Preparation Of Angelica Essential Oils/β-cyclodextrin Inclusion Complexes And Their Inhibitory Mechanism Against Fusarium Oxysporum From Fresh Bulbs Of Stored Lanzhou Lily

Objective:To investigate the effect of angelica essential oils（AEOs）and angelica essential oils/β-cyclodextrin（AEOs/β-CD）on the inhibition of Fusarium oxysporum.In this study,AEOs/β-CD inclusion complexes were prepared and their properties were characterized,assassessed the toxicological effects of AEOs and AEOs/β-CD inclusion complexes,the inhibitory effect of AEOs and AEOs/β-CD inclusion complexes on F.oxysporum in vitro and vivo and the preservation effect of fresh bulbs of Lanzhou lily was also observed.By studying the influence of AEOs and AEOs/β-CD inclusion complexes treatment on the inhibitory effect on the activities of related enzymes and the content of substances,discusses on AEOs and AEOs/β-CD inclusion complexes inhibition and molecular mechanism of F.oxysporum which as the main pathogen of bulb rot disease in Lanzhou lily,compare AEOs and AEOs/β-CD inclusion complexes in terms of antibacterial preservation effects and antibacterial time,lays the theoretical basis for the storage and preservation of fresh bulbs in Lanzhou lily,and provides the scientific basis for the resource utilization and development of AEOs.Methods:Prepare AEOs/β-CD inclusion under different process conditions（core and wall material quality,cyclodextrin concentration,inclusion temperature,inclusion time）,and define the optimal process conditions by determining the yield,inclusion and oil content;SEM,TLC,FT-IR,DSC and HPLC to determine the morphology and structure of the inclusion prepared under the optimal process conditions.Using C.elegans as a toxicology evaluation model,regard body length and body width as nematode ontogeny indicators,The effects of AEOs and AEOs/β-CD inclusion compounds on the body length and body width of nematodes;using longevity as an indicator of nematode aging,the effects of AEOs and AEOs/β-CD inclusion compounds on the survival rate and lifespan of C.elegans were observed;with the gonads and spawning number as indicators of nematode reproductive ability,the effects of AEOs and AEOs/β-CD inclusion on gonad morphology and egg laying number in C.elegans;using the number of head swings and the number of body bends as indicators of the motor behavior of the nematodes,the effects of AEOs and AEOs/β-CD inclusion complexes on the frequency of head swinging and body bending.AEOs and AEOs/β-CD inclusion compounds were screened by direct bacterial inhibition and direct fumigation;the MIC and MFC values of AEOs and AEOs/β-CD inclusion compounds against F.oxysporum were determined by microdilution;the antibacterial activities of AEOs and AEOs/β-CD inclusions were evaluated by growth rate inhibition;the incidence and decay of AEOs and AEOs/β-CD inclusion complex were observed in vivo,the sensory indexes and nutritional quality indexes of Lanzhou lily fresh bulbs were determined.Evaluated the anti-fungal effect of AEOs and AEOs/β-CD inclusion complexes and their preservation effect on fresh bulbs by PCA,to screen for the optimal concentration for the use of AEOs and AEOs/β-CD inclusion complexes.SEM observed the ultrastructural changes of F.oxysporum mycelium before and after treatment with AEOs and AEOs/β-CD inclusion complexes;the structural integrity of the cytoplasmic membrane of F.oxysporum 12h after treatment with AEOs and AEOs/β-CD inclusion complexes was observed by fluorescence microscopy;chitinase activity,SOD enzyme activity,POD enzyme activity,ATP enzyme activity,HK enzyme activity,SDH enzyme activity,MDH enzyme activity,and MDA content of AEOs and AEOs/β-CD inclusion complexes were measured and H₂O₂protein content,soluble sugar content,ATP content;LC-MS/MS method detected the contents in the central carbon metabolism pathway of F.oxysporum cells 12h after treatment with AEOs and AEOs/β-CD inclusion complexes.Results:（1）AEOs/β-CD inclusion complexes was successfully prepared by saturated aqueous solution.The best preparation process was:core material and wall material mass ratio 1:10,cyclodextrin concentration 10%,inclusion temperature 45℃,and coating time 180min.Under this process condition,the yield of the inclusion compound is 84.37%,the inclusion rate is 54.25%,and the oil content is 7.14%.SEM results showed that,different fromβ-CD,AEOs/β-CD inclusion complexes with homogeneous diamond crystals,and no obvious cracks were observed on the surface,cracks or holes;TLC the results show that,the inclusion compound was prepared successfully and the inclusion technology had no effect on the composition of angelica oil;FT-IR and DSC,also indicating that the AEOs/β-CD inclusion complexes with the AEOs,there are obvious differences in the molecular structure andβ-CD and physical properties;besides,The HPLC results also suggest that the inclusion process is a physical process,and there are no changes in the structure of the inclusion complexes,AEOs/β-CD inclusion complexes did not change the structure of the main component in AEOs.（2）Using C.elegans as a toxicological model,at each dose exposure condition of AEOs and AEOs/β-CD inclusion complexes,significant changes in body length and body width in C.elegans,compared with the control group,the body size and width of nematodes of AEOs（P<0.05）,AEOs/β-CD inclusion complexes can increase body length and body width but not statistically significant（P>0.05）;compared with the control group,Both AEOs and AEOs/β-CD inclusion complexes increased the lifespan of C.elegans but no significant difference（P>0.05）,the nematodes in the middle dose group of AEOs had a maximum longevity of 28 days,the maximum life span of nematodes in the dose group of AEOs/β-CD inclusion compound reached 30d;at the same time,AEOs and AEOs/β-CD inclusion complexes had no effect on C.elegans gonad morphology,AEOs can reduce the number of eggs laid in C.elegans,but the difference was not statistically significant（P>0.05）,AEOs/β-CD inclusion complexes mator increase in C.elegans（P>0.05）;in addition,there was no significant difference in the number of head swings and body bends of AEOs and AEOs/β-CD inclusion complexes in different doses compared with the control group（P>0.05）.（3）AEOs and AEOs/β-CD inclusion complexes produced significant inhibition of F.oxysporum（P<0.05）with a MIC of 8μL/m L and 12mg/g with an MFC of 32μL/m L and48mg/g;F.oxysporum can cause the incidence of fresh bulbs of Lanzhou lily,compared with the control group,60%and 67%reduction in medium and high dose AEOs respectively（P<0.01）,AEOs/β-CD inclusion complexes reduced bulb decay but were not statistically significant（P>0.05）;AEOs significantly increased the weight,water content,content of soluble sugar,soluble protein,crude fiber,Vc and flavonoids of fresh bulbs（P<0.05）,effect on total acid content and concentration（P<0.01）,and there is no obvious effect on the starch content（P>0.05）;AEOs/β-CD inclusion complexes significantly increased the weight,water content and flavonoids content of Lanzhou lily fresh bulbs（P<0.05）,the content of soluble sugar and soluble protein decreased significantly（P<0.05）,there was no significant effect on the total acid,starch,crude fiber,and Vc content（P>0.05）.（4）AEOs medium dose group and AEOs/β-CD inclusion complexes low dose group were the best concentrations for antifungal and preservation.（5）The AEOs and AEOs/β-CD inclusion complexes produced MDA and H₂O₂by inducing the tested organism（P<0.05）,causes oxidative damage to cell wall and cell membrane,destroys the integrity of cell wall and cell membrane,affects the synthesis of macromolecular substances such as sugar and protein（P<0.05）,inhibiting the activity of key enzymes HK,SDH and MDH in respiratory metabolism（P<0.05）,and disturbing the synthesis of ATP and key substances of energy metabolism（P<0.05）.Metabolomics showed that AEOs and AEOs/β-CD inclusion complexes inhibited F.oxysporum through D-amino acid metabolism and ABC transporter metabolism pathway.Conclusion:（1）AEOs and AEOs/β-CD inclusion complexes have no toxic side effects on C.elegans and can be used safely.（2）AEOs and AEOs/β-CD inclusion complexes had a strong inhibitory effect on F.oxysporum and could improve the sensory index and nutritional quality indexes of fresh bulbs of Lanzhou lily.（3）AEOs and AEOs/β-CD inclusion complexes inhibits the activity of Fusarium oxysporum by disrupting cell structural integrity,interfering with intracellular substance synthesis,and affecting the D-amino acid and ABC transporter pathways（4）As antibacterial agents,the antibacterial effect of AEOs is better than that of AEOs/β-CD inclusion complexes,but its antibacterial effect is time-dependent.In contrast,AEOs/β-CD inclusion complexes is safer and has a sustained release effect,reducing the volatilization of AEOs and prolonging the antibacterial time.