Metabolites 2022, 12, 656. https://doi.org/10.3390/metabo12070656 www.mdpi.com/journal/metabolites Article HS‐SPME‐GC/MS Analysis for Revealing Carob’s Ripening Panagiota Fella, Kyriaki Kaikiti, Marinos Stylianou and Agapios Agapiou * Department of Chemistry, University of Cyprus, P.O. Box 20537, Nicosia 1678, Cyprus; fella.panagiota@ucy.ac.cy (P.F.); kaikiti.kyriaki@ucy.ac.cy (K.K.); stylianou.a.marinos@ucy.ac.cy (M.S.) * Correspondence: agapiou.agapios@ucy.ac.cy; Tel.: +357‐22‐895432; Fax: +357‐22‐895088 Abstract: Carob’s recognized nutritional and medicinal value next to its unique agriculture im‐ portance 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 composi‐ tion 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 imple‐ mented during weeks 26–36 from five different locations in Cyprus. Additionally, the gaseous emis‐ sions 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‐ (isobu‐ tyric acid), 2‐heptanone, propanoic acid, 2‐methyl‐,2‐methylbutyl ester, acetic acid, methyl isobu‐ tyrate, 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. Keywords: carob fruit; VOCs; ripening process; odor; smell 1. Introduction The carob tree (Ceratonia siliqua L.) belongs to the family of Leguminosae, in the ge‐ nus 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 prod‐ ucts, 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. Fur‐ thermore, 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 [5–7]. 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 Citation: Fella, P.; Kaikiti, K.; Stylianou, M.; Agapiou, A. HS‐SPME‐GC/MS Analysis for Revealing Carobs Ripening. Metabolites 2022, 12, 656. https://doi.org/10.3390/ metabo12070656 Academic Editor: Do Yup Lee Received: 10 June 2022 Accepted: 11 July 2022 Published: 15 July 2022 Publisher’s Note: MDPI stays neu‐ tral with regard to jurisdictional claims in published maps and institu‐ tional affiliations. Copyright: © 2022 by the authors. Li‐ censee MDPI, Basel, Switzerland. This article is an open access article distrib‐ uted under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecom‐ mons.org/licenses/by/4.0/).