendering it a non-invasive tool for the determination of the ripening period and freshness of the fruit. Therefore, headspace solid-phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC/MS) was applied to reveal the respective gaseous signal molecules related to fruit maturity. The sampling was implemented during weeks 26–36 from five different locations in Cyprus. Additionally, the gaseous emissions of total VOCs (TVOCs) and carbon dioxide (CO2) were recorded next to the moisture content of the fruit. The major chemical classes in the ripening are acids, followed by esters, and ketones. More specifically, the most abundant VOCs during ripening are propanoic acid, 2-methyl-(isobutyric acid), 2-heptanone, propanoic acid, 2-methyl-, 2-methylbutyl ester, acetic acid, methyl isobutyrate, propanoic acid, 2-methyl-, 3-methylbutyl ester, 2-pentanone, butanoic acid and propanoic acid, 2-methyl-ethyl ester. Finally, CO2 emissions and moisture content showed a rapid decline until the 31st week and then stabilized for all examined areas. The methodology revealed variations in VOCs’ profile during the ripening process.


Introduction
The carob tree (Ceratonia siliqua L.) belongs to the family of Leguminosae, in the genus of Ceratonia. The thermophilus nature of the tree allows it to thrive in environments with mild and drought climate conditions, such as in most Mediterranean countries [1]. Carob cultivation in Cyprus dates back to the first centuries and is inextricably linked with the culture and society of the island. As one of the most important exportable products, it also plays an important role in the agricultural economy of the country [2].
Biogenic volatile organic compounds (BVOCs) are major chemical components of the natural environment, as they contribute to the communication between plants, insects, hosts, soil organisms, etc. The part of the earth system that includes all ecosystems and living organisms in the atmosphere and on land, acts as the head source of BVOCs. Furthermore, BVOCs are released in the rhizosphere at low concentrations to regulate plants' growth, well-being, resistance, and nutrient uptake [3,4]. In general, flowers and fruits release a wide variety of BVOCs, with the emission rates peaking during ripening. On the other hand, BVOCs contribute to troposphere chemistry triggering (directly or indirectly) the production of air pollutants and greenhouse gases, as well as increasing acidity and aerosol production [5][6][7].
Fruit color and other organoleptic characteristics are evidence of fruit maturity. The ripening of the carob fruit takes almost a year and maturity is judged by the color of the carob pod (from green bean to dark brown with hard texture pulp) and the respective dehydration. According to Kyriacou et al. [8], the fruit respiration rate declines during the ripening process of the carobs; the same applies also to the phenolic content, tannins, Table 1. Studies focusing on fruit VOCs ripening.

Sample Type Analytical Method Ripening Biomarkers References
Purple passion fruit (Passiflora edulis Sims) Headspace solid-phase micro extraction gas chromatography/mass spectrometry (HS-SPME-GC/MS) 85 volatile organic compounds (VOCs) (e.g., esters, ketones, alcohols, hydrocarbons, aldehydes, and terpenes) increased at the intermediate and ripe stages (purple color); 49 VOCs were not detected in the immature stage (green color); 11 VOCs (9 esters and 2 alcohols) were found in the ripe stage (symbols for ripe fruits). [25] Tamarillo (Solanum betaceum Cav.) HS-SPME-GC/MS Increase in acids, esters, and terpenoids/decrease in alcohols, phenols; Increase in color index (CI) values and sugars; Color changes from green to red; Softening of the flesh. [12] Grape berry HS-SPME-GC/MS During the three developmental stages: Increase in aldehydes; Alcohols, ketones, and hydrocarbons compounds did not change; Increase in esters (bourbonene, ethyl geranate, (Z)-butanoic acid, 3-hexenyl ester, and dodecanoic acid ethyl ester are the most dominant esters at ripening stage); Increase in monoterpenes at veraison and ripening stage; Decrease in sesquiterpenes after fruit-set stage. [26]

Results and Discussion
Fruit coloring was previously employed to describe carobs' different ripening stages. In particular, Kyriacou et al. [8], divided the maturity of carobs into six critical stages (RSI-RS6: fully developed green, dull green, breaker, green pedicel, ripe fully dark, and late ripe fruit), whereas Ben Othmen et al. [31] reported only three stages of carob pods' maturation (unripe, mid-ripe, and ripe stage). Figure 1 shows the carob fruit samples each week from the five different orchards. The dark brown color of the carob pods indicates the ripening of the fruit. For almost all orchards (O1, O3, O4, O5), between the 26th and 28th week of the examined period, the carobs were almost immature due to the green color except in the case of the orchard O2. From the 29th week of the examined period, the examined samples began ripening, reaching a dark brown color in the last week of the examination period.
As the main compounds (greater abundance), some of the respective VOCs were selected for further study of their ripeness profile in each week of the analysis. Figure 2 presents the selected VOC trends per week for different villages. Overall, in the cases of the six selected VOCs, there were some fluctuations in the examined periods, with the highest abundance occurring around the 31st week. More specifically, propanoic acid, 2-methyl-(isobutyric acid) increased significantly in the 31st and 34th week of the examination period. Regarding 2-heptanone, no fluctuations were observed, reaching its maximum value in the 31st week, with the O5 area showing the greatest abundance. Furthermore, carobs showed the highest abundance of butanoic acid and hexanoic acid methyl ester in the 31st week. Similarly, propanoic acid, 2-methyl-and 2-methyl butyl esters reached their maximum abundance in the 31st week. However, the highest abundance of propanoic acid, 2-methyl-, ethyl ester was observed earlier (29th week).
The different chemical groups play an important role in the determination of carob odor and flavor. Figure 3 shows the distribution of VOCs chemical groups per week of maturation and orchard. The VOCs emitted from the carob pods were classified into chemical classes, with acids (42.6-65.4%) (total area under the curve, AUC) and esters (22.0-32.0%) being the most abundant compounds, followed by ketones (5.1-29.8%). Acids were the main abundant group in the last week of ripening study for all 5 regions, while the percentages of ketones and esters fluctuated in almost all orchards. The other chemical groups, such as aldehydes (<2.6%), alcohols (<0.2%), and furans/pyrans (<0.2%), were present in much lower percentages. Overall, in the cases of the six selected VOCs, there were some fluctuations in the examined periods, with the highest abundance occurring around the 31st week. More specifically, propanoic acid, 2-methyl-(isobutyric acid) increased significantly in the 31st and 34th week of the examination period. Regarding 2-heptanone, no fluctuations were observed, reaching its maximum value in the 31st week, with the O5 area showing the greatest abundance. Furthermore, carobs showed the highest abundance of butanoic acid and hexanoic acid methyl ester in the 31st week. Similarly, propanoic acid, 2-methyl-and 2-methyl butyl esters reached their maximum abundance in the 31st week. However, the highest abundance of propanoic acid, 2-methyl-, ethyl ester was observed earlier (29th week).
The different chemical groups play an important role in the determination of carob odor and flavor. Figure 3 shows the distribution of VOCs chemical groups per week of maturation and orchard. The VOCs emitted from the carob pods were classified into chemical classes, with acids (42.6-65.4%) (total area under the curve, AUC) and esters (22.0-32.0%) being the most abundant compounds, followed by ketones (5.1-29.8%). Acids were the main abundant group in the last week of ripening study for all 5 regions, while the percentages of ketones and esters fluctuated in almost all orchards. The other chemical groups, such as aldehydes (<2.6%), alcohols (<0.2%), and furans/pyrans (<0.2%), were present in much lower percentages.   Figure 4 illustrates the overall HS-SPME-GC/MS procedure of carob ripening from Livadia (O1) with the respective chromatograms of the five study periods of ripening. VOCs fluctuations were observed during ripening, with the maximum emissions emitted at weeks 34-36, as shown in the respective chromatograms. Additionally, the color of carobs during these weeks was dark brown, indicating ripening. Butanoic acid ethyl ester (12), propanoic acid, 2-methyl- (13), butanoic acid (14), 2-heptanone (15), propanoic acid, 2-methyl-, 3-methyl butyl ester (17), and 2-nonanone (19) were significantly increased in the final stage of the examined period. After being stored in the carob mills, carob fruits were processed to produce several related products (e.g., small-and medium-size kibble nibbles, cubes, seeds, powder, etc.). Figure 4 illustrates the overall HS-SPME-GC/MS procedure of carob ripening from Livadia (O1) with the respective chromatograms of the five study periods of ripening. VOCs fluctuations were observed during ripening, with the maximum emissions emitted at weeks 34-36, as shown in the respective chromatograms. Additionally, the color of carobs during these weeks was dark brown, indicating ripening. Βutanoic acid ethyl ester (12), propanoic acid, 2-methyl- (13), butanoic acid (14), 2-heptanone (15), propanoic acid, 2-methyl-, 3-methyl butyl ester (17), and 2-nonanone (19) were significantly increased in the final stage of the examined period. After being stored in the carob mills, carob fruits were processed to produce several related products (e.g., small-and medium-size kibble nibbles, cubes, seeds, powder, etc.). It is worth noting that the listed BVOCs in Table 2 along with the corresponding average values of the five regions (O1, O2, O3, O4, and O5) for each examined ripening period were identified in non-roasted carob fruits. Roasting is a thermal procedure that takes place in carob mills to produce carob powder. Some VOCs are released in both the raw carob fruit and carob powder, such as hexanoic acid [33], propanoic acid, 2-methyl-, hexanal, benzaldehyde, o-cymene, limonene [34], as well as furans, and their derivatives (furan, 2-methyl) [35]. Furan levels are raised by roasting and need to be monitored and regulated since they are considered toxic [36]. More specifically, a previous study by Krokou et al. [18] showed that the main chemical group of VOCs in carob fruits are acids, followed by esters, aldehydes/ketones, furans, hydrocarbons, and alcohols. Thus, the present results on non-roasted carob (carob fruit) from the five orchards confirm the earlier findings, as the acids (65.4%) remained the main chemical group (Table 2, Figure 3), followed by esters (32%). It is worth noting that the listed BVOCs in Table 2 along with the corresponding average values of the five regions (O1, O2, O3, O4, and O5) for each examined ripening period were identified in non-roasted carob fruits. Roasting is a thermal procedure that takes place in carob mills to produce carob powder. Some VOCs are released in both the raw carob fruit and carob powder, such as hexanoic acid [33], propanoic acid, 2-methyl-, hexanal, benzaldehyde, o-cymene, limonene [34], as well as furans, and their derivatives (furan, 2-methyl) [35]. Furan levels are raised by roasting and need to be monitored and regulated since they are considered toxic [36]. More specifically, a previous study by Krokou et al. [18] showed that the main chemical group of VOCs in carob fruits are acids, followed by esters, aldehydes/ketones, furans, hydrocarbons, and alcohols. Thus, the present results on non-roasted carob (carob fruit) from the five orchards confirm the earlier findings, as the acids (65.4%) remained the main chemical group (Table 2, Figure 3), followed by esters (32%). Figure 5 shows the trends of TVOCs and CO 2 emissions per week and for each orchard. Total VOCs show an increasing trend in the first weeks of sampling. After the maximum value was reached, a gradual decline started for all orchards. Additionally, CO 2 emissions and humidity presented a rapidly declining trend until the 31st week, when they eventually stabilized for all sampling areas. According to Burg et al. [37], CO 2 as a by-product of the ripening process controls the characteristics of the fruit and can delay ripening. Figure 5 shows the trends of TVOCs and CO2 emissions per week and for each orchard. Total VOCs show an increasing trend in the first weeks of sampling. After the maximum value was reached, a gradual decline started for all orchards. Additionally, CO2 emissions and humidity presented a rapidly declining trend until the 31st week, when they eventually stabilized for all sampling areas. According to Burg et al. [37], CO2 as a by-product of the ripening process controls the characteristics of the fruit and can delay ripening.

Ripening and Influence on TVOCs, CO2, and Humidity Values
According to Figure 5, the carobs from regions O1 and O2 presented the highest concentrations of emitted TVOCs in the 29th week, with region O1 having the highest value, followed by O2. In general, for all areas (28th-31st week), a decreasing trend in the TVOCs was noticed. This is attributed to the maturation conditions during the last weeks of the experiment. The potential differences in the observed TVOCs profiles originate from the micro-climate of each region, the altitude, rainfall, and sunshine.

Samples
Carob pods were harvested from five different orchards in the regions of Larnaca and Nicosia (Cyprus), between June and September 2020 (26th to 36th week). More information about the sampling locations is provided in Table 3. After harvesting, the fruits were transferred to the laboratory and carefully cleaned with paper (no water) to remove potential impurities.  According to Figure 5, the carobs from regions O1 and O2 presented the highest concentrations of emitted TVOCs in the 29th week, with region O1 having the highest value, followed by O2. In general, for all areas (28th-31st week), a decreasing trend in the TVOCs was noticed. This is attributed to the maturation conditions during the last weeks of the experiment. The potential differences in the observed TVOCs profiles originate from the micro-climate of each region, the altitude, rainfall, and sunshine.

Samples
Carob pods were harvested from five different orchards in the regions of Larnaca and Nicosia (Cyprus), between June and September 2020 (26th to 36th week). More information about the sampling locations is provided in Table 3. After harvesting, the fruits were transferred to the laboratory and carefully cleaned with paper (no water) to remove potential impurities.

HS-SPME-GC/MS Analysis
The HS-SPME technique was applied to extract the VOCs emitted from carob fruits. Carob pods were weighed (315 g), placed in a 1.5 L in-house-made glass jar, and sealed hermetically with a Teflon cap. The sample was left in the jar for 24 h at room temperature prior to the analysis. Then, the 75 µm Carboxen/Polydimethylsiloxane (CAR/PDMS, Supelco) fiber was exposed to the headspace of the jar for 30 min to extract the VOCs. Next, the fiber was thermally desorbed into the GC injector for 1 min at 280 • C in split mode 1:10. Before sampling, the fiber was conditioned according to the instructions of the supplier. Therefore, the injector was set at 300 • C, and the CAR/PDMS fiber was exposed for 30 min. The optimization of the SPME parameters (fiber type, extraction time, desorption time, and temperature) were examined in a previous study [18].
The determination of VOCs was performed using a 7890B GC coupled with a 5977B mass detector (MS) (Agilent, Santa Clara, CA, USA). The VOCs were separated on an SPB-624 capillary column (60 m × 0.25 mm × 1.4 µm film thickness, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany). Helium was used as the carrier gas at a flow rate of 1.7 mL/min. The initial oven temperature was held at 35 • C for 5 min, then raised to 180 • C with a rate of 4 • C/min, and maintained for 20 min. The MS detector operated under the electron impact (EI) ionization mode at 70 eV with a scan range of 35-350 m/z. The temperatures of the quadrupole, ion source, and transfer line were set at 150 • C, 230 • C, and 250 • C, respectively. The identification of compounds was carried out by using the retention times relative to those of analytical standards [18], and a mass spectrum matching the NIST 17 library database.
Each sample was analyzed in triplicate. The respective chromatograms were processed using the AUC (ChemStation software, F.01.03.23.57, Agilent Technologies Inc., Santa Clara, CA, USA). The mean value of AUC was used as displayed in Figure 2 (3 replicates, 5 carob orchards), and Figure 3 (% sum of AUCs per chemical class per sampling week), as well as in Table 2 (% mean of total AUC per VOC from the five examined carob fields).

Determination of Moisture Content
The determination of moisture content in carob fruits was measured following the oven-drying method. The calculation of moisture content was based on the loss of weight; the carobs were weighed and then placed in a forced-air oven (J.P. Selecta, Barcelona, Spain) and heated at 105 • C for 12 h.

Monitoring of Other Gaseous Emissions
A portable photoionization gas detector (PID, Dragger Xam 8000, Dräger Safety AG & Co. KGaA, Lübeck, Germany) was used to monitor the TVOCs and CO 2 emissions by carobs, after being left for 24 h in a hermetically sealed glass jar (1.5 L).

Conclusions
A simple, non-invasive, analytical method (HS-SPME-GC/MS) that extracts carob aroma helped to highlight the optimum time of carob ripening. An HS-SPME-GC/MS analysis was applied to reveal carob ripening; variations of VOCs emitted during the ripening stages were noted. This can be attributed to the respective terrain (e.g., temperature, climate, soil composition, topography, cultivar, etc.) of each carob field. Among all the compounds, acids were found to be the major contributors to the perceived carob aroma followed by esters; the abundant presence of isobutyric acid was verified. The methodology is directly applicable to any fruit, even in the field. Thus, unripe and over-ripe carob accumulation in mills can be potentially avoided. The latter contributes to the fruit's quality and freshness.