# Optimization of Chinese Chive Juice as a Functional Feed Additive
## 3 Results
### Biochemical Changes with Optimized Drying Conditions  
In order to evaluate the heating effect on the composition of the active compounds in the non-heated and heated CC juice, untargeted metabolite profiling using UHPLC-LTQ-Orbitrap-MS/MS platform was performed. The PCA score plot presented a clearly separate pattern for the non-heated (CC) and heated CC (CC60 and CC80) metabolomic datasets across PC1 (40.6%) and PC2 (26.1%) (Figure 2A). Similar clustered patterns were observed among the datasets in supervised PLS-DA forPLS1 (40.6%) and PLS2 (26.1%) coupled with high predictive ability (Q2 = 0.942), high predictive variations (R2X = 0.667 and R2Y = 0.999), and a considerable significance metric (\(p\) = 0.0235) (Figure 2B). Overall, there were fifteen different metabolites tentatively identified in the CC juice extracts, which were mainly composed of flavonoids and lipids (Table 6). Based on the VIP (variable importance for projection) value (\(>\)0.7) and \(p\)\(<\) 0.05, significantly discriminated metabolites among the groups were selected, and a total of 14 metabolites were annotated (Figure 3). In the fresh CC juice extract, the relative content of quercetin-glucosylgalactoside-glucoside (or quercetin-triglucoside), kaempfer-di-glucoside (or kaempfer-sophoroside), kaempfer-sophoroside-glucoside, feruloyl-galactaric acid, kaempfer diglucoside-(feruloylglucose), lysoPE(18:2), lysoPC(18:2), lysoPE(16:0), and lysoPC(16:0) was significantly higher than that in the heated CC juice extracts. Conversely, the relative content of several metabolites such as N-(1-deoxy-1-fructosyl) leucine, quercetin-di-glucoside, sinapinic acid-O-glucuronide isomer, quercetin-hexoside, and kaempfer-glucoside was significantly higher in the CC60 group compared to that of other groups. Figure 1: Effect of different drying conditions on moisture content and antibacterial activity of CC products. \begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Drying Time (h)**} & \multirow{2}{*}{**Moisture Content (\%)**} & \multicolumn{2}{c}{**Zone of Inhibition (mm)**} \\ \cline{3-4}  & & _E. coli_ & _S. pullorum_ \\ \hline
6 & 25.71 \({}^{\text{c}}\) & 12.2 & 14.0 \\
7 & 14.96 \({}^{\text{b}}\) & 13.7 & 15.0 \\
8 & 4.68 \({}^{\text{a}}\) & 12.7 & 13.8 \\
9 & 5.51 \({}^{\text{a}}\) & 12.0 & 13.0 \\
10 & 2.70 \({}^{\text{a}}\) & 11.7 & 13.8 \\ SEM & 2.45 & 0.27 & 0.24 \\ \(p\)-value & \textless{}0.001 & 0.122 & 0.118 \\ \hline \hline \end{tabular} Different superscripts in the same column mean they are significantly different (\(p\)\(<\) 0.05). SEM: Standard error of mean.  
\end{table} 
Table 5: Effect of different drying durations (h) at 60 \({}^{\text{o}}\)C on moisture content and antibacterial activity of the CC products with 20% SBM content. Figure 3: Box and whisker plots of metabolites that were significantly different between the fresh and heated CC juice with dedicated conditions analyzed by UHPLC-LTQ-Orbitrap-MS/MS. The Y-axis of the box and whisker plots indicates the peak area of each metabolite (Square, mean; box, standard error; whisker, standard deviation). (**A**) N-(1-Deoxy-1-fructosyl)leucine, (**B**) Quercetin-glucosylgalactoside-glucose or Quercetin-triglucoside, (**C**) Kaempfer-di-glucose or Kaempfer-sophoroside, (**D**) Feruloyl-galactaric acid, (**E**) Kaempfer-sophoroside-glucose, (**F**) Quercetin-di-glucose, (**G**) Sinapinic acid-O-glucuronide isomer, (**H**) Kaempfer-loglucose-(feruloylglucoside), (**I**) Quercetin-hexoside, (**J**) Kaempfer-glucose, (**K**) LysoPE (18:2), (**L**) LysoPC (18:2), (**M**) LysoPE (16:0), and (**N**) LysoPC (16:0). Figure 2: Multivariate statistical analysis of LC-MS datasets for fresh and heated CC juice extracts with dedicated conditions. (**A**) Principal component analysis (PCA) score plot. (**B**) Partial least squares discriminant analysis (PLS-DA) score plot. **C**, fresh CC juice; **CC60**, CC juice heated at 60 \({}^{\circ}\)C for 8 h; and **CC80**, CC juice heated at 80 \({}^{\circ}\)C for 3 h.