# Study on the Kinetic Model of Mixed Fermentation by Adding Glutathione-Enriched Inactive Dry Yeast
## 2 Materials and Methods
### Analytical Method  
The yeast quantity was measured using a Countstar Rigel S2, purchased from Shanghai Rui Yu Biotech Co., Ltd. (Shanghai, China). Every 24 h, a 20 \(\upmu\)L sample of the fermentation broth was mixed with 20 \(\upmu\)L of 0.4% [PERSON]'s alkaline methylene blue staining solution in a centrifuge tube. After thorough shaking, 20 \(\upmu\)L of this mixture was transferred to a counting template after 3 min. The cell number was then determined using the cell counting mode, with a dilution ratio of 1:1.  
The content of reducing sugar in the fermentation process was determined by 3.5-dinitrosalicylic acid (DNS). The regression equation of glucose standard curve was \(\mathrm{y=13.721x-0.0645}\), and the correlation coefficient was \(\mathrm{R^{2}=0.9944}\).  
The alcohol content was determined by alcohol meter ([PERSON] et al., 2016).  
The aroma components were determined by headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS). Wine sample (8 mL), internal standard (0.45 mg/L 2-octanol), and 2 g NaCl were added to a 15 mL headspace bottle. The HS-SPME conditions were as follows: extraction head, DVB/CAR/PDMS (50/30 \(\upmu\)m, 1 cm). GC-MS analysis was performed with an Agilent 7890A Series gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) connected to an Agilent 5975B mass selective detector. The procedure involved preheating the wine sample at 50 \({}^{\circ}\)C for 10 min. Then, an extraction head was inserted into the headspace bottle to allow for adsorption for 35 min, with the fiber head positioned 1.5 cm from the liquid level. After adsorption, the fiber head was retrieved and quickly introduced into the GC injection port, followed by thermal analysis at 250 \({}^{\circ}\)C for 3 min.  
GC conditions included the use of a DB-WAX capillary column (60 m \(\times\) 0.25 mm \(\times\) 0.25 \(\upmu\)m, Agilent Technology, Santa Clara, CA, USA), an injection port temperature of 250 \({}^{\circ}\)C in non-shunt injection mode. The programmed temperature profile was as follows: initial temperature 40 \({}^{\circ}\)C, held for 5 min, increased at a rate of 2 \({}^{\circ}\)C/min to 60 \({}^{\circ}\)C, further increased at 5 \({}^{\circ}\)C/min to 180 \({}^{\circ}\)C, held for 5 min, and finally increased at 10 \({}^{\circ}\)C/min to 230 \({}^{\circ}\)C, where it was held for 10 min. High-purity helium served as the carrier gas with a constant flow rate of 1.2 mL/min.  
MS conditions included an electron bombardment ionization source (EI) with an ionization source temperature of 230 \({}^{\circ}\)C, an electron energy of 70 eV, full scanning acquisition mode, an MS quadrupole temperature of 150 \({}^{\circ}\)C, and 3 min solvent delay.  
For qualitative and semi-quantitative analysis of volatile substances, mass spectra corresponding to the chromatographic peaks were compared with the NIST/Wiley Database. Identification results with a matching degree of more than 80% were retained. The relative amount of each component was determined by comparing the peak area of the internal standard (2-octanol) with the peak area of each component in the wine. The calculation formula of Kovats index is as follows:  
\[\text{RI}=100\times(\text{N}+(\text{t}-\text{t}_{\text{n}})/(\text{t}_{\text{n +1}}-\text{t}_{\text{n}}))\]  
where RI is the chromatographic retention index, n is the carbon number of the compound, t is the retention time of the compound under experimental conditions, and t\({}_{\text{n}}\) and t\({}_{\text{n+1}}\) are the retention times of two adjacent known standard samples.