Study On The Growth And Riboflavin Production Mechanism Of Ashbya Gossypii In Ultrasonic Field

Ultrasound technology is used more and more widely in the fermentation industry,and the proliferation and metabolism of microorganisms will inevitably be affected by the ultrasonic field.Therefore,understanding the changes of microbial proliferation or metabolism in ultrasonic field is of great significance to improve the yield of fermentation products.In this study,through the basic research on the growth and riboflavin production of Ashbya gossypii in the low-intensity ultrasonic field,the mechanism of A.gossypii growth and riboflavin production was explored from the cellular and genetic levels,and the fermentation process..The main results are as follows:（1）The favorable low-intensity ultrasound with a power of 210 W was applied to stimulate A.gossypii for 45 min at 33 k Hz during the logarithmic phase of 48 h.Compared with those of the untreated A.gossypii,the extracellular riboflavin,total riboflavin and biomass of A.gossypii increased by23.5%,26.95%and 71.99%respectively at 168 h,while the intracellular riboflavin decreased by 17.34%.（2）Microscopic observation showed that after optimized ultrasonic field treatment,the number of A.gossypii mycelium increased significantly,the mycelium became longer and the diameter of mycelium became smaller.After ultrasonic field treatment,the Ca²⁺fluorescence intensity of the ultrasonic treatment group was significantly lower than that of the untreated control group.Scanning electron microscopy showed that the surface of A.gossypii cells treated with ultrasound was smoother than that of untreated A.gossypii cells.The increase of extracellular nucleic acid and protein in A.gossypii after ultrasonic field treatment and ONPG hydrolysis experiments showed that the effect of ultrasonic field increased the cell membrane permeability of A.gossypii.After ultrasonic field treatment,a large amount of reactive oxygen species（ROS）accumulated in the mycelium.ROS produced during ultrasonic stimulation can increase the biosynthesis and secretion of riboflavin.（3）A total of 2178 differential genes were detected in A.gossypii after low intensity ultrasound field,including 1079 up-regulated genes and 596 down-regulated genes.KEGG enrichment analysis shows that the main function of secondary metabolites in fungi is to respond to external pressure.Riboflavin is a kind of secondary metabolites.Relevant studies also show that the excessive production of riboflavin by A.gossypii is related to its stress defense ROS.The increase of A.gossypii cell membrane permeability under ultrasonic field is related to the up-regulation of related genes encoding ABC transporters on the cell membrane,and the increase of glucose consumption in fermentation broth is related to the up-regulation of most of the genes related to the tricarboxylic acid cycle.The enhanced ability to synthesize riboflavin was associated with up-regulation of riboflavin synthesis-related gene expression.（4）The kinetic models of microorganism cell growth,production of extracellular riboflavin and total riboflavin and glucose consumption in the fermentation process of A.gossypii treated with ultrasonic field and without ultrasonic field were established respectively.The R²values of the models were greater than 0.95,indicating that the fitting was good and can be used to analyze the fermentation process of A.gossypii.It was found that the maximum specific growth rate（μ_m）and the product synthesis constant associated with cell growth（α）of ultrasonic group were lower than those of non ultrasonic group,and the yield constant(Y_X/S)of substrate glucose for cell growth of ultrasonic group was higher than that of non ultrasonic group,which could partly explain that ultrasonic field treatment was more beneficial to the production of riboflavin.And it is more conducive to maintain the production rate of riboflavin at a high level;The overproduction of riboflavin by A.gossypii under the action of ultrasonic field is a manifestation of its stress defense mechanism.Ultrasonic field treatment can accelerate the consumption of glucose in fermentation broth,improve fermentation efficiency and facilitate the fermentation process.