Inoculated raw wheat Qu (IRWQ), which can be produced with high efficiency and low cost while maintaining stable quality, is the new Qu who have applied for huangjiu brewing. In this study, a single molecule real-time DNA sequencing technology and culture dependent method combined for the first time to study the microbiota and function of microorganisms principle in IRWQ. The glucoamylase, amylase and protease IRWQ contents are 1.17, 1.55 and 2.87 times greater, respectively, than traditional wheat Qu. As traditional wheat Qu, the main volatile flavor compounds in IRWQ is alcohol; However, the ester content is much higher and much lower acid content. Real-time single molecule DNA sequencing technology identified 18 species of fungi and 59 species of bacteria in IRWQ. Then, 30 species were isolated by culture dependent method.
These species represent approximately 80% of total fungal microbiota and 35% of the total bacterial microbiota. Mold is a major contributor to the activities of glucoamylase, amylase and protease. Aspergillus flavus has the highest glucoamylase and protease activity. Bacillus subtilis and Bacillus amyloliquefaciens also produce high hydrolytic enzyme activity. Saccharomyces cerevisiae produces the largest amount of aromatic compounds, alcohols, esters and acids. Aldehydes and ketones were mainly produced by molds.
Sequencing by synthesis is a significant method for high-throughput DNA sequencing. Here, we synthesized terminal-induced-emission collected luminogen (AIEgen) labeled nucleotides (dNTP-HCAP) that can serve as a substrate for a polymerase and is applied to the sequencing of small DNA fragments. In the process of DNA amplification, ratiometric AIEgens released from dNTP-HCAP and aggregates through the effect of phosphatase, which produces a change in the ratiometric fluorescent signal.
With AIEgen-labeled nucleotides, we accomplished a small DNA fragment sequences through a double change in fluorescence. In addition, we achieved a single nucleotide polymorphism differentiation through rolling circle amplification reaction without the addition of a signal probe, rapid and cost-effective. The introduction of DNA synthesis AIEgens ratiometric to make the detection of a DNA sequence that is more efficient and accurate. Therefore, the development of AIEgen-labeled nucleotides meaningful for the study of DNA sequencing method.
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