Journal of microbiology and biotechnology最新文献

The following study aimed to characterize the biological potential of the purified compounds of Pseudomonas aurantiaca PB-St2. Optimization of temperature and incubation time of 32oC and 72 h yielded the highest crude extract weight and optical density of bacterial culture. HPLC analysis of the crude metabolite extract (purified using gravitational column chromatography) showed three fractions named as PC1, PC2, and PC3. HPLC-purified fractions were subjected to LC-MS/MS analysis and the data was compared using reference library. Fraction PC1 was identified as mupirocin, PC2 as phenazine-1-carboxylic acid (PCA), and PC3 as the mixture of three compounds including pyoluteorin, PCA and 2-hydroxyphenazine (2-OH-phz). Fungicidal potential of the purified compounds was assessed against phytopathogens including Fusarium equiseti, Fusarium incarnatum, Alternaria alternata, and Colletotrichum falcatum. Fraction PC3 showed the highest fungicidal activity of ~89%, whereas, the least antifungal activity (~27%) was noted for mupirocin. Antibacterial activity of the purified compounds against Gram-positive pathogen Bacillus cereus, and Gram-negative pathogens Pseudomonas aeruginosa, Salmonella enterica, and Klebsiella oxytoca was also assessed. Fraction PC3 demonstrated the highest antibacterial activity against B. cereus and P. aeruginosa showing 1.8 cm, and 0.9 cm zones of inhibition, respectively. Against K. oxytoca and S. enterica, the antibacterial activity of PB-St2 crude extract was slightly higher than the fraction PC3. The fraction PC3 also demonstrated the highest IC₅₀ against HepG-2 and SF767 cancer cell lines at 25 μg and 20 μg concentrations, respectively. The multifaceted attributes of P. aurantiaca PB-St2 make it an ideal candidate for agricultural and pharmacological applications.

Phytosterols are naturally found in lipid-rich plant foods and oils. These compounds exhibit various pharmacological effects, including anti-inflammatory, antimicrobial, antioxidant, and cholesterol-lowering properties. However, their industrial application has been limited due to their high melting points and poor solubility in both water and oil. Some unsaturated fatty acids can enhance phytosterols' oil solubility while exhibiting antimicrobial activities against various bacterial strains. In this study, we synthesized a linolenic acid stigmasterol ester (LASE) to improve oil and emulsion solubility of stigmasterol, a phytosterol known for its beneficial physiological effects in humans. LASE was synthesized with Candida antarctica lipase A through a transesterification reaction using stigmasterol and α-linolenic acid-rich linseed oil as substrates. The reaction was conducted at 50°C in isooctane with 20 mM of both stigmasterol and linseed oil. Following synthesis, LASE was purified using preparative liquid chromatography. The purified LASE demonstrated a 20-fold improvement in solubility in tricaprylin (TCN) compared to stigmasterol. Additionally, its antibacterial activities against specific food spoilage bacteria were confirmed using a TCN-based emulsion system.

Necrotizing enterocolitis (NEC) is a life-threatening inflammatory bowel disease linked to gut microbiome dysbiosis. This study evaluates the efficacy of Limosilactobacillus reuteri strain DS0384, which secretes N-carbamyl glutamic acid (NCG), in modulating lipid peroxidation and inflammatory pathways in NEC. After pretreatment with DS0384, NEC mouse model was induced by gavage with bacteria-containing formula. NCG levels in the ileum were measured via CE-TOFMS metabolomic analysis. Additionally, rat small intestinal epithelial IEC-6 cells were exposed to lipopolysaccharide (LPS), treated with DS0384 DNA (D-DNA), and/or transfected to overexpress fatty acid synthase (FASN) and Toll-like receptor 4 (TLR4). Lipid peroxidation, peroxidation and inflammatory factors and NF-κB pathways were analysed. Immunofluorescence was used to measure the expression levels of ZO-1 and TLR4 in the ileum. DS0384 treatment significantly reduced more histological abnormalities, apoptosis, and TLR4 expression in NEC mice, while restoring NCG levels, downregulating FASN and inhibiting lipid peroxidation and inflammation. Pre-treatment with D-DNA maintained cell vitality, reduced apoptosis, and suppressed TLR4/NF-κB-mediated inflammasome activation. Overexpression of FASN or TLR4 reversed these effects. DS0384 is a promising therapeutic against NEC, enhancing gut barrier integrity and modulating inflammatory and oxidative responses, suggesting potential clinical benefits in preventing NEC progression.

This study investigates the effects of casticin on osteoclastogenesis, aiming to elucidate its underlying mechanisms for potential clinical applications. We assessed the cytotoxicity of casticin using a CCK assay in RAW 264.7cell (murine cell line, from ATCC), which differentiate into osteoclasts upon RANKL treatment. Various concentrations (0.125, 0.25, 0.50 μM) were tested to establish a dose-dependent response. The effects of casticin on osteoclast differentiation and actin filament organization were evaluated through TRAP and F-actin staining. Additionally, qPCR and Western blot analyses were performed to assess gene expression. Concentrations exceeding 1.00 μM caused significant cytotoxicity. Notably, casticin at 0.50 μM significantly inhibited osteoclast differentiation and function, reducing marker gene expression, including c-FOS, NFATc1, CtsK, and MMP-9. Furthermore, casticin decreased phosphorylation levels of NF-κB and IκBα and downregulated BCL-2 expression. Our findings highlight casticin's potent regulatory effects on osteoclasts via the NF-κB/BCL-2 signaling pathways, suggesting potential therapeutic applications in bone-related disorders.

Inonotus obliquus is a large, medicinal and edible fungus known for its extremely slow growth. It has garnered significant attention due to its high socioeconomic and pharmaceutical value. Our research group previously isolated an endophytic fungus, Acremonium sp. MEP2000, from I. obliquus, and a fermentation broth of MEP2000 enhanced both the growth and bioactive compound accumulation in I. obliquus. However, the activity of MEP2000 gradually declined after successive subcultures on PDA medium. We therefore revitalized MEP2000 by supplementing the PDA medium with birch bark powder (H medium), I. obliquus fruiting body powder (Z medium), and a combination of both (H&Z medium). MEP2000 was cultured on the PDA medium as the unrejuvenated control (PDA medium). The rejuvenated MEP2000 broth exerted significant effects on the secondary metabolite production, colony morphology, and growth rate of I. obliquus, with the H&Z medium inducing the most uniform mycelial morphology and fastest growth. Similarly, liquid shake-flask fermentation experiments showed that the H&Z inducer had the strongest growth-promoting effect. I. obliquus intracellular polysaccharide content was also highest in the H&Z treatment, with polysaccharide synthesis-related gene expression following a similar trend. GC-MS analysis confirmed that the H&Z medium had the highest concentration of triterpenoid bioactive compounds, such as inotodiol. These findings suggest that the MEP2000 broth, revitalized using the H&Z medium, effectively promotes I. obliquus mycelial growth and bioactive compound production, providing a valuable reference for large-scale production of active endophytic fungi.

Levan is a fructose polymer with unique physicochemical properties and physiological activities, making it useful in various industries. It is biologically synthesized from sucrose by the enzyme levansucrase, which does not require metal ions for its fructosylation activity. However, during the molecular characterization of a Pseudomonas chlororaphis levansucrase (PcLscA), it was observed that several divalent metal ions stimulate fructosylation activity by over 2.5-fold. To understand the structural basis of this action, a 3D model of PcLscA was constructed using the artificial intelligence-based modeling software (RoseTTAFold). This analysis revealed a novel metal-binding loop motif. Interestingly, this newly identified loop motif was also found in six known fructansucrase crystal structures. Furthermore, metal-stimulated levan synthesis was confirmed in the other two levansucrases. By identifying this loop motif, this study suggests an efficient approach for the enzymatic synthesis of levan, which traditionally is not known to require metal ions as activators.

Matrix metalloproteinases-2 (MMP-2) is crucial for collagen degradation at the dermal-epidermal junction, contributing to skin aging and photoaging. This study presents a series of hydroxamate-based inhibitors selectively targeting MMP-2. Through structure-activity relationship analysis, we systematically modified the N-arylsulfonyl group and amino acid backbone to enhance MMP-2 selectivity. Compounds 1ad, 1af, and 4an showed strong MMP-2 inhibition, with 1ad demonstrating nanomolar-level selectivity. Zymogram assays revealed 30-60% MMP-2 activity reduction, while gene expression analysis confirmed post-transcriptional inhibition. These hydroxamate-based inhibitors are promising candidates for anti-photoaging applications, combining potent MMP-2 inhibition with simplified synthesis, supporting their potential for large-scale cosmetic formulations aimed at improving skin firmness and reducing wrinkles.

Non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis and hepatitis, is the most frequently encountered complication of type 2 diabetes mellitus (T2DM). Due to its hepatoprotective, anti-obesity, antioxidant, and anti-inflammatory effects, Protaetia brevitarsis (P. brevitarsis) larvae have been used as traditional medicine to treat liver diseases since ancient times. Therefore, this study was conducted to confirm the positive effect of fermented P. brevitarsis larvae (FPB) on NAFLD. The results showed that high-fat diet (HFD)-induced dysglycemia was improved by treatment with FPB as determined by testing for fasting blood glucose and oral glucose tolerance. The weight of liver and white adipose tissue and the levels of serum lipid, hepatotoxicity, and nephrotoxicity indicators were reduced by FPB. In addition, oxidative stress and mitochondrial dysfunction caused by HFD were improved by FPB. In a similar manner, HFD-induced hepatic steatosis was prevented by FPB through regulation of the AMP-activated protein kinase pathway and serum lipid profile. HFD-induced hepatitis and apoptosis were ameliorated by FPB via the nuclear factor-kappa B pathway and the B-cell lymphoma 2 protein family. In conclusion, this study suggests the potential for application of FPB as a prophylactic agent for treatment of NAFLD through suppression of lipid accumulation and inflammation in the liver.

The roots of Rubia spp. (Rubiaceae) have been employed to treat hematemesis, inflammatory disease, and tumor. Cyclohexapeptides derived from Rubia spp. have been reported to have antitumor potential; however, the mechanism of action for their antitumor activity remains unclear. We aimed to examine the antitumor effect of deoxybouvardin glucoside (DBG), a cyclohexapeptide from Rubia spp. on oxaliplatin (Ox)-resistant human HCT116 colorectal cancer (CRC) cells. Cell viability in the presence of DBG was monitored using an MTT viability assay, and flow cytometry was used to analyze changes in apoptosis, cell cycle, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) activity. The antiproliferative activity involved apoptosis and phosphorylation of JNK and p38 MAPK. Inhibition of JNK and p38 MAPK by specific inhibitors prevented DBG-induced apoptosis, underscoring the close involvement of these kinases. Further, DBG induced cell cycle arrest in CRC cells at the G2/M phase by regulating the p21, p27, cyclin B1, and cdc2 proteins. DBG-induced apoptosis was accompanied mitochondrial membrane depolarization, resulting in cytochrome c release into the cytoplasm and caspase activation. Remarkably, DBG induced apoptosis by generating high ROS levels. The mediation of apoptosis by increased ROS generation was confirmed by pretreatment with the ROS scavenger N-acetyl cysteine (NAC). Collectively, DBG exhibited anticancer activity against both Ox-sensitive and Ox-resistant CRC cells by targeting JNK and p38 MAPK, inducing cell cycle arrest, elevating cellular ROS levels, and disrupting MMP. This study suggests that DBG has the potential to be utilized as a therapeutic agent for treating Ox-resistant CRC.

There is growing interest in pentose-based prebiotic oligosaccharides as alternatives to traditional hexose-based prebiotics. Among these, arabino-oligosaccharides (AOS), derived from the enzymatic hydrolysis of arabinan polymers, have gained significant attention. AOS can selectively stimulate the growth of beneficial gut bacteria, including Bifidobacterium and Bacteroides species, and contribute to health-benefit functions such as blood sugar control, positioning AOS as a promising synbiotic candidate. For the industrial production of AOS, the development of efficient enzymatic processes is essential, with exo- and endo-1,5-α-L-arabinanases (exo- and endo-ABNs) playing a crucial catalytic role. Most ABNs belong to the glycoside hydrolase (GH) family 43, characterized by a five-bladed β-propeller fold structure. These enzymes hydrolyze internal α-1,5-L-arabinofuranosidic linkages, producing AOS with varying degrees of polymerization. Some ABNs GH43 were known to exhibit exo-type hydrolytic modes of action, producing specific AOS products such as arabinotriose. Additionally, exo-ABNs from GH93, which feature a six-bladed β-propeller fold, exclusively release arabinobiose through their exo-type catalytic mechanism. This review represents the first comprehensive analysis of exo- and endo-ABNs, offering scientific insights into their biotechnological potential for AOS production. It systematically compares enzyme classification, structural differences, catalytic mechanisms, paving the way for innovative applications in health, food, and pharmaceutical industries.