## 4 Discussion  
In this study, the three different strains _Lactobacillus bulgaricus_, _Saccharomyces cerevisiae_ and _Lactobacillus reuteri_ were used to ferment Coix seed, and the differences in active substance content and in vitro free radical scavenging ability in the fermentation broths were investigated. _Lactobacillus reuteri_ was finally selected as the best fermenting strain, and its activity in CLRF and CW in reducing skin aging caused by oxidative stress and differences were investigated at the biochemical, cellular and molecular levels. Studies have proven that Coix seed are rich in a variety of active ingredients that can prevent cellular oxidative stress and have anti-oxidant effects [13, 27]. Fermentation of Coix seed can increase the content of active ingredients and enhance antioxidant and other activities [23]. The results of the biochemical investigation show that all three Coix seed fermentation solutions can effectively increase the active substance content of Coix seed, including total sugar, total phenol, and total protein, probably because the fermentation treatment of Coix seed is accompanied by structural or molecular weight changes that increase the active substance content. However, the specific changes of active substances in the fermentation process need further study. The three fermentation solutions also showed different degrees of antioxidant activity, with CLRF having a higher content of active substances and a superior ability to scavenge free radicals. In this study, we established a model of H\({}_{2}\)O\({}_{2}\)-induced HSF damage to explore the protective effects and mechanism of CLRF and CW against oxidative stress-induced skin aging damage. Under normal physiological conditions, ROS maintain a dynamic balance between the oxidation and antioxidant systems, and are important regulators of intercellular signalling. The increase in ROS levels leads to oxidative stress in skin cells, which is believed to be precursor to premature skin aging [13, 23]. Excessive accumulation of ROS disturbs the balance between oxidative and antioxidant systems and enhances the synthesis of MMP-1, which breaks down collagen type I (COL-I) and causes the accompanying degradation of HA in ECM [28, 29]. Studies have shown that Coix lacryma has a protective effect  
Figure 5: Expression of PI3K/AKT pathway genes in cells at 2% concentration of CLRF and CW: (**A**) Relative expression of PI3K in HSF cells; (**B**) Relative expression of AKT mRNA in HSF cells. CW: Coix seed water extract; CLRF: Coix seed _Lactobacillus reuteri_ fermentation broth; C: control group; M: H\({}_{2}\)O\({}_{2}\) damage model group. Each value is expressed as mean \(\pm\) SD (\(n\) = 3). (*: \(p\) < 0.05, **: \(p\) < 0.01, ***: \(p\) < 0.001 versus model group, ***: \(p\) < 0.001, versus control group). against oxidative stress, reducing oxidative stress markers in rat serum and improving its antioxidant capacity [30]. It was found that after H2O2-induced damage, the content of ROS and the enzymatic activities of SOD and CAT were significantly increased in HSF cells. However, HSF pretreated with CW and CLRF, with reduced ROS content, had a protective effect against cellular oxidative stress. The activities of intracellular antioxidant enzymes and protein hydrolases were increased, with the effect of CLRF being more significant. In addition, compared to CW, CLRF were able to inhibit the synthesis of MMP-1, protect COL-I and HA from catabolism more effectively, maintain the stability of extracellular matrix structure, and reduce the decomposition and adhesion of collagen, alleviate the disintegration of dermal support structure, and effectively slow down the dermal structure damage and skin aging caused by H2O2 stimulation. To further investigate the protective mechanisms of CLRF and CW against antioxidant in HSF, we examined the effects of CLRF and CW on the transcriptional levels of key genes in the PI3K/AKT signaling pathway.