Morphological and Anatomical Alterations During Ripening
Visual observation indicated the change of coloration and loss of firmness at three different stages (pre-ripe, ripe and over-ripe) of guava fruit. A gradual reduction of the green color and an increase in the yellow coloration from the pre-ripe to over-ripe (Fig. 1) was noted. Softening is one of the important characteristics of fruit ripening. We observed an accentuated firmness decrease in three different ripening stages of guava pulp. Scanning electron microscopy (SEM) is routinely used for studying the anatomical characteristics in different plants. Recently, Majeed et al., (2022) [11] used this technique to study morpho-palynological and anatomical characteristics in desert cacti. Multiple researchers utilized this sophisticated microscopy technique to study fruit anatomy as well [12]. For guava, Abreu et al., (2012) [13] reported beehives-like cellular anatomy in the mature guava fruit by using SEM. In agreement with the authors, we also observed beehive-shaped preserved cell structures at the beginning (pre-ripe) and gradual formation of the uneven cell masses in the subsequent ripening (ripe and over-ripe) stages (Fig. 2).
The spectrophotometric analysis of the guava fruit peel showed a gradual decrease in the chlorophyll content (chlorophyll a, b and total chlorophyll Content) as it progressed towards maturation (Fig. 3). We observed 35.92 and 2.63% decreases in the chlorophyll a content from pre-ripe to ripe and ripe to over-ripe stages, respectively. For the chlorophyll b content, a decrease of 24.38 and 2.89% were noted from the pre-ripe to ripe and ripe to over-ripe respectively. Total chlorophyll content, also showed a similar pattern across the maturation stages (29.85 and 2.13% from pre-ripe to ripe and ripe to over-ripe respectively) (Table S1). The results indicated that the major change in the chlorophyll content was found during pre-ripe to ripe transition only. Our finding was consistent with the literature Trong et al., (2021) [14]. Upregulation of the chlorophyll degrading enzymes such as peroxidase, chlorophyllase, and chlorophyll oxidase, might be attributed [15] for the decrease of the chlorophyll content of the guava peel. Plant Senescence Reflectance Index (PSRI) is an important parameter to assess the carotenoid content in the plant. Increase in the PSRI reflects the increase in the bulk carotenoid content [16]. Degradation of chlorophyll and subsequent synthesis of carotenoid is a common process in the development of fruit ripening. Rojas-Garbanzo et al., (2017) [17] showed that Criolla cultivar of guava fruit accumulated a large fraction of carotenoids as it attained maturity. Similarly, we observed marked increase (>70%) in the carotenoid content of the fruit peel in terms of PSRI, as the maturation progressed. The quantitative change of chlorophyll and carotenoid pigmentation in the guava peel, across three ripening stages, is shown in Fig. 3.
Phenolic and antioxidant profiling of fruit pulp at different ripening stages
We observed a statistically significant difference in the Total Phenolic Content (TPC) of guava fruit across the three different stages. TPC showed a decrease at the ripe phase (4174.00 to 3349.67 mg GAE/100 g DW) followed by an increase at the over-ripe (5086.33 mg GAE/100 g DW) (Fig. S1 and Table 1). Phenolic compounds are secondary metabolites of plants with multiple functions, including attraction to pollinators, protection against pathogens, pigmentation and antioxidant activities. Various researchers earlier demonstrated the influence of phenolic compounds and their corresponding antioxidant properties during the guava fruit ripening process. Mahmood et al., (2012) [18] demonstrated a consistent increase in the phenolic content from the un-ripening to the full-ripened stage for the korona variety of strawberry. However, there was a decrease in the phenolic content in the full ripened stage for the other variety (tufts). Guofang et al., (2019) [19] also reported high phenolic content in the post-ripening stage for different cultivars of blueberries. On the other hand, blackberry species Rubus adenotrichus Schltdl showed a decrease in the total phenolic compounds during ripening [20]. The trend in the decrease of phenolics during ripening and subsequent increase in the post-ripening (over-ripe) as seen in our data, could be attributed to the participation of phenolics to the biosynthesis of other molecules. Further, polymerization or association of phenolics with other compounds, during maturation of the fruit, might also be the contributing factor [21]. In an earlier study, Thaipong et al., (2006) [6] showed a linear correlation between antioxidant assays (FRAP, DPPH and ABTS) and total phenolic content in the guava fruit extract. The powerful correlation between the phenolic content and antioxidant parameters was also demonstrated in other fruits. Differences in the total flavonoid content (TFC) of the guava fruit at different stages of maturity were also statistically significant. Similar to the TPC, we observed a decrease in the TFC from pre-ripening (259.67 mg QUE/100 g DW) to ripening (217.67 mg QUE/100 g DW) and a subsequent increase towards over-ripening (276.67 mg QUE/100 g DW).
Table 1 Phytochemical and antioxidant parameters of guava pulp at different maturation stages
|
Pre-ripe
|
Ripe
|
Over-ripe
|
TPC (mg GAE/100 g DW)
|
4174±198.40
|
3349.67±129.14
|
5086.33±143.05
|
TFC (mg QUE/100 g DW)
|
259.67±7.51
|
217.67±5.69
|
276.67±22.19
|
FRAP (mg AAE/100 g DW)
|
3913.33±98.03
|
3474±71.23
|
4246±165.58
|
DPPH (mg AAE/100 g DW)
|
3730.67±29.14
|
3439±11.79
|
3974.33±4.62
|
ABTS (mg AAE/100 g DW)
|
162.33±6.33
|
165.0±5.57
|
160.57±8.50
|
GAE: Gallic acid equivalent, QUE: Quercetin equivalent, AAE: Ascorbic acid equivalent
Antioxidant assays like FRAP (3913.33 to 4246 mg AAE/100 DW) and DPPH (3730.67 to 3974.33 mg AAE/100 g DW) showed similar trend as of TPC and TFC, however there was no significant change in the ABTS activity (162.33 to 165 mg AAE/100 DW) across three maturation stages (Fig. S2 and Table 1).
It was noted in the literature that multiple factors, such as biosynthesis, degradation, cultivars, climate etc might affect the complex changes in phenolics, thereby impacting their profile and concentration at different stages of fruit ripening [22].
In general, the patterns of phenolic/flavonoid synthesis and antioxidant properties were found to be inconsistent as discussed above. However, it should be noted that most of these studies were performed at different stages of maturation while fruits were still borne in the plants. Nevertheless, our research was focused on the post-harvest (pre-ripe) storage of guava cultivars in an unregulated condition. Recently, Thapa et al., (2022) [23] observed that applying exogenous Putrescine could delay fruit ripening and maintain phenolic and antioxidant levels. In general, the pattern of phenolic synthesis is comparable to the post-harvest storage to some extent other than the exogenous application of ripening inhibitors. Our experiments showed that in terms of firmness and phenolic-antioxidant content, the fruit quality of the Arka mridula guava cultivar, were maintained during room temperature storage within 8 (eight) day window from the harvest of the pre-ripe stage, without any external control of physical parameters and/or exogenous chemical applications.
Principal Component Analysis
PCA, a statistical tool witnessed frequent applications in fruit metabolomics for grouping various inherent characteristics. Recently, Omayio et al., (2022) [24] used PCA to cluster the nutrient composition of three fruits, namely strawberry, white and red guavas. The variables of our data were loaded into thy PCA, and two components PC1 (50.5%) and PC2 (49.5%) were obtained (Fig. 2). We observed that parameters TFC-DPPH-FRAP and ABTS-TPC into two separate clusters based on their loading into the PC1 (Principal Component 1) and PC2, respectively. Further, based on the loading in both the components, TPC-TFC and DPPH-FRAP formed two different clusters. Each of the clusters represented correlated variables. Overall, a strong correlation among various parameters was established by the multivariate analysis in agreement with the literature Chen et al., (2020) [25]; however, possible influence of non-phenolic and non-flavonoid compounds on the antioxidant properties of the guava, might be the basis of differential loadings of variables across the PCs.
Analytical Characterization at Different Ripening Stages
Analytical characterization of guava fruit extract at different maturation stages showed fifty-five (55) compounds in the LC-MS and GC-MS analysis. LC-MS and GC-MS techniques identified thirty-four (34) and twenty-one (21) compounds, respectively. As determined by the GC-MS, a large number of volatile compounds explained their presumptive influence on the guava antioxidant parameters as hypothesized during PCA in the previous section (Table S2-S5).
In the LC-MS analysis, several classes of phytochemicals such as flavonoids, terpenoids, carboxylic acids and vitamins, were identified. GC-MS analysis revealed the dominance of alkane hydrocarbon and fatty acid molecules. Among these molecules, n-hexadecanoic acid showed marked change through the maturation stages of the guava. Overall, it was observed that all the major classes of compounds generally had a decrease in the ripe stage and a subsequent increase in the post-ripening stage to the pre-ripening level of the fruit. Such a trend could be well correlated with the result obtained in the biochemical assays for phytochemical and antioxidant power estimation.
We used multi-color heat map and principal component analysis (PCA) tools (Fig. 3) to discern the contribution of phytochemicals towards the ripening process. Previous researchers suggested that different compounds present in the fruits show varied concentrations during the ripening process depending on their nature of synthesis, structure, availability and other chemical properties [22]. Our observation during PCA analysis also supported the view. PCA revealed four clusters based on their contribution towards the maturation process of the guava fruit. Among these four clusters, the fourth cluster containing seven compounds (Table S5) showed gradual increase in the ripe stage, followed by decrease or no change at the post-ripe stage. The major compound in this cluster was Catechin, reaching as high as 26.52% at the ripe stage from 10.73% of the pre-ripe. However, it showed marked decrease in the over-ripe stage accounting only 1.43% among seven compounds. Although we observed a narrow spectrum of literature on the phytochemical identification throughout the maturation process of guava, several studies indicated various phytochemicals involved in the natural ripening progress in guava. Catechin is regarded as one of the principal compounds in guava by various authors in the literature, especially at the mature stage [26]. Although cluster 1 and 2 showed a similar trend in terms of having similar values in the pre-ripe and post-ripe stages, and increased percentage in the ripe phase with a few exceptions (Table S2 and S3), the second cluster was dominated by the alkane hydrocarbons, as determined by the GC-MS. Cluster 1 and 2 contained fourteen and sixteen compounds respectively. The major compounds in the first and second clusters were Glycine derivative (4.61% at over-ripe), Kaempferol-4'-glucoside (3.70% at over-ripe), Myricetin (3.25% at over-ripe) and Myricitrin (4.46% at over-ripe), n-Hexadecanoic acid (9.44% at pre-ripe), Gamma-Sitosterol (5.39% at overripe) & Octacosane (7.75% at over-ripe) respectively. Betta et al., (2018) [26] and Dhianawaty et al., (2022) [27] reported higher amounts of kaempferol in the guava fruit over-ripe stage than the ripe one consistent with our report. Myricetin was another major flavonoid reported in the literature from guava fruit [28]. Changes in the percentage of compounds in the third cluster (Table S4) were comparatively much lower than the other three clusters. The contribution of these 18 compounds in the ripening was minimal (≤2% on average).