Lactiplantibacillus plantarum RS20D Alleviates Male Reproductive Toxicity Induced by Pubertal Exposure to Di-n-butyl Phthalate and Mono-n-butyl Phthalate.

Phthalate acid esters (PAEs) and their metabolites, such as di-n-butyl phthalate (DBP) and mono-n-butyl phthalate (MBP), are known to cause male reproductive damage. Lactiplantibacillus plantarum RS20D has demonstrated the ability to remove both DBP and MBP in vitro, suggesting its potential as a detoxifying agent against these compounds. This study aimed to investigate the protective effects of RS20D on DBP or MBP-induced male reproductive toxicity in adolescent rats. Oral administration of RS20D significantly mitigated the histological damage to the testes caused by MBP or DBP, restored sperm concentration, morphological abnormalities, and the proliferation index in MBP-exposed rats, and partially reversed spermatogenic damage in DBP-exposed rats. Furthermore, RS20D restored serum levels of estradiol (E2) and testosterone, and superoxide dismutase (SOD) activity in DBP-exposed rats, significantly increased testosterone levels in MBP-exposed rats, and restored copper (Cu) concentrations in the testes after exposure to DBP or MBP. Additionally, RS20D effectively modulated the intestinal microbiota in DBP-exposed rats and partially ameliorated dysbiosis induced by MBP, which may be associated with the alleviation of reproductive toxic effects induced by DBP or MBP. In conclusion, this study demonstrates that RS20D administration can alleviate male reproductive toxicity and gut dysbacteriosis induced by DBP or MBP exposure, providing a dietary strategy for the bioremediation of PAEs and their metabolites.

Phenotypic and genomic analysis of inulin consumption by Lactiplantibacillus plantarum strains from Sichuan pickle.

AIMS: To investigate the capability, properties, and molecular mechanism of inulin fermentation by lactic acid bacteria (LAB) from Sichuan pickle. METHODS AND RESULTS: A total of 79 LAB strains were purified from 30 aged Sichuan pickle brine samples, and only 21 Lactiplantibacillus plantarum strains (26.58%, 21/79) derived from 15 samples grew well through utilizing inulin as a carbon source. The fermentation tests through using long-chain inulin (lc-inulin) as a carbon source showed that only 6 L. plantarum strains grew well, while other 15 strains could only utilize short-chain oligofructose (FOS), and thin-layer chromatography analysis evidenced a strain specificity of inulin consumption patterns. Lactiplantibacillus plantarum YT041 is a vigorous inulin fermenter, and whole genome sequencing data revealed that sacPTS1 and fosRABCDXE operons might be associated with the fermentation of FOS and lc-inulin, respectively. CONCLUSIONS: The phenotype of inulin consumption is commonly present in LAB from Sichuan pickle, which is strain-specific and largely depends on their specific ecological niche and degree of polymerization.

Quantification of heavy metals and health risk assessment in Sichuan pickle.

Sichuan pickle is one of popular traditional fermented foods in China. However, the contamination of heavy metals in Sichuan pickle, particularly home-made Sichuan pickle and aged pickle brine, is little known. Therefore, the content of trace (Cr, Cu, and Zn) and toxic elements (As, Pb, and Cd) in Sichuan industrial pickle (SIP), Sichuan home-made pickle (SHP), and aged pickle brine collected from local markets and families in Sichuan province, respectively, was detected by inductively coupled plasma-tandem mass spectrometry (ICP-MS/MS) and the health risk was assessed by target hazard quotients including target hazard quotient (THQ) and total target hazard quotient (TTHQ). Consequently, the mean concentrations of Cr, Cu, Zn, As, Pb, and Cd were 0.122, 0.540, 2.516, 0.023, 0.015, and 0.106 mg/kg in SIP and 0.071, 0.364, 2.698, 0.014, 0.015, and 0.289 mg/kg in SHP, respectively, lower than the maximum allowable concentrations set by Chinese regulations, except for Cr and Cd in few samples. Principal component analysis of the heavy metal content could obviously distinguish between SIP and SHP. The content of As, Pb, and Cd in leaf pickles was significantly higher than that in pickles fermented with other types of vegetables. A significant enrichment of heavy metals in aged pickle brine over 10 years was observed, but pickle jars had no significant effect on heavy metal content in aged pickle brine. The intake of heavy metals through daily consumption of SIP and SHP was at a safe level, whereas the TTHQ of leaf pickle was 1.006, indicating a potential health risk. In conclusion, this study provided fundamental data for food safety assurance of Sichuan pickle. PRACTICAL APPLICATION: Sichuan pickle is one of popular traditional fermented foods in China. In the present study, we investigated the contamination of heavy metals in Sichuan pickles by detecting the content of Cr, Cu, Zn, As, Cd, and Pb in Sichuan industrial pickle, Sichuan home-made pickle, and aged pickle brine, and estimated the health risk to local residents. This study can provide a reference for the safety risk of Sichuan industrial and home-made pickle in terms of heavy metal contamination, and enhance the food safety in the processing, production, and consumption of Sichuan pickle in local families and pickle industry.

Global genome and comparative transcriptomic analysis reveal the inulin consumption strategy of Lactiplantibacillus plantarum QS7T.

Sichuan pickle is a natural combination of probiotics and dietary fibers, in which a strain Lactiplantibacillus plantarum QS7T was found to be capable of efficiently metabolizing inulin. However, the underlying molecular mechanism of inulin consumption by the strain QS7T is unclear. Therefore, this study firstly investigated the metabolic characteristics of inulin in the strain QS7T, and the results showed it could grow very well on the medium containing inulin as a carbon source (maximum OD600 nm, 1.891 ± 0.028) and degrade both short-chain oligofructose and long-chain fructan components through thin layer chromatography analysis. Genomic sequencing and analysis revealed a high percentage of functional genes associated with carbohydrate transport and metabolism, particularly glycoside hydrolase (GH) genes responsible for hydrolysing carbohydrates, within the genome of the strain QS7T. Furthermore, comparative transcriptomic analysis of L. plantarum QS7T in response to inulin or glucose indicated that functional genes associated with inulin consumption including several genes encoding PTS sugar transporters and two predicted GH32 family genes encoding beta-fructofuranosidase and beta-fructosidase were significantly up-regulated by inulin compared to the gene expression on glucose. In conclusion, we obtained a mechanistic understanding of interplay between probiotic L. plantarum QS7T derived from Sichuan pickle and natural dietary fiber, inulin; totally two operons including a sacPTS1 operon responsible for metabolizing short-chain oligofructose primarily in the cytoplasm and a fos operon responsible for extracellularly degrading all moderate and long-chain fructan components linked to inulin consumption by L. plantarum QS7T.

Prebiotics Inulin Metabolism by Lactic Acid Bacteria From Young Rabbits.

Inulin as a commercial prebiotic could selectively promote the growth of beneficial gut microbes such as lactic acid bacteria (LAB). Whether LAB in rabbit gut possesses the capability to metabolize and utilize inulin is little known. Therefore, this study recovered 94 LAB strains from neonate rabbits and found that only 29% (28/94) could metabolize inulin with both species- and strain-specificity. The most vigorous inulin-degrading strain, Lacticaseibacillus paracasei YT170, could efficiently utilize both short-chain and long-chain components through thin-layer chromatography analysis. From genomic analysis, a predicted fosRABCDXE operon encoding putative cell wall-anchored fructan ß-fructosidase, five fructose-transporting proteins and a pts1BCA operon encoding putative ß-fructofuranosidase and sucrose-specific IIBCA components were linked to long-chain and short-chain inulin utilization respectively. This study provides a mechanistic rationale for effect of inulin administration on rabbits and lays a foundation for synbiotic applications aimed at modulating the intestinal microbiota of young rabbits.

Plasma Choline as a Diagnostic Biomarker in Slow Coronary Flow.

AIM: The slow coronary flow (SCF) phenomenon was characterized by delayed perfusion of epicardial arteries, and no obvious coronary artery lesion in coronary angiography. The prognosis of patients with slow coronary flow was poor. However, there is lack of rapid, simple, and accurate method for SCF diagnosis. This study aimed to explore the utility of plasma choline as a diagnostic biomarker for SCF. METHODS: Patients with coronary artery stenosis <40% evaluated by the coronary angiogram method were recruited in this study and were grouped into normal coronary flow (NCF) and SCF by thrombolysis in myocardial infarction frame count (TFC). Plasma choline concentrations of patients with NCF and SCF were quantified by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry. Correlation analysis was performed between plasma choline concentration and TFC. Receiver operating characteristic (ROC) curve analysis with or without confounding factor adjustment was applied to predict the diagnostic power of plasma choline in SCF. RESULTS: Forty-four patients with SCF and 21 patients with NCF were included in this study. TFC in LAD, LCX, and RCA and mean TFC were significantly higher in patients with SCF in comparison with patients with NCF (32.67 ± 8.37 vs. 20.66 ± 3.41, P < 0.01). Plasma choline level was obviously higher in patients with SCF when compared with patients with NCF (754.65 ± 238.18 vs. 635.79 ± 108.25, P=0.007). Plasma choline level had significantly positive correlation with Mean TFC (r = 0.364, P=0.002). Receiver operating characteristic (ROC) analysis showed that choline with or without confounding factor adjustment had an AUC score of 0.65 and 0.77, respectively. CONCLUSIONS: TFC were closely related with plasma choline level, and plasma choline can be a suitable and stable diagnostic biomarker for SCF.

Overexpression of long noncoding RNA ANRIL inhibits phenotypic switching of vascular smooth muscle cells to prevent atherosclerotic plaque development in vivo.

BACKGROUND: Phenotypic switching of vascular smooth muscle cells (VSMCs) plays a key role in atherosclerosis. Long noncoding RNA ANRIL (lncRNA-ANRIL) is critical in vascular homeostasis. Metformin produces multiple beneficial effects in atherosclerosis. However, the underlying mechanisms need to be elucidated. METHODS AND RESULTS: Metformin increased lncRNA-ANRIL expression and AMPK activity in cultured VSMCs, and inhibited the phenotypic switching of VSMCs to the synthetic phenotype induced by platelet-derived growth factor (PDGF). Overexpression of lncRNA-ANRIL inhibited phenotypic switching and reversed the reduction of AMPK activity in PDGF-treated VSMCs. While, gene knockdown of lncRNA-ANRIL by adenovirus or silence of AMPKγ through siRNA abolished AMPK activation induced by metformin in VSMCs. RNA-immunoprecipitation analysis indicated that the affinity of lncRNA-ANRIL to AMPKγ subunit was increased by metformin. In vivo, administration of metformin increased the levels of lncRNA-ANRIL, suppressed VSMC phenotypic switching, and prevented the development of atherosclerotic plaque in Apoe-/- mice fed with western diet. These protective effects of metformin were abolished by infecting Apoe-/- mice with adenovirus expressing lncRNA-ANRIL shRNA. The levels of AMPK phosphorylation, AMPK activity, and lncRNA-ANRIL expression were decreased in human atherosclerotic lesions. CONCLUSION: Metformin activates AMPK to suppress the formation of atherosclerotic plaque through upregulation of lncRNA-ANRIL.

Fast identification of lipase inhibitors in oolong tea by using lipase functionalised Fe3O4 magnetic nanoparticles coupled with UPLC-MS/MS.

Oolong tea is an important member in tea family, which claims for various health benefits such as preventing obesity and improving lipid metabolism. In this work, using pancreatic lipase (PL) functionalised magnetic nanoparticles (PL-MNPs) as solid phase extraction absorbent in combination with ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS), we developed a method for rapid screening and identification of lipase inhibitors from oolong tea. Three PL ligands were selectively extracted and identified as (-)-epigallocatechin-3-O-gallate (EGCG), (-)-gallocatechin-3-O-gallate (GCG) and (-)-epicatechin-3-O-gallate (ECG). Their lipase inhibitory activities were significantly higher than those non-ligands. Structure-activity analysis revealed that the presence of a galloyl moiety in the structure was required for binding to PL-MNPs, and therefore, exhibiting a strong inhibition on the enzyme. Taking advantages of the specificity in enzyme binding and the convenience of magnetic separation, this method has great potential for fast screening of lipase inhibitors from natural resources.

Lipase ligands in Nelumbo nucifera leaves and study of their binding mechanism.

Lotus (Nelumbo nucifera) leaves have been widely used in weight-loss foods to prevent obesity in China. In this work, a facile procedure based on ligand fishing was developed to isolate and identify lipase inhibitors present in lotus leaves. Highly stable and active lipase-Fe3O4 superparamagnetic nanoparticle conjugates (LMNPs) were prepared and used as baits. Two flavonoids in lotus leaf extract were found to bind to the baits and were identified as quercetin-3-O-ß-d-arabinopyranosyl-(1→2)- ß-d-galactopyranoside (1) and quercetin-3-O-ß-d-glucuronide (4) based on electrospray ionization-mass spectrometric analyses. Their 50% inhibitory concentrations on lipase (IC50) were 52.9 ± 3.2 and 17.1 ± 1.5 µg/mL, respectively. In addition, they were found to significantly quench the fluorescence of lipase, suggesting their strong affinities with this enzyme, which was further evidenced by molecular docking. Ligand fishing based on LMNPs shows great power for fast screening and identification of lipase inhibitors present in edible and medicinal plants.

Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: characterization and application for enzymatic inhibition assays.

Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe3O4-SiO2) possessed three dimensional core-shell structures with an average diameter of ~20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g(-1). The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The Km and the Vmax values (0.02 mM, 6.40 U·mg(-1) enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg(-1) enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds.

Selective extraction of berberine from Cortex Phellodendri using polydopamine-coated magnetic nanoparticles.

A new extraction agent featuring dopamine self-polymerized on magnetic Fe3 O4 nanoparticles has been successfully synthesized and evaluated for the SPE of berberine from the extract of the traditional Chinese medicinal plant, Cortex Phellodendri. The nanoparticles prepared possessed a core-shell structure and showed super-paramagnetism. It was found that these polydopamine-coated nanoparticles exhibited strong and selective adsorption for berberine. Among the chemical components present in C. Phellodendri, only berberine was adsorbed by the nanoparticles and extracted by a following SPE procedure. Various conditions such as the amount of polydopamine-coated nanoparticles, desorption solvent, desorption time and equilibrium time were optimized for the SPE of berberine. The purity of berberine extracted from C. Phellodendri was determined to be as high as 91.3% compared with that of 9.5% in the extract. The established SPE protocol combined advantages of highly selective enrichment with easy magnetic separation, and proved to be a facile efficient procedure for the isolation of berberine. Further, the prepared polydopamine-coated magnetic nanoparticles could be reused for multiple times, reducing operational cost. The applicability and reliability of the developed SPE method were demonstrated by isolating berberine from three different C. Phellodendri extracts. Recoveries of 85.4-111.2% were obtained with relative standard deviations ranging from 0.27-2.05%.