## 2 Materials and Methods
### Analytical Methods  
3-Met concentration were determined using a 1260 series HPLC (Agilent, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse Plus C18 column (4.6 \(\times\) 250 mm, 5 \(\upmu\)m) and a UV detector [16]. After the injection of 10 \(\upmu\)L culture sample, the column was eluted with methanol-water (30: 70, \(\upmu\)v) at 30 \({}^{\circ}\)C at a flow rate of 0.7 mL/min for 15 min. The UV-detector was used at a fixed wavelength of 215 nm. The chromatogram grade 3-Met (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) was prepared by adding 0, 1, 2, 3, 4 and 5 mg of the standard solutions (0-5 g/L) in 1.0 mL of ultrapure water into a serial concentration to set up the calibration curve. The standard calibration curve working solution was analyzed by HPLC. The calibration curve was obtained and used to calculate of 3-Met in the sample. The analysis was performed in triplicate. The aroma compounds were analyzed by headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) with a TraceGC TSQ 8000 Evo instrument (Thermo \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Code** & **Variable** & \
\begin{tabular}{c} **Low Level** \\ **(\(-\)1)** \\ \end{tabular} &
\begin{tabular}{c} **High Level** \\ **(\(+\)1)** \\ \end{tabular} & **Effect (Exi)** & **F Values** & **\(p\) Values** & **Rank** & **Significance** \\ \hline X\({}_{1}\) & Shaking speed (rpm) & 90 & 180 & 0.11338 & 180.80 & 0.0055 & 7 & **\(\ast\)** \\ X\({}_{2}\) & Temperature (\({}^{\circ}\)C) & 28 & 36 & \(-\)0.247247 & 859.76 & 0.0012 & 6 & **\(\ast\)** \\ X\({}_{3}\) & Glucose concentration (g/L) & 20 & 40 & 0.44919 & 2837.78 & 0.0004 & 2 & **\(\ast\)** \\ X\({}_{4}\) & Initial pH & 4.0 & 5.0 & 0.257057 & 929.34 & 0.0011 & 4 & **\(\ast\)** \\ X\({}_{5}\) & Loading volume (mL) & 50 & 100 & \(-\)0.0476967 & 78.47 & 0.0125 & 8 & \(\ast\) \\ X\({}_{6}\) & Inoculum size (\%) & 0.1 & 0.4 & 0.03717 & 19.43 & 0.0478 & 11 & \(\ast\) \\ X\({}_{7}\) & Time point of L-Met addition (h) & 12 & 36 & \(-\)0.49425 & 3435.67 & 0.0003 & 1 & **\(\ast\)** \\ X\({}_{8}\) & Culture time (h) & 36 & 60 & \(-\)0.0403607 & 22.85 & 0.0411 & 10 & \(\ast\) \\ X\({}_{9}\) & Yeast extract concentration (g/L) & 1.6 & 2.4 & \(-\)0.27358 & 1052.66 & 0.0009 & 3 & **\(\ast\)** \\ X\({}_{10}\) & L-Met concentration (g/L) & 2 & 6 & 0.254017 & 907.49 & 0.0011 & 5 & **\(\ast\)** \\ X\({}_{11}\) & Tween-80 concentration (g/L) & 1 & 3 & 0.0456267 & 29.28 & 0.0325 & 9 & \(\ast\) \\ \hline \hline \end{tabular} Note: \({}^{\ast\ast\ast\ast}\) Significant at 5% level (\(p<\) 0.05); \({}^{\ast\ast\ast}\) Significant at 1% level (\(p<\) 0.01); \({}^{\ast\ast\ast\ast}\) Significant at 0. 1% level (\(p<\) 0.001).  
\end{table} 
Table 2: Levels of the variables and statistical analysis in PB design for 3-Met production. \begin{table}
\begin{tabular}{c c c c c c c c c c c c} \hline \hline \multirow{2}{*}{**Congo**} & \multirow{2}{*}{ \begin{tabular}{c} **Shaling** \\ **Speed (rpm)** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **Temperature** \\ **Comet** \\ **(\(-\)0)** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **Clineineine{2-16}  \\ **Consortions** \\ **(\(-\)1)** \\ \end{tabular}  } & \multirow{2}{*}{ \begin{tabular}{c} **Cline{2-16}  \\ **Total pH** \\ \end{tabular}  } & \multirow{2}{*}{\ \begin{tabular}{c} **Leading** \\ **Total pH** \\ \end{tabular}  } & \multirow{2}{*}{
\begin{tabular}{c} **Time Point** \\ **Comet** \\ **Comet** \\ **Addineine{2-16}  \\ **Addine{2 Fisher Scientific, Waltham, MA, USA) according to our previous report [25]. Briefly, the aroma compounds in samples were extracted by 50/30-\(\upmu\)m divinylbenzene/carbox on polydimethylsiloxane (DVB/CAR on PDMS)-coated fibers. After centrifuge, 5 mL supernatant, 2 g NaCl and 10 \(\upmu\)L of 0.5 g/L tetraoctanol solution were placed into a 15-mL headspace vial.