To understand the mechanism of tolerance of lactic acid bacteria (LAB) during cold storage of fermented milk, 31 LAB strains were isolated from traditional fermented products, and Lactiplantibacillus plantarum NMGL2 was identified with good tolerance to both cold and acid stresses. Data-independent acquisition proteomics method was employed to analyze the response of Lpb. plantarum NMGL2 to the combinational cold and acid stresses during storage of the fermented milk made with the strain at 4 °C for 21 days. Among the differentially expressed proteins identified, 20 low temperature-resistant proteins and 10 acid-resistant proteins were found. Protein interaction analysis showed that the low temperature-resistant proteins associated with acid-resistant proteins were Hsp1, Hsp2, Hsp3, CspC, MurA1, MurC, MurD, MurE1, and MurI, while the acid-resistant proteins associated with low temperature-resistant proteins were DnaA, DnaK, GrpE, GroEL, and RbfA. The overall metabolic pathways of Lpb. plantarum NMGL2 in response to the stresses were determined including increased cell wall component biosynthesis, extracellular production of abundant glycolipids and glycoproteins, increased expression of F1Fo-ATPase, activation of glutamate deacidification system, enhanced expression of proteins and chaperones associated with cell repairing caused by the acidic and cold environment into the correct proteins. The present study for the first time provides further understanding of the proteomic pattern and metabolic changes of Lpb. plantarum in response to combinational cold and acid stresses in fermented milk, which facilitates potential application of Lpb. plantarum in fermented foods with enhanced survivability.
There has been an increased application of exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) in fermented dairy products, but interactions between EPS and casein (CAS), and bioactivities of their complex are poorly studied. In this study, EPS produced by Lactobacillus plantarum YW11 (EPS-YW11) was studied for interactions with CAS in a simulated fermentation system acidified by D-(+)-gluconic acid δ-lactone. The results showed that there was interaction between EPS-YW11 and CAS when EPS (up to 1%, w/v) was added to the casein solution (3%, w/v) as observed with increased viscoelasticity, water holding capacity, ζ-potential and particle size of EPS-YW11/CAS complex compared with CAS alone. Microstructural analysis showed that a higher concentration of EPS facilitated more even distribution of CAS particles that were connected through the polysaccharide chains. Infrared spectroscopy further confirmed interactions between EPS and CAS by intermolecular hydrogen bonding, electrostatic and hydrophobic contacts. Further evaluation of the bioactivities of EPS-YW11/CAS complex revealed significantly increased antibiofilm, antioxidation, and bile acids binding capacity. The present study provides further understanding on the mechanism of interactions between EPS produced by LAB and CAS, which would benefit potential applications of EPS in fermented dairy products with enhanced functionality.
The exopolysaccharide (EPS) produced by Lactiplantibacillus plantarum NMGL2 isolated from traditional fermented dairy cheese was purified chromatographically with DEAE-Sepharose and Sepharose CL-6B columns. The purified EPS was characterized by various physicochemical methods and in vitro fecal microbiota regulation assay. The results showed that the EPS had a relatively low molecular weight of 3.03 × 104 Da, and it had a relatively high degradation temperature of 245 °C as determined by differential scanning calorimetry. Observation of the EPS by scanning electron microscopy, transmission electron microscopy, and atomic force microscopy revealed a highly branched and tangled fibrous network microstructure with many hollow microtubules and spherical particles. Structural study by 1H NMR spectroscopy suggested that the EPS contained a tetrasaccharide repeating unit with monosaccharide components of β-galactose (4.6%), α-glucose (20.6%), and α-mannose (74.8%). The EPS was highly resistant to hydrolysis of simulated human saliva, gastric, and intestinal juices. Moreover, the EPS beneficially affected the composition and diversity of the fecal microbiota, e.g., increasing the relative abundance of Firmicutes and inhibiting that of Proteobacteria. The results of this study indicated significant bioactivity of this novel low-molecular-weight EPS produced by Lpb. plantarum NMGL2, which could serve as a bioactive agent for potential applications in the food and health care industry.
Background: Recent studies have shown that amphoteric regulatory protein (AREG), a member of the epidermal growth factor (EGF) family, is expressed in many cancers and is an independent prognostic indicator for patients with pancreatic cancer, but whether AREG is regulated at the epigenetic level to promote the development of pancreatic cancer (PC) has not been elucidated.
Methods: The expression level and role of AREG in PC cell tissues were investigated by GEPIA database, quantitative PCR (qPCR), Western blotting, CCK8 and Transwell assays. Methylated RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing indicated that METTL3 regulates the m6A modification of AREG and affects its expression. RIP, MeRIP were used to explore its regulatory role and potential mechanisms. The association between AREG expression and clinical classification parameters was analyzed by Fisher’s exact test. In addition, we predicted that miR-33a-3p regulates METTL3 expression by miRDB and RNAinter. The intrinsic mechanisms and regulatory roles were explored using dual luciferase reporter assays and rescue experiments.
Results: Our results support the notion that AREG is overexpressed in pancreatic cancer tissues and cell lines. Functionally, the deletion of AREG impedes pancreatic cancer (PC) cell proliferation, migration, and invasion. In addition, we identified and validated that methyltransferase-like 3 (METTL3) induced the m6A modification on AREG and facilitated the stability of AREG mRNA after sequencing. Additionally, we obtained experimental evidence that miR-33a-3p targets and inhibits METTL3 from taking action. Remediation experiments showed that miR-33a-3p inhibits PC progression through METTL3.
Conclusions: this research reveals that miR-33a-3p inhibits m6A-induced stabilization of AREG by targeting METTL3, which plays a key role in the aggressive progression of PC. AREG could be a potential target for PC treatment.
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