Isolation And Identification Of Yunnan Cigarette Molds And Study On The Antifungal Effect Of Cinnamaldehyde

With the improvement of people's living standard,more attention has been paid to natural antifungal agents.The mildew of tobacco and cigarettes is the major factor of tobacco storage diseases,causing significant economic loss.The natural essential oil has the characteristics of broad-spectrum bacteriostasis,and can be used as an ideal new type,low-toxicity,and highly effective antifungal agent for cigarette.However,the active ingredients of essential oils such as cinnamaldehyde are easy to volatilize and oxidie,which limits their application.The microcapsulation technology is one of the effective ways to improve the above problems.In this paper,the main moldy microorganisms were isolated and identified from Yunnan cigarettes.The differences in antifungal activity of four representative essential oil active ingredients,such as cinnamaldehyde,were compared,and the antifungal mechanism of cinnamaldehyde was preliminarily explored.The stability and antifungal properties of cinnamaldehyde were improved with microencapsulation by composite coacervation.The antifungal stability of encapsulated cinnamaldehyde was investigated and its antifungal effect on cigarettes was evaluated.The moldy microorganisms in cigarettes produced in Yunnan were isolated,purified and identified through the methods of plate coating,fungal morphology observation,microscopic examination and 18SrDNA-ITS sequence determination.Fifteen strains of cigarette mildew microorganisms were identified from mildew cigarettes.It was found that the main microorganisms causing cigarette mildew were Aspergillus,Penicillium,Cladosporium and Alternaria.Among them,Aspergillus brasiliensis was the most active,with the colony diameter of 75.67 ± 0.89 mm.Comparing the antifungal characteristics of the dominant strains and the mixed fungal,A.brasiliensis was established as the indicator of the antifungal activity of cigarette antifungal agents.The antifungal activity of four representative essential oil active ingredients on the dominant and mixed strains were studied with the inhibition zone and the minimum inhibitory concentration(MIC)as indicators.The results showed that the antifungal activity of four plant essential oil active components on A.brasiliensis was: cinnamaldehyde> eugenol> citral> menthol.With the measurement of extracellular protein content,propidium iodide fluorescent staining labeling,and scanning electron microscopy,the antifungal mechanism of essential oil active ingredients against A.brasiliensis was investigated.The results showed that all four essential oils could inhibit mold by destroying the mycelium cell membrane and causing the leak of intracellular proteins.However,the antifungal activities of the four essential oils all decreased in varying degrees after accelerated storage.With cinnamaldehyde as the core material and with the encapsulation rate and the inhibition zone diameter as the index,the microcapsule preparation conditions by complex coacervation were optimized.When the gelatin / gum arabic was usded as the wall material,the ratio of core and wall was 2:1,and using tannic acid(0.3%)was used as the crosslinking agent,the microencapsulation efficiency of cinnamaldehyde was 92.53±5.20%,and the inhibition zone diameter was 37.82 ± 0.63 mm.After accelerated storage at 37°C for 30 d,cinnamaldehyde microcapsules showed more obvious antifungal and thermal stability than cinnamaldehyde.The microencapsulation of cinnamaldehyde also destroyed the integrity of mycelia cell membrane,resulting in the leakage of intracellular protein,and increased the degradation temperature to 350? ~ 400?.The microencapsulated cinnamaldehyde were applied to cigarettes,and the effect of different dosage on the control of cigarette mildew were compared.The optimal microcapsule dosage in cigarettes was determined to be 0.5%(w/w).The microcapsules were prepared by spray drying,and applied to cigarette products to detect the changes of microbial colonies during storage under different humidity conditions.The results showed that the microencapsulated cinnamaldehyde could effectively inhibit the cigarette mildew during storage,especially under high humidity condition.