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.

[Effects of tannins in Galla Chinensis on pharmacokinetics of rifampicin in vivo and in vitro].

This study was aimed to explore the effects of tannins in Galla Chinensis on rifampicin in vivo. In the experiment in vitro, UV spectrophotometry and high performance liquid chromatography (HPLC) were used to investigate the solubility of rifampin in pH 1.3, 6.8, artificial gastric juice environment and artificial intestinal fluid environment as well as the effects of tannins on solubility of rifampin in the above conditions. In the experiment in vivo, the process of rifampicin was studied after intragastric administration of rifampicin and rifampicin+ tannins in Galla Chinensis, and then the pharmacokinetic parameters were calculated. The results showed that rifampicin was constantly precipitated in the artificial gastric juice environment over time, and nearly 85% of the rifampicin was precipitated after 6 hours; it showed a good solubility in the artificial intestinal juice environment. After adding the said tannins, the concentration of rifampicin was decreased significantly in both environments, and the concentration of rifampicin in artificial intestinal juice remained relatively stable, while that in artificial gastric juice remained the original downward trend. The pharmacokinetic parameters displayed that as compared with rifampicin alone, AUC0-t and Cmax were decreased significantly, MRT0-t slowed down significantly, Tmax doubled to 7.0 h and the bioavailability was only 31.65% in rifampicin + tannins in Galla Chinensis group. The experiment indicated rifampicin had a poor solubility in acidic environment and the decrease of bioavailability of rifampicin when in combination with tannin was mainly due to the reduction of rifampicin solubility in intestinal tract by complexation of rifampicin with tannin, thus affecting its absorption in intestinal tract. Therefore, rifampicin and the Chinese herbal medicines or Chinese patent medicines rich in tannin should not be taken simultaneously.

Evaluation of a rat meibomian gland dysfunction model induced by closure of meibomian gland orifices.

AIM: To find a stable, inexpensive, and reliable method to produce a rat meibomian gland dysfunction (MGD) model. METHODS: We inserted slim guidewires into the meibomian gland orifices of twelve Brown Norway rats and fulgurized every guidewire to destroy part of the meibomian gland. We then observed the morphological changes in the eyelid margin, and compared the data of tear breakup time (TBUT), Schirmer I test, and the corneal fluorescence staining scores at different times (1, 2, 4, and 6wk). We observed pathological changes of the cornea, conjunctiva and meibomian gland, and we used real-time polymerase chain reaction to analyze epithelial growth factor (EGF), interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and Ki67. RESULTS: In the fourth week, compared with the control group, the TBUT of the model group began to decreased (P<0.05). The tear secretion remained stable (P>0.05). The corneal dots were significantly increased in the fourth week when the fusion stain began to appear (P<0.05). In the fourth week, partial meibomian gland openings had hoary secretions blocked, orifices were expanded, and there was a partial convex deformation. In the sixth week, the tissue section showed that the number of conjunctival goblet cells was decreased, epithelial cells were irregular, the epithelium was detached and rough, and meibomian glands were lost. The expressions of EGF, IL-6, IL-8, and TNF-α in corneal, conjunctival, and meibomian tissues were highly increased (P<0.05), but no statistical difference was found in the expression of Ki67 in corneal and conjunctival tissues (P>0.05). CONCLUSION: The MGD rat model, produced via electrocauterization of meibomian gland orifices, matched clinical manifestations and cytokine levels. Our research provides a new method of achieving an MGD animal model.

[LC-LTQ-Orbitrap analysis on chemical constituents in Scrophulariae Radix extract and their metabolites in rat plasma].

Chemical constituents in extract of Scrophulariae Radix and their metabolites in rat plasma after oral administration were identified by HPLC-LTQ-Orbitrap. Samples were separated by a Venusil MP C18 column using a binary gradient elution. The information on the total ion chromatogram, the extraction chromatogram and the mass spectrogram in a negative mode were synthetically analyzed by comparing the retention time, MS and MS/MS spectra with literature data and some of reference standards to conduct a qualitative study on constituents of Radix Scrophulariae extract in vivo and in vitro. Totally 37 compounds from Scrophularia ningpoensis extract were detected including 12 iridoid glycosides, 20 phenylpropanoids and 5 unknown compounds. In vivo, harpagide, harpagoside and angoroside C were confirmed to enter into the blood in prototype forms. Besides, another 2 prototype compounds and 2 metabolites were detected in rat plasma after oral administration of S. ningpoensis extract. The results are beneficial for the determination of bioactive substances of S. ningpoensis and significant for further studies on S. ningpoensis.