The Study On The Mechanism For The Cinnamaldehyde-caused Inhibition Of Fungal Growth And Aflatoxin Biosynthesis Of Aspergillus Flavus

Cinnamon essential oil is primarily derived from a plant scientifically named as Cinnamomum spp.and is mainly made up of cinnamaldehyde?CIN?or cinnamic aldehyde.They have been applied to protect food from microbiological infection for several centuries and generally recognized as safe for commodities as well as consumers.However,there is scanty information on physiology of the filamentous fungi and biological mechanisms governing the process of fungal growth and mycotoxins biosynthesis at cell level.We herein innovatively fabricated an optical-fiber nanosensor system for evaluating endogenous reactive oxygen species?ROS?dynamics of Aspergillus flavus at the single cell level.Meanwhile,this work thus seted the basis of electrochemical biosensors modified by two different nano-composite materials for monitoring extracellular ROS dynamics of filamentous fungi during the CIN-treated period.We could summarize the following conclusions from the results achieved in this work.1.By applying a laboratory-built high-sensitivity optical-fiber nanosensor system based on the principle of the evanescent wave and the use of the fluorescent probe,we herein reported the single cell analysis of endogenous ROS dynamics with a temporal and spatial resolution in different hyphal compartments and cellular sections.Meanwhile,this work thus seted the basis of a new methodology for quantitative,temporally and spatially resolved monitoring endogenous ROS dynamics of filamentous fungi during the long-term culture period.Moreover,the exceptional sensitivity of the optical-fiber nanosensor system enabled us to study the apical dominance as well as polarized growth at cellular and subcellular level for filamentous fungi.Statistical analysis of intracellular ROS detection results demonstrated that endogenous ROS levels were different in specific compartments of hyphal cell.The apical stem of filamentous fungi is dominant over other side stems.It was obvious that the hyphae is characterized by the apical dominance in the early and middle stages of fungal growth?24-72h?.However,the mentioned advantage gradually became less obvious and there was no significant difference between the apical compartment and non-apical compartments.2.The natural CIN is characterized by antifungal as well as antiaflatoxigenic properties.The present investigation is of value as it demonstrates the inhibitory effects of CIN mainly depend on its concentration as well as treatment duration,and the tested fungal strains.The CIN in the test exhibited a capacity to delay or inhibit fungal growth.The results obtained in batch cultivation assay demonstrated that mycelia dry weight of A.flavus in 0.4 mmol/L CIN-treated group was no significant difference when compared to the control.But CIN could effectively reduce their pellet diameter at the end of incubation.At this concentration,however,CIN displayed the ability to apparently reduce AFB₁production.On the other hand,A.flavus treated with 0.6 mmol/L CIN were more susceptible than fungus in the control,in terms of both mycelium formation and AFB₁ biosynthesis.This concentration also exhibited maximal inhibitory effect in the end of incubation,including 19.7%for mycelia growth and 70.8%for AFB₁ production.In addition,no visible growth of mycelia or measurable amount of AFB₁ was detected during the entire incubation by 0.8 mmol/L CIN.The inhibition of mycelia growth,including spore germination,pellet evolution as well as biomass content,was obviously detrimental to mycotoxin formation and there may be a positive correlation between the formation of mycotoxins and the hyphae differentiation.However,the inhibition of fungal growth is not the major mechanism for CIN to inhibit AFB₁ biosynthesis by toxigenic A.flavus.Our results also showed that ROS may play a vital role in the mechanism for CIN-caused inhibition of A.flavus.The CIN treatment could affect the process of ROS metabolism in A.flavus.Transient stimulation of CIN could result in the decrease of intracellular ROS content but the increase of extracellular superoxide anion(O₂^·-)and nitric oxide?NO?levels.Both intracellular and extracellular ROS contents of A.flavus were decreased in the process of short-term CIN treatment,which could be proportional to the concentration of the plant essential oil as well as the re-duration time.Nevertheless,the production of intracellular ROS and the release of extracellular NO increased dramatically when the tested fungi were exposed to the long-term CIN stimulation.3.The short-term CIN treatment caused inhibition of physiological metabolic rate of A.flavus could be ascribed to the alleviation of oxidative stress as well as the reduction of ROS.On the one hand,the short-term stimulation of CIN could activate the activities of antioxidant enzymes such as total superoxide dismutase?T-SOD?,catalase?CAT?and glutathione peroxidase?GPx?in A.flavus.And the increase of the total antioxidant capacity could result in the decrease of ROS.On the other hand,transcription processes of core genes such as atfB,ve A,noxA and aflR,which are closely related to the fungal growth or metabolism activities,were down-regulated by the reduction of ROS.4.The excessive oxidative stress caused by the increase of ROS was found in long-term treatment of CIN,which could be responsible for the inhibition of fungal growth and aflatoxin B₁ production.On the one hand,the antioxidant system in hyphae cells was not enough to eliminate excessive ROS produced during the long-term stimulation of CIN.The over-existence of ROS would result in cell organelles and lipid oxidative damage.It also increased cell membrane permeability.On the other hand,the destructions of cell structure as well as biological tissue would further disturb the energy metabolism and the regulation of redox level,and down-regulate the normal expression of key genes.It also would interfere with the essential physiological processes mediated by cell signaling pathway,and ultimately affect fungal growth and AFB₁ metabolism.