Genetic identification and expression optimization of a novel protease HapR from Bacillus velezensis.

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-SPyfkD-hapR, increased by 167% than that of initial recombinant strain HZ/pHY-P43-SPhapR-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.

Control of tobacco-specific nitrosamines by the Bacillus siamensis: Strain isolation, genome sequencing, mechanism analysis and genetic engineering.

Nitrosamines are considered carcinogens that threaten human health and environment. Especially, high contents of Tobacco-specific nitrosamines (TSNAs) are generated during the fermentation process of cigar tobacco. To control the accumulation of TSNAs, one novel strain WD-32 was isolated by comprehensively evaluating the reduction characteristics of nitrate, nitrite, and TSNAs, and this strain was identified as Bacillus siamensis by 16 S rRNA gene analysis and MALDI-TOF MS evaluation. Subsequently, whole genome sequencing of B. siamensis WD-32 was carried out to excavate important genes and enzymes involved, and the possible reduction mechanism of TSNAs was explored. More importantly, the reduction of TSNAs by B. siamensis was significantly promoted by knockout of narG gene. During the practical agricultural fermentation process of the cigar tobacco leaves, the treatment by the WD-32∆narG cells resulted in a 60% reduction of the total TSNAs content compared with the control, and the concentrations of the NNN and NNK were decreased by 69% and 59%, respectively. In summary, this study offers efficient strains for reduction of the TSNAs in cigar tobacco, and provides new insights into the reduction mechanism of TSNAs, which will promote the application of microbial methods in control of TSNAs and nitrite.

Isolation of starch and protein degrading strain Bacillus subtilis FYZ1-3 from tobacco waste and genomic analysis of its tolerance to nicotine and inhibition of fungal growth.

Aerobic fermentation is an effective technique for the large-scale processing of tobacco waste. However, the specificity of the structure and composition of tobacco-derived organic matter and the toxic alkaloids in the material make it currently difficult to directly use microbial agents. In this study, a functional strain FYZ1-3 was isolated and screened from thermophilic phase samples of tobacco waste composting. This strain could withstand temperatures as high as 80°C and grow normally at 0.6% nicotine content. Furthermore, it had a strong decomposition capacity of tobacco-derived starch and protein, with amylase activity of 122.3 U/mL and protease activity and 52.3 U/mL, respectively. To further understand the mechanism of the metabolic transformation of the target, whole genome sequencing was used and the secondary metabolite gene cluster was predicted. The inhibitory effect of the strain on common tobacco fungi was verified using the plate confrontation and agar column methods. The results showed that the strain FYZ1-3 was Bacillus subtilis, with a genome size of 4.17 Mb and GC content of 43.68%; 4,338 coding genes were predicted. The genome was annotated and analyzed using multiple databases to determine its ability to efficiently degrade starch proteins at the molecular level. Moreover, 14 functional genes related to nicotine metabolism were identified, primarily located on the distinct genomic island of FYZ1-3, giving a speculation for its nicotine tolerance capability on the molecular mechanism. By mining the secondary metabolite gene cluster prediction, we found potential synthetic bacteriocin, antimicrobial peptide, and other gene clusters on its chromosome, which may have certain antibacterial properties. Further experiments confirmed that the FYZ1-3 strain was a potent growth inhibitor of Penicillium chrysogenum, Aspergillus sydowii, A. fumigatus, and Talaromyces funiculosus. The creation and industrial use of the functional strains obtained in this study provide a theoretical basis for its industrial use, where it would be of great significance to improve the utilization rate of tobacco waste.

The lipid digestion behavior of oil-in-water emulsions stabilized by different particle-sized insoluble dietary fiber from citrus peel.

In this study, oil-in-water emulsions stabilized by insoluble dietary fibre from citrus peel (CIDF) exhibited an obviously delayed lipid digestion property through gastrointestinal tract (GIT) model. Our results suggested that the rate and extent of lipid digestion greatly relied on particle sizes and concentrations of CIDF, and the inhibition effect of lipolysis was markedly enhanced with decreasing particle sizes and increasing CIDF levels. Furthermore, compared with Tween80-stabilized emulsion, the maximum inhibition extent of lipolysis was 38.77% for CIDF400-stabilized one at 0.4 wt% concentration. Effects of CIDFs on lipid digestion was mainly due to the formation of protective layers around oil droplets, further blocking the entry of lipase to the internal lipids, and/or attributed to the increasing viscosity of emulsions caused by CIDFs, finally limiting the transportation of some substances in the simulated small intestine digestion. Our research would provide useful references for the application of CIDF-stabilized emulsions in low-calorie food.

Inhibition of lignification of Zizania latifolia with radio frequency treatments during postharvest.

Zizania latifolia is easily lignified after harvesting, leading to the degradation of food quality and commercial value. Thus, this study evaluated the effect of radio frequency (RF) treatments on lignification inhibition of Zizania latifolia. The results showed that the lignin content of Zizania latifolia treated with RF decreased significantly compared with the control group. At the 7th day of storage, the phenylalanine ammonia lyase activity of the 90 W RF treatment group decreased by 52.9% compared with the initial value. The activities of peroxidase and polyphenol oxidase in the stems of Zizania latifolia were significantly (p < 0.05) decreased after RF treatments. Besides, a decrease in conversion rate of O2 - and H2O2 to downstream products was observed, indicating that the related invertases were inhibited by RF treatment. All of these showed that RF treatments contribute to inhibit or delay the lignification of Zizania latifolia, providing a better taste and quality for products.

Study on controlling of cadmium pollution with fly ash-bentonite blocking wall.

A novel blocking wall was synthesized using fly ash and bentonite with strong adsorption capability. The optimized material ratio of the blocking wall was determined by penetration tests, shear tests and adsorption tests. The morphologies and stabilities of blocking wall samples with optimized material ratio were investigated by scanning electron microscopy (SEM), energy spectrum analysis (EDS), and erosion tests, the adsorption capability of blocking wall with optimized material ratio was investigated by adsorption kinetic and isotherm tests, the migration and penetration time of Cd pollutants in blocking wall with optimized material ratio were investigated by penetration tests and numerical simulations. Results indicated that the optimized fly ash/bentonite ratio (in mass) of blocking wall was 5:1. The permeability coefficient was 1.11 × 10-8 m s-1 and the maximum adsorption rate was 98.38%. Meanwhile, Cd2+ adsorbed on the blocking wall homogeneously; the alkali corrosion resistance of the blocking wall was higher than its acid corrosion resistance, and its resistance to organic pollutants and solutions with high Cd2+ concentrations were high. The adsorption process can be described by the Quasi-first-order kinetics and the Freundlich equation. Specifically, the overall adsorption efficiency was simultaneously affected by liquid membrane diffusion and particle diffusion and the adsorption process is an endothermic one dominated by physical adsorption. Additionally, Cd2+ penetration through the wall was dominated by molecular diffusion. The variation of permeability coefficient was inversely proportional to the initial concentration of Cd2+. The penetration of Cd2+ at initial concentration of 500 mg L-1 through the blocking wall needs 40 years.

[Multiple headspace solid-phase microextraction after matrix modification for the determination of ethyl carbamate in alcoholic beverages using gas chromatography].

A method based on multiple headspace solid-phase microextraction (MHS-SPME) combined with gas chromatography for determining ethyl carbamate (EC) in various alcoholic beverages was established. A novel polyethylene glycol/hydroxy-terminated silicone oil fiber was used instead of commercial ones because of its high extraction ability. Anhydrous sodium sulphate was added to modify the matrix and the extraction efficiency of EC was greatly improved. The optimum conditions for MHS-SPME were as follows: extraction time, 10 min; extraction temperature, 35 degrees C; Na2SO4 addition, 4.0 mg Na2SO4 per microliter of sample; volume of sample, 20 microL. The proposed method was linear in the range of 0.04 to 100 mg/L with a correlation coefficient of 0.999 7. The limit of detection was 34 microg/L and the repeatability of six replicates was 2.19%. The method was used to determine EC in various alcoholic beverages. The concentrations obtained were compared with those obtained by standard addition method and no statistically significant differences were observed. The application of MHS-SPME avoids the matrix effect, which commonly appears in SPME-based analysis. The results indicate that MHS-SPME has a great potential for EC quantification of complex samples due to its simplicity, sensitivity, reliability, ease of operation and environmental protection, especially for the analysis of a large number of samples in different matrices.

[Determination of trace phthalates in beer by gas chromatography coupled with solid-phase microextraction using a calix[6] arene fiber].

A method based on headspace solid-phase microextraction/gas chromatography (HS-SPME/GC) for the determination of phthalates (PAEs) in beer using benzoxy-C[6]/OH-TSO coated fiber was developed. A Taguchi's L25 (5(6)) orthogonal array design was employed to evaluate potentially significant factors and screen the optimum conditions for each analyte to ensure the highest extraction efficiency. The extraction for the determination of dimethyl phthalate (DMP) and diethyl phthalate (DEP) was carried out at 65 degrees C for 50 min with a constant stirring speed of 1,250 r/min, that of dibutyl phthalate (DBP) and diamyl phthalate (DAP) was at 95 degrees C for 50 min with a speed of 624 r/min, and that of the other PAEs was at 105 degrees C for 60 min without agitation. Owing to the good selectivity and high sensitivity of this new calixarene fiber, the method enabled the quantification of PAEs at low microg/L level in beer media with good precisions and recoveries. The survey of three bottled beer samples showed that DBP and bis(2-ethylhexyl)-phthalate (DEHP) were the main PAEs found in beer and total phthalates concentrations were between 6.22 and 7.76 microg/L. The migration tests revealed that the high content of DEHP incorporated in PVC gaskets in the lids could be a potential source of PAEs contamination in bottled beer during transportation and storage.