Changes with time of a population of Bacillus weihenstephanensis KBAB4 and Bacillus licheniformis AD978 dormant spores into germinated spores and vegetative cells were followed by flow cytometry, at pH ranges from 4.7 to 7.4, and temperature from 10°C to 37°C for B. weihenstephanensis and from 18°C to 59°C for B. licheniformis.

Incubation conditions lower than optimal temperatures or pH led to lower proportions of dormant spores able to germinate and extended time of germination, lower proportion of germinated spores able to outgrow, an extension of their times of outgrowth, and an increase of the heterogeneity of spore outgrowth time. A model based on the strain growth limits, was proposed to quantify the impact of incubation temperature and pH on the passage through each physiological stage.

The heat-treatment temperature or time acted independently on spore recovery. Indeed, a treatment at 85°C during 12 min or at 95°C during 2 min did not have the same impact on spore germination and outgrowth kinetics of B. weihenstephanensis despite they both led to a tenfold reduction of the population. Moreover, acidic sporulation pH increased the time of outgrowth by 1.2 fold and lowered the proportion of spores able to germinate and outgrow by 1.4 fold. Interestingly, we showed by a proteomic analysis that some proteins involved in germination and outgrowth were detected at a lower abundance in spores produced at pH 5.5 compared to those produced at pH 7.0, maybe at the origin of germination and outgrowth behavior of spores produced at suboptimal pH.

Importance

Sporulation and incubation conditions have an impact on the numbers of spores able to recover after exposure to sub-lethal heat-treatment. Using flow cytometry we were able to follow at a single cell level the changes in the physiological states of heat-stressed spores of Bacillus sp. and to discriminate between dormant spores, germinated spores and outgrowing vegetative cells. We developed original mathematical models that describe (i) the changes with time of the proportion of cells in their different states during germination and outgrowth, and (ii) the influence of temperature and pH on the kinetics of spore recovery using the growth limits of the tested strains as model parameters. We think that these models better predict spore recovery after a sub-lethal heat-treatment, a common situation in food processing and a concern for food preservation and safety.

魏氏史氏杆菌KBAB4和地衣芽孢杆菌AD978休眠孢子随时间的变化变成发芽孢子和营养细胞，然后用流式细胞仪测定其pH值在4.7至7.4之间，温度为10°C至37°C 。地衣芽孢杆菌从18°C到59°C 。

低于最佳温度或pH的孵化条件会导致休眠的孢子能够发芽的比例降低，发芽时间延长，发芽的孢子能够消长的比例降低，发芽时间的延长，孢子伸出异质性的增加时间。提出了一个基于菌株生长极限的模型，以量化孵育温度和pH对通过每个生理阶段的影响。

热处理温度或时间独立于孢子回收。事实上，在12分钟或期间，2分钟95℃，在85℃的治疗并没有对孢子萌发和生长动力学的同种冲击的B. weihenstephanensis尽管它们都导致减少十倍的人口。而且，酸性孢子形成的pH使生长时间延长了1.2倍，而能够发芽和生长的孢子比例降低了1.4倍。有趣的是，我们通过蛋白质组学分析表明，与pH 7.0相比，在pH 5.5产生的孢子中检测到一些参与发芽和生长的蛋白质的丰度较低，可能是在次优孢子的萌发和生长行为的起源pH值

重要性

孢子形成和培养条件对暴露于亚致死性热处理后能够恢复的孢子数量产生影响。使用流式细胞仪，我们能够在单个细胞水平上追踪芽孢杆菌热应激孢子的生理状态变化sp。并区分休眠孢子，发芽孢子和营养细胞生长。我们开发了原始的数学模型，这些模型描述了（i）在发芽和长出过程中处于不同状态的细胞比例随时间的变化，以及（ii）使用细菌的生长极限，温度和pH对孢子恢复动力学的影响。测试菌株作为模型参数。我们认为这些模型可以更好地预测亚致死性热处理后的孢子恢复，食品加工中的普遍情况以及对食品保存和安全的关注。