Bacillus thuringiensis has been used as a bioinsecticide to control agricultural insects. Bacillus cereus group genomes were found to have a Bacillus enhancin-like (bel) gene, encoding a peptide with 20 to 30% identity to viral enhancin protein, which can enhance viral infection by degradation of the peritrophic matrix (PM) of the insect midgut. In this study, the bel gene was found to have an activity similar to that of the viral enhancin gene. A bel knockout mutant was constructed by using a plasmid-free B. thuringiensis derivative, BMB171. The 50% lethal concentrations of this mutant plus the cry1Ac insecticidal protein gene were about 5.8-fold higher than those of the BMB171 strain. When purified Bel was mixed with the Cry1Ac protein and fed to Helicoverpa armigera larvae, 3 g/ml Cry1Ac alone induced 34.2% mortality. Meanwhile, the mortality rate rose to 74.4% when the same amount of Cry1Ac was mixed with 0.8 g/ml of Bel. Microscopic observation showed a significant disruption detected on the midgut PM of H. armigera larvae after they were fed Bel. In vitro degradation assays showed that Bel digested the intestinal mucin (IIM) of Trichoplusia ni and H. armigera larvae to various degrading products, similar to findings for viral enhancin. These results imply Bel toxicity enhancement depends on the destruction of midgut PM and IIM, similar to the case with viral enhancin. This discovery showed that Bel has the potential to enhance insecticidal activity of B. thuringiensis-based biopesticides and transgenic crops.

The purpose of the study was to reveal the differences of the flavor compounds among five Baiyunbian aged liquors by liquid-liquid extraction-gas chromatography-mass spectrometry (LLE-GC-MS) and headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS). In optimizing the LLE parameters, an extractant, methyl tert-butyl ether, was found which has a good extract effect and has never been used for the extraction of liquor flavor substances. Then the optimized LLE method has been applied to comprehensively analyze flavor compounds in 3-year-storage liquors (3Y), 5Y, 12Y, 15Y, and 20Y of Baiyunbian liquors combined with GC-MS. The results showed that the number and concentration of total flavor compounds also enhanced with the increase of cellaring ages. The total concentration of flavor compounds in 20Y was the highest (4543.23 mg/L), and the 3Y was the lowest (3984.96 mg/L). Among them, the significant differences among five samples were esters, alcohols, acids and nitrogen-containing compounds. Cluster analysis was used to analyze the aromas profiles by LLE-GC-MS, which revealed relationship among five samples. The results showed that the similarity of the samples was highest between 15Y and 20Y, followed by 3Y and 5Y. The characteristic flavors fingerprints of five kinds of Baiyunbian aged liquors were established by HS-GC-IMS. The results showed that the characteristic peaks in GC-IMS 3D spectra corresponding to flavor compounds can effectively characterize the sample information areas. The sectional intensities of 60 characteristic peaks in the corresponding three-dimensional spectra were selected as variables. After the principal components analysis (PCA) was used to reduce information dimensionality, it was further distinguished by HS-GC-IMS that 3Y and 5Y can be completely separated, but 15Y and 20Y were very similar and cannot be completely distinguished. The obtained results are valuable for the in-depth understanding and further study of flavors of Baiyunbian liquors.

Background 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. Results A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. Conclusions The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable.