# 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
In the case of the cultivation time (Figure 5b) after which yeast cells were subjected to PEF, the greatest decrease in biomass was observed after 16 h and it was lower by 23% compared to the control sample C2. It is assumed that the main mechanism responsible for the inactivation of yeast cells is electroporation, which is a consequence of an increase in transmembrane voltage. The transmembrane voltage threshold value correlates with the cell size. Therefore, the critical electric field required for yeast electroporation is lower than that of bacteria, which are smaller in size, and higher than that required for electroporation of eukaryotic (plant and animal) cells, which are larger in size [32]. Previous studies have shown that PEF treatment can be used to stabilize vitamin C in food products such as juices [33; 34] and to increase their antioxidant activity [34]. Application of this technology does not damage vitamin C and even enhances its antioxidant properties [16]. So far, no studies have been published on the use of PEF treatment to increase vitamin C accumulation in yeast cells. This vitamin is a highly unstable compound that poses technological challenges for its incorporation into various food systems. Therefore, microencapsulation is a promising approach to ensure the stability of ascorbic acid and at the same time, improve consumer acceptance of the food carrier [35]. Yeast does not have an endogenous biochemical pathway for vitamin C synthesis. However, appropriately recombinant yeast strains gain the ability to accumulate the vitamin in the culture medium. Budding yeast cells with overexpression of the enzymes d-arabinose dehydrogenase and d-arabinone-1,4-lactone oxidase are able to produce about 100 mg of L-ascorbic acid/L, converting 40% (\(w/v\)) of L-galactose [36]. In a study by [PERSON] et al. [37], _S. cerevisiae_ cells were used as a new carrier for vitamin D3 encapsulation. The influence of the initial concentration of cholecyst (100.000 and 500.000 IU/g yeast) on the properties of the microcapsules was investigated. Vitamin D3 concentration was found to have a significant influence on the encapsulation efficiency. The study showed that yeast cells can be used to extend the stability of compounds such as vitamins. The Effect of Storage on the Vitamin C Content in Yeast Cells and Antioxidant Activity of Yeast Extract  
The yeast with vitamin C accumulated in the cells was stored for 7, 14, and 28 days under different temperature conditions, and the content of this vitamin was compared with that of fresh yeast (Figure 6). A significant decrease in vitamin C concentration was observed in yeast cells stored at 4 \({}^{\circ}\)C; after 28 days its content was 93% lower than that in fresh yeast. The lowest loss of vitamin C was observed in freeze-dried yeast stored at 20 \({}^{\circ}\)C. After 28 days of storage, the vitamin C content in cells was lowered by approx. 53%. Vitamins are reactive chemicals. Their stability during production and storage processes is influenced by many chemical and physical factors, e.g., light, temperature, pH, and oxygen levels. These factors can cause a significant loss of vitamins in food products. Therefore, to prevent loss of vitamin C, microencapsulation is used. Microencapsulation integrates bioactive substances (e.g., vitamins or also enzymes, phenols, or other molecules) in a specific shell to protect these substances against external factors [38,39]. There are no studies in which vitamin C is encapsulated in yeast cells, but studies using a different encapsulating material for vitamin C are available. In the work of [PERSON] et al. [40], the content of vitamin C in encapsulated passion fruit juice stored at temperatures 7 and 25 \({}^{\circ}\)C was investigated. At 7 \({}^{\circ}\)C, the samples retained 77.1% of vitamin C, while at 25 \({}^{\circ}\)C it was 71.5%. The storage time was 77 days. [PERSON] et al. [41] investigated the stabilization of ascorbic acid by microencapsulation in liposomes.