Pseudomonas fluorescens is the main spoilage bacterium in milk, and its proliferation is one of the factors leading to the deterioration of the quality of raw milk. In this study, a rapid detection system for P. fluorescens was developed based on recombinase-aided amplification combined with a test strip (RAA-TS), which contained a double test line (DTL) targeting the virulence gene aprX of P. fluorescens and the housekeeping gene gyrB of Pseudomonas. Visual observation could detect gyrB (50 CFU/mL) and aprX (250 CFU/mL) within 90 min, including sample pretreatment and RAA reaction and detection steps. No cross-reactions were observed with Pseudomonas or other bacteria (n = 19). The quantitative detection limits (LOD) of gyrB and aprX for P. fluorescens in milk were 37 CFU/mL and 233 CFU/mL, respectively. Compared with polymerase chain reaction-agarose gel electrophoresis (PCR-AGE), the sensitivity of the developed RAA-TS-DTL system was increased by approximately four times. Furthermore, it could detect live P. fluorescens in milk when combined with optimized sample pretreatment by propidium monoazide (PMAxx). Its consistency with the traditional culture method in the detection of P. fluorescens spiked in milk samples (n = 25) was 100%. The developed RAA-TS-DTL had the advantages of high accuracy and short time consumption. Thus, it provides a new way or tool for the rapid screening or detection of P. fluorescens in milk.

The by-products that may contain tetrodotoxin (TTX) produced during the processing of farmed pufferfish have caused food safety risks and environmental pollution. Peptidoglycan (PG) of lactic acid bacteria could adsorb TTX; however, its complex structure and poor solubility limited adsorption efficiency. In this study, hydroxyl modifications of three PGs (A3α, A1γ and A4α) were realized via TEMPO-mediated selective oxidation of the primary hydroxyl group. Compared with PGs, it was found that the carboxyl density of hydroxyl-modified PGs (HM-PGs) increased from 1.66 mmol/g to 3.33 mmol/g and the surface electronegativity increased from −36 mV to −59 mV. The adsorption capacity of HM-PGs to TTX reached 1.48 μg/mg, which was comparable to the adsorption of the conventional adsorbent chitosan for aflatoxin B1 (1.39 μg/mg). Moreover, HM-PGs decreased the toxicity of TTX from strong toxic to nearly non-toxic, with the toxicity reduction rate reached 99.85%. After treatment with HM-PGs, the mouse hippocampus and neuronal cell model confirmed that lower neural injury and sodium channel blocking effects were observed in the residual TTX, whose neurotoxicity was lower. Molecular docking simulation and physicochemical analysis revealed that the adsorption of TTX by HM-PGs was a complex adsorption mode driven by the synergy of physicochemical interaction. There were both physical adsorptions based on electrostatic and hydrophobic interactions and chemical binding with strong hydrogen bonding (1.46 Å) and Mayer bond order (0.1229). This study not only developed a new, efficient and safe tool for TTX removal, but also provided a theoretical basis for the development of biological toxin removal material.