This study evaluated Natrix (R) Q anion membrane chromatography for the purification of Streptococcus pneumoniae serotype 5 capsular polysaccharide (CPS) as an alternative to conventional organic solvent-based purification and bead-based ion-exchange chromatography. The purification process was optimized and characterized using a Design of Experiments (DoE) approach to establish a robust operating space for key process parameters. Under the optimized conditions, residual DNA was reduced to below 2%, while CPS recovery remained above 80%. The membrane process achieved a loading capacity of 45-60 mg CPS/mL membrane, approximately 4-6-fold higher than that of conventional anion-exchange resins (<10mg/mL), and operated at a residence time of approximately 6s, substantially shorter than the approximately 5 min typically required for bead-based ion-exchange chromatography. The CPS recovery was also higher than the previously reported range of 50%-73%. Scale-up studies further demonstrated that the optimized process could be successfully transferred to scale production. Product quality was evaluated by double immunodiffusion, proton nuclear magnetic resonance (H-1-NMR) spectroscopy, and relevant biochemical and physicochemical assays. The CPS purified by membrane chromatography met the predefined quality requirements. These results demonstrate that Natrix (R) Q anion membrane chromatography provides an efficient, high-throughput, and scalable approach for the purification of serotype 5 pneumococcal CPS and has potential for application in large-scale pneumococcal polysaccharide vaccine manufacturing.
Pediocin PA-1 is a Class IIa bacteriocin with potent activity against foodborne pathogens, but its application is limited by the lack of efficient purification methods. In this study, pediocin PA-1 was purified from Pediococcus acidilactici LH31 using a two-step procedure consisting of pH-mediated cell adsorption-desorption followed by semi-preparative reversed-phase high-performance liquid chromatography. The purified bacteriocin showed a single band at approximately 5.0 kDa on Tricine-SDS-PAGE. It exhibited optimal antimicrobial activity against Enterococcus faecalis at 25 degrees C and pH 6.0. The bacteriocin retained significant activity after boiling for 20 min and remained stable across pH 4.0-9.0. The minimum inhibitory concentration (MIC) was determined to be 50 mu g/mL. Time-kill kinetics demonstrated concentration-dependent bactericidal activity, with >3 log(10) reduction in viable counts within 10 h at 2 & times; MIC. All experiments were performed in triplicate using three independent bacterial culture batches. These findings provide an efficient purification protocol for pediocin PA-1 and comprehensively characterize its stability and antimicrobial kinetics, supporting its potential as a natural food preservative.
Peanuts (Arachis hypogaea L.) are among the richest plant sources of superoxide dismutase (SOD), with seed activity reaching 1,400–2,200 U/g fresh weight. This study established a systematically optimized, laboratory-to-pilot-scale workflow for preparing high-purity SOD from kilogram-scale peanut kernels. Because SOD is consumed rather than synthesized during germination, overnight-soaked seeds were directly homogenized for extraction. Single-factor optimization identified the key extraction conditions: 2.5 mM sodium phosphate buffer (pH 7.8), a 2:1 (v/w) liquid-to-solid ratio, and 35 °C for three 60-min rounds. Impurity removal combined n-hexane defatting (1:1, v/v, 30 min), heat treatment (55 °C, 40 min), and ammonium sulfate fractionation (50% (w/v) for impurities; 85% for SOD). Sequential Sephadex G-100 size-exclusion and DEAE-52 anion-exchange chromatography yielded a specific activity of approximately 506 U/mg protein with 79.5-fold purification and 26% overall recovery. The freeze-dried SOD powder exhibited robust thermal stability (no significant loss at 55 °C for 60 min), broad pH tolerance (5.0–11.0), and room-temperature stability (48 h in 2.5 mM phosphate buffer, pH 7.8). Notably, the enzyme retained up to 81% activity after 120 min in simulated gastrointestinal fluid, suggesting favorable oral bioavailability. This work provides a comprehensive process engineering foundation for the pilot-scale production of plant-derived SOD for oral applications.
