Research Of Camphor Endophytes Metabolism’s Facilitation On The Antibacterial And Antioxidant Activity Of Camphor Oil

Cinnamomum camphor is an important special economic tree species in southern China.It is widely used in furniture,food,daily cosmetics,agriculture,medicine and other industries because its wood contains special resistant components.Camphor oil is a multi-component aromatic substance extracted from the tissue of Cinnamomum camphor.The main components of camphor oil include linalool,camphor,1,8-cineole,borneol,terpineol,caryophyllene and limonene,which are the main active substances that form the biological resistance of Cinnamomum camphorthe,such as anti-microbial,antioxidant,anti-inflammatory,insecticidal and mosquito repellent.Camphor endophyte is an important part of the microecosystem in the living tree of Cinnamomum camphora.The endophyte in camphor can participate in the secondary metabolic process of Cinnamomum camphora tissues,assist in the synthesis or modification of metabolites of Cinnamomum camphora through synergistic metabolism,and thus change the structure and properties of metabolites in Cinnamomum camphora tissues.This work aims to explore the effects of camphor endophyte metabolites on the anti-microbial and antioxidant activity of camphor oil components,and reveal the modification methods and mechanism.Trying to establish a research strategy,which can be manipulated artificially,for improving the activity of camphor oil components,in order to obtain the productive processe of natural resistance substances with better performance,and provide important scientific basis for deep development of camphor and camphor oil products.The main results of the study are as follows:1.A compound disinfection method was established to isolate endophytes from the perennial living woody tissues which were difficult to disinfect.Eleven camphora endophytic fungi were isolated and purified from the fibrous root,wood or leaf tissues.An in vivo disinfection method for root,leaf tissue and wood was established.2.The antioxidant activity,anti-bacterial and-fungal activity of camphor oil was quantitatively analyzed,and the resistance activity index of camphor oil was established.The experimental results showed that the minimun inhibitory concentration(MIC)of camphor oil against sensitive fungi is 6.25%,and the MIC against Staphylococcus aureus and Escherichia coli are 12.5%and 6.25%,respectively.The scavenging rate of camphor oil on DPPH free radical is as high as50%at 6.25%concentration,while the camphor oil with a concentration of more than12.5% could effectively scavenging?OH radical.3.The modification methods and mechanism of endophytic fungi promoting the transformation of resistance of camphor oil components were explored,which provides operational basis for the improvement of resistance of camphor oil components.Among the 11 strains of endophytic fungi isolated from camphor tissues,the effect of Endophyte X4 on enhancing the antioxidant activity,anti-bacterial and-fungal activity of camphor oil is through the transformation or modification effects of its metabolite?-D-Glucosidase on the structure of camphor resistance components.Endophyte X4 was identified to be a Trichoderma viride strain.4.The fermentation medium and fermentation conditions for endophytic fungi Endophyte X4 to produce?-D-Glucosidase were optimized by response surface analysis and other experiments,and the efficiency of effect of endophytic metabolites on camphor oil components was enhanced.The experimental results showed that the optimal fermentation medium is as follows:beef extract 22.5 g/L,dipotassium hydrogen phosphate 0.998 g/L,yeast powder 1.231 g/L,glucose 30 g/L,zinc sulfate0.03 g/L,ammonium sulfate 3 g/L,ferrous sulfate 0.06 g/L.The optimal fermentation condition is as follows:the incubation temperature is 25℃,the inoculation volume is60 mm~2fungal moss per 250 ml,the seed age is 3 days,and the liquid volume is 50m L.The activity of?-D-Glucosidase is increased by 33.2%after optimization.5.The differences in improvement effects of?-D-Glucosidase produced by Endophyte X4 and the common commercial glucosidase on the resistance of camphor oil components were verified,and the reaction condition of transformation effect of?-D-Glucosidase produced by endophytic fungi on the changes in resistance of camphor oil was optimized.The results showed that the anti-DPPH free radical activity of 10%camphor oil treated with crude enzyme solution at 45℃for 15 min increased by 42%;the anti-·OH activity of 7.5%camphor oil treated with crude enzyme solution at 50℃for 30 min increased by 29%;the anti-fungal rate of 12.5%camphor oil treated with crude enzyme solution at 50℃for 30 min increased by 76%;the inhibition zone against S.aureu of 12.5%camphor oil treated with crude enzyme solution at 50℃for 30 min increased by 7.3 mm,and the MIC decreased by 62.6%;the inhibition zone against Escherichia coli of 10%camphor oil treated with crude enzyme solution at 45℃for 45 min increased by 13.9 mm,and the MIC decreased by61.9%.The preliminary mechanism of the enzyme produced by endophytic fungi on the main components of camphora oil was also studied.In conclusion,this work can significantly improve the anti-microbial and antioxidant effects of camphor oil through modifying and transforming the components of camphor oil by the selected specific camphora endophytic fungi.According to the optimized experimental conditions,the synergistic effect of endophytes and camphor metabolites could better performe the biological resistance of camphor components and improve the resistance effect of camphor oil,which provides a technical route for the development of natural resistant products with better performance.