# Functional Characterization of _Saccharomyces_ Yeasts from Cider Produced in Hardanger
## 3 Results and Discussion
### Behavior of Saccharomyces Strains under Fermentative Conditions  
The total AA content in apple juice from the cultivar Aroma averaged 303.97 mg/L. The evaluation of the AA content revealed that asparagine (42.2%) was the most important AA in juice, followed by tyrosine (13.1%), aspartic acid (8.2%), arginine (6.7%), and glutamine (5.8%) (Table S5 in Supplementary Materials). The AA utilization profile of both species showed a similar utilization pattern of AAs present in apple juice, and the pattern of AAs utilized also reflected the strain effect (Figure 2, Table S6 in Supplementary Materials). The major AA source for all strains tested was asparagine, which provided an average of 57.7% of the total AAs utilized. Note that the initial asparagine concentration in apple juice was 3-65 times higher than that of the other AAs (Table S5). Aspartic acid, arginine, and glutamine were the next most utilized AAs, followed by glutamic acid, serine, proline, and then valine and tyrosine (Figure 2, Table S6). The remaining AAs were present in very low concentrations in apple juice and were mostly utilized during fermentation. For methionine, histidine, and alanine, some residual amounts were still detected at the end of fermentation (Figure 2, Table S6). The good utilization of asparagine and glutamine by the _Saccharomyces_ strains tested in this study is consistent with what has been reported in the literature, as these two AAs, along with ammonium, have often been reported as preferred nitrogen sources for _S. cerevisiae_[26; 27]. Aspartic acid, arginine, glutamic acid, and serine were also included among the preferred nitrogen sources [26; 27], which we also observed in our study. Although tyrosine was the second most abundant AA in apple juice, less than a quarter of it was utilized by the strains, which is consistent with the literature where tyrosine is considered a non-preferred nitrogen source [26; 27]. Figure 1: HPLC–UV chromatogram of simultaneously separated 19 AAs and 4 BAs (each at 25 mg/L on-centration level) with prior derivatization using dansyl chloride agent. Cysteine, lysine, and especially tryptophan were found in higher concentrations at the end of fermentation compared to what was present in the apple juice. This could be explained by the release of these AAs into the medium during fermentation (Tables S5 and S6). Tryptophane and methionine were also previously classified as non-preferred nitrogen sources [26; 27]. However, the data in the literature on the classification of nitrogen sources were mostly based on their ability to support yeast growth of laboratory strains under non-fermentative conditions. The HPLC-UV method enabled us to determine AAs and BAs simultaneously. Thus, we were able to detect strains with BA-producing ability using this method (Table S6). At the end of fermentation, strains of both species formed two BAs, putrescine and tyramine. Putrescine was detected at relatively low concentrations in all strains tested, whereas the highest concentrations for tyramine averaged 3.9 mg/L in the high-producing strains 2104, 2186, and 2402 (Table S6). The BA production capability is another characteristic that is important for the selection of starter yeasts for cider production, especially when substrates for the production of BA are present in apple juice, such as tyrosine, which can be further decarboxylated to tyramine. The initial sugar content of apple juice was 101.6 g/L, and the main sugar was fructose (52.9%), followed by sucrose (35.2%) and glucose (11.9%), as shown in Table S7 (in Supplementary Materials). At the end of fermentation, most of the sugar was consumed (98.6% on average), and the residual sugar consisted mainly of remaining glucose, which varied in very low concentrations from 1.3 to 1.6 g/L (Table S7). Hence, studies showed that _Saccharomyces_ yeasts display a clear preference for glucose over fructose [29; 30; 31].