Dynamics of Phyllosphere Microbiota and Chemical Parameters at Various Growth Stages and Their Contribution to Anaerobic Fermentation of Pennisetum giganteum.

This work evaluated the dynamic changes of phyllosphere microbiota and chemical parameters at various growth stages of Pennisetum giganteum and their effects on the bacterial community, cooccurrence networks, and functional properties during anaerobic fermentation. P. giganteum was collected at two growth stages (early vegetative stage [PA] and late vegetative stage [PB]) and was naturally fermented (NPA and NPB) for 1, 3, 7, 15, 30, and 60 days, respectively. At each time point, NPA or NPB was randomly sampled for the analysis of chemical composition, fermentation parameter, and microbial number. In addition, the fresh, 3-day, and 60-day NPA and NPB were subjected to high-throughput sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional prediction analyses. Growth stage obviously affected the phyllosphere microbiota and chemical parameters of P. giganteum. After 60 days of fermentation, NPB had a higher lactic acid concentration and ratio of lactic acid to acetic acid but a lower pH value and ammonia nitrogen concentration than NPA. Weissella and Enterobacter were dominant in 3-day NPA and Weissella was dominant in 3-day NPB, while Lactobacillus was the most abundant genus in both 60-day NPA and NPB. The complexity of bacterial cooccurrence networks in the phyllosphere decreased with P. giganteum growth. The ensiling process further decreased the complexity of bacterial networks, with the simplest bacterial correlation structures in NPB. There were great differences in the KEGG functional profiles of PA and PB. Ensiling promoted the metabolism of lipid, cofactors, vitamins, energy, and amino acids but suppressed the metabolism of carbohydrates and nucleotides. Storage time had a greater influence than growth stage on bacterial community diversity, cooccurrence networks, and functional profiles of P. giganteum silage. Differences in bacterial diversity and functionality of P. giganteum silage caused by growth stage appear to be offset by long-term storage. IMPORTANCE The phyllosphere microbiota consists of various and complex microbes, including bacteria with crucial relevance to the quality and safety of fermented food and feed. It initially derives from soil and becomes specific to its host after interaction with plants and climate. Bacteria associated with the phyllosphere are highly abundant and diverse, but we know little about their succession. Here, the phyllospheric microbiota structure was analyzed within the growth of P. giganteum. We also evaluated the effects of phyllosphere microbiota and chemical parameter changes on the anaerobic fermentation of P. giganteum. We observed remarkable differences in bacterial diversity, cooccurrence, and functionality of P. giganteum at various growth stages and storage times. The obtained results are important for understanding the fermentation mechanism and may contribute to high-efficient production without additional cost.

Fast screening of tyrosinase inhibitors from traditional Chinese medicinal plants by ligand fishing in combination with in situ fluorescent assay.

A simple and rapid method for screening of tyrosinase (TYR) inhibitors present in traditional Chinese medicines (TCMs) was developed by combining ligand fishing and the fluorescent enzymatic assay based on dopamine-functionalized carbon quantum dots (CQDs-Dopa). Ligands of the enzyme present in the TCM extractions were firstly adsorbed on the enzyme-modified magnetic beads, and then the beads were magnetically separated and subjected directly to the CQDs-Dopa-based fluorescent assay. Finally, compounds were desorbed from the "active" beads and identified with ultra-performance liquid chromatography-triple quadrupole mass spectrometry. A known natural TYR inhibitor quercetin was selected to assess the feasibility and quantification performance of this method, and good linearity in the range of 0.01-0.16 mM (R2 = 0.992) with a low detection limit of 0.004 mM was obtained. This method was then applied to screen TYR inhibitors present in Scutellaria baicalensis and Sophora flavescens. Six TYR inhibitors including baicalin (1), baicalein (2), wogonin (3), oroxylin A (4), kurarinone (5), and sophoraflavanone G (6) were found, among which 1-4 were firstly discovered in this work. This is the first report on the in situ assessment of the target compounds obtained by ligand fishing in the form of a mixture, which exhibited the combined advantages of specific extraction ability of ligand fishing and the high sensitivity of CQDs-based fluorescent assay, showing great potential for fast screening of enzyme inhibitors from TCMs.