Screening,Identification And Characterization Of 1-deoxynojirimycin Strain PD04 From Silkworm Excrement

1-Deoxynojirimycin(DNJ)is a piperidine alkaloid with inhibition of α-glucosidase activity,and studies have shown that DNJ has antiviral,hypoglycemic,antitumor and other application effects.Due to the low content of 1-deoxynojirimycin in known natural biological sources,the extraction process is more complicated,and it is difficult to meet the needs of large-scale production,combined with microbial fermentation engineering technology,largescale DNJ application production has become an important research direction in this field.Studies have shown that mulberry trees are the main source of natural DNJ,and silkworms enrich DNJ by eating mulberry leaves,and the excreted silkworm excrement also contains a certain amount of DNJ.Previous studies have shown that the DNJ content in silkworm excrement after natural fermentation is higher than that in unfermented silkworm excrement,and it is speculated that microorganisms in the fermentation system play an important role.In this study,DNJ-producing strains were screened from silkworm excrement to identify their morphological characteristics,biological characteristics and molecular biological characteristics.To study the process conditions under which mulberry extract induced this strain to increase DNJ yield;To study the process conditions under which glycolysis inhibitors promote DNJ yield in strains;The study of the influence of glycolysis inhibitors on the expression of genes related to DNJ synthesis in this microorganism provides a technical basis for the development of DNJ production by microorganisms and its application,and the specific results are as follows:1.Screening,identification and biological characteristics of DNJ strains in s silkworm excrementMicrobial culture method A DNJ-producing strain PD04 was screened from silkworm excrement and identified as Enterobacter hormaechei by morphology and molecular biology.The strain is an acidophilic bacterium with a certain degree of salt tolerance.The optimum growth temperature is 40℃.After 12 hours of cultivation,the strain reaches the fastest growth period.The optimum growth conditions for this strain are: culture temperature 40℃,p H 6.0,inoculum size 4% and salt content lower than 3%.2.Study on process conditions of mulberry extract induced DNJ yield of PD04 strainAdding mulberry leaf alkaloid alcohol extract,mulberry seed alkaloid alcohol extract and mulberry leaf flavonoid alcohol extract at the initial stage of fermentation of PD04 strain can significantly promote the production of DNJ in the bacterial liquid.When the alcoholic extract of mulberry leaf alkaloids was added in an amount of 0.10 mg/m L,the DNJ content reached the highest value of 17.61 μg/m L;when the alcoholic extract of mulberry seed alkaloids was added in an amount of 0.20 mg/m L,the DNJ content reached the highest value The DNJ content was 20.28 μg/m L,and when the mulberry leaf flavonoid alcohol extract was added at 0.80 mg/m L,the DNJ content reached the highest value of 26.37 μg/m L,which were6.31,7.27 and 9.45 times the DNJ production of the corresponding control groups,respectively.The results showed that the flavonoid alcohol extract of mulberry leaves had the best effect on improving the DNJ production by fermentation of PD04 strain.3.Study on process conditions for glycolysis inhibitors to induce DNJ yield of PD04strainOxaloacetic acid,iodoacetic acid and sodium citrate were added in the initial fermentation stage of PD04 strain to study the change of DNJ production in the bacterial liquid.When oxaloacetic acid was added at 10 mmol/L,the DNJ content reached the highest value of 6.06 μg/m L;when iodoacetic acid was added at 3 mmol/L,the DNJ content reached the highest value of 6.53 μg/m L,sodium citrate When the addition amount was 20 mmol/L,the DNJ content reached the highest value of 9.37 μg/m L,which were 2.17,2.34 and 3.35 times the DNJ production of the corresponding control groups,respectively.The results showed that oxaloacetic acid,iodoacetic acid and sodium citrate could significantly reduce the content of DNJ in the bacterial liquid,and sodium citrate had the best effect on increasing the DNJ production by fermentation of PD04 strain.4.Glycolysis inhibitors affect the expression of genes related to DNJ synthesis in PD04strainsTaking the DNJ synthesis-related gene ytkc1 and glycolysis pathway regulatory genes fruk,pgi and gapa1 in PD04 strain as the research objects,the effects of different glycolysis inhibitors on the expression of related genes were studied,and it was found that the influence of the three inhibitors on the expression of each gene was in line with the predicted trend: 10mmol/L oxaloacetic acid fermentation and culture for 10 h,and the expression of DNJ synthetic gene yktc1 gene and glycolysis pathway regulatory genes fruk and pgi were downregulated.The relative expression was 0.04,0.21 and 0.21 times that of the control group,respectively.After 3 mmol/L iodoacetic acid fermentation and culture for 12 h,the gene expression of yktc1,fruk,pgi and gapa1 was down-regulated,and the relative expression levels were 0.26,0.01,0.01 and 0.03 times that of the control group,respectively.After 20mmol/L sodium citrate fermentation and culture for 12 h,the expression of yktc1 gene of strain was up-regulated,and the relative expression was 2.06 times that of the control group,and the expression of fruk and pgi genes was down-regulated,and the relative expression was0.20 and 0.23 times that of the control group.The results showed that oxaloacetic acid,iodoacetic acid and sodium citrate could all affect the synthesis of DNJ by influencing the expression of DNJ synthesis-related genes and glycolysis regulatory genes in PD04 strains,and sodium citrate upregulated the expression of yktc1 gene in strains,so that PD04 strains had the highest DNJ yield at 12 h while still having the ability to continue rapid growth,with the best effect.