Bacterial spores commonly co-exist with vegetative cells, presenting challenges in spore separation and detection. The separation of spores is a crucial process for laboratory research and the detection of spore mechanisms. This study introduced a novel method that leverages the high binding affinity of vancomycin (Van) and ampicillin sodium (Amp) to vegetative cells, integrated with magnetic separation technology, to selectively collect spores from complex environments by eliminating vegetative cells. First, Van/Amp-modified magnetic Fe₃O₄ nanoparticles (Fe₃O₄ NPs) were synthesized and characterized. Subsequently, these NPs bound vegetative cells, forming magnetic conjugates that could be efficiently removed using a magnetic field. Concurrently, spores were collected with an efficiency exceeding 95%, completing the entire process within 30 min and achieving a spore separation efficiency of up to 10⁵ CFU/mL. This method was successfully applied to actual samples, including tap water and milk. The state of the collected spores was confirmed using Raman spectroscopy and microscopic techniques, verifying that their characteristics matched those of typical spores. The proposed novel method for rapid spore separation, leveraging the "remove bacterial effect" facilitated by Van/Amp-Fe₃O₄ NPs, showed outstanding spore collection capabilities while preserving the excellent physiological state of spores.

In order to study the effect of heat stress on the inner membrane proteins of Clostridium perfringens spores, the physicochemcial changes in the inner membrane proteins after heat treatment at different temperatures (25, 37, 75 or 95 ℃) for 20 minutes were characterized by measuring particle size distribution, surface hydrophobicity, ultraviolet absorption spectrum, endogenous fluorescence spectrum and Raman spectrum. The results showed that heating temperature had a significant effect on C. perfringens spore inner membrane proteins. Compared with the control group (treated at 25 ℃), treatment at 37 ℃ had no significant effect on the inner membrane proteins, and did not cause any significant changes in the particle size distribution, amino acid microenvironment, surface hydrophobicity or secondary structure. After heat stress at 75 ℃, the particle size distribution was uniform and stable, the UV absorption and fluorescence intensity was significantly enhanced, and the surface hydrophobicity was significantly increased. The percentage of α-helix decreased by 3.17%, the percentage of β-sheet decreased by 3.94%, and the proportion of random coil increased by 8.31%. After heat stress at 95 ℃, the proteins’ structure was damaged, the proteins were obviously aggregated or denatured, and the particle size distribution moved significantly to larger particle size. The above results indicated that heat stress at 75 ℃ could effectively affect the physicochemical properties of C. perfringens spore inner membrane proteins, leading to the exposure of hydrophobic sites and amino acid residues and significant secondary structural changes. This study provides a theoretical basis for further research on the effect of heat stress on the functional properties of C. perfringens spore inner membrane proteins.

In this study, using a one-step dynamic numerical analysis and optimization method, a tertiary model describing the growth of total viable count in sauced pig head meat was developed for real-time freshness prediction under fluctuating temperature (2–35 ℃). The validation results showed that the root mean square error (RMSE) of the prediction model for total viable count was 0.26 (lg(CFU/g)), and the residual mean and standard deviation were –0.047 (lg(CFU/g)) and 0.125 (lg(CFU/g)), respectively, which were within the normal experimental error range. The results of this study can be used to predict the changes of total viable count during cold chain and temperature abuse, and obtain a real-time freshness prediction model for pre-packaged sauced pig head at 2–35 ℃, thereby providing a theoretical basis for the real-time monitoring of the freshness of prepackaged sauced pig head at fluctuating temperatures.