3.1. Content of the Main Metabolites of Microwave-Dehydrated Saffron
To determine the effect of microwave dehydration on saffron’s main metabolites, the stigmas were dehydrated at different powers and time lapses, and subsequently analysed through HPLC-DAD, which is the only way to determine these compounds [
11].
Picrocrocin, safranal, and crocetin esters concentrations are shown in
Table 2. With regards to picrocrocin, the first metabolite to be detected in the analysis, control values were significantly higher in traditional dehydration than the microwave treatments. Safranal was not quantified in traditional “toasting” because its content was below the limit of quantification. However, all microwave dehydration treatments showed safranal. Tong et al. [
6] observed that an increase in the time of microwave dehydration of fresh stigmas (from 3 to 6 min) at 600 W registered a decrease of safranal concentration, while at 450 W a longer time (from 6 to 10 min) obtained a higher concentration. In our study, it is essential to supply an energy of the same order by combining powers and times (providing different time lapses), so that within each power studied there is no great difference in total time to be able to compare our results with those showed by these authors. Another study described the effect on different dehydration methods, including microwave dehydration [
14], in which safranal reported the highest concentration in compared to electric oven dehydration and vacuum oven dehydration; however, the results in the mentioned study cannot be compared to ours as the dehydration time employed was excessive (1.9 h).
The total content of crocetin esters showed significant variances between the control and the other microwave treatments, except for 440-36, 440-130, 616-39, 616-52, and 800-20. This also happened in the sum of
trans crocetin esters with the exception for the treatment 440-130, which also showed significant differences to the control. The
cis isomers showed significant differences in microwave-treated samples against the control, and those samples which showed the highest concentration of
cis isomers were also the ones that had the highest safranal concentration. In this sense, Carmona et al. [
23] reported that a high temperature for the dehydration process promotes the isomerization of
trans crocetin esters to
cis, as well as safranal synthesis from these carotenoids and picrocrocin. Speranza and Dadá [
32] observed the formation of 13-
cis-crocin, after 1 h of exposition to light. On the other hand, this is the first time that the concentration of the main metabolites of stigmas dehydrated by microwave is evaluated in detail. There is no previous study showing the concentration of
trans and
cis isomers, which could be used to compare our results, but it seems that the energy supplied by microwave dehydration of stigmas may be involved in the formation of
cis crocetin esters. Thus, all these results could indicate that different energy sources could influence the isomerization process and the formation of safranal from picrocrocin and the cycling of
cis isomers.
The proportion between
trans and
cis crocetin esters was analysed, showing that
trans crocetin esters are the predominant form of crocins. All the microwave-treated samples showed significant variance with the control, the values of which ranged from 20 in 800-40 to 48 in 616-83. Therefore, the saffron obtained by microwave dehydration contains less content of
trans crocetin esters than those obtained from traditional “toasting”, resulting in saffron with less bioactive capacity, as
trans crocetin esters compounds are more bioactive than
cis isomers [
3,
12,
33].
Crocins are a wide group of glycosyl esters, of which the predominant ones are
trans-4-GG and
trans-3-Gg. Concentration values of the crocetin esters are shown in
Table 3. One of the predominant crocetin esters is
trans-4-GG, which showed differences to the control in all microwave dehydration treatments, except for 440-73, 616-83, 616-90, and 800-40. The other main crocetin ester is
trans-3-Gg, which showed the highest concentration values of all the reported glycosides and obtained significant differences between the control and all the microwave dehydration treatments studied. Previous studies showed that
trans-4-GG concentration is higher than
trans-3-Gg in traditionally dehydrated stigmas [
11,
16,
34,
35]. Other works are in accordance with our results as a greater value of
trans-3-Gg concentration than
trans-4-GG is also registered when stigmas are dried in the shade or freeze-dried [
35,
36]. However, under these different methods of dehydration, contrary results have also been obtained [
36,
37]. Therefore, it can be mentioned that these two compounds are the main crocetin esters of saffron.
In the other crocetin esters studied, significant differences to the control were shown for
trans-5-nG,
trans-2-gg,
trans-2-G and
trans-1-g. All these compounds showed higher content in control except for
trans-1-g, whose content was higher in all the microwave treatments [
3].
Cis-4-GG and cis-3-Gg were the cis-crocetin esters identified and quantified in this work. Both showed significant differences to the control with higher content in the treatments studied. These crocetin esters are known for being less bioactive than the trans esters, which results in a saffron with lower bioactive capacity.
Saffron aroma is enhanced after at least a month of its storage in stigmas traditionally dehydrated, the reason why saffron is not sold immediately after dehydration. In addition, previous studies observed that saffron’s main compounds’ content evolves over time [
4,
35]. Thus, microwave dehydrated saffron was stored for three months in order to study the evolution of the main metabolites, after which these compounds were analysed by HPLC-DAD again.
Picrocrocin, safranal, and total crocetin esters concentration after three months of storage are shown in
Table 4. Both picrocrocin and safranal maintained the significant differences shown between the control and the treatments studied before the storage. In practically all dehydration parameters studied, safranal values were higher after storage, which matches with the previous results of storage effect on safranal according to Maggi et al. [
38]. Moreover, Sereshti et al. [
39] reported that safranal content is lower in freshly dried stigmas, and at least a month of storage is known to be necessary for the development of saffron aroma [
22,
23].
The content of total crocetin esters was less in the control and in some microwave dehydration treatments compared to the results obtained before its storage. The treatment 616-83 showed significant variances with the rest of the microwave dehydration treatments and with the control for total crocetin esters and for the sum of trans crocetin esters. All dehydration treatments showed significantly higher cis content than the control, resulting in a lower trans/cis proportion. The treatments that showed the highest concentration of total cis isomers also registered the highest safranal content, which was also observed before the storage, reinforcing the previously mentioned relationship between safranal and cis crocetin esters.
Crocetin esters were analysed after three months of storage to analyse whether deterioration of these compounds had taken place. Main crocetin esters’ concentration values are shown in
Table 5.
The two main crocetin esters, trans-4-GG and trans-3-Gg kept the previous proportion, trans-3-Gg concentration being higher than trans-4-GG in all microwave dehydration treatments studied and in the control. The concentration value of trans-4-GG compared to the content obtained before storage decreased slightly in the control, while it increased in some of the microwave dehydration treatments. For trans-3-Gg, the control also obtained lower value compared to its content before storage, and the treatments increased their concentration only in some of them. Regarding trans-4-GG, at 440 W for 36 s, 55 s, and 73 s, along with 616-90 and 800-20, there was significant variance compared to the rest of the treatments. Trans-3-Gg showed similar significant differences to those described for trans-4-GG in relation to the microwave dehydration treatments. As before the storage of the saffron, cis-4-GG and cis-3-Gg were identified and quantified. Regarding cis-4-GG, all microwave treated samples showed significant variances with the control, which obtained the lowest concentration (1.04 g/kg). Cis-3-Gg, however, could not be detected in the control and in the treatments performed at 440 W.
Therefore, after storage, new significant variances were observed. Some of the microwave treated samples presented different trends compared to the control. It is noteworthy that microwave dehydrated saffron showed higher total crocetin esters content in some treatments after storage (at 440 W for 36 s, 55 s and 73 s, 616-90 and at 800 W for 20 s, 40 s and 60 s) and safranal content decreased in 440-73, although the storage is known for improving safranal content but also diminishing carotenoids due to its oxidation [
23,
24,
40]. Considering the main metabolites’ content of saffron obtained from the different microwave dehydration treatments studied, and compared to the control (traditionally obtained saffron), the treatments that obtained an increase in total crocetin esters after storage also showed high contents of picrocrocin and safranal. These treatments were able to dehydrate stigmas of
C. sativus L. and obtain saffron with a content of main metabolites equal to or superior to those obtained by traditionally dehydrated saffron. Among them, treatment 800-20 stands out for obtaining saffron with the highest content of picrocrocin, safranal, and total crocetin esters. The 440-130 treatment would stand out for obtaining saffron with a great bioactive capacity. In addition, it would have high content of the main metabolites. On the other hand, 616-83 would not be recommended for use due to the lowest content of all evaluated compounds in the saffron obtained from this treatment.
Discriminant function analysis was performed on results from HPLC-DAD grouped together according to the power used in the dehydration process, in order to identify a relationship between the compounds during the dehydration process and determine canonical functions that separate samples within two functions (
Figure 1).
After the dehydration process, the control sample was separated from microwave dehydrated stigmas by function 1 (71.4% of variance) and function 2 (92.8% of cumulative variance) (
Figure 1a). Function 1 depended on
trans-4-GG,
trans-3-Gg, and
trans-5-nG mainly, and function 2 depended on
trans-4-GG,
trans-5-tG, and
trans-2-G primarily. Similar analysis was performed after three months of storage, and in this case, control sample and stigmas dehydrated at 440 W were separated from the rest of the treatments by function 1 (82.9% of variance) and function 2 (99.5% of cumulative variance) (
Figure 1b). In this case, function 1 depended on
cis-3-Gg,
trans-4-GG, and
trans-2-G mainly, and function 2 depended on
trans-3-Gg,
trans-5-tG, and
trans-5-nG, principally. After three months of storage, a higher separation of the treatments at 440 W due to
cis-3-Gg, mainly, was observed. Considering
Table 3 and
Table 5,
cis-3-Gg content decreased at 440 W after storage, while the isomer
trans-3-Gg increased. It seems that the dehydration process at this power produces a less stable isomerization than the rest of the powers studied.
Therefore, the microwave dehydration process separated saffron obtained from the stigmas dehydrated by “toasting”, and this separation was more pronounced after three months of storage, due mainly to the content of crocetin esters and more specifically to the most abundant crocins.
3.2. Commercial Quality of Microwave-Dehydrated Saffron
The commercial quality of saffron is classified into three categories established by ISO 3632 [
7]. This standard indicates that the highest quality category must be composed of saffron with a minimum value of 200 of
440 nm, 70 of
257 nm, and a
330 nm value between 20 and 50. Results of UV-vis spectrophotometric analyses of the control and saffron dehydrated at different powers and time lapses are shown in
Table 6.
The initial results showed that the control could not be classified as saffron according to ISO 3632 [
7] due to its low value of
330 nm, while all the microwave treatments produced saffron belonging to Category I. Color strength (
440 nm) results showed that there were several differences across different treatments, although saffron obtained in 440-73, 616-83, 616-90, 800-30, 800-40, and 800-60 did not show significant differences between traditional and microwave dehydration. Values of ISO parameters obtained in microwave dehydration treatments are in concordance with those reported by Maghsoodi [
5] in a previous essay, taking into account their results obtained at 200 W for 720 s (in total), since the same order of energy (144,000 J) as our work was applied. The values of
257 nm were significantly lower for all the microwave dehydration treatments compared to the control.
After storage, the treatments 616-83 and 800-60 showed a 440 nm value below 200, which relegated them to Category III (≤170) and II (≤200), respectively. These treatments did not maintain the quality previously observed before storage, which shows that they are not suitable for producing high quality saffron.
Saffron value is mainly determined by its 440 nm value, therefore, producers want the saffron with the highest 440 nm value as possible. Between all the treatments analyzed, those that showed the highest 440 nm value were 440-55, 440-73, 616-90, and 800-20. The last mentioned showed high crocetin esters content in HPLC-DAD analysis, proving to be a good alternative to traditional “toasting” for obtaining high quality saffron.