# Functional Characterization of _Saccharomyces_ Yeasts from Cider Produced in Hardanger
## 3 Results and Discussion
### HPLC-UV Determination of Amino Acids and Biogenic Amines  
Determination of AAs and BAs is challenging when using the HPLC-UV system since both AAs and BAs lack chromophores for detection. Therefore, if HPLC coupled with mass spectrometric detection (MS) is not used, a sample derivatization step requiring HPLC coupled with a fluorescence detector (FLD) is necessary before analysis, especially if quantitation below 1 mg/L is required [26]. The HPLC-FLD system has more than a 10-fold higher sensitivity for such analyses compared to HPLC-UV [27]. All in all, only two methods for simultaneous determination of AAs and BAs have been published to the best of our knowledge, one based on the HPLC-MS system [28] and the second based on ultra-performance liquid chromatography (UPLC) coupled with a diode array detector (DAD) [29]. Here, we present a newly developed method for the simultaneous determination of nineteen AAs and four BAs based on the HPLC-UV system using dansyl chloride as a derivatizing agent for AAs and BAs. The method was validated and applied for the analysis of fermented beverages based on apple juice (cider) and can also be used for wine samples or, as in our study, for the in-depth characterization of AA utilization and BA production of a larger number of yeasts. The separation system is based on the C18 reverse phase (RP) system with 3 um particles, which allows better resolution as well as higher sensitivity for the analyzed compounds (Figure 1). Together with the previous derivatization with dansyl chloride, we were able to obtain adequate LODs and LOQs for the determination of AAs and BAs in the analyzed samples. These LODs and LOQs are \begin{table}
\begin{tabular}{l c c} \hline \hline
**Yeast Species and** & **Colony Color on** & **H\({}_{2}\)S Detector Tubes 2** \\
**Strain Code** & **BiGGy Agar 1** & **(ppm)** \\ \hline _S. uvarum_ 2046 & white & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2071 & white & 35 \(\pm\) 8.7 BC \\ _S. uvarum_ 2120 & brown & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2186 & brown & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2401 & white & 13.3 \(\pm\) 5.8 DE \\ _S. cerevisiae_ 2003 & brown & 10 \(\pm\) 0.00 DE \\ _S. cerevisiae_ 2095 & brown & 0 \(\pm\) 0.00 E \\ _S. cerevisiae_ 2265 & white, light brown edge & 0 \(\pm\) 0.00 E \\ _S. cerevisiae_ 2273 & white & 0 \(\pm\) 0.00 E \\ _S. cerevisiae_ 2303 & brown & 0 \(\pm\) 0.00 E \\ _S. cerevisiae_ 2349 & brown & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2128 & brown & 36.7 \(\pm\) 10.4 BC \\ _S. uvarum_ 2204 & brown & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2216 & white & 23.3 \(\pm\) 2.9 CD \\ _S. uvarum_ 2376 & white & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2083 & white, light brown edge & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2104 & brown & 0 \(\pm\) 0.00 E \\ _S. uvarum_ 2176 & white & 58.3 \(\pm\) 18.9 A \\ _S. uvarum_ 2402 & brown & 48.3 \(\pm\) 2.9 AB \\ _S. uvarum_ 2061 & brown & 0 \(\pm\) 0.00 E \\ \hline \hline \end{tabular}  
* White colony color = no H\({}_{2}\)S production; White colony color with light brown edge = moderate H\({}_{2}\)S production; Brown colony color = strong H\({}_{2}\)S production. 2 H\({}_{2}\)S was measured with gas detector tubes (120 SF; Komyo Kitagawa, Kawasaki-City, Japan). Values are reported as mean \(\pm\) SD of three replicates. Values not connected by the same letter are significantly different (ANOVA, [PERSON]’s method).  
\end{table} 
Table 1: Results of color staining on BiGGY agar and measured H\({}_{2}\)S production during fermentation for 20 _Saccharomyces_ strains tested. comparable to the published method [29], although the UPLC system with 1.8 \(\upmu\)m particles was used. The linearity range of the method for each compound is between 0.5 mg/L and 200 mg/L, which corresponds to the actual range of occurrence of the analyzed compounds in juices and fermented beverages (the detection limits are between 0.03 and 0.3 mg/L).