Evolution of phytochemical and antioxidant activity of Tunisian carob (Ceratonia siliqua L.) pods during maturation

Carob trees (Ceratonia siliqua L.), belong to the Caesalpinaceae sub family of the leguminosae family. For a long time, they have grown in Mediterranean areas (1,2), including Maghreb countries in Nord-Africa, for forestry and consumption (3). They have been posteriorly planted in USA and Australia as an ornamental and shade tree (4). The carob fruit has been widely investigated due to the presence of various compounds as well as an important level of carbohydrates, minerals and phenolic compounds, which contribute to the great nutritive and functions potential (1,3,5). Therefore, it has been mainly used in human nutrition, including syrups, popular drink and Arabic confectionery (1). In addition, as a feed for livestock (6) and especially used in traditional medicine to prevent or/and control health diseases (3). Besides, it has been qualified as an important raw material in food and non-food industries and afforded valuable products (2,7), especially locust bean gum obtained from endosperm and marketed E410, which has been used as a stabilizer in emulsions (1,8). Furthermore, the pods are used as cacao substitute(9), also as a raw material for alcohol and sugars production, in addition, it was applied in pharmaceutical, cosmetic and textile industries (1,5,7).

Sugars and bioactive phenolic compounds are the main components of carob tree pulps (10). Indeed, pulps are considered as an interesting source of sugars which determine its organoleptic quality and its commercial value (1,11). They present a significant concentration of bioactive principles that often reflect a considerable antioxidant potential (12) and confer an important medicinal and functional properties (3,4). Besides, it is proven that the carob pulps contain a reasonable amount of mineral elements (2) which contribute a considerable nutritional value. In addition, it is known that geographical origin influences this biochemical composition. Indeed, the genetic and environmental expressions were known by its considerable effect on enzymatic activities that are involved in the biosynthesis and biodegradation of several primary and secondary metabolites (15). Therefore, pulps collected from Portugal (16) contain higher concentrations of total polyphenols than those collected from Algeria (12) and Tunisia (17). Besides, it has been also proved that the ripening process alters significantly this composition. Indeed, during the increasing of ripening level several primary and secondary metabolites are activated, then each compound will serve to an appropriate function to determine the final fruit characteristics. Accordingly, it was showed that the carob pods achieved the highest sucrose content at the ripe stage (11), consequently the industries producing the carob syrup, carob powder and bioethanol could to be interested in mature carob pods. However, in nutritional and functional term, the immature fruits are the preferred, since they contained the highest levels of phenolic content at the unripe stage (12).Therefore, it is necessary to study the biochemical variation of the carob pods harvested from different regions of Tunisia and evaluate its susceptibility to ripening process. This study aims to classify different carob provenance at different harvest dates based on their sugar content and bioactive phenolic compounds.

Previous published data were revealed that carob pods contained considerable amounts of minerals (2,13), important levels of bioactive compounds (12) and was considered as interesting source of carbohydrates(1,11) as well antioxidant principles (12). However, its valuation is mainly depends on their nutritional and functional compounds which are accumulated and degraded during ripening process (3,12). Indeed, the users are always looking for a well-finished quality to satisfy their needs, maybe it can be the astringency, the sweetness or the abundance of nutritive or functional principles (24). In this term the present investigation focused to determine the changes in a significant range of photochemical compounds of carob pods as influenced by ripening stages and provenance in order to extend the use of carob pods.

The storage conditions was correlated with water content (25) which affected the sensorial parameters and nutritional quality of fruits. However, the decline pattern of moisture recorded by selected provenances was necessary to maintain the nutritional quality of mature fruits during storage. This behavior would also enhance the percentage of solid compounds which explains the hardness character of mature pulps. Therefore, we could guess that the “SK” and “GB” provenances are characterized by the hardest texture and predict they will maintain its sensorial and nutritional properties for a longer period. Therefore, fruit that contain considerable amounts of mineral elements could be found as a good source of mineral supplement in food diets. However, several investigation reported the mineral composition of the pods (2,13,26), but no available information about its variation during ripening.

It was seen that ash and mineral elements contents revealed a downward trend, which was related to the mobility of mineral elements in phloem from pods to another parts of fruits such as seeds which are known by their highest ash content (2,27,28). Their participation in various metabolic pathways justify also this behavior (29). In our investigation, the changes that were produced during ripening conferred significant mineral yields at the end of maturity, but it remains lower than those recorded by the wild and grafted pods harvested from Antalya (2). Besides, it was shown that the ash and minerals contents are susceptible to climate and environmental change which introduced by geographical origin variation. This behavior might be explained by the fact that the transfer of mineral salts in the phloem depends mainly on the environment where the carob trees are grown and the plant state (28). Indeed, the minerals convey from the soil to the plant depends on their availability in the soil, the presence of water and its quality that promotes their absorption and affects the roots state; growth and activity (30). Moreover, the sugars confer particular organoleptic properties and defines the commercial value for carob pods (24). Again, the sugars contents were reported previously by Ayaz et al(26), Biner et al.(1) and Vekiari et al(11), who revealed that sucrose, fructose and glucose were the main sugars qualified and quantified in carob pods. The same results are displayed in our investigation. The considerable influence of ripening stage was also demonstrated by others investigations (11,32), where it was indicated that glucose and fructose content showed a decreasing trend during ripening of carob pods, since it would mainly contribute to the biosynthesis of polysaccharides and might be involved in certain physical and chemical reactions (33,34). During fruit development, the sucrose content exhibited an increment which was in agreement with the evolutional pattern exhibited by carob pods harvested from Turkey (11). This behavior is mainly controlled by the profile of sucrose phosphate synthase (SPS) activity. Indeed, Schaffer et al.(35) and Gomez et al. (36) revealed, during ripening of melon and papaya, a synchronization between increased SPS activity and sucrose accumulation. During maturation, the fruits receive a regular portion of sucrose from photosynthesis which occurs in the leaves and found to be the main biosynthesis pathway of sucrose (36). The accumulation of sucrose could be also explained by the breakdown of the different carbohydrates present in the other organs of the plant (15). Indeed, Hubbard et al. (37) found that partial decomposition of stachyose in leaves is relatively related to sucrose synthesis. Schaffer et al. (35) detected significant amounts of raffinose and stachyose, which can be transported to the fruit, while sucrose is the only polysaccharide present in the melon.

Besides, the sucrose was found as a discriminant factor between provenances, since it was present as the predominant sugar in the mature stage and its concentrations varied significantly between the provenances. This variation was generally due to genetic and environmental exhibition that could modify the activity of the enzymes involved in biosynthesis and decomposition of sugars including sucrose phosphate synthase and acid invertase (15,37). Moreover, in sensorial and nutritional term the mature pods obtained from “KE” would be the most appreciated by consumers as a delicious and energetic fruit, since it revealed the highest sugar content. It deserves an industrial development to enhance the yield of syrup, juice and carob flour preparation as well bioethanol production.

The polyphenols were the most important secondary metabolites and considered as an interesting compound define the nutritional and functional fruits quality (12,38). As the primary metabolites, this groups of molecules recorded important variation during ripening(12), since some authors revealed that during ripening of fruits the evolutionary behavior of secondary metabolisms was highly associated with that of primary ones (28,39). Indeed, the biosynthesis and biodegradation of these latest would generate the abundance or the deficiency of substrates which contribute to the biosynthesis of secondary metabolisms. The analysis of carob TP content showed a gradual decrease during ripening to attain the lowest levels at the end of the maturity. However, they remain higher than those demonstrated by El-sherif et al. (40) and Benchikh et al (12) whose investigated the ethanolic extract (186.07 mg GAE/ 100 g) and aqueous extract of carob pods (1.35 g GAE/ 100 g DW). This significant variation could be related to the difference of harvest date, climatic and geographical conditions, the methods of extraction, the solvent polarity and genetic characters (12).

The observed progressive patterns were in agreement with the results reported by Benchikh et al(12) and Howard et al(41) whose studied respectively carob pods harvested from Algeria and pepper fruits. Once more, this tendency was probably explained by the activation of polyphenol oxidase (PPO) which contribute to the oxidation of phenolic compounds (42). The changes in their solubility in water might justify our observations. In fact, at the beginning of maturity the phenolics compounds exhibited weak polar character and then their condensation during ripening coffered an important polarity (12,43). Their participation in enzymatic browning confirm also this pattern. As for condensed tannins, its downward trend is crucial to reduce the severity of astringent taste (39). The “GB” provenance is the most profitable for the users who’s looking for the curative properties, since the flavonoids compounds were the main controller of the anti-inflammatory, anti-allergic and anti-cancer activities. The high temperatures in the southern regions might justify these important flavonoids amounts, since it was shown that the lower temperature prevent the accumulation of flavonoids compounds (44). Besides, the rainfall reduced the total phenolic levels (45), therefore the “TN” provenance which grown in northern areas that probably present the highest rainfall level, showed the lowest phenolics compounds content. Moreover, the yields of the dry extracts obtained from studied provenances showed upward tendency during ripening to attain the highest amounts at the ripe stage, which were close to those found by Roseiroet al. (46) for aqueous extract of mature carob pods (53.17%). This behavior seems reasonable since throughout ripening the percentage of solid compounds showed an increasing trend.

The antioxidant capacity followed the same change pattern shown by the bioactive compounds content which confirms the high correlation between the amounts of bioactive compounds and antioxidant capacity assessed by DPPH test (28,47). The same results were reported by Benchikh et al (12) whose investigated carob fruits harvest from Algeria. Furthermore, the origin effect was also showed for all determined parameters, since each provenance has its each provenance has its own climatic, geographical and pedological natures, such as rainfall levels, sun exposure and temperature that affect the biosynthesis of phenolic compounds. The interesting content of bioactive compounds and the considerable antioxidant capacity confirmed the great nutritional and functional potential of this fruits, notably at the unripe stage and especially to “GB” provenance.

Our findings showed that biochemical composition and organoleptic properties were considerably influenced by the ripening and the geographic origin. However, in the studied provenances, ripening was accompanied by a remarkable regression in phenolic compounds correlated with downtrend of antioxidant activity, an interesting decrease in minerals content and a considerable accumulation of sucrose. The behavior exhibited by each component justified the fact that carob fruit could be used largely in all ripening stages depending in targeted nutrient. Therefore, the green carob pods were considered as an interesting source of natural antioxidant for pharmaceutical uses, while the mature pods should be selected as a good sugar source for juice extraction, cacao substitution and bioethanol production.