## 3 Results
### Changes in the Volatile Compounds of Co-Fermented Steamed Bread during the Steaming Process  
To analyze the effect of the steaming process on the changes in the volatile components, the volatile substances in steamed bread co-fermented by _S. cerevisiae_ SQJ20 with _W. anomalous_ GZJ2 were compared with those in the co-fermented dough (Figure 6). As shown in the score chart of the OPLS-DA (Figure 6A), the two groups have obvious discrimination on the \(t1\) axis, indicating that the steaming had a great influence on the volatile substances. Figure 6B shows the visual volcano map of the univariate statistical analysis. Compared with co-fermented dough, 20 volatile metabolites were significantly upregulated (red dots) and 26 were significantly downregulated (blue dots) (\(p<0.05\)) in co-fermented steamed bread, indicating that the steaming process significantly affected the content of the volatile metabolites. Compared with co-fermented dough, the differential volatile metabolites in co-fermented steamed bread were screened for the cluster analysis. As shown in Figure 6C and Table 3, 35 differential volatile metabolites were screened in steamed bread, including 16 upregulated and 19 downregulated differential metabolites. Among them, the upregulated \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Metabolites**} & \multirow{2}{*}{**Sub Class**} & **FC** & **FC** \\  & & **(SQJ20/Control)** & **(SQJ20 + GZJ2/Control)** \\ \hline
1-(2-Carboxy-4,4-Dimethylcyclobutenyl)-1-Buten-3-One & Ketones & 10.75 & 7.28 \\ Benzaldehyde & Aldehydes & / & 7.47 \\
2-Ethyl-Hexanoic Acid & Acid & 2.32 & 6.08 \\
4,5-Dimethyl-2-Hepten-3-Ol & Alcohols & 7.10 & 5.68 \\ Benzoic Acid, Ethyl Ester & Esters & / & 4.67 \\
3-Methylene-Tridecane & Hydrocarbons & / & 4.33 \\
1-Butanol & Alcohols & / & 3.41 \\ Dihydro-5-Pentyl-2(3h)-Furanone & Heterocycle & 3.10 & 3.29 \\ Hexanoic Acid & Acid & 2.01 & 3.19 \\
1-Nonanol & Alcohols & 2.09 & 3.06 \\
1-Heptanol & Alcohols & / & 2.66 \\ Benzothiazole & Heterocycle & 2.75 & 2.55 \\ \hline \hline \end{tabular} Note: FC means fold change. SQJ20 stands for _S. cerevisiae_ SQJ20; SQJ20 + GZJ2 stands for _S. cerevisiae_ SQJ20 and _W. anomalous_ GZJ2.  
\end{table} 
Table 2: _Cont._volatile substances included six alkanes, three esters, two heterocycles, one ketone, one aldehyde, and three other substances, and the downregulated volatile substances included seven esters, five alcohols, two aldehydes, two heterocycles, one acid, one ketone, and one alkane, with more downregulated volatile substances. Due to the instability of heat, some metabolites were transformed or decomposed to form other volatile compounds, in particular, alcohols and esters were more affected by heat. However, they did not form the characteristic flavor compounds such as pyrazines and pyrrole in bread, because the temperature of steaming did not reach the temperature of the Maillard reaction and the caramelization reaction. Previous studies have confirmed the transformation of substances during heating. [PERSON] et al. (2018) proposed that alcohols, such as 2-methyl-1-propanol and 3-methyl-1-butanol, were largely lost during bread baking, mainly due to the low boiling point of alcohols. [PERSON] et al. (2019) also proposed that esters in bread were extremely volatile during baking. In this study, the content of five alcohols ((Z)-3-Nonen-1-OI, 1-Octanol, 2-Ethyl-1-Hexanol, 1-Nonanol, and 1-Heptanol) in co-cermented steamed bread significantly decreased after steaming, indicating that alcohols were volatile in the heating process. Meanwhile, the contents of seven esters decreased and only three esters showed an increase after steaming, which showed that esters were greatly affected by heating. In the increased esters, the contents of 3-methyl-1-butanol acetate and acetic acid 2-phenylethyl ester increased after steaming, likely due to their higher boiling points (142 \({}^{\circ}\)C and 232 \({}^{\circ}\)C, respectively) than the steaming temperature (100 \({}^{\circ}\)C).