# Accumulation of Vitamin C in Yeast under Pulsed Electric Field (PEF) Conditions
## 3 Results and Discussion  
### Optimal PEF Conditions for Vitamin C Accumulation in Yeast  
Yeast is mainly used in the brewing and baking industries. Its cell structure--a thick and mechanically strong wall and a lipid plasma membrane, enables encapsulation of hydrophobic and hydrophilic compounds and ensures high load capacity [20]. Yeast cell wall materials are biodegradable, they protect bioactive compounds against external factors, and the microencapsulation process is relatively simple. PEF treatment can lead to an improvement in encapsulation efficiency [21]. The first stage of the research concerned the optimization of PEF parameters for the effective accumulation of vitamin C in yeast cells and the assessment of their impact on biomass production and cell viability. The results are shown in Figures 2-5. The control sample C1 contained only small amount of vitamin C: 0.04 mg/g dry mass. Yeast cells do not have the ability to produce L-ascorbic acid (L-AA), but contain small amounts of erythroascorbic acid (similar in properties to L-ascorbic acid) [22]. The results of our previous experiments show that PEF treatment significantly increases the accumulation of minerals in yeast cells, e.g., iron increased by 157% compared to the control sample without PEF [11], selenium by 65%, zinc by 100% [23]. So far, the influence of the pulsedelectric field treatment on the accumulation of vitamins in the cells of microorganisms has not been studied due to the high instability of these compounds [24]. The application of a low-voltage electric field (Figure 2a) with a value of 300 V did not cause a significant increase in the amount of vitamin C in yeast cells compared to the control sample C2. The highest accumulation of vitamin C was observed at 1000 V (amounting to 1.15 mg/g of dry mass), and it was 1.72 times higher than in the control sample C2. Higher voltages above 1000 V caused a significant decrease in the content of vitamin C in yeast cells. The optimal value of pulse width was 10 us (Figure 3a). In this case, the accumulation of vitamin C was two times higher than in the control sample C2. Increasing the pulse width value in the range of 20-100 us caused a significant and gradual decrease in vitamin C accumulation. In the studies by [PERSON] and [PERSON] [17; 25], the highest accumulation of magnesium in _S. cerevisiae_ cells was noted at a voltage of 2000 V and a pulse width of 20 us, while in the case of zinc, it was at 1500 V and 10 us. The differences in the optimal PEF parameters may result from different sizes of particles introduced into the yeast cells. Figure 4a shows the effect of treatment time on the accumulation of vitamin C in yeast cells. Treatment time studies were conducted over a range of 5-20 min. Vitamin C concentration in _S. cerevisiae_ increased with the treatment time, reaching the maximum value after 20 min of PEF action. This value was 1.45 times higher that of the control C2. In our previous study on iron accumulation in _S. cerevisiae_, the optimal treatment time was also 20 min [11], whereas in the studies by [PERSON] and [PERSON] [25], the highest accumulation of zinc in yeast cells was achieved after 10 min of treatment. The final step in optimizing the parameters was determining the optimal cultivation time after which yeast cells would be subjected to PEF (Figure 5a). We took into account the production of biomass and the accumulation of vitamin C in yeast cells. In our study, the optimal cultivation time was 20 h. This is in line with the studies of [PERSON] et al. who also obtained the optimal cultivation time of 20 h for the highest accumulation of selenium and zinc [23] as well as magnesium and zinc [26] in yeast. Figure 2: Effect of voltage on vitamin C accumulation in yeast cells (**a**). Effect of voltage on the number of dead yeast cells and on biomass (**b**).