# Enhancing the Content of Hesperidin and Nobiletin in Citrus Wines through Multi-Strain Fermentation
## 2 Materials and Methods
### Determination of Organic Acids and Polyphenolic Compounds  
Nanfeng tangerine wine was analyzed for organic acids using HPLC (Agilent Technologies, 1260 Infinity, Palo Alto, CA, USA) and an Ultimate AQ-C18 column (4.6 mm \(\times\) 250 mm, 5 \(\upmu\)m), following a modified version of a previously established research protocol [17]. The mobile phase was a mixture of 0.025% trifluoroacetic acid solution and methanol in a ratio of 95:5 (\(V\)/\(V\)). A flow rate of 0.8 mL/min was established. The gradient elution protocol is described in Table 2, using a detection wavelength of 210 nm and injecting 10 \(\upmu\)L. Nine organic acid standards--oxalic acid, malic acid, vitamin C, lactic acid, acetic acid, citric acid, succinic acid, and fumaric acid--were used to create standard curves for measuring organic acids in Nanfeng tangerine wine through the external standard technique.  
Phenolic acid levels in Nanfeng tangerine wine were examined by utilizing an Ultimate AQ-C18 column (4.6 mm \(\times\) 250 mm, 5 \(\upmu\)m), following a previously established procedure with slight adjustments [17]. The HPLC flow rate was adjusted to 1.0 mL/min using a gradient elution program outlined in Table 2. The temperature of the column was kept at 40 degrees Celsius, with an injection volume of 10 microliters. Quantification wavelengths were 260 nm and 320 nm, with a scanning wavelength range of 200 to 400 nm. Retention time was utilized for qualitative analysis, while quantification was achieved through the external standard method. Protocatechuic acid and p-hydroxybenzoic acid were quantified at 260 nm, whereas chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid were quantified at 320 nm for their peak areas. HPLC chromatograms and standard curve equations for the phenolic acid standards were obtained using HPLC.  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Time** & **0** & **2** & **8** & **14** & **20** & **25** & **30** \\ \hline
0.3\% acetic acid solution & 80 & 80 & 75 & 70 & 60 & 50 & 80 \\ Acetonitrile & 20 & 20 & 25 & 30 & 40 & 50 & 20 \\ \hline \hline \end{tabular}
\end{table}
Table 2: HPLC mobile phase gradient elution procedure.  
The level of flavonoids in Nanfeng tangerine wine was analyzed by HPLC using an Agilent Eclipse XDB-C18 column (4.6 mm \(\times\) 250 mm, 5 \(\upmu\)m), following a modified version of a previously established research protocol [17]. Table 3 indicates a gradient elution program was established with a flow rate of 1.0 mL/min. The temperature of the column was held constant at 30 \({}^{\circ}\)C, with measurement wavelengths set at 283 nm and 330 nm and a scan range from 200 to 400 nm. A 10 \(\upmu\)L volume was injected for analysis. Retention time was used for qualitative analysis, while quantification was performed through the external standard method to generate chromatograms and standard curve equations for the flavonoid standards. At 283 nm, analysis included naringin, hesperidin, neohesperidin, eriocitrin, didymin, poncirin, and hesperetin, while at 330 nm, analysis included nobiletin, tangeretin, and sinensisetin.