| Nowadays,a large proportion of polypores have become a research hotspot due to their widespread use.The strain LH-1 in our laboratory is obtained by field acquisition,which can produce a large amount of secondary metabolites during the fermentation,and hasn't been reported so far.In addition,previous studies in our lab showed that this strain could synthesize y-decanolactone and its analogues,which is widely used in the spice industry,and has very wide application prospects.After consulting literatures materials,I find many polypores can produce secondary metabolites with medical value,especially terpenoids,fungal polysaccharides,mannitol,phenols and other substances.In view of this,in order to comprehensive exploitate the strain LH-1,in this paper I studied its metabolites under the condition of flask fermentation.Including the whole-genome sequencing;and in the shaking flask fermentation of bacterial strain,the yield of terpenoids in the supernatant of fermentation liquor was taken as the response value to optimize the culture conditions;optimization of the extraction methods of terpenoids from the cells of LH-1 strain;explore the biological activity and the purification of crude extracts from supernatant of fermentation broth.The main research results are as follows:Extraction,amplification and sequencing were performed on the 18s rRNA of LH-1 strain and a medicinal polypore strain F.betulina.After comparison,it was found that their sequence similarity reached 98.75%.The establishment of evolutionary tree based on 18s rRNA also indicated that the two strains are closely related.After the whole-genome sequencing and genomic analysis,I found the gene clusters related to the production of y-decanolactone and some key genes related to the synthesis of terpenoids in LH-1,so I speculated that LH-1 can produce terpenoids.The basic shaking bottle fermentation of LH-1 strain showed that the strain could indeed synthesize terpenoids,and during the process,the terpenoids in the supernatant reached the maximum accumulation amount by the sixth day of fermentation,and the growth of the bacteria also entered a medium-late growth stage,after which the accumulation rate of the biomass decreased greatly.Therefore,the comprehensive cost and other factors determined that the best time to harvest LH-1 terpenoids from shaking bottle fermentation was the sixth day.The the carbon and nitrogen sources of terpenoids from LH-1 shaker fermentation were screened,the results showed that the best carbon source was sucrose,and the best nitrogen source was a mixture of peptone and yeast extract.Then explored the ultrasonic assisted extraction method to extract terpenoids from LH-1 strain.According to the literature search and the comprehensive consideration of the cost and safety of the experiment,I decided to use ethanol solution as the extraction agent and use ultrasound-assisted method to extract terpenoids from the bacteria.The factors such as liquid-material ratio,ethanol concentration,ultrasonic time and ultrasonic power were optimized.The results showed that the extraction rate was the highest when the ratio of liquid to material was 30:1,the ethanol concentration was 70%,the ultrasonic time was 30 min,and the ultrasonic power was 360 W,reaching about 6.4%.It was proved that the crude terpene extraction of the supernatant and the bacteria had certain anticancer activity.In the experiment,the anti-cancer activity of crude extracts was screened,and the final results showed that the extraction of LH-1 had very little inhibitory effect on solid tumor,but had a good inhibitory effect on hematoma U937 and HL60.And if grinding the biomass in a low temperature,the inhibitory effect of the final extract will increase. |
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