Evaluation the Effect of Wheat Germ Fermentation Using Yeast and Lactic Acid Bacteria on the Bioactive Compounds

Introduction Wheat germ is a valuable nutritional supplement and a by-product of the flour milling industry used for animal feed and oil extraction. Quinone compounds found in wheat germ have anti-cancer properties that are abundantly found in wheat germ. The aim of this study was to investigate the effect of fermentation conditions on the bioactive compounds in wheat germ with anti-cancer properties. For this purpose, the Saccharomyces cerevisiae 5022 and Lactobacillus plantarum strain 1058 were used for fermentation of wheat germ under different pH levels (4.5, 6, and 7.5) over different time (24, 48, and 72h). Response Surface Methodology was used to find the optimal fermentation conditions and to investigate the effects of above-mentioned conditions on DPPH radical scavenging activity, total phenolics and dimethoxy benzoquinone (DMBQ) content. Moreover, the amounts of bio-peptides and gamma aminobutric acid (GABA) were also determined under optimum conditions.   Materials and Methods To accomplish the fermentation process, 10 g of wheat germ was suspended in 200 mL of sodium phosphate buffer solution. Bacterial and yeast cells were then separated from the culture medium by a centrifugation at 6,000×g for 5 min at room temperature. The harvested cells were then washed with sterile phosphate buffer multiple times, resuspended in water to achieve a cell population of 108 CFU/mL, and finally homogenized using a vortex unit. The yeast and bacterial cells were incubated at 28° C and 37° C, respectively, for 24, 48, and 72 h at pH levels of 4.5, 6.0, and 7.5. Upon the completion of each fermentation process, the obtained samples were lyophilized. Total phenolic content (TPC) was measured using the method adapted  by Liu et al. (2017). Briefly, the Folin-Ciocalteu phenol reagent was diluted ten times using distilled water. Subsequently, 0.1 mL of the extract was mixed with 0.75 mL of the diluted reagent. After 10 min, 0.75 mL sodium carbonate solution (2% w/v) was added to the mixture and vortexed. The absorbance was measured at 765 nm by a spectrophotometer. The antioxidant activity of fermented wheat germs was assessed using the free radical scavenging activity of the samples evaluated through a DPPH radical assay. Briefly, 2 mL of wheat germ extract was diluted with 100 mL 90% methanol aqueous solution. The methanol extract was then mixed with 4 mL of DPPH stock solution. The tube was subsequently kept in the dark for 45 min. The absorbance of each sample was then read using a spectrophotometer at 517 nm (Adedoyin et al., 2013). Dimethoxy benzoquinone (DMBQ) content was measured by an HPLC system. Briefly, 10 g of lyophilized wheat germ sample was dissolved in 250 mL of distilled water and extracted three times by shaking with 200 mL of chloroform. The chloroform layers were collected, washed three times with distilled water, and exposed to sodium sulfate solution to induce drying of the sample. The filtrate was then evaporated using a vacuum evaporator at 30° C to achieve a stable dry material. The dried sample was thereafter dissolved in the mobile phase and injected into the HPLC column to determine the DMBQ content. The HPLC system was equipped with a C-18 column and a UV detector operating at 245 nm. The mobile phase consisted of 20% acetonitrile-80% water (v/v) mixture at a flow rate of 0.5 mL/min and a temperature of 25° C.   Results and Conclusion The highest biological activity was found when fermentation proceeded by L. plantarum under pH 6 for 48 h. Under these optimal conditions, total phenol content (3.33 mg of GAE/g), free DPPH radical scavenging (86.49%), dimethoxy benzoquinone content (DMBQ) (0.56 mg/g), peptide content (607 μg/mL) and gamma aminobutyric acid (GABA) (19983.88 mg/kg) were significantly higher than those of raw non-fermented samples. During the fermentation process, increasing the pH levels led to enhancement of antioxidant activity, total phenolic and DMBQ contents up to 48 h followed by a decline. Also, the fermentation time had a positive effect in the amount of the antioxidant activity, while it allowed an increased followed by a decrease in the contents of total phenolic and DMBQ. These findings underscore the importance of fermentation conditions of wheat germ by L. plantarum and Saccharomyces cerevisiae and can potentially serve as a promising way for the development of valuable products with anti-cancer and antioxidant functions.