The initial assessment of the bioreaction with citronella oil at different concentrations demonstrated that the total conversion of the major alcohols (citronellol and geraniol) was completed within 24 h for the test solutions containing 0.1% to 10% w/v of the oil. When 50% oil was used, the same analytes had an average conversion of about 95% after 24 h and 100% after 48 h reaction. No significant changes were observed for the 90% w/v solution within 48 h of reaction, suggesting that the enzyme efficiency is much reduced at such a high sample concentration. Thus, a 10% w/v essential oil sample concentration and 48 h reaction time were chosen as the most suitable working condition for further experiments, taking into consideration that there are differences in the composition and analyte abundance in the other samples, such as the presence of secondary and tertiary alcohols, which may take longer to react.

Additionally, these results indicate that the previously optimised reaction conditions [7] are similarly efficient for an essential oil sample at a concentration (mg/mL) around 167 times higher than that of the enzyme, and it may continue to produce the desired reaction products slightly above this level, provided the reaction is left to proceed for a longer duration. This is important information for future scale-up studies, even though the essential oil sample concentrations are not directly equivalent to the concentration of the total substrate alcohols within each sample.

The GC×GC‒MS method enabled the tentative identification of 125 target analytes, including 79 alcohols and 46 esters (Table 1), within the 35 essential oil samples studied, before and after enzyme bioprocessing. The compounds that did not satisfactorily match the reference mass spectrum and retention indices were not included in the table.

Thus, the occurrence of a given substrate alcohol in multiple samples was frequently observed, which means that many of these specific analytes were tested multiple times within samples of different compositions and complexities. In general, a consistent efficiency was observed for the conversion of primary alcohols identified in multiple samples, such as citronellol and geraniol, indicating that neither the number of analytes, their chemical diversity, nor their abundance in the samples seems to adversely affect or inhibit the enzyme activity towards these substrates.

A scatter diagram of the GC×GC‒MS chromatogram (Fig. 1) illustrates the retention time coordinates of the target analytes (i.e. alcohols and esters) and demonstrates the superior separation capacity through additional separation on the 2D column, and easy visual assessment of chemical changes that this technique offers. Generally, the relative position of a given analyte peak in the 2D space does not significantly vary from one sample to another, provided that the analytical conditions are kept the same for all the sample sets. This attribute allows an analyst to instantly locate the target analytes across the sample set once they are identified in a sample, to recognise clustering groups with similar chemical class and/or molecular features (e.g. monoterpene alcohols and monoterpene esters), and to determine any differences in the overall chemical profile of the samples, such as the changes resulting from the enzymatic process.

Scatter diagram of the GC×GC‒MS results, demonstrating the relative position of the target analyte peaks in the 2D space (1tR and 2tR according to Table 1) and clustering of groups of monoterpene alcohols (blue), monoterpene esters (orange), sesquiterpene alcohols (purple), and sequiterpene esters (green). Other alcohols (grey) and esters (black) are also included, with clustering of groups of phenyl (area I) and diterpenoid (area II) compounds highlighted. GC×GC‒MS analysis was performed using a DB-5/SLB-IL60 (non-polar/polar) column set (see details in the “Experimental” section); hence, generally more polar analytes elute at later retention times in the second dimension

A total of 42 out of the 79 alcohols identified were successfully converted into their respective acyl esters. The lipase-catalysed esterification reactions were generally quite efficient for primary and secondary alcohols, such as β-citronellol, geraniol, menthol, (Z)-carveol, β-santalol, (E)-farnesol, (E)-nuciferol, 1-octen-3-ol, and benzyl alcohol (Fig. 2), with conversions (C%) of 80 to 100% within 48 h reaction time. Some secondary alcohols, such as fenchol, isopulegol, isoborneol, and borneol (Fig. 2), were only partially converted (C% = 30–60) within the same time. No significant conversion was observed in the tested conditions for tertiary alcohols and phenols, which are major components in many of the samples investigated (e.g. linalool in lavender and eugenol in clove). Despite that, for completeness, the GC×GC‒MS chromatograms of all the tested E.O. have been included in the Appendix S2 of the Supplementary Information.

Chemical structures of some of the alcohols esterified by CALA lipase

As demonstrated in our previous study [7], large conversions (C% ≥ 90) can be quickly achieved (within ~ 1 h) for primary alcohol reaction with CALA enzyme, in the tested conditions. Secondary alcohols had around 20% conversion within the same time and around 70% within 24 h in the same study. Thus, in the present work, the reaction was allowed to proceed for 48 h to obtain the highest possible C% for all types of alcohol substrates across all the samples tested. This also seems to have contributed to less variations for most part of the compounds with high C% and identified in multiple samples, such as (Z)-carveol (C% = 90–96), as well as citronellol and geraniol which were 100% converted in all of the samples containing them. The highest variations were observed for a few compounds, such as isopulegol (C% = 29–49) and borneol (C% = 34–61). However, it is important to reiterate that these C% are estimated from the difference in the indicative chromatographic areas of these alcohols before and after the reaction, but this was not a quantitative measurement, which was not the aim of this study.

By comparison of all the E.O. investigated, the most significant bioprocessing changes were observed for vetiver and sandalwood, which contain a large number of sesquiterpene alcohols that were successfully esterified. GC×GC‒MS enabled the tentative identification of 18 of those alcohols and their respective esters, as well as other alcohols that were not esterified or are from other groups. The samples showing fewer changes contained lower amounts (in concentration and number) of the enzyme’s preferred substrates (i.e. primary and secondary alcohols).

The comparison of the 2D chromatograms of the original and bioprocessed vetiver (Fig. 3A and B, respectively) and sandalwood (Fig. 3C and D) samples allows the location of clustering areas in the 2D space corresponding to alcohols (Fig. 3, area I) and esters (Fig. 3, area II), and to clearly observe the chemical changes in the samples obtained with enzymatic processing of the original oil.

GC×GC‒MS chromatograms of vetiver (A and B; sample VTV1) and sandalwood (C and D; sample SDWA) essential oils, illustrating the chemical changes achieved after the lipase-catalysed esterification processing (B and D), in comparison with the original samples (A and C). The highlighted areas I and II represent the location in the 2D space where most of the alcohols and esters are found, respectively

Sandalwood samples had conversions of 92% and 91% for the major alcohols α- and β-santalol (Fig. 2) within 48 h, respectively. Other sesquiterpene alcohols, such as (E)-farnesol and (E)-nuciferol (Fig. 2), were also present in high abundance and were efficiently esterified (90–100%). A number of high-abundance compounds can be observed in sandalwood chromatograms, indicated by large peaks in the contour plot, with some apparent extended tailing. However, to adequately study the effects of lower abundance peaks, some overloading of high-abundance peaks is required (as opposed to diluting the sample).

Specifically for vetiver samples, the enzymatic esterification of secondary terpene alcohols, such as eudesma-4,11-dien-2-ol, and ziza-6(13)-en-3-α-ol, as well as the full conversion of the major alcohol khusimol (Fig. 2), outperforms the biocatalysis results reported by Notar Francesco et al. [9], being closely comparable to their observations for chemical esterification of vetiver alcohols. According to these authors, the acetylation processing can introduce grapefruit, sandalwood, and cedarwood undertones to vetiver oil, as opposed to the usual earthy notes, which can increase the value of this fine fragrance ingredient to almost double the price of the original oil [9].

The use of such chemically diverse natural samples as lipase substrates, aligned with the resolution power of GC×GC‒MS analysis, has enabled the assessment of the enzyme’s performance towards specific compounds, such as the aforementioned vetiver alcohols, for which the pure standards are very expensive or not commercially available.

It is important to highlight that a positive overall odour change arising from the esterification of such complex samples could be related to either the pleasant aroma of the new esters produced, or the suppression of the off-flavour character of the initial alcohols. A lower odour activity of these new esters in comparison to their respective alcohols could also bring other types of odorants present in the original oil to the spotlight as the new key aroma impact compounds in the processed sample. In the case of vetiver, for example, there are different compounds (unrelated to the esterification process) with grapefruit notes present in the raw oil, such as nootkatone, β-vetivone, and valencene, which could also play a more significant role to the overall aroma of the sample after processing. In fact, Tissandié et al. [14] observed in the olfactory assessment of the esterified vetiver oil that the main esters produced were generally odourless or with low odour impact to the overall sample, except for 12-norziza-6(13)-en-2α-yl acetate, which apparently has a stronger vetiver-like note. Thus, the accurate aroma assessment of the overall esterified samples and the identification of their key odorants are complex tasks that require additional analytical steps, which are not covered in the present study.