Integration of food raw materials, food microbiology, and food additives: systematic research and comprehensive insights into sweet sorghum juice, Clostridium tyrobutyricum TGL-A236 and bio-butyric acid.

Introduction: Sweet sorghum juice is a typical production feedstock for natural, eco-friendly sweeteners and beverages. Clostridium tyrobutyricum is one of the widely used microorganisms in the food industry, and its principal product, bio-butyric acid is an important food additive. There are no published reports of Clostridium tyrobutyricum producing butyric acid using SSJ as the sole substrate without adding exogenous substances, which could reach a food-additive grade. This study focuses on tailoring a cost-effective, safe, and sustainable process and strategy for their production and application. Methods: This study modeled the enzymolysis of non-reducing sugars via the first/second-order kinetics and added food-grade diatomite to the hydrolysate. Qualitative and quantitative analysis were performed using high-performance liquid chromatography, gas chromatography-mass spectrometer, full-scale laser diffraction method, ultra-performance liquid chromatography-tandem mass spectrometry, the cell double-staining assay, transmission electron microscopy, and Oxford nanopore technology sequencing. Quantitative real-time polymerase chain reaction, pathway and process enrichment analysis, and homology modeling were conducted for mutant genes. Results: The treated sweet sorghum juice showed promising results, containing 70.60 g/L glucose and 63.09 g/L fructose, with a sucrose hydrolysis rate of 98.29% and a minimal sucrose loss rate of 0.87%. Furthermore, 99.62% of the colloidal particles and 82.13% of the starch particles were removed, and the concentrations of hazardous substances were effectively reduced. A food microorganism Clostridium tyrobutyricum TGL-A236 with deep utilization value was developed, which showed superior performance by converting 30.65% glucose and 37.22% fructose to 24.1364 g/L bio-butyric acid in a treated sweet sorghum juice (1:1 dilution) fermentation broth. This titer was 2.12 times higher than that of the original strain, with a butyric acid selectivity of 86.36%. Finally, the Genome atlas view, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and evolutionary genealogy of genes: Non-supervised Orthologous (eggNOG) functional annotations, three-dimensional structure and protein cavity prediction of five non-synonymous variant genes were obtained. Conclusion: This study not only includes a systematic process flow and in-depth elucidation of relevant mechanisms but also provides a new strategy for green processing of food raw materials, improving food microbial performance, and ensuring the safe production of food additives.

Physiological and biochemical characteristics of the carbon ion beam irradiation-generated mutant strain Clostridium butyricum FZM 240 in vitro and in vivo.

Clostridium butyricum (C. butyricum) represents a new generation of probiotics, which is beneficial because of its good tolerance and ability to produce beneficial metabolites, such as short-chain fatty acids and enzymes; however, its low enzyme activity limits its probiotic efficacy. In this study, a mutant strain, C. butyricum FZM 240 was obtained using carbon ion beam irradiation, which exhibited greatly improved enzyme production and tolerance. The highest filter paper, endoglucanase, and amylase activities produced by C. butyricum FZM 240 were 125.69 U/mL, 225.82 U/ mL, and 252.28 U/mL, which were 2.58, 1.95, and 2.21-fold higher, respectively, than those of the original strain. The survival rate of the strain increased by 11.40 % and 5.60 % after incubation at 90 °C for 5 min and with simulated gastric fluid at pH 2.5 for 2 h, respectively, compared with that of the original strain. Whole-genome resequencing and quantitative real-time PCR(qRT-PCR) analysis showed that the expression of genes related to enzyme synthesis (GE000348, GE001963 and GE003123) and tolerance (GE001114) was significantly up-regulated, while that of genes related to acid metabolism (GE003450) was significantly down-regulated. On this basis, homology modeling and functional prediction of the proteins encoded by the mutated genes were performed. According to the results, the properties related to the efficacy of C. butyricum as a probiotic were significantly enhanced by carbon ion beam irradiation, which is a novel strategy for the application of Clostridium spp. as feed additives.

Strategies to improve the efficiency and quality of mutant breeding using heavy-ion beam irradiation.

Heavy-ion beam irradiation (HIBI) is useful for generating new germplasm in plants and microorganisms due to its ability to induce high mutagenesis rate, broad mutagenesis spectrum, and excellent stability of mutants. However, due to the random mutagenesis and associated mutant breeding modalities, it is imperative to improve HIBI-based mutant breeding efficiency and quality. This review discusses and summarizes the findings of existing theoretical and technical studies and presents a set of tandem strategies to enable efficient and high-quality HIBI-based mutant breeding practices. These strategies: adjust the mutation-inducing techniques, regulate cellular response states, formulate high-throughput screening schemes, and apply the generated superior genetic elements to genetic engineering approaches, thereby, improving the implications and expanding the scope of HIBI-based mutant breeding. These strategies aim to improve the mutagenesis rate, screening efficiency, and utilization of positive mutations. Here, we propose a model based on the integration of these strategies that would leverage the advantages of HIBI while compensating for its present shortcomings. Owing to the unique advantages of HIBI in creating high-quality genetic resources, we believe this review will contribute toward improving HIBI-based breeding.

Theoretical investigations on P-stabilized boryl cation radicals: from the Aufbau principle to SOMO-HOMO conversion.

We report the characterization of a series of novel phosphinidene-stabilized (P-stabilized) boryl cation radicals, in which the phosphinidene and boryl are stabilised by iPrNHC (iPrNHC[:C{N(iPr)C(H)}2]), and the P-stabilized boryl (P → B) moieties are linked by 1,8-naphthalene (1PB-a), 1,10-biphenyl (1PB-b), 1,2-perylene (1PB-c), and 4,5-perylene (1PB-d), to form a series of 1PB compounds. The 2PB series is designed by adding another P-stabilized boryl (P → B) unit into the 1PB series, in which the two P-stabilized boryl (P → B) moieties for each 2PB compound are linked by 1,4,5,8-naphthalene, (2PB-a), 1,5,6,10-biphenyl (2PB-b), 1,2,7,8-perylene (2PB-c), and 4,5,10,11-perylene (2PB-d), respectively. Theoretical calculations demonstrate that for all the studied molecules, the spin density mainly locates on the B atoms. Interestingly, the series of 2PB(a-d) compounds possess SOMO-HOMO conversion properties, while 1PB(a-d) compounds obey the Aufbau principle, resulting from the difference in the number of the P-stabilized boryl (P → B) moieties and an increase of the π-conjugation bridge that lead to the significantly increased HOMO energy in 2PB(a-d) compounds, which should be responsible for the different structural properties of compounds 1PB(a-d) and 2PB(a-d). The natural bond orbital (NBO) and atoms in molecules (AIM) analysis reveal how the interactions contribute to the covalent bond between P and B atoms. Moreover, the absorption properties show that the spectra of the 2PB(a-d) compounds are red-shifted relative to those of the corresponding 1PB(a-d) compounds in the near infrared region. We hope this work can provide new insights into tuning the electronic structures of the well-defined forms of P-stabilized boryl cation radicals and expand their potential application in organic optoelectronics.

High expression and purification of amino-terminal fragment of human amyloid precursor protein in Pichia pastoris and partial analysis of its properties.

The cleaved amino-terminal fragment of human amyloid precursor protein (N-APP) binds death receptor 6 (DR6) and triggers a caspase-dependent self-destruction process, which was suggested to contribute to Alzheimer's disease. To investigate the N-APP-DR6-induced degeneration pathway at the molecular level, obtaining abundant and purified N-APP is fundamental and critical. The recombinant N-APP has been produced in mammalian expression system. However, the cost and yield disadvantages of mammalian expression system make it less ideal for protein mass production. Here, we successfully expressed and purified recombinant N-terminal 18-285 amino acid residues of human amyloid precursor protein from the methylotrophic yeast Pichia pastoris with a high yield of 50 mg/L. Flow cytometry indicated the purified N-APP-induced obvious apoptosis of human neuroblastoma SHEP cells.

[Expression and purification of fusion protein CTP-SOD in Pichia pastoris and antioxidant capacity analysis].

Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide, by which special molecules can be carried out and localized into cytoplasmic compartment. Superoxide dismutase (SOD) is a protein that is difficult to go into cytoplasm. In this study, CTP-SOD fusion gene was amplified from human cDNA by PCR, and the active recombinant protein was successfully expressed in Pichia pastoris. HeLa cells pretreated with CTP-SOD showed a significantly improved survival against the pyrogallol-induced oxidative stress, suggesting CTP-SOD could cross the cell membrane more efficiently and protect cells from oxidative stress.