关键词: 蜂蜜, 芽孢杆菌, 热处理, 污染特征, MALDI-TOF MS, 二代测序, 风险评估, 加工工艺

Abstract:

【Objective】The thermostability and potential safety risks of Bacillus may pose a threat to the quality of honey products. Therefore, it is necessary to optimize the pre-treatment process for Bacillus detection in honey, to clarify the contamination characteristics of Bacillus, and to evaluate the risks in the processing chain, aiming to provide a theoretical basis for establishing a scientific and effective quality and safety control system.【Method】To improve the accuracy of Bacillus detection results in honey, this study selected a temperature range of 60-80 ℃ to carry out heat treatment experiments, and systematically explored the optimal process conditions for simultaneously achieving efficient inactivation of miscellaneous bacteria and retention of Bacillus spore concentration. Quantitative counting of Bacillus was performed on 150 commercial honey samples and 150 raw honey samples, followed by further strain isolation and purification. Meanwhile, Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) and next-generation sequencing based bacterial genome sequencing were combined to identify the species of Bacillus in honey. The species and detection rates of Bacillus in each honey sample were statistically analyzed, and food safety risk assessment was conducted accordingly. Furthermore, by simulating the honey processing procedures, the study investigated the effect of heat treatment at 40-60 ℃ on the growth of Bacillus cereus spores in honey and clarified its variation trend.【Result】Heat treatment at 80 ℃ for 10 min could completely inactivate the interfering bacteria (Escherichia coli and Saccharomyces cerevisiae) in honey, while maintaining the spore viability at the same order of magnitude. Statistical analysis was conducted on the results of Bacillus quantification and identification in 150 commercial honey samples and 150 raw honey samples. The results showed that the total detection rate of Bacillus in commercial honey was 38.7%, dominated by Bacillus cereus (20.7%), Bacillus subtilis (12.7%) and Bacillus pumilus (11.3%). By contrast, the total detection rate of Bacillus in raw honey was 23.3%, mainly including Bacillus cereus (16.7%), Bacillus pumilus (8.0%) and Bacillus licheniformis (3.3%). A comparison of the samples with the highest Bacillus concentration in commercial and raw honey indicated that all of these samples were subject to single contamination by Bacillus cereus. In addition, experiments simulating the heat treatment conditions (40-60 ℃) during honey processing demonstrated that the number of Bacillus cereus spores was positively correlated with temperature-time, namely, the spore count exhibited a significant upward trend with the increase of treatment temperature and extension of treatment duration. Correlation analysis of Bacillus species indicated that Bacillus contamination in commercial honey was primarily derived from raw honey. The detection of environmental strains indirectly demonstrated that honey was susceptible to microbial contamination throughout the whole chain, including honey collection by bees, raw honey harvesting and subsequent processing procedures.【Conclusion】Heat treatment at 80 ℃ for 10 min was identified as the optimal pre-treatment protocol for Bacillus detection in honey. Bacillus cereus was confirmed as the primary hazard bacterium in honey. It was recommended that honey production enterprises listed Bacillus as one of the core monitoring indicators for the quality control of raw honey.

Key words: honey, Bacillus, heat treatment, pollution characteristics, MALDI-TOF MS, second-generation sequencing, risk assessment, processing technology