# Changes in Biochemical and Volatile Flavor Compounds of Shine Muscat at Different Ripening Stages
## 2 Materials and Methods
### Sugar Content, Acidity, Color, and Pigments  
The total sugar content in the extracts of the grape berries was measured using a digital refractometer (Palete Digital Refractometer PR-32 Alpha; ATAGO Co. Ltd., Tokyo, Japan). Twenty berries from each group were crushed to collect free flowing juice. After filtering the juice, the total soluble solid content was measured at the constant temperature of 2 \({}^{\circ}\)C. The total sugar content was expressed as \({}^{\circ}\)Bx. The tittatable acidity was determined using an automatic titrator (TitroLine Easy; SI Analytics GmbH, Mainz, Germany). Briefly, 5 mL juice was mixed with 20 mL distilled water. Then, 0.1 M NaOH standard solution was titrated until the pH of the sample solution reached 8.2. The tittatable acidity was expressed as tartaric acid equivalent and calculated using the following Equation:  
\[\text{Titrtatable acidity}\left(\%\right)=\frac{\text{mL NaOH}\times 0.1\times \text{Mw. tartaric acid}}{\text{S}\times 10} \tag{1}\]  
The \({}^{\circ}\)Brix/acid ratio is known to be a better attribute to describe the consumers acceptability, such as sweetness, sourness, and flavor attributes, as compared with individual \({}^{\circ}\)Brix or acidity value [13]. Thus, the \({}^{\circ}\)Brix/acid ratio was calculated by dividing the \({}^{\circ}\)Brix value of the grape juice samples by the tittatable acidity (\({}^{\circ}\)Brix/% acidity). For color measurement, the color values L, a, and b of twenty randomly selected berries were measured using a colorimeter (DP-400; Konica Minolta, Tokyo, Japan). Standard illuminant C was used as a reference. The hunter color indices L, a, and b were converted into their corresponding X, Y, and Y values. The yellowness index (YI) of the berries was calculated using the equation below [14].  
\[\text{YI}=100(1.28\text{X}-1.06Z)/Y \tag{2}\]  
Figure 2: Visual appearances of Shine Muscat berries at different ripening stages: **RS1**, ripening stage 1; **RS2**, ripening stage 2; and **RS3**, ripening stage 3. The increasing numbers behind the abbreviation RS indicate the degree of ripening.  
The concentrations of pigments, including chlorophyll a and b (Chl\({}_{\text{a}}\) and Chl\({}_{\text{b}}\), respectively) and total carotenoids (x+c) (C\({}_{\text{x+c}}\)), were estimated by spectrophotometry [15]. For the extraction of pigments, 0.15 g of grape skin was added to 1.5 mL of 0.1% CaCO\({}_{\text{3}}\) in acetone solution, vortexed, and centrifuged at 4500 rpm for 5 min. The supernatant was isolated from the sediment. Residual pigments were further extracted using fresh extraction solution. The resulting supernatants were merged and filtered using a 0.2 \(\upmu\)m membrane filter. The absorbances of the extracted pigment samples were recorded at 661.6 nm, 644.8 nm, and 470 nm on a microplate reader (Multiscan GO; Thermo Fisher Scientific, Waltham, MA, USA). The whole experimental process was performed under dim light to prevent pigment loss. The concentrations of Chl\({}_{\text{a}}\), Chl\({}_{\text{b}}\), and C\({}_{\text{x+c}}\) were calculated using the equations below [15]:  
Chlorophyll a (Chl\({}_{\text{a}}\)) = 11.24 Abs\({}_{661.6}-2.04\)Abs\({}_{644.8}\) (3) Chlorophyll b (Chl\({}_{\text{b}}\)) = 20.13 Abs\({}_{644.8}-4.19\)Abs\({}_{6661.6}\) (4) Carotenoids (x+c) (C\({}_{\text{x+c}}\)) = (1000 Abs\({}_{470}-1.09\) Chl\({}_{\text{a}}-63.14\) Chl\({}_{\text{b}}\))/214 (5)