## 3 Results
### Determination of Amino Acids  
Many herbs are rich in essential amino acids and can be used as an alternative source of amino acids in animal feed supply [25]. An ideal combination of essential and non-essential amino acids caters to different animal varieties at their corresponding growth stages. The complete amino acid profile of the herbal samples from different treatments is reported in Figure 1.  
The total concentration of essential amino acids differed significantly between all treatments (\(p\) < 0.01). Groups #2 and #4 had a richer essential amino acid profile than the other two groups. Group #1 had the fewest essential amino acids, reaching only 6.47 \(\pm\) 0.51 ug/mL. Group #2 showed the highest content of essential amino acids (222.33 \(\pm\) 43.47 ug/mL). Group #3, with autochthonous microorganisms (75.86 \(\pm\) 1.27 ug/mL), and group #4, with inoculated fermentation (184.01 \(\pm\) 20.71 ug/mL), showed a reduction in essential amino acids compared to group #2. This is not surprising since free amino acids act as substrates for microorganism growth and can also be converted into aroma compounds by lactic acid bacteria [26]. Figure 1: Compositions of essential (**a**) and nonessential amino acids (**b**) in the four treatment groups. **, \(p\) < 0.01, *, \(p\) < 0.05. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Sample ID** & **Flavonoids (g/kg)** & **Saponins Re (\%)** & **Crude Polysaccharide (g/L)** \\ \hline \#1 & 0.56 \(\pm\) 0.12 & 5.50 \(\pm\) 0.70 & 0.93 \(\pm\) 0.10 \\ \#2 & 4.27 \(\pm\) 0.33 ** & 39.02 \(\pm\) 2.19 ** & 6.54 \(\pm\) 0.34 ** \\ \#3 & 1.77 \(\pm\) 0.18 ** & 1.61 \(\pm\) 0.21 ** & 3.91 \(\pm\) 0.11 ** \\ \#4 & 4.07 \(\pm\) 1.05 ** & 25.30 \(\pm\) 2.26 ** & 7.50 \(\pm\) 0.85 ** \\ \hline \hline \end{tabular} Note: The data represent the means \(\pm\) SD (\(n\) = 3); **, \(p\) < 0.01.  
\end{table} 
Table 3: Contents of functional components in herbal broth from the four different treatments. Compositionally, the main essential amino acids in group #2 were leucine, lysine, tryptophan, phenylalanine, and valine. The main amino acids in group #4, from highest to lowest content, were lysine, leucine, valine, tryptophan, isoleucine, phenylalanine, histidine, and methionine. The total concentration of non-essential amino acids was 11.35 \(\pm\) 0.71 \(\upmu\)g/mL in group #1, 400.60\(\pm\) 35.57 \(\upmu\)g/mL in group #2, 140.06 \(\pm\) 16.66 \(\upmu\)g/mL in group #3, and 210.06 \(\pm\) 24.39 \(\upmu\)g/mL in group #4 (\(p<\) 0.01). The amount of asparagine in group #2 was 112.57 \(\pm\) 8.40 \(\upmu\)g/mL, 26.68 times higher than in group #4, in which it was only 4.22 \(\pm\) 0.21 \(\upmu\)g/mL. In addition to asparagine, glutamate (78.23 \(\pm\) 8.30 \(\upmu\)g/mL), alanine (51.00 \(\pm\) 4.10 \(\upmu\)g/mL), and proline (45.29 \(\pm\) 3.40 \(\upmu\)g/mL) were the top three most abundant non-essential amino acids in group #2. The same order of abundant non-essential amino acids was found in group #3, but at lower concentrations. In group #4, the top three non-essential amino acids were alanine (51.25 \(\pm\) 7.20 \(\upmu\)g/mL), proline (38.97 \(\pm\) 5.40 \(\upmu\)g/mL), and glutamate (32.38 \(\pm\) 2.50 \(\upmu\)g/mL). The amino acid requirements for different farm animals are not identical. Lysine, threonine, and methionine are generally considered to be the key limiting amino acids for the postprandial muscle protein synthesis response [25]. Methionine was not detected in group #2, but it was detectable in group #4. The ratios of essential to total amino acids in this study were 0.36 (group #1), 0.36 (group #2), 0.35 (group #3), and 0.47 (group #4). The ratios of essential to non-essential amino acids were 0.57 (group #1), 0.55 (group #2), 0.54 (group #3), and 0.88 (group #4). The ratio of essential to non-essential amino acids (0.88) and essential to total amino acids (0.47) was highest in group #4. It is also higher than the FAO/WHO standard [9; 27]. The optimal ratio of essential to non-essential amino acids is approximately 1.0 in feeds [28].