## 3 Results
### Selection of Factor Levels  
Both ED-F1 fraction yield and HMF concentration in the extraction liquor showed a positive linear relationship affected by extraction temperature from 95 \({}^{\circ}\)C to 135 \({}^{\circ}\)C (Figure 3a) with the yield of ED-F1 fraction increased by 6.52 g/100 g at extraction temperature of 125 \({}^{\circ}\)C compared to 95 \({}^{\circ}\)C. It has been shown previously that water at subcritical level (>100 \({}^{\circ}\)C) has the tendency to produce hydronium ions (H\({}_{3}\)O\({}^{+}\)), which resulted in the rapid hydrolysis of macromolecules into smaller molecules [40]. It was observed that the increment of temperature in the hydrolysis of passion fruit peel and oat is proportionate to the gradual increase in solid loss percentage of the raw material as well as the increase in detectable smaller carbohydrates such as oligosaccharides and monosaccharides as well as acids and HMF by-products [21,40]. For optimization, a shorter temperature range from 115 \({}^{\circ}\)C to 135 \({}^{\circ}\)C was selected based on significance increase in the yield of ED-F1 fraction at temperatures 125 \({}^{\circ}\)C to 135 \({}^{\circ}\)C. The temperatures 115 \({}^{\circ}\)C and 135 \({}^{\circ}\)C were selected for the lower and upper level, respectively, to be applied in the optimization design using RSM. When the temperature of 125 \({}^{\circ}\)C was brought down for further application in varying the time of hydrolysis, the results were observed showing an inverse linear relationship between the yield of ED-F1 fraction and hydrolysis time 25 to 55 min while HMF increased gradually from 15 to 55 min. [PERSON] et al. (2020) [41], showed that the degree of hydrolysis of fucosylated glycosaminoglycan using mild acid increase exponentially against time at temperatures below 100 \({}^{\circ}\)C over the span from 1 to 36 h while [PERSON] et al. (2018) [42], reported that oligosaccharides can be produced within 30 to 60 min at temperatures between 100-121 \({}^{\circ}\)C. In the current study, a gradual decrease of ED-F1 yield observed at temperature 125 \({}^{\circ}\)C against a timespan of 35 to 55 min suggested a preferable shorter hydrolysis time. The gradual decrease of ED-F1 could be due to the hydrolysis of  
Figure 3: The yield of ED-F1 fraction and HMF by-product as affected by (**a**) hydrolysis temperature, (**b**) hydrolysis time and (**c**) H\({}_{2}\)SO\({}_{4}\) concentration. Figure 2: Prebiotic activity score of _E. denticulatum_ fractions obtained through anion exchange chromatography (ED-F1, ED-F2, ED-F3) compared against commercial prebiotic Fírulose (F97) and a negative control (Cellulose). the oligosaccharide components generating more HMF as shown in (Figure 3b). It is also important to note that time of hydrolysis may also be affected by the plant matrix itself made up of different glycan backbones between the current and previous studies. Time of hydrolysis at 15 min to 35 min was selected based on the observed peak increase of the ED-F1 fraction yield at 25 min. Incrementing the H\({}_{2}\)SO\({}_{4}\) concentration in the hydrolysis solvent showed an increasing trend of ED-F1 fraction yield with a plateauing trend at 0.2 M-0.25 M concentration at constant 25 min time (Figure 3c). In contrast, [PERSON] et al. (2019) [PERSON] et al. (2019), demonstrated that mild sulfuric acid concentration (0.3-0.7 M) had a significantly increasing trend in the hydrolysis of press-lye waste hemicellulose to xylo-oligosaccharides. This could be explained by the difference in the other factors used such as hydrolysis time and temperature as well as the difference in the type of carbohydrates in the raw material in the current study. It is also shown in the previous study by [PERSON] et al. (2018), acid concentration beyond a certain range with the effect of time and temperature favours the production of monosaccharide over the oligosaccharide fraction with the oligosaccharide completely undetectable at hydrolysis using sulfuric acid concentration at 2 M for 1 h [PERSON] et al. (2019).