Modelling the influence of pH and organic acid types on thermal inactivation of Bacillus cereus spores

A model is proposed to describe the influence pH on the heat resistance of Bacillus cereus spores. In addition to the conventional z value, the effect of pH on the thermal resistance of spores is characterised by a z(pH) value (z(pH) is the distance of pH from a reference pH*, which leads to a 10-fold reduction of D value). The type of organic acid used for acidifying the heating medium, influences the z(pH) value. For nine organic acids, a linear relationship between the calculated z(pH) value and its lower acid pKa is observed. This relationship showed that the acid form (dissociated or undissociated) modifies the thermal spore resistance in addition to the H+ ion. The influence of acetic acid concentration on the D value at pH 7 shows the protective effect of the dissociated acid form on the heat resistance of spores. The acid concentration in the medium modified the heat resistance of spore and the z(pH) value.     

Modeling the inactivation of Bacillus subtilis spores during cold plasma sterilization

Cold plasma sterilization is an emerging non-thermal technology that is receiving great attention in the food processing area. Plasma is a neutral ionized gas composed of reactive gas species that inactivate bacteria or spores in a variety of food materials without compromising the main physico-chemical characteristics of the food. Survival curves of Bacillus subtilis spores were obtained after spore strip samples containing an initial spore population of 1.5–2.5 × 10⁶ cfu/strip were subjected to plasma treatment. The shape of the survival curves was clearly not linear indicating that spores exhibited a spectrum of inactivation resistances to the plasma treatment. A Weibull model was used to describe these curves. In order to capture the effects of the typical variability in the concentration of the inactivating reactive gas species during plasma processing, time-varying concentrations were incorporated in the calculating approach. The result was an ordinary differential equation (ODE) that was numerically solved using MATLAB. This approach was successfully applied to describe the survival of B. subtilis spores during plasma processing as well as data obtained from the literature for B. atrophaeus. Ozone was assumed the lethal reactive gas species responsible for spore inactivation.Industrial relevanceModeling plasma processing is of great interest because it may provide an accurate estimation of time and conditions required for a complete plasma-based sterilization process.     

Comparison of the thermal inactivation of Bacillus subtilis spores in foods using the modified Weibull and Bigelow equations

The association of a modified Weibull model and Bigelow model was applied to the thermal inactivation of Bacillus subtilis spores heated in phosphate buffer, milk, kayu (a Japanese style rice porridge) and soy sauce as well. The inactivation kinetics presented a light downward concave profile, the acidic pH increased the efficiency of the heat treatment but on the opposite, lesser the water activity, weaker was the efficiency. The heat treatment kinetics observed in milk, soy sauce and kayu were greatly different from each other, while no large difference between sterilized whole milk, UHT whole milk, sterilized skim milk and UHT skim milk, were observed. The model established in buffer system allowed heat treatment in milk products to be simulated although it could not be employed to describe the inactivation of B. subtilis spores in soy sauce and kayu. For these two latter products, the food itself had to be introduced in the model as a parameter. Finally, this approach combining primary model (to simulate inactivation kinetics) and secondary model (to introduce temperature, pH, a_w and food matrix effect) seemed available for food application, nevertheless validations of results such as challenge-tests, must be performed before it is put to routine use.     

不同抑菌剂对枯草芽孢杆菌芽孢动态生长的影响

目的 研究不同抑菌剂对枯草芽孢杆菌(Bacillus subtilis)芽孢生长的影响及作用阶段。方法 本研究选取枯草芽孢杆菌芽孢为研究对象,考查了茶多酚、Nisin、亚硝酸盐、双乙酸钠、乙二胺四乙酸盐5种抑菌剂对枯草芽孢杆菌芽孢存活量、萌发特性、内容物泄漏量、结构变化的影响。结果 5种抑菌剂均能降低枯草芽孢杆菌芽孢的存活率,且不同抑菌剂对枯草芽孢杆菌芽孢生长的主要抑制阶段存在差异。其中乙二胺四乙酸盐、茶多酚在芽孢萌发阶段的作用效果相对较为显著,与芽孢悬浊液共孵育60 min后吸光度分别下降至90.61%、90.74%, 2,6-吡啶二羧酸释放量分别为21.18%、21.92%,孵育12 h后仍有部分芽孢保持“相亮”状态。亚硝酸盐、双乙酸钠、Nisin主要在萌发后发挥抑制作用,其中亚硝酸盐、双乙酸钠能使芽孢在后期生长过程中内膜通透性增大,并导致芽孢核内物质发生泄漏。拉曼光谱显示茶多酚、亚硝酸盐能够使得芽孢生长过程中蛋白质、脂质等成分及其结构发生变化,使萌发后的芽孢无法继续生长形成营养体。结论 乙二胺四乙酸盐、茶多酚在芽孢萌发阶段发挥主要作用,亚硝酸盐、双乙酸钠、Nisin在萌发后及营养...     

Thermal inactivation of Bacillus cereus spores formed at different temperatures

The effects of the sporulation temperature in the range 20-45 degrees C on the D and z values of three isolates of Bacillus cereus (ATCC 4342, 7004 and 9818) were investigated. The strains were found to differ in their response. Higher D100 values (around 10-fold) were obtained with isolates 4342 and 9818 when the sporulation temperature increased from 20 to 45 degrees C. With isolate 7004 (the least heat resistant of the three strains), however, the most thermal tolerant spores were obtained at 35 degrees C. The z values were not significantly modified (P > 0.05) by the sporulation temperature. Mean z values of 7.46+/-0.22 degrees C for isolate 4342, 7.80+/-0.40 degrees C for 7004 and 8.09+/-0.33 degrees C for 9818 were obtained.     

Thermosonication versus thermal processing of skim milk and beef slurry: Modeling the inactivation kinetics of psychrotrophic Bacillus cereus spores

Non-thermal processed foods are generally cold stored and distributed. The use of ultrasound for food preservation has attracted the interest of many research groups. In the current study, the thermosonication (TS, simultaneous ultrasound and thermal process) inactivation of psychrotrophic Bacillus cereus spores was investigated (24 kHz, 210 μm, 0.33 W/mL or W/g). First, the effectiveness of a 1.5 min TS process at 70 °C in skim milk, beef slurry, cheese slurry, and rice porridge was investigated. The TS was more effective than sole thermal treatment in reducing B. cereus spores in rice porridge, beef slurry and cheese slurry by 7, 6, and 4 fold, respectively. Then, the first-order D- and z-values for TS and thermal processing in skim milk and beef slurry, and the best model to fit TS inactivation of B. cereus spores in beef slurry were determined. The D_70 °C-values in skim milk were 2.9 min for TS and 8.6 min for the thermal treatment. And in beef slurry, values of 0.4 min for TS and 2.3 min for thermal were estimated. It was found that the Log-logistic model better described the TS spore inactivation in beef slurry. The ultrasound technology required 20–30 °C lower temperatures for the same spore inactivation, which resulted in better food quality and energy saving gains.     

Quantitative evaluation of the synergistic sequential inactivation of Bacillus subtilis spores with ozone followed by chlorine

This investigation of sequential disinfection, with ozone followed by free chlorine, was carried out using Bacillus subtilis spores, to make a quantitative evaluation and to improve the mechanistic understanding of their synergistic effect. This study shows that the extent of the synergistic effect in the inactivation of Bacillus subtilis spores appears to be dependent upon the level of preozonation. However, when the ozone pretreatment level exceeded the lag phase of the ozone inactivation curve, the chlorine inactivation curves were almost identical regardless of the level of preozonation. When this sequential disinfection was performed in the reverse order, no enhanced disinfection was observed. This difference, depending on the order of disinfectant application in sequential disinfection, was explained in terms of the enhanced disinfection being the result of the greater intracellular diffusion of free chlorine, caused by the cell surface disruption induced by ozone. The practical implications of this synergistic sequential inactivation with ozone followed by free chlorine were discussed, along with the issue of selecting the amount of each oxidant to use in water treatment plants, to achieve a specific level of microorganism inactivation.     

Surfactin对蜡样芽孢杆菌芽孢灭活作用途径研究

目的研究Surfactin对蜡样芽孢杆菌芽孢灭活机制。方法将不同浓度Surfactin和芽孢相互作用后,利用紫外分光光度计检测芽孢内大分子组分(如蛋白质、核酸等)的泄漏情况。结果研究发现300和450μg/mL的Surfactin处理后的菌悬液中可明显检测到核酸和蛋白质的泄露。随着时间的延长,加入Surfactin后可使其DNA紫外吸收光谱发生变化,导致DNA最大吸收峰发生红移现象,最初的最大吸收峰253.5 nm移至了257 nm,产生了明显的增色效应。流式细胞的结果说明在最低抑菌浓度(minimal inhibitory concentration,MIC)的情况下,芽孢的膜结构没有明显破损,核内遗传物质没有明显泄露,随着时间延长,泄漏明显;在2～3 MIC浓度的情况下,芽孢的膜结构有明显破损,核内遗传物质有明显泄露。结论 Surfactin通过破坏芽孢壁的完整性、与DNA结合对蜡样芽孢杆菌芽孢发挥灭活效应。     

High-pressure inactivation of Bacillus cereus spores in the presence of argon

We investigated the high-pressure inactivation of Bacillus cereus spores in water containing argon. At the pressure of 600 MPa, addition of argon accelerated the inactivation of spores at 20 degrees C, but had no effect on the inactivation at 40 degrees C. The influence of added argon on inactivation of the spores was marked under conditions with a strong 'water ordering' effect. The pressure resistance of B. cereus spores was thus shown to be affected by 'water ordering'.     

The impact of high pressure and temperature on bacterial spores: inactivation mechanisms of Bacillus subtilis above 500 MPa

Abstract: High‐pressure thermal sterilization (HPTS) is an emerging technology to produce shelf stable low acid foods. Pressures below 300 MPa can induce spore germination by triggering germination receptors. Pressures above 500 MPa could directly induce a Ca⁺²‐dipicolinic acid (DPA) release, which triggers the cortex‐lytic enzymes (CLEs). It has been argued that the activated CLEs could be inactivated under HPTS conditions. To test this claim, a wild‐type strain and 2 strains of Bacillus subtilis spores lacking germinant receptors and one of 2 CLEs were treated simultaneously from 550 to 700 MPa and 37 to 80 C (slow compression) and at 60 to 80 C up to 1 GPa (fast compression). Besides, an additional heat treatment to determine the amount of germinated cells, we added TbCl₃ to detect the amount of DPA released from the spore core via fluorescent measurement. After pressure treatment for 120 min at 550 MPa and 37 °C, no inactivation was observed for the wild‐type strain. The amount of released DPA correlated to the amount of germinated spores, but always higher compared to the belonging cell count after pressure treatment. The release of DPA and the increase of heat‐sensitive spores confirm that the inactivation mechanism during HPTS passes through the physiological states: (1) dormancy, (2) activation, and (3) inactivation. As the intensity of treatment increased, inactivation of all spore strains also strongly increased (up to ?5.7 log₁₀), and we found only a slight increase in the inactivation of one of the CLE (sleB). Furthermore, above a certain threshold pressure, temperature became the dominant influence on germination rate. Practical Application: The continuous increase of high‐pressure (HP) research over the last several decades has already generated an impressive number of commercially available HP pasteurized products. Furthermore, research helped to provoke the certification of a pressure‐assisted thermal sterilization process by the U.S. FDA in February 2009. However, this promising sterilization technology has not yet been applied in industrial settings. An improved understanding of spore inactivation mechanisms and the ability to calculate desired inactivation levels will help to make this technology available for pilot studies and commercialization at an industrial scale. Moreover, if the synergy between pressure and elevated temperature on the inactivation rate could be identified, clarification of the underlying inactivation mechanism during HP thermal sterilization could help to further optimize the process of this emerging technology.     

超声波对液态奶中枯草芽孢杆菌的杀菌作用

利用超声波协同热处理对液态奶中的枯草芽孢杆菌进行杀菌实验,并与传统方法做比较。研究了经不同功率、时间、温度的超声波处理后液态奶的杀菌效果。结果表明:经1400 W,180 s,60℃条件下杀菌率达到了97.96%,经1400 W,60 s,60℃条件下杀菌率达到了96.77%,达到了巴氏杀菌的效果。同时,该条件下超声作用后再经巴氏处理,杀菌率达到98.71%,对比于巴氏杀菌,其菌落数降低了60.24%。与热杀菌相比,超声波杀菌温度低,时间短,效率高。     

Effect of heat activation and inactivation conditions on germination and thermal resistance parameters of Bacillus cereus spores

The effect of isothermal and non-isothermal heat activation on germination and thermoresistance of two strains of Bacillus cereus spores was studied. Results indicated that the germination after isothermal activation was lower than after non-isothermal heating. The activation rate affected the z value, which increased with faster heating rates. For each temperature and inactivation rate, the non-isothermal activation at rate of 2 degrees C/min resulted in larger D values (D90 = 4.70 min) than isothermal activation (D90 = 4.04 min). The two mathematical equations used to analyse non-isothermal data produced similar predicted D and z values, nevertheless the Hayakawa equation modified in this work for non-linear regression analysis, requires less computational effort.     

蛋清溶菌酶对枯草芽孢杆菌芽孢萌发的影响

研究了在不同培养温度下溶菌酶对枯草芽孢杆菌芽孢萌发的抑制作用。溶菌酶浓度为0.5mg/mL,采用胰蛋白胨大豆营养肉汤培养基进行培养,用稀释平板计数法测定36h内的生长曲线。结果表明:在33℃条件下添加溶菌酶的处理组的迟滞期比对照组延长了8h,传代时间延长了15min;在25℃条件下,处理组的迟滞期比对照组延长了10h,传代时间延长了12min。到达稳定期后,处理组的菌落总数始终低于对照组水平。在10℃条件下,对照组芽孢的萌发也经历了较长的迟滞期,传代时间延长至194min;但在36h内处理组始终没有观察到芽孢萌发后的指数生长期,芽孢一直处于被抑制的状态。研究结果表明,溶菌酶对枯草芽孢杆菌芽孢的萌发具有明显的抑制作用,而且在低温条件下溶菌酶仍然具有抑菌活性。      

Comparative effect of different test methodologies on Bacillus coagulans spores inactivation kinetics in tomato pulp under isothermal conditions

Heat resistant micro‐organisms are an ongoing challenge to the food industry. Various factors may influence the heat resistance of micro‐organisms including type and strain; the environmental influences during cell and spore formations and during heat exposure; and the equipment and test tools used to perform the experimental process. In an attempt to analyse the influence of different test tools used on the heat inactivation processes, this study aimed to define the isothermal inactivation kinetics of Bacillus coagulans spores in tomato pulp at different temperatures and compare the inactivation of this bacterium when thermal death time (TDT) and capillary tube methods were used. Temperature ranges from 95 °C to 120 °C were studied, and inactivation kinetic parameters were estimated through the application of primary models. TDT inactivation curves consisted of shoulder and linear decline, while capillary method inactivation curves consisted of shoulder, linear decline and long tail. A secondary model was used to describe the influence of the temperature on spore inactivation parameters. The results showed test methods are at least as important in determining thermal processes as the micro‐organisms and media used.     

Minimizing the level of Bacillus cereus spores in farm tank milk

In a year-long survey on 24 Dutch farms, Bacillus cereus spore concentrations were measured in farm tank milk (FTM), feces, bedding material, mixed grass and corn silage, and soil from the pasture. The aim of this study was to determine, in practice, factors affecting the concentration of B. cereus spores in FTM throughout the year. In addition, the results of the survey were used in combination with a previously published modeling study to determine requirements for a strategy to control B. cereus spore concentrations in FTM below the MSL of 3 log₁₀ spores/L. The B. cereus spore concentration in FTM was 1.2 ± 0.05 log₁₀ spores/L and in none of samples was the concentration above the MSL. The spore concentration in soil (4.9 ± 0.04 log₁₀ spores/g) was more than 100-fold higher than the concentration in feces (2.2 ± 0.05 log₁₀ spores/g), bedding material (2.8 ± 0.07 log₁₀ spores/g), and mixed silage (2.4 ± 0.07 log₁₀ spores/g). The spore concentration in FTM increased between July and September compared with the rest of the year (0.5 ± 0.02 log₁₀ spores/L difference). In this period, comparable increases of the concentrations in feces (0.4 ± 0.03 log₁₀ spores/g), bedding material (0.5 ± 0.05 log₁₀ spores/g), and mixed silage (0.4 ± 0.05 log₁₀ spores/g) were found. The increased B. cereus spore concentration in FTM was not related to the grazing of cows. Significant correlations were found between the spore concentrations in FTM and feces (r = 0.51) and in feces and mixed silage (r = 0.43) when the cows grazed. The increased concentrations during summer could be explained by an increased growth of B. cereus due to the higher temperatures. We concluded that year-round B. cereus spores were predominantly transmitted from feeds, via feces, to FTM. Farmers should take measures that minimize the transmission of spores via this route by ensuring low initial contamination levels in the feeds (<3 log₁₀ spores/g) and by preventing growth of B. cereus in the farm environment. In addition, because of the extremely high B. cereus spore concentrations in soil, the contamination of teats with soil needs to be prevented.     

The impact of different process gas compositions on the inactivation effect of an atmospheric pressure plasma jet on Bacillus spores

Atmospheric plasma provides the advantages of high microbial inactivation that can be performed under ambient conditions. It is consequently regarded as potential alternative to traditional food preservation methods.In this study we systematically tested the influence of argon as plasma carrier gas with admixtures of oxygen (0–0.34 vol.%) and nitrogen (0–0.3 vol.%) towards its emission intensity of UV-C light, excited OH and N₂-species and atomic oxygen. A mixture of argon, 0.135 vol.% oxygen and 0.2 vol.% nitrogen emitted four fold more UV photons than pure argon. However, sporicidal effects on Bacillus atrophaeus (3.1 log₁₀) and Bacillus subtilis spores (2.4 log₁₀) were found for pure argon plasma, which were similar as compared to the sporicidal effect of the plasma with highest UV-emission. To distinguish lethal effects caused by emitted UV-light and reactive species, UV-sensitive mutant spore strains (PS578 and FB122) were exposed to plasmas with different UV-emission intensities and a significant impact of UV-light on the first phase of spore inactivation was confirmed.Industrial relevanceAs an efficient method for the inactivation of microorganisms at low temperatures and atmospheric pressure, plasma is already commercially used for the sterilization of medical devices. The results presented in this study could be useful for a process optimization regardless if the plasma is applied for food preservation or surface decontamination. Especially the impact of emitted UV photons from the plasma on the first inactivation phase of endospores attached to surfaces, depicts a high potential of such plasmas for a rapid spore inactivation.     

Effect of heat treatment of milk on activation of Bacillus spores

The quality and shelf life of fluid milk products are dependent on the amount and type of microorganisms present following pasteurization. This study evaluated the effects of different pasteurization processes on the microbial populations in fluid milk. The objective was to determine whether certain pasteurization processes lead to an increase in the amount of bacteria present in pasteurized milk by activating Bacillus spores. Samples of raw milk were collected on the day of arrival at the dairy plant. The samples were pasteurized at 63 degrees C for 30 min (low temperature, long time), 72 degrees C for 15 s (high temperature, short time), 76 degrees C for 15 s, and 82 degrees C for 30 min. The pasteurized samples were then stored at 6 and 10 degrees C for 14 days. The samples were analyzed for standard plate count and Bacillus count immediately after pasteurization and after 14 days of storage. Pasteurization of milk at 72 and 76 degrees C significantly (P < 0.05) increased the amount of Bacillus spore activation over that of 63 degrees C. There was no detection of Bacillus in initial samples pasteurized at 82 degrees C for 30 min, but Bacillus was present in samples after storage for 14 days, indicating that injury and recovery time preceded growth. The majority of isolates were characterized as Bacillus mycoides and not Bacillus cereus, suggesting that this organism might be more a cause of sweet curdling of fluid milk than previously reported.     

Glassy state in Bacillus subtilis spores analyzed by differential scanning calorimetry

We report four cases of blood cultures testing positive for yeast strains belonging to the species Saccharomyces cerevisiae. Using molecular techniques, RFLP of mtDNA and δ-PCR amplification, we show the association of two of the isolates with non-clinical strains. Specifically, with two commercial bread-making strains and the therapeutic S. boulardii strain. The association of S. boulardii with cases of fungemia has been reported previously. Nevertheless, this is the first time that a baker's yeast has been isolated from blood.