The Improved Resistance Of Bacillus Subtilis And Metabolomics Analysis By Fig Leaf Fermentation

Fig is a widely grown plant in China,which is known for its medicinal use as well as being edible fruit.,How to improve the utilization of the fig leave,which is a common medicinal material,is an important issue worthy of attention.In this paper,the endophytic bacteria in fig leaves were isolated,the fig leaves were fermented with endophytic Bacillus subtilis.The bacteriostatic activity and related biologically active substances of the fermentation broth were determined to evaluate the stress resistance of Bacillus subtilis..Combined with metabolomics analysis of the changes of active substances in fermentation broth,the research is expected to provide some theoretical support for the utilization of fig leaves and the application of Bacillus subtilis.The main contents and results of the paper are as follows:1 Isolation of endophytic bacteria from fig leaves and optimization of fermentation mediumThe endophytic bacteria of fig leaves were isolated by disinfecting the surface of fig leaves and other methods in total 6 strains of endophytic bacteria were obtained:Brevundimonas naejiangsanensis,Ochrobactrum intermediate,Bacillus subtilis,Acinetobacter lwoffii,Acinetobacter baumannii,Chryseobacterium sp.Filter paper was used to determine the bacteriostasis of these endophytic bacteria,and the results showed that Acinetobacter baumannii and Bacillus subtilis had bacteriostasis.The six strains of endophytic bacteria were inoculated into the medium with fig leaves as carbon source.Experiments found that Bacillus subtilis had the best performance in fermentation and the broth also showed antibacterial activity.As for the effect of nitrogen source on fig leaf fermentation and antibacterial activity,results showed that the diameter of the bacteriostatic circle of organic nitrogen fermentation broth is 5mm larger than that of inorganic nitrogen fermentation broth,which means that organic nitrogen fermentation broth produced more active substances.2 Detection and component analysis of fig leaf fermentation brothTo further explore the active substances in fig leaves and its fermentation broth,the research used thin-layer experiment to analyze the main sugar components in fig leaves and the fermentation broth and found that the fig leaves mainly contained two kinds of monosaccharides.The research assessed total amino acids,total sugars,flavonoids and coumarins in four kinds of fig leaf fermentation broth: Fermentation liquid of Bacillus subtilis plus organic nitrogen,fermentation liquid of Bacillus subtilis plus inorganic nitrogen,fermentation liquid of Bacillus subtilis enzyme plus organic nitrogen,and fermentation liquid of Bacillus subtilis enzyme plus inorganic nitrogen The total amino acid content in fermentation broth decreased gradually with time,except for that in the enzyme organic nitrogen fermentation broth which showed a first increasing and then slowly decreasing trend..The total sugar content in organic nitrogen fermentation broth decreased gradually with the progress of fermentation,while in the other three fermentation broth the total sugar content increased slightly at first and then decreased gradually.The content of flavonoids first decreased,then slightly increased and then decreased with the progress of fermentation.In Bacillus subtilis organic nitrogen fermentation broth and Bacillus subtilis enzyme organic nitrogen fermentation broth,the content of coumarin decreased first and then increased over time;in Bacillus subtilis inorganic nitrogen fermentation broth and Bacillus subtilis enzyme inorganic nitrogen fermentation broth,the content of coumarin increased gradually over time.3 Study on stress resistance of Bacillus subtilis fermented fig leavesBy culturing Bacillus subtilis with fig leaves,we studied the stress resistance of Bacillus subtilis.It is found that the survival rate of Bacillus subtilis cultured by fig leaves was 1.0～5.8 times higher than that of beef-protein medium.It can be inferred that fig leaf,as a medicinal substrate,could induce it to produce new metabolic active substances or increase the yield of subtilis metabolites,therefore enhanced its tolerance.4 Metabonomic analysis of fig leaf fermentation brothThe fermentation broth of fig leaves was analyzed by metabonomics,and eight specific substances rich in content were screened out,including Alanine l ysine tyrosine,cysteine aspartic acid,bradykinin hydroxyproline,tirofiban,salbu tamol,1-lauroyl-sn-glycerophosphatidylethanolamine,（2S,5S）-2-（pent-4-alkyne-1-yl）-5-（pent-3,4-diene-1-yl）decahydroquinoline and thiamine triphosphate.Meanwhil e,20 substances were screened out for considerable increase,including thiafuram ide,surfactant,C-2 ceramide,doxaammonium chloride,etc.Through screening,it was found that epicatechins,myricetin,chlorogenic acid,aesculin,aesculin an d some other substances had inhibitory effects on Staphylococcus aureus,whichwas consistent with the antibacterial effect results of fig leaf fermentation brot h and Bacillus subtilis suspension.The research also analyzed flavonoids,organ ic acids,coumarins,peptides and other substances in the fermentation broth to further learn the changes of active substances before and after fermentation.In conclusion,to isolate Endophytic Bacillus subtilis from fig leaves and then fermented with fig leaves as carbon source,can improve the stress resistance of Bacillus subtilis and promote the production of active substances,which provides the theoretical basis for the further research on Bacillus subtilis and the utilization of fig leaves.