# Effects of Growth Parameters on the Analysis of _Aspergillus flavus_ Volatile Metabolites
## 3 Results and Discussion
### Effect of the Growth Substrates on MVOCs' Production  
Growth factors affecting aflatoxin production by _Aspergillus parasitic_ have been reported by [PERSON] _et al._[54]. They found the amino acid asparagine to be essential for aflatoxin production. A number of authors have reported how substrate composition influences volatile production by fungi. For example, [PERSON] and [PERSON] [58] found that while volatile profiles from _Penicillium_ isolates were generally similar when the isolates were grown on either yeast extract sucrose agar or malt extract agar, fewer VOCs were produced by the same isolates when grown on Czapek yeast autolyaste agar. [PERSON] _et al._[59] found that VOCs produced by the brown rot fungus _Gloeophyllum odoratum_ varied, depending on the presence in the media of different growth elicitors, indicating the importance of media in studies of this nature. Hence, precedence exists for expecting MVOC variability due to growth media. The relationship between growth medium and _A. flavus_ MVOC profiles is explored in the section below. Five growth media substrates (Table 1) were evaluated. CMA contains the least amount of organic nutrients (around two grams); MEA is most commonly used in the MVOC studies [60; 31; 61]; while CSA is typically used for _A. flavus_ cultures. CDA and CDL are chemically-defined media that have previously been used for an aflatoxin production study [54]. The fungal culture was incubated for seven days at 30 \({}^{\circ}\)C with an initial inoculation spore concentration of \(1\times 10^{6}\) spores/mL. CAR/PDMS fiber was used for extracting the MVOCs for 5 h at 30 \({}^{\circ}\)C. Table 4 lists a subset of the identified MVOCs and their relative quantity (expressed in peak area) produced by _A. flavus_ isolate 5-3B on the five different incubation media (CMA, CSA, CDA, CDL and MEA). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & & & \multicolumn{3}{c}{**Peak Area RSD\%**} & \multicolumn{3}{c}{**Peak Area\% RSD\%**} \\ \cline{3-10}
**No.** & **Compound Name** & **R.T. (min) b & \multicolumn{3}{c}{**CAR**} & \multicolumn{1}{c}{**DVB**} & \multicolumn{1}{c}{**CAR**} & \multicolumn{1}{c}{**DVB**} & \multicolumn{1}{c}{**CAR**} & \multicolumn{1}{c}{**DVB**} \\ \cline{3-10}  & & & **CAR** & **CVB** & **CVB** & **CAR** & **CVB** & **CVB** \\ \hline
4 & 1.4-Pentalene & 5.543 & 48.8 & 61.9 & - & 40.4 & 40.0 & - \\
9 & Propanoic acid, 2-methyl-, & 8.313 & 68.0 & 31.1 & - & 64.1 & 65.4 & - \\
10 & Furan, 2-methyl- & 8.568 & 38.4 & 20.3 & - & 30.1 & 22.4 & - \\
11 & 1-Propanol, 2-methyl- & 9.046 & 30.4 & 32.6 & - & 29.7 & 30.8 & - \\
15 & 1-Butanol, 3-methyl- & 13.825 & 52.6 & 51.3 & - & 57.7 & 74.6 & - \\
16 & 1-Butanol, 2-methyl- & 13.943 & 37.8 & 34.3 & - & 41.8 & 32.3 & - \\
18 & Toluene & 15.501 & 13.8 & 15.9 & - & 25.1 & 26.2 & - \\
22 & Styrene & 21.901 & 23.8 & 17.3 & 19.3 & 35.7 & 24.5 & 19.7 \\
36 & Undecane, 2,6-dimethyl- & 38.682 & 73.5 & 37.2 & 43.5 & 48.0 & 37.0 & 20.0 \\
52 & 2,4,4,6,6,8,8-Hepttamethyl-2-nonene & 43.784 & 26.0 & 27.0 & 31.5 & 15.3 & 40.0 & 11.7 \\
56 & \(\beta\)-Elemene & 44.256 & 26.8 & 24.7 & 30.2 & 27.1 & 23.8 & 7.8 \\
64 & \(\alpha\)-Selienne & 45.951 & 34.7 & 46.5 & 44.5 & 39.6 & 0.6 & 42.7 \\
68 & Cedrene & 46.060 & 29.5 & 37.6 & 28.4 & 14.6 & 20.4 & 29.0 \\
70 & Calamenene & 46.338 & 27.3 & 52.4 & 48.8 & 23.7 & 45.1 & 29.0 \\
72 & \(\pi\)-Calacorene & 46.627 & 17.0 & 32.6 & 36.5 & 22.8 & 21.7 & 23.1 \\  & Average RSD\% a & - & 56.7 & 40.9 & 49.8 & 56.7 & 52.08 & 39.5 \\ \hline \hline \end{tabular}  
* Total RSD% is the average RSD% of 133 MVOCs; b R.T. is Retention time.  
\end{table} 
Table 3: Fifteen selected MVOCs and their RSD% (using both peak area and peak area percentage) obtained using three types of SPME fibers, CAR/PDMS, DVB/PDMS, and DVB/CAR/ PDMS, with 6 replications.