• Different particle-sized CIDF-stabilized emulsions were fabricated under various CIDF levels. • A simulated three-stage gastrointestinal tract model was used to study the lipid digestion of CIDF-stabilized emulsions. • Lipid digestion rate and extent depended on particle sizes and concentrations of CIDF. • The initial lipid digestion rate and extent were retarded in the CIDF-stabilized emulsions.

Due to the broad application and substantial market demand for proteases, it was vital to explore the novel and efficient protease resources. The aim of this study was to identify the novel protease for tobacco protein degradation and optimize the expression levels. Firstly, the tobacco protein was used as the sole nitrogen resource for isolation of protease-producing strains, and a strain with high protease production ability was obtained, identified as Bacillus velezensis WH-7. Then, the whole genome sequencing was conducted on the strain B. velezensis WH-7, and 7 proteases genes were mined by gene annotation analysis. By further heterologous expression of the 7 protease genes, the key protease HapR was identified with the highest protease activity (144.19 U/mL). Moreover, the catalysis mechanism of HapR was explained by amino acid sequence analysis. The expression levels of protease HapR were further improved through optimization of promoter, signal peptide and host strain, and the maximum protease activity reaced 384.27 U/mL in WX-02/pHY-P43-SP_yfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SP_hapR-hapR. This study identified a novel protease HapR and the expression level was significantly improved, which provided an important enzyme resource for the development of enzyme preparations in tobacco protein degradation.

The combustion of excessive starch in tobacco leaves leads to more harmful substances, adversely affecting the sensory properties of tobacco and posing significant risks to human health. Therefore, there is an urgent need to develop specific amylases targeting tobacco starch to address these issues. In this study, 5 different α-amylase genes were selected for recombinant expression in Bacillus amyloliquefaciens BAX-5, and the amyA(LC) (derived from Bacillus amyloliquefaciens MK10163) was confirmed to be the optimal gene. Then, the α-amylase activity was further increased by screening host bacteria BAX-5 and signal peptides SP₀₀₃ (derived from the dacB gene of Bacillus subtilis 168). Subsequently, the α-amylase properties were characterized, such as temperature tolerance, pH tolerance and metal ion. Through replacement of culture medium, the recombinant strain BAX-5/PT-17SP₀₀₃amyA(LC) produced the maximum α-amylase activity of 904.91 IU/mL, which was about 4 times higher than that of the original culture medium. Finally, the α-amylase Amy (LC) was applied to the enzyme treatment of tobacco leaves, and the evaluation results showed that α-amylase Amy (LC) could play a positive role in reducing damage and enhancing quality of cigarettes. This research provides a novel enzymatic resource for the development of amylases, and it has enormous market potential and application value.

Vinyl acetate is a restricted substance in food products. The quantification of the organic impurities in vinyl acetate is a major problem due to its activity, instability, and volatility. In this paper, while using the mass balance method to determine the purity of vinyl acetate, an improved method was established for the determination of the content of three impurities in vinyl acetate reference material, and the GC-FID peak area normalization for vinyl acetate was calibrated. The three trace organic impurities were identified by gas chromatography tandem high-resolution mass spectrometry to be methyl acetate, ethyl acetate, and vinyl propionate. The content and relative correction factors for the three organic impurities were measured. The purity of vinyl acetate determined by the mass balance method was 99.90% with an expanded uncertainty of 0.30%, and the total content of organic impurities was 0.08% with a relative correction factor of 1.23%. The vinyl acetate reference material has been approved as a national certified reference material in China as GBW (E) 062710.