| Nowadays,traditional Chinese medicine has attracted more and more attention from the international community.With the increasing demand for traditional Chinese medicine in the world,potential hidden dangers of quality and safety of traditional Chinese medicine are increasingly prominent.Because of its rich oil,platycladi semen is easy to produce Aspergillus flavus under improper storage conditions,which leads to the over standard of aflatoxin,and seriously threatens people’s life and health.Therefore,it is particularly important to solve the problem of Aspergillus flavus and its toxin pollution in the storage process of platycladi semen.Traditional chemical fungicides are used in the storage of Chinese medicine,but they are easy to pollute the environment,difficult to degrade and a series of problems have not been used.Plant volatile oil has been widely concerned in the field of bacteriostasis due to its advantages of safety,green,environmental protection and high efficiency.However,there is no study on the application of plant volatile oil in the storage of platycladi semen.The purpose of this study is to provide early risk warning for the infection of Aspergillus flavus and the growth of aflatoxin in platycladi semen,and to provide technical solutions to solve the problem of Aspergillus flavus and its toxin pollution in the storage of platycladi semen,so as to ensure the clinical medication safety of platycladi semen.In this study,high-throughput sequencing technology combined with the traditional culture method was used to analyze the contamination degree of Aspergillus flavus and fungal structure on the surface of platycladi semen.Then,the antimicrobial activity of 16 kinds of plant volatile oil was studied,so as to determine the plant volatile oil that can effectively inhibit Aspergillus flavus as the volatile oil of Zanthoxylum armatum DC.,and to identify and analyze the chemical components of the volatile oil.Finally,the volatile oil of Zanthoxylum armatum DC.was applied to platycladi semen to study its effect on the morphology,colony number and toxin content of Aspergillus flavus.The specific contents and conclusions are as follows:1.Analysis on the fungal diversity and the pollution degree of Aspergillus flavus on the surface of platycladi semenHigh throughput sequencing technology was used to analyze the diversity of fungi on the surface of 13 batches of platycladi semen and the contamination degree of Aspergillus flavus.High throughput sequencing results showed that a total of 729948 sequences with a length of 248-282 bp were obtained,which were distributed in 4 phyla,19 classes,46 orders,89 families,132 genera and 208 species of fungi.The percentage of Aspergillus flavus on the surface of 13 batches of platycladi semen was 7.12%,14.58%,15.98%,17.60%,9.12%,4.2%,23.39%,16.44%,9.89%,18.86%,33.03%,0.97%and 10.65%,respectively.The percentage of Aspergillus flavus from Dongping,Shandong Province was the highest,reaching 33.03%.Aspergillus niger and Aspergillus fumigatus are the most abundant fungi on the surface of platycladi semen.The traditional culture method was used to culture and isolate the culturable fungi,and the surface fungi were identified by character,microscope and DNA molecular technology.The results of traditional culture showed that the main fungi on the surface of platycladi semen were Aspergillus flavus,Aspergillus niger,Penicillium and Aspergillus fumigatus.13 batches of samples were polluted by Aspergillus flavus in varying degrees,and the number of colonies were 13.33 cfu·g-1,20 cfu·g-1,180 cfu·g-1,13.33 cfu·g-1,0 cfu·g-1,26.67 cfu·g-1,66.67 cfu·g-1,0 cfu·g-1,60 cfu·g-1,60 cfu·g-1,400 cfu·g-1,26.67 cfu·g-1,13.33 cfu·g-1,among which the number of colonies of No.11 samples from Dongping,Shandong Province was the largest,400 cfu·g-1.The results of high-throughput sequencing were basically consistent with that of traditional culture method,and complementary to each other.All the above results showed that Aspergillus flavus was seriously polluted and other fungi were existed.2.Screening of volatile oil of plants inhibiting Aspergillus flavus and identification and analysis of medicinal materials16 kinds of plant volatile oils were extracted by steam distillation,and the degree of inhibition of 16 kinds of plant volatile oils against Aspergillus flavus was studied by filter paper diffusion method,and then inhibition of Aspergillus flavus plant volatile oils was determined and the minimum inhibition concentration was studied.The results showed that the bacteriostatic activity of the volatile oil of Zanthoxylum armatum DC.was the highest,and the bacteriostatic rate reached 100%.The minimum bacteriostatic concentration was 0.8μL·mL-1.Accurate identification of Zanthoxylum armatum DC.by character,microscope and DNA molecule.GC-MS technology was used to analyze the chemical constituents of the essential oil of Zanthoxylum armatum DC..The results showed that the main constituents were linalool(41.73%),D-limonene(13.24%),β-phellandrene(7.53%),which were significantly different from those of Zanthoxylum bungeanum Maxim.(estragole 45.97%,piperitone 13.95%,anethole 7.18%),so it can be inferred that the three main chemical components of volatile oil of Zanthoxylum armatum DC.may play an antibacterial role.The above results laid a solid foundation for the study of the application of the volatile oil of Zanthoxylum armatum DC.in the storage process of platycladi semen.3.Effect of volatile oil of Zanthoxylum armatum DC.on the morphology and colony number of Aspergillus flavus and aflatoxin content in platycladi semenThe morphological changes of Aspergillus flavus after fumigation with volatile oil of Zanthoxylum armatum DC.were studied by means of stereomicroscope and scanning electron microscope.The colony number of Aspergillus flavus fumigated with volatile oil of Zanthoxylum armatum DC.at room temperature and cold storage was studied by means of plate counting method.The results showed that the growth rate of Aspergillus flavus slowed down after fumigation with volatile oil,mycelium shrunk like a destructive sample;under the scanning electron microscope,the mycelium of Aspergillus flavus shrunk extremely.The results of plate count experiment showed that with the increase of storage time,the colony number of Aspergillus flavus in the volatile oil group was 550 cfu·g-1 on the 8th day at room temperature(20℃),which was less than that in the rest of the control group;the colony number of Aspergillus flavus in the volatile oil group was 44.44 cfu·g-1 on the 8th day at cold storage(4℃),which was less than that in the rest of the control group.The results show that the volatile oil of Zanthoxylum armatum DC.can effectively inhibit Aspergillus flavus,and the inhibition is better under cold storage conditions.The volatile oil of Zanthoxylum armatum DC.was fumigated to simulate the storage conditions of platycladi semen,and the content of aflatoxin in platycladi semen during storage was determined by HPLC-FLD.The results showed that the initial content of AFB1 was 0.4509 μg·kg-1,and the total amount of toxin was 0.5710 μg·kg-1.After fumigation of volatile oil of Zanthoxylum armatum DC.for 10 days,the content of AFB1 was 0.4442 μg·kg-1,the total amount of toxin was 0.5730 μg·kg-1,the content of AFB1 in control group was 1.4549μg·kg-1,the total amount of toxin was 2.6535 μg·kg-1;after 100 days of fumigation of the volatile oil of Zanthoxylum amatum DC.,the content of AFB1 was 2.2762 μg·kg-1,the total amount of toxin is 2.7705 μg·kg-1,the content of AFB1 in control group is 6.8380 μg·kg-1(the Pharmacopoeia stipulates that the total amount of toxin should not exceed 5 μg·kg-1),the total amount of toxin is 8.8572 μg·kg-1.These results show that the volatile oil of Zanthoxylum armatum DC.can ensure that the toxin content of platycladi semen does not exceed the standard within 100 days,and it can be used as a natural bacteriostatic agent in storage.Conclusion:in this study,we found that there was a serious problem of Aspergillus flavus pollution in platycladi semen.The volatile oil of Zanthoxylum armatum DC.could effectively inhibit Aspergillus flavus,and the inhibition rate reached 100%.The method of fumigation with the volatile oil of Zanthoxylum armatum DC.can effectively inhibit the growth of Aspergillus flavus in the storage process and then slow down the accumulation of aflatoxin,which can ensure that the content of aflatoxin does not exceed the standard within 100 days.Therefore,the volatile oil of Zanthoxylum armatum DC.as a natural and green antibacterial agent in storage has a very broad application prospect. |
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