## 3 Results and Discussions
### Differences in Cell Morphology and Cell Membrane Fatty Acid Composition
The high ATP content in liquid state-fermented bacteria and the further increased intracellular ATP content induced by acid stress can maintain sufficient energy to cope with the adverse environment. Figure 3: Heat map of fatty acid composition in cell membrane. G, solid state-fermented cells; Y, liquid state-fermented cells. GP, solid state-fermented cells treated with pH 2 for 10 min; YP, liquid state-fermented cells treated with pH 2 for 10 min. The numbers on the right side indicate the fatty acid content (\(\mu\)g/g). Results are based on three technical replicates. The change in intracellular microenvironment has most direct effect on the activity of various intracellular metabolic enzymes. Therefore, we measured the activities of intracellular H\({}^{+}\)-ATPase and glutathione reductase involved in environmental stress response. There are many kinds of ATPase in cells, of which H\({}^{+}\)-ATPase is a very important one, and it could maintain the pH balance in cells. Before acid stress treatment, H\({}^{+}\)-ATPase activity (1.05 U/g protein) of the bacteria from solid state fermentation was higher than that from liquid-state fermentation (0.54 U/g protein) (Figure 4c). After acid stress treatment, in solid-state fermented cells, H\({}^{+}\)-ATPase activity decreased significantly to 0.04 U/g protein, which was close to a complete activity loss, whereas in liquid-state fermented cells, H\({}^{+}\)-ATPase activity decreased slightly from 0.54 U/g protein to 0.45 U/g protein. Although H\({}^{+}\)-ATPase activity in solid-state fermented cells was higher than that in liquid-state fermented cell, H\({}^{+}\)-ATPase stability in solid-state fermented cells was obviously lower than that in liquid-state fermented cells under acid stress. Glutathione reductase (GR) can catalyze NADPH-dependent reduction of GSH disulfide. Similarly, our data indicated that although before acid stress, the GSH reductase activity in solid-state fermented cells (0.35 U/g protein) was higher than that in liquid-state fermented cells (0.11 U/g protein), the enzyme activity of solid-state fermentation bacteria significantly decreased to 0.16 U/g protein under acid stress, while the enzyme activity in the liquid-state fermented cells basically remained unchanged (0.12 U/g protein) (Figure 4d). The stability of the intracellular environment, especially the stability of pH, is the prerequisite of the normal physiological responses of cells. If the intracellular pH cannot be  
Figure 4: Physiological characteristic differences of cells cultured from solid-state fermentation and liquid-state fermentation. (**a**) Changes in intracellular pH. (**b**) Intracellular ATP concentration. (**c**) Activity of ATPase. (**d**) Activity of glutathione reductase. In bar chart, blue bar represents the cells without acid stress treatment, red bar indicates the cell exposed to pH 2.0 acid stress for 10 min. The results represent the mean of three duplicate experiments for each sample. Error bar indicates the mean \(\pm\) standard deviation of three biological and three technical replicates. * statistically significant differences (\(p<0.05\)) *, statistically significant differences (\(p<0.01\)). maintained at a level close to neutral, the activity of important metabolic enzymes will be affected [56; 57], resulting in the decreased cell activity or even cell death [58]. In this study, liquid-state fermented cells exhibited a better ability to regulate intracellular pH; thus, the activity of many important metabolic enzymes could be maintained better, including H\({}^{+}\)-ATPase and glutathione reductase, to ensure the normal intracellular physiological metabolism under acid stress. Some studies have shown that when confronted with acid stress conditions, LAB will resist acid stress by increasing the expression level of H\({}^{+}\)-ATPase or increasing the activity of H\({}^{+}\)-ATPase [59].