Effects of sporulation media and strain on thermal resistance of Bacillus cereus spores

Spores of Bacillus cereus strains ATCC 7004, ATCC 4342 and ATCC 9818 were produced in four sporulation media (Nutrient Agar supplemented with 1 ppm Mn²⁺, Fortified Nutrient Agar, Angelotti Medium and Milk Agar) and their percentages of sporulation and heat resistance parameters obtained in a wide temperature range were compared. In all conditions studied, high rates of sporulation were obtained. Clear differences among D-values for spores produced in the four media were observed. the medium which yielded the most resistant spores and the magnitude with which the sporulation medium affected D-values was different for each strain. z-Values of the three strains were not influenced by the medium used to obtain spores.     

Modeling the influence of electron beam irradiation on the heat resistance of Bacillus cereus spores

The effect of electron beam irradiation (EBI) on Bacillus cereus spore heat resistance was investigated. Irradiation with accelerated electrons had an important heat-sensitizing effect on distilled-water spore suspensions. After irradiation doses of 1.3, 3.1, or 5.7 kGy followed by heating at 90 degrees C, calculated D(90)-values for strains Escuela Politécnica Superior de Orihuela (EPSO)-41WR and EPSO-50UR were reduced more than 1.3, 2.4, and 4.6 times, respectively. Plots of calculated log D(T)-values versus irradiation doses (1.3, 3.1, and 5.7 kGy) yielded straight parallel lines for the 85-100 degrees C heating temperature range, which made it possible to develop an equation to predict the changes in heat sensitivity of B. cereus spores that occurred with changing irradiation dose. Radiation-induced heat-sensitivity was characterized by a z(EBI)-value which was determined as the irradiation dose that should be required to reduce the decimal reduction time (D(T)) by one log(10) cycle when log(10)D(T) was plotted against irradiation treatment. A model is proposed to describe the influence of a pre-irradiation treatment with electron beams followed by heating on the heat resistance of B. cereus spores. This study also suggests the potential use of EBI followed by heating for food preservation.     

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.     

The effect of nisin on growth kinetics from activated Bacillus cereus spores in cooked rice and in milk

The growth kinetics of germinated cells from activated spores of Bacillus cereus in cooked white rice and in milk were evaluated at different temperatures for control samples and for samples with 25 microg of nisin per ml added. Nisin was applied in the form of Nisaplin (10(6) IU/g), which contained 25,000 microg of nisin per g. The length of the lag phase for cooked white rice controls was 120 h at 10 degrees C, 8 h at 25 degrees C, and 2.5 h at 33 degrees C. The generation times for cooked rice were 327.7 min at 10 degrees C, 59.0 min at 25 degrees C, and 42.3 min at 33 degrees C; those for milk without nisin were 297.0 min at 20 degrees C, 31.2 min at 30 degrees C, 28.6 min at 35 degrees C, and 33.7 min at 40 degrees C; and those for milk with nisin added were 277.2 min at 20 degrees C, 66.9 min at 30 degrees C, and 66.4 min at 35 degrees C. No development of B. cereus was observed for milk with nisin added at 40 degrees C for 12 h, in which germinated cells decreased by a decimal reduction time (D) of 4.7 h. A temperature of 45 degrees C was shown to be harmful to B. cereus, decreasing the germinated cells in both formulations with D-values of 4.3 to 4.6 h. Similar inhibition of cell growth at 40 degrees C was not observed with lower nisin concentrations.     

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.     

Antimicrobial effect of nisin and carvacrol and competition between Bacillus cereus and Bacillus circulans in vacuum-packed potato puree

A study was undertaken to investigate the growth and competition between Bacillus cereus and B. circulans strains in both BHI and ready-to-eat potato puree. In addition, antimicrobial potential of supplemented nisin and carvacrol was evaluated against inoculated B. cereus and B. circulans strains. The accomplished growth inhibition was observed for both B. cereus and B. circulans, where B. circulans strains were more sensitive. MIC values were decreased by lowering the incubation temperature in separate applications of nisin and carvacrol, while the effect of combined application of nisin and carvacrol appeared to be more obvious at higher temperatures. The overall effect of interactions between spoilage and pathogenic Bacillus spp. was dependent on the psychrotrophic character of both cultures. The complete inhibition of B. circulans in the co-culture experiments corresponded to B. cereus population density of approximately 6 log units. Microbial challenge testing in potato puree showed that, at both 7 and 10 °C, B. cereus TZ415 and B. circulans 4.1 could grow to high counts when no antimicrobial substances (nisin and carvacrol) were applied, while no visible spoilage occurred. A more pronounced antimicrobial activity of nisin and nisin–carvacrol combination was observed in potato puree compared to the BHI medium.     

Inactivation of spores of Bacillus cereus in cheese by high hydrostatic pressure with the addition of nisin or lysozyme

The objective of this work was to study high hydrostatic pressure (HHP) inactivation of spores of Bacillus cereus ATCC 9139 inoculated in model cheeses made of raw milk, together with the effects of the addition of nisin or lysozyme. The concentration of spores in model cheeses was approximately 6-log10 cfu/g of cheese. Cheeses were vacuum packed and stored at 8 degrees C. All samples except controls were submitted to a germination cycle of 60 MPa at 30 degrees C for 210 min, to a vegetative cells destruction cycle of 300 or 400 MPa at 30 degrees C for 15 min, or to both treatments. Bacillus cereus counts were measured 24 h and 15 d after HHP treatment. The combination of both cycles improved the efficiency of the whole treatment. When the second pressure-cycle was of 400 MPa, the highest inactivation (2.4 +/- 0.1 log10 cfu/g) was obtained with the presence of nisin (1.56 mg/L of milk), whereas lysozyme (22.4 mg/L of milk) did not increase sensitivity of the spores to HHP. For nisin (0.05 and 1.56 mg/L of milk), no significant differences were found between counts at 24 h and 15 d after treatment. Considering that mesophilic spore counts usually range from 2.6 to 3.0 log10 cfu/ml in raw milk, HHP at mild temperatures with the addition of nisin may be useful for improving safety and preservation of soft curd cheeses made from raw milk.     

Influence of nisin on the resistance of Bacillus anthracis sterne spores to heat and hydrostatic pressure

The influence of nisin on the heat and pressure resistance of Bacillus anthracis Sterne spores was examined. The decimal reduction times (D-value) of spores in milk (2% fat) at 80, 85, and 90 degrees C were determined. In the absence of nisin, the D-values were 30.09, 9.30, and 3.86 min, respectively. The D-values of spores heated in the presence of nisin (1 mg/ml) were not significantly different (P = 0.05). However, spores heated in the presence of nisin had a 1- to 2-log reduction in viability, after which the death kinetics became similar to those of spores in the absence of nisin. The z-values all were 11.2 degrees C regardless of the presence or absence of nisin. The pressure sensitivity of B. anthracis Sterne spores in the presence and absence of nisin also was determined. Spores treated with nisin were 10 times more pressure sensitive than were spores subjected to pressure in the absence of nisin under the conditions used in this study.     

Combined effect of nisin and carvacrol at different pH and temperature levels on the viability of different strains of Bacillus cereus.

The influence of pH and temperature on the bactericidal action of nisin and carvacrol on vegetative cells of different Bacillus cereus strains was studied. The five strains tested showed significant differences in sensitivity towards nisin, at pH 7.0 and 30 degrees C. Carvacrol concentrations of 0.3 mmol l(-1) had no effect on viability of B. cereus cells. When the same carvacrol concentration was combined with nisin, however, it resulted in a greater loss of viability of cells than when nisin was applied alone. The concentration of carvacrol played an important role on the bactericidal effect of nisin and, therefore, on the synergistic action of both compounds combined. At lower pH values (6.30 and 5.75), nisin was more active against B. cereus cells than at pH 7.0 at 30 degrees C, with a different sensitivity of the strains tested. The combined effect of nisin and carvacrol was found to be significantly different at pH 7.0 and 5.75. When the temperature was 8 degrees C, nisin was significantly less active against B. cereus IFR-NL 94-25 than at 30 degrees C, both at pH 7.0 and 6.30. At 8 degrees C, there was a significant increased effect of nisin at lower pH values. Also at this low temperature, a synergistic effect between nisin and carvacrol on B. cereus cells was observed at the pHs tested. This study indicates the potential of nisin and carvacrol at lower pHs to be used for preservation of minimally processed foods.     

Emetic toxin-producing strains of Bacillus cereus show distinct characteristics within the Bacillus cereus group

One hundred representative strains of Bacillus cereus were selected from a total collection of 372 B. cereus strains using two typing methods (RAPD and FT-IR) to investigate if emetic toxin-producing hazardous B. cereus strains possess characteristic growth and heat resistance profiles. The strains were classified into three groups: emetic toxin (cereulide)-producing strains (n=17), strains connected to diarrheal foodborne outbreaks (n=40) and food-environment strains (n=43), these latter not producing the emetic toxin. Our study revealed a shift in growth limits towards higher temperatures for the emetic strains, regardless of their origin. None of the emetic toxin-producing strains were able to grow below 10 degrees Celsius. In contrast, 11% (9 food-environment strains) out of the 83 non-emetic toxin-producing strains were able to grow at 4 degrees Celsius and 49% at 7 degrees Celsius (28 diarrheal and 13 food-environment strains). non-emetic toxin-producing strains. All emetic toxin-producing strains were able to grow at 48 degrees Celsius, but only 39% (16 diarrheal and 16 food-environment strains) of the non-emetic toxin-producing strains grew at this temperature. Spores from the emetic toxin-producing strains showed, on average, a higher heat resistance at 90 degrees Celsius and a lower germination, particularly at 7 degrees Celsius, than spores from the other strains. No difference between the three groups in their growth kinetics at 24 degrees Celsius, 37 degrees Celsius, and pH 5.0, 7.0, and 8.0 was observed. Our survey shows that emetic toxin-producing strains of B. cereus have distinct characteristics, which could have important implication for the risk assessment of the emetic type of B. cereus caused food poisoning. For instance, emetic strains still represent a special risk in heat-processed foods or preheated foods that are kept warm (in restaurants and cafeterias), but should not pose a risk in refrigerated foods.     

Modelling the overall effect of pH on the apparent heat resistance of Bacillus cereus spores.

A simple overall model is proposed to describe the effect of both the pH of the heating menstruum and the pH of the recovery medium on the apparent spore heat resistance of Bacillus cereus. Applied to foods making up both heating and recovery media, the model can be reduced to only two parameters. Its goodness of fit and its robustness enable it to be applied to the optimisation of heat treatments. However. further experiments should be undertaken to validate the model for other species and to determine the parameters related to reference species such as Clostridium botulinum.     

Empirical model building based on Weibull distribution to describe the joint effect of pH and temperature on the thermal resistance of Bacillus cereus in vegetable substrate

A total of 296 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged 'gravad' rainbow trout stored at 3 and 8 degrees C were characterised and identified using a molecular approach. The isolates were initially grouped according to their HindIII restriction endonuclease profiles and further identified to species level using an rRNA gene restriction pattern (ribotype) identification database. Lactobacillus sakei, L. curvatus and Carnobacterium piscicola were the three main species detected. Only one isolate was identified as C. divergens. Most of the carnobacteria were found in the samples stored at 3 degrees C. The relative proportion of L. sakei was higher in the samples stored at 8 degrees C.     

蜡样芽胞杆菌生长特性及大蒜精油对其抑菌活性的研究

主要研究了37℃条件下3种蜡样芽胞杆菌在营养肉汤中的生长状况,建立了37℃下蜡样芽胞杆菌在营养肉汤中的Boltzmann牛长模犁,3条牛长曲线相关系数鼯均大于0．97;检测了不同培养时间蜡样芽胞杆菌的产芽胞情况,结果表明1号菌株和14号菌株较早产芽胞,培养相同时间,产芽胞数：1号菌株,4号菌株〉标准菌株;采用牛沣杯法和平板计数法研究了大蒜精油对蜡样芽胞杆菌的抑制效果,结果表明浓度为lO。的人蒜精油对3种蜡样芽胞杆菌都有很好的抑制效果,3种菌株中l号菌株最难抑制。     

Influence of food matrix on inactivation of Bacillus cereus by combinations of nisin, pulsed electric field treatment, and carvacrol.

Carvacrol was used as a third preservative factor to enhance further the synergy between nisin and pulsed electric field (PEF) treatment against vegetative cells of Bacillus cereus. When applied simultaneously with nisin (0.04 microg/ml), carvacrol (0.5 mM) enhanced the synergy found between nisin and PEF treatment (16.7 kV/cm, 30 pulses) in potassium-N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) buffer. The influence of food ingredients on bactericidal activity was tested using skimmed milk that was diluted to 20% with sterile demineralized water. The efficacy of PEF treatment was not affected by the presence of proteins, and results found in HEPES buffer correlated well with results in milk (20%). Nisin showed less activity against B. cereus in milk. Carvacrol was not able to enhance the synergy between nisin and PEF treatment in milk, unless used in high concentrations (1.2 mM). This concentration in itself did not influence the viable count. Carvacrol did act synergistically with PEF treatment in milk, however not in HEPES buffer. This synergy was not influenced by proteins in milk, as 5% milk still allows synergy between carvacrol and PEF treatment to the same extent as 20% milk.     

Combined effect of lysozyme and nisin at different incubation temperature and mild heat treatment on the probability of time to growth of Bacillus cereus

Stochastic models can be useful to predict the risk of foodborne illness. The presence of Bacillus cereus in liquid egg can pose a serious hazard to the food industry, since a mild heat treatment cannot guarantee its complete inactivation. However, most of the information available in the scientific literature is deterministic, including growth of B. cereus. In this paper, a stochastic approach to evaluate growth of B. cereus cells influenced by different stresses (presence of nisin and lysozyme separately or in combination) was performed, using an individual-based approach of growth through OD measurements. Lag phase duration was derived from the growth curves obtained. From results obtained, histograms of the lag phase were generated and distributions were fitted. Normal and Weibull distributions were ranked as the bestfit distributions in experiments performed at 25 °C. At 16 °C, lag values (obtained in presence of combinations of both antimicrobials) were also fitted by a Gamma distribution. Predictions were compared with growth curves obtained in liquid egg exposed to mild heat, nisin and/or lysozyme to assess their validity.     

食品组分与天然抑菌剂对芽孢耐热性的影响

研究了不同浓度的NaCl、乳清蛋白、蔗糖、表没食子儿茶素没食子酸酯（EGCG）、乳酸钠和乳酸链球菌素（Nisin）以及不同pH对于嗜热脂肪芽孢杆菌芽孢耐热性的影响。结果显示,pH对芽孢耐热性有显著影响,D值随着pH的降低而逐渐减低;乳清蛋白在115℃和118℃条件下对芽孢耐热性有保护作用,但在121℃时无显著保护性;在115℃条件下,8%以内NaCl对芽孢耐热性无显著影响,但在118℃和121℃条件下,NaCl浓度在4%～8%之间时,对芽孢耐热性有降低效应;蔗糖只在10%浓度时对芽孢的耐热性有保护作用外,其余浓度对芽孢耐热性无显著影响。在三种报道的具有抑菌物质中,0.4mg/mLEGCG和4%乳酸钠对芽孢耐热性无显著影响,而Nisin则可以显著降低芽孢D值,而且随着浓度提高,对芽孢耐热性的降低效果越明显。     

大蒜提取物对蜡样芽孢杆菌抑制效果的研究

该文采用抑菌圈法研究了大蒜的水提取物、乙醇提取物和酸性水溶液的提取物对蜡样芽孢杆菌的抑制作用.结果表明大蒜的乙醇提取物的抑菌效果好过水提取物,酸性的水溶液提取物效果最好,可以较好地控制蜡样芽孢杆菌的生长,有较好的应用前景.     

The inhibition of the growth of Bacillus cereus in liver sausage

The growth of Bacillus cereus is a problem in liver sausage especially when the sausages are stored at high temperatures. Even concentrations of greater than 10(6)/g have been detected. In this study we found that when combining glucono-delta-lactone, sodium erythorbate and citric acid with sodium nitrite and salt the growth of B. cereus could be delayed or totally inhibited.     

保健食品中检出蜡样芽孢杆菌的情况分析

目的了解保健食品的卫生状况。方法依照国家标准GB/T 4789.14—2003和进出口商品检验行业标准SN 0176—92,对市场随机抽检的12个品种225批保健食品检出的芽孢杆菌采用全自动微生物分析系统仪进行鉴定研究。结果确认有12批保健食品被检出蜡样芽孢杆菌,其中检出率较高的有螺旋藻片和钙片,检出率分别为60.0%和21.1%。结论分析结果可为保健食品的生产、消毒灭菌、卫生学检验标准及监督管理等方面提供参考。