Study On The Inhibitory Effect And Mechanism Of EGCG Disinfectant On Bacillus Subtilis

The disinfection process of drinking water is essential to ensure the safety of drinking water quality and human health.Traditional chlorine-based disinfectants have made important contributions in inhibiting microbial regeneration in water distribution system and ensuring the biological stability of pipe network.However,the wide application of chlorine-based disinfectants has also brought about a large number of chlorine-resistant bacteria and the risk of disinfection by-products,which seriously threatens public health.Therefore,it is of great significance to explore new effective measures to control the risk of chlorine-resistant bacteria and disinfection byproducts in drinking water.The good bacteriostatic effect and a variety of beneficial biological activities make tea polyphenols the most likely plant polyphenols to be used in drinking water disinfection.Its main bacteriostatic component,EGCG,has been widely demonstrated to have a sustained bacteriostatic ability and has a distinct advantage in reducing disinfection by-products.In order to further explore the effectiveness and potential use of EGCG against Bacillus chlorophilus in water,and provide new ideas for the control of chlorine-resistant bacteria and the improvement of biological stability of pipe network.In this paper,EGCG was used as a disinfectant and Bacillus subtilis ATCC 9372 was used as the representative bacterium of Bacillus chlorophilus to explore the inactivation effect and mechanism of EGCG on Bacillus chlorophilus.In order to explore the influence of various factors on the sterilization effect of EGCG,the inactivation rates of Bacillus subtilis and its spores were tested under different conditions.The results showed that when the concentration of EGCG was less than 800 mg/L,the inactivation rate of Bacillus subtilis increased with the increase of the concentration of EGCG,but when the dosage of EGCG was more than800 mg/L,the solubility of Bacillus subtilis was decreased,resulting in the decrease of sterilization effect.Acidic conditions can significantly improve the inactivation effect of EGCG on Bacillus subtilis.EGCG has the potential to continuously inhibit Bacillus chlorophilus in pipe network water,and can effectively improve the biological stability of pipe network.EGCG has no obvious inactivation effect on Bacillus subtilis spores,and it is more suitable to be used as an auxiliary disinfectant for ozone,ultraviolet or ultrafiltration processes with strong spores removal ability.In order to explore the bactericidal mechanism of EGCG on Bacillus subtilis at the cellular level,the changes of cell structure,metabolic process and antioxidant system of Bacillus subtilis after disinfection with different concentrations of EGCG were monitored.The results showed that the morphology of Bacillus subtilis cells changed significantly under EGCG.EGCG can effectively inhibit the respiration of Bacillus subtilis,which leads to the obstruction of glucose metabolism,the decrease of intracellular ATP content and the decrease of cell activity.The higher the concentration of EGCG,the stronger the damage effect on the cell wall and membrane of Bacillus subtilis,the stronger the inhibition effect on respiration and antioxidant capacity.In order to further explore the mechanism of EGCG inactivation of Bacillus subtilis at the gene level,the gene expression of Bacillus subtilis was studied.The results showed that EGCG could inhibit the growth,development,reproduction,metabolic activity,stress response,biofilm formation and other biological processes of Bacillus subtilis by inhibiting the expression of related genes.EGCG can block peptidoglycan and fatty acid synthesis and metabolism pathway,and decrease the synthesis ability of cell wall and membrane.In the presence of EGCG,most of the differential genes involved in Bacillus subtilis glucose metabolism were significantly down-regulated,resulting in the obstruction of Bacillus subtilis glucose metabolism,the weakening of respiration,and the decrease of ATP synthesis ability.EGCG led to the down-regulation of genes related to the synthesis of antioxidant enzymes and the metabolism of glutathione in Bacillus subtilis,which resulted in the decrease of the defense ability of the bacterial antioxidant system.