## 3 Results and Discussion
### Multivariate Lipid Signature  
To explore changes in the milk lipidome due to SCG supplementation, we analyzed, using the UHPLC-QTOF/MS technique, the three groups of milk samples (C, LD, and HD). Using this analytical platform, we were able to detect, in both the positive and negative ionization modes, hundreds of milk metabolites and in particular lipid species with a total average number of 5000 mass spectrometry features [22]. Only samples collected in the last week of the treatment were studied. In fact, although the caffeine levels in milk did not accumulate during the five weeks of treatment (Figure 1), over time, a daily dose of SCG can induce changes in the overall lipid profile. These changes can be better highlighted in the last stage of SCG supplementation. This choice was also driven by previous observations [22], where we noticed that changes in the lipid profile of sheep's milk due to the diet supplemented with cocoa husks were detectable only after five weeks of treatment. Attempts to differentiate samples based on the diet supplementation were carried out by performing three classes of PLS-DA. In the PLS-DA score plot of data collected in positive ionization mode (Figure 2a), samples clustered into three well-defined groups, while in the negative ionization mode (Figure 2b), HD and LD classes overlapped. \begin{table}
\begin{tabular}{c c c c c c} \hline  & **HD** & **LD** & \multicolumn{2}{c}{**Random Effect**} \\  & **(_n = 20_)** & **(_n = 20_)** & **Doses** & **Weeks** & **Animals** \\ \hline  & \multicolumn{2}{c}{Mean \(\pm\) SD} & \multicolumn{2}{c}{\(p\)1  } \\ Conc (\(\mu\)g/100 mL) & 33 \(\pm\) 16 & 59 \(\pm\) 20 & *** & n.s. & n.s. \\ Carryover (\%) & 3.0 \(\pm\) 1.5 & 3.1 \(\pm\) 1.2 & n.s. & n.s. & n.s. \\ \hline \end{tabular}  
* *** \(p\) < 0.001, \(n.s.\)\(p\) > 0.05.  
\end{table} 
Table 1: Milk caffeine concentrations (mean \(\pm\) SD) and milk caffeine carryover after 12 h from the last administration. Subsequently, with the purpose of finding discriminant metabolites, pairwise OPLS-DA was carried out comparing the control group vs. either the LD or HD groups. The list of discriminant lipid metabolites between the control group and those on diets supplemented with SCG is reported in Tables 2 and 3. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Chemical Class** & **Metabolite** & **Regulation b** \\ \hline Catechins & 8,8\({}^{\prime}\)-Methylenebiscatechin & Up \\ Ceramides phosphate & CerP(d18:1/26:1) & Up \\ CerP(d18:0/16:0) & Up \\  & DG(18:4/20:5) & Up \\ Diacylglycerols & DG(20:5/20:5) & Up \\  & DG(12:0/22:6) & Up \\ Flavonoids & Quercetin 3-(6\({}^{\prime\prime\prime}\)–ferullyophorotrioside) & Down \\ Glucosylcermamides & Glc(d18:1/24:0) & Up \\  & Glc(d15:1/18:0) & Down \\ Glycerophosphocholines & PC(24:0/P:18:1) & Up \\  & PC(O-17:0/20:4) & Up \\  & PE(12:0/15:1) & Up \\ Glycerophosphoethanolamines & PE(15:0/18:2) & Up \\  & PE(13:0/20:0) & Up \\  & PE(18:0/24:0) & Up \\ Glycerophosphoethanolamines & PE-CerP(d14:1/25:0) & Up \\ ceramides & PE-CerP(d15:2/20:0(2 CH)) & Up \\ Glycerophosphoglycerols & PG(20:0/16:1) & Up \\ Glycerophosphoserine & PS(16:0/16:0) & Down \\ Monoacylglycerols & MG(22:2) & Up \\ Monogalactosyldicacyglicerols & MGDG(18:2/18:2) & Up \\ N-Acylamides & N-docosahexaenyl glutamine & Up \\ Polyprenols & Aracishenpenol 12 & Down \\ Secosteroids & \(\alpha\),25-dihydroxy-2\(\beta\)-(6-hydroxyhexyl) & \\ vitamin D3 & Up \\ Sesquiterpenoids & 1,2-Epoxy-1,2,7,7,8,8\({}^{\prime}\),11,12-octahydro-\(\psi\), & \\  & \(\psi\) -carotene & Up \\ Sphingomyelins & SM(d16:1/24:1) & Up \\  & SM(d16:1/22:1) & Up \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Main discriminant metabolites a in the milk of goats fed SCG vs. control detected in the positive ionization mode. Figure 2: Partial least square discriminant analysis (PLS-DA) score plots of the milk lipidome of control, low-dose and high-dose samples.