## 3 Results and Discussion
### Differential Volatile Aroma Screening and OPLS-DA Analysis of VOCs during Ripening of R. roxburghii
In this study, we observed that the majority of hydrocarbons, particularly long-chain alkanes, were predominantly present in the IV and V maturity stages. The specific cause underlying this phenomenon warrants further investigation. Through cluster heat map analysis, the fruit of _R. roxburghii_ exhibited a distinctive distribution of VOCs across five different ripening stages, which could serve as a landmark chemical fingerprint for each stage. It is worth noting that at stages IV and V, a large amount of alkene accumulation acted as an effective biomarker, distinguishing these two stages from the previous stages. In contrast, the content of volatile esters exhibited a sharp increase at stages II and III, with these changes closely correlating with the enhancement of fruit, flower, and pleasant aromas. This correlation further corroborated the consistency of the data presented in Table 1, with the aroma profiles depicted in Figures 2 and 3. It was important to highlight that the clustering pattern revealed through the abundance model of volatile substances aligned with the results of the OPLS-DA analysis (Figure 4B), thereby enhancing our understanding of the changes in volatile substances during the ripening process of _R. roxburghii_ fruit. However, further in-depth exploration and verification are required to determine whether the specific volatile substances at each stage are adequate as characteristic markers for that stage. To elucidate the trend of VOCs during the ripening of _R. roxburghii_ and to identify the VOCs responsible for changes in aroma, OPLS-DA was employed (Figure 4B). Concurrently, 200 permutation tests were conducted to assess the model's accuracy. The results indicated that R\({}^{2}\)X was 0.995, R\({}^{2}\)Y was 0.998, and Q\({}^{2}\) was 0.996. Both R\({}^{2}\) and Q\({}^{2}\) values exceeding 0.5 suggest that the model fitting results were acceptable, demonstrating that the established model possessed strong interpretive and predictive capabilities [67]. Consequently, the model effectively elucidated the relationship of VOCs in _R. roxburghii_ fruit across different ripening stages. Following the 200 permutation tests, as illustrated in Figure 4C, the intersection of the blue regression line with the vertical axis of Q\({}^{2}\) was below zero, indicating that the model was not overfitted and that the validation was robust; thus, the results were deemed suitable for the identification and analysis of various ripening stages of _R. roxburghii_. The OPLS-DA diagram clearly differentiated the distinct ripening stages of _R. roxburghii_ fruit, particularly stages IV and V. Stages I, II, and III were relatively close to one another, all situated in the fourth quadrant, suggesting that their volatile compounds were similar. Furthermore, Figure 2 revealed that stage IV was characterized by a richness in aldehydes, alkenes, and phenols, while stage V represented the most mature stage, abundant in alcohols, acids, ketones, alkenes, and alkanes, thereby making these two stages highly distinguishable in the figure. In contrast, stages I, II, and III were predominantly composed of aldehydes, alcohols, and esters. In addition, the VIP was employed to identify the primary volatile organic compounds responsible for aroma changes during the ripening process of _R. roxburghii_. A VIP value greater than 1 indicated that the corresponding variable was a key component of the discriminant model; furthermore, a larger VIP value signified a more significant difference between the variables at various maturity stages [68]. Utilizing the screening criteria of \(p\) < 0.05 and VIP > 1, we identified 30 key differentially volatile aroma components across different ripening stages of _R. roxburghii_ (Figure 4D). For more details of VOCs with VIP > 1, please refer to Figure S2 in the Supplementary File. These components comprised six types of aldehydes (such as 2- (E)-hexenal, (E,E)-2,4-hexadienal, 1-nonanal, etc.