# Effect of Ultrasonic Treatment on the Quality of Pumpkin Juice Fermented by Yeast
## 2 Materials and Methods
### Experimental Methods
#### 2.2.6 Determination of the Color of Pumpkin Juice Fermented by Yeast with Ultrasonic Assistance  
The method for determining the color referred to the approach proposed by [PERSON] (1964) et al. The color of the pumpkin juice was determined via spectrophotometric colorimetry (X-Rite, Color i7, US). The test conditions were as follows: total transmission, LAV, D65/10\({}^{\circ}\), and SPIN. The values of \(L^{*}\) (lightness/darkness), \(a^{*}\) (redness/greenness), and \(b^{*}\) (yellowness/blueness) were measured. According to the following formulas, the chroma (C*), hue angle (h\({}^{0}\)), total color difference (\(\Delta E\)), browning index (BI), yellowness index (YI), and color index (CI) were calculated:  
\[c^{*}=\sqrt{\left(a^{*}\right)^{2}+\left(b^{*}\right)^{2}} \tag{2}\]  
\[\mathrm{h}^{0}=\arctan(b^{*}/a^{*}) \tag{3}\]  
\[\Delta E=\sqrt{\left(\Delta L^{*}\right)^{2}+\left(\Delta a*\right)^{2}+\left( \Delta b*\right)^{2}} \tag{4}\]  
\[\mathrm{BI}=100(x-0.31)/0.172,\,x=(a^{*}+1.75b^{*})/(5.645L^{*}+a^{*}-3.012b^{ *}) \tag{5}\]  
\[\mathrm{YI}=142.86\,b^{*}/L^{*} \tag{6}\]  
\[\mathrm{CI}=(180-\mathrm{h}^{0})/(L^{*}-C^{*}) \tag{7}\]  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Project** & **Sample** & **Score/(Points)** \\ \hline \multirow{8}{*}{Pumpkin Juice Content} & 10 mL & 7.3 \(\pm\) 0.6 \\  & 15 mL & 7.1 \(\pm\) 0.6 \\  & 20 mL & 9.8 \(\pm\) 0.8 \\  & 25 mL & 6.9 \(\pm\) 0.6 \\  & 30 mL & 7.5 \(\pm\) 0.7 \\  & 1\% & 6.8 \(\pm\) 0.6 \\  & 2\% & 7.3 \(\pm\) 0.6 \\ Yeast Inoculation Amount & 3\% & 8.8 \(\pm\) 0.8 \\  & 4\% & 7.5 \(\pm\) 0.6 \\  & 5\% & 6.3 \(\pm\) 0.6 \\  & 0 d & 8.0 \(\pm\) 0.7 \\  & 1 d & 8.8 \(\pm\) 0.8 \\ Fermentation Time & 2 d & 7.8 \(\pm\) 0.7 \\  & 3 d & 7.3 \(\pm\) 0.7 \\  & 4 d & 6.1 \(\pm\) 0.6 \\  & 26 \({}^{\circ}\)C & 7.0 \(\pm\) 0.6 \\  & 28 \({}^{\circ}\)C & 8.1 \(\pm\) 0.8 \\ Fermentation Temperature & 30 \({}^{\circ}\)C & 8.3 \(\pm\) 0.8 \\  & 32 \({}^{\circ}\)C & 7.3 \(\pm\) 0.7 \\  & 34 \({}^{\circ}\)C & 6.8\(\pm\) 0.6 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Sensory evaluation of ultrasonic-assisted yeast fermentation of pumpkin juice under different fermentation conditions.  
2.7 Determination of the Caroenoid Content in Pumpkin Juice Fermented by Yeast with Ultrasonic Assistance  
The method for determining the carotenoid content referred to the approach proposed by [PERSON] et al. [7] First, 0.2 g of pumpkin juice was weighed and 8 mL of ethanol along with 6 mL of n-hexane were added. The test tube was covered with aluminum foil. Then, the test tube was placed in crushed ice and shaken for 1 h, after which 2 mL of distilled water was added, and the mixture was left to stand for 5 min. All samples were allowed to stand for 10 min, after which the samples in the n-hexane phase were measured using a spectrophotometer at 444, 50, 451, and 472 nm. The calculation formula for the content of carotenoids was:  
\[\text{ug/g}=(A_{\text{nm}}\ \times\ \text{M.wt}\ \times\ 3)/(0.2\ \times\ E_{\text{1 cm}}^{ \text{1\%}}) \tag{8}\]  
The absorbance value was represented by \(A_{\text{nm}}\), the molecular weight of the carotenoids was denoted by M.wt, the volume of the hexane layer was measured as 3 mL, the pumpkin juice was weighed at 0.2 g, and the extinction coefficient of carotenoids in hexane was represented by \(E_{\text{1 cm}}^{\text{1\%}}\). We used the extinction coefficients \(E_{\text{1 cm}}^{\text{1\%}}\) of \(\beta\)-carotene, \(\alpha\)-carotene, \(\beta\)-cryptoxanthin, zeaxanthin, and lycopene in n-hexane, which were 2560, 2800, 2460, 2480, and 3540, respectively. To convert the result from ug/g to mg/L, appropriate unit conversion factors needed to be applied according to the density of the solution and other relevant parameters. After conversion, the concentration (mg/L) could be calculated.