## 3 Materials and Methods
### Isolation and Identification of the Novel Yeast Strain from fermented Apple Vinegar  
The novel strain was isolated from traditionally fermented apple vinegar in Yongin Province (Gyeonggi, Korea). The stock solution of vinegar was diluted one in ten with sterilized saline (Seoul, Korea), followed by serial dilution to \(10^{-6}\). Each solution was plated onto YPD agar plates, using the general streak plate method, and then incubated in an incubator at 25 \({}^{\circ}\)C for 48 h. After incubation, major colonies were selected based on colony morphology, and were subsequently transferred and separated from the colonies of other microorganisms on the agar. The isolated strain was diluted in YPD broth medium, containing 30% (\(v/v\)) glycerol, and stored at \(-80\)\({}^{\circ}\)C for future study. To identify the yeast strain, ITS region sequences were analyzed using Genocell (Daejeon, Korea). The associated taxa were searched using the GenBank database at the National Center for Biotechnology Information (NCBI; [[https://www.ncbi.nlm.nih.gov/taxonomy](https://www.ncbi.nlm.nih.gov/taxonomy)]([https://www.ncbi.nlm.nih.gov/taxonomy](https://www.ncbi.nlm.nih.gov/taxonomy)), accessed on 23 August 2021), and multiple alignments from the related taxa database were organized using cluster software. Phylogenetic trees were constructed with the bootstrap test (1000 replicates) in the Molecular Evolutionary Genetics Analysis (Mega)-X website  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**NO.**} & \multirow{2}{*}{**Chemical Compound**} & \multirow{2}{*}{**Formula**} & \multicolumn{2}{c}{**Area (\%)**} \\ \cline{3-4}  & & & **SP** & **SP-BD** \\ \hline
1 & Hexane & C\({}_{6}\)H\({}_{14}\) & 11.0 & 12.1 \\
2 & Methylcyclopentane & C\({}_{6}\)H\({}_{12}\) & n.d. & 7.8 \\
3 & 1,3-dioxolane & C\({}_{3}\)H\({}_{6}\)O\({}_{2}\) & 10.4 & n.d. \\
4 & 4-Methylheptane & C\({}_{8}\)H\({}_{18}\) & n.d. & 4.0 \\
5 & 2,4-Dimethylheptane & C\({}_{9}\)H\({}_{20}\) & n.d. & 2.6 \\
6 & Acetone & C\({}_{3}\)H\({}_{6}\)O & 10.5 & 6.2 \\
7 & 2,4-Dimethyl-1-heptene & C\({}_{9}\)H\({}_{18}\) & 8.9 & 3.0 \\
8 & 2-Butanone & C\({}_{4}\)H\({}_{8}\)O & 2.8 & n.d. \\
9 & 2-Pentanone & C\({}_{9}\)H\({}_{10}\)O & 1.5 & n.d. \\
10 & Dimethyl disulfide & C\({}_{2}\)H\({}_{6}\)S\({}_{2}\) & 13.5 & n.d. \\
11 & Ammonia & NH3 & 16.7 & n.d. \\
12 & 2-Isobutyl-4,4-dimethyl-1,3-dioxan & C\({}_{10}\)H\({}_{20}\)O\({}_{2}\) & n.d. & 26.0 \\
13 & Decamethyl-cyclopentasiloxane & C\({}_{10}\)H\({}_{30}\)O\({}_{5}\)Si\({}_{5}\) & 0.5 & n.d. \\
14 & 4-Methyl-2-heptanone & C\({}_{8}\)H\({}_{16}\)O & 11.7 & 1.1 \\
15 & 3-Methyl-1-butanol & C\({}_{5}\)H\({}_{12}\)O & n.d. & 16.6 \\
16 & 2-Hexanol & C\({}_{6}\)H\({}_{14}\)O & 11.6 & n.d. \\
17 & \(\alpha\)-Methyl-\(\beta\)-oxo-2-pyridinpropanoic acid ethyl ester & C\({}_{9}\)H\({}_{9}\)NO\({}_{3}\) & 1.3 & n.d. \\
18 & 4,6-Dimethyl-2-heptanone & C\({}_{9}\)H\({}_{18}\)O & 1.6 & 1.3 \\
19 & 1,3-Bis(1,1-dimethylethyl) benzene & C\({}_{14}\)H\({}_{22}\) & 2.7 & 3.5 \\
20 & Benzene-ethanol & C\({}_{8}\)H\({}_{12}\)O & n.d. & 7.5 \\
21 & Trace materials & - & 6.3 & 6.6 \\ \hline \hline \end{tabular} Various volatile compounds were determined using SPME-GC analysis, and area (\(\%\)) was calculated as percentage of area of target compounds against the area of total compounds shown in the GC-MS chromatogram. SP, sheep placenta; SP-BD, sheep placenta fermented with _B. demaine_ kh3; n.d., not determined.  
\end{table}
Table 4: Determination and identification of volatile compounds in the fermented placenta via SPME-GC analysis.  
([[https://www.megasoftware.net](https://www.megasoftware.net)]([https://www.megasoftware.net](https://www.megasoftware.net)), accessed on 23 August 2021) using the neighbor-joining method [30].