# Effect of Cation Influx on the Viability of Freeze-Dried _Lactobacillus brevis_ WiKim0069
## 4 Discussion  
In our previous study, food-grade soy powder was shown to have a protective effect on LAB cell viability during freeze-drying and subsequent storage [4]. Soy powder contains plenty of amino acids, sugars, and minerals, which could play a role in their cryoprotective effect. However, it remained unclear which component in the protective agent is critical for its effect. In this study, we found that the improved viability of _L. brevis_ WiKim0069 cells freeze-dried in the presence of soy powder could be explained by enhanced cation influx induced by the protective agent. It was clear that increased intracellular Ca\({}^{2+}\) and Mg\({}^{2+}\) showed a protective effect against detrimental effects of freeze-drying; however, cation influx alone did not explain the difference in viability of _L. brevis_ WiKim0069 when soy powder was used as the protective agent. Further study is required to investigate which soy powder component other than divalent cations plays a crucial role in the protection of _L. brevis_ WiKim0069 during freeze-drying. Considering the changes observed in the viability of _L. brevis_ WiKim0069 upon pretreatment of the agent with a cation chelator (i.e., EDTA, EGTA, or a resin filter), the amount of cations has a critical impact on cell viability during freeze-drying. Of the two cations tested, calcium ions had a stronger effect than magnesium ions. EDTA has a high affinity for metal ions, and accordingly, the amount of  
Figure 4: Cold adaption enhances cation influx into the bacterial cells by enhancing cation channel activity in the bacterial cells. Fluorescence polarization of _L. brevis_ WiKim0069 in the presence or absence of cold adaptation. cations in soy powder was reduced by half. On the other hand, EGTA is selective for calcium ions and thus, had less effect on the content of magnesium ions. Considering the differential effects of EDTA and EGTA on the amount of cations remaining in the bacterial cells, the viability of _L. brevis_ WiKim0069 was more improved by calcium ions than by magnesium ions. It is well known that cation influx can enhance the viability of microorganisms through various mechanisms. Increased intracellular calcium ions induce the activation of calcineurin, which is a Ca\({}^{2+}\)/calmodulin-dependent serine/threonine protein phosphatase [28; 29]. [PERSON] et al. [21] reported that intracellular Mg\({}^{2+}\) homeostasis is involved in resistance to external stress. The presence of Mg\({}^{2+}\) minimizes the damage caused by osmotic pressure [30; 31]. To the best of our knowledge, this study is the first to describe LAB resistance to freeze-drying caused by cation influx, resulting in increased viability of freeze-dried cells. Confocal microscopic observations supported the protective effect of cation influx on freeze-dried LAB. We reason that the accumulation of intracellular cations contributed to the extension of the shelf life of freeze-dried LAB. Cold adaptation has been extensively studied and found to improve cryotolerance in various bacterial species. Cold stress prior to freezing induces a higher proportion of unsaturated fatty acids in the plasma membrane, which leads to a decrease in the solid-liquid phase transition temperature [32]. Certain fatty acids are closely related to stress responses: C16:0 and C18:2 fatty acids were observed in _L. acidophilus_ grown at low temperature, and C18:1 fatty acids were increased in _L. plantarum_ as a result of low temperature [33; 34]. In this study, we focused on the effect of cold adaptation on cation influx because intracellular cation concentration was closely related to the viability of _L. brevis_ WiKim0069 cells. Considering the decrease in the fluorescence polarization value after cold adaptation, the membrane fluidity increased with cold stress adaptation, resulting in enhanced cation channel activity. The increased cell viability might thus have resulted from the enhancement of intracellular cation influx. In conclusion, cation influx enhanced the resistance of _L.