Monte Carlo Simulation to establish the effect of pH, temperature and heating time on the final load of Bacillus stearothermophilus spores

Bacillus stearothermophilus heat treatment studies at different pH levels were carried out in order to predict the final microbiological load as affected by the temperature and pH treatment. For that, distribution functions were obtained for the initial number of microorganisms, the decimal reduction coefficient, the z _T and the z _pH parameters, and Monte Carlo Simulation was used as a tool to determine this microbial load after a heat treatment introducing in this way the variability and uncertainty. The mean value of the predictions made by Monte Carlo Simulation, when compared with independent data, rendered Accuracy Factors close to 1, values close to 1 indicated good predictions. Bias values were close to 1, but some values were below 1 and others above 1. The tornado graphs indicated that the main factor contributing to the final load after the heat treatment was the initial number of microorganisms followed by the temperature and pH.     

The effect of inoculum size on the lag phase of Listeria monocytogenes.

The effect of inoculum size on population lag times of Listeria monocytogenes was investigated using the Bioscreen automated microtitre plate incubator and reader. Under optimum conditions, lag times were little affected by inoculum size and there was little variation between replicate inocula even at very low cell numbers. However, in media containing inhibitory concentrations of NaCl, both the mean lag time and variation between replicate inocula increased as the inoculum size became smaller. The variation in lag time of cells within a population was investigated in more detail by measuring the distribution of detection times from 64 replicate inocula containing only one or two cells capable of initiating growth. The variance of the lag time distribution increased with increasing salt concentration and was greater in exponential than in stationary phase inocula. The number of cells required to initiate growth increased from one cell under optimum conditions to 10(5) cells in medium with 1.8 M NaCl. The addition of spent medium from a stationary phase culture reduced the variance and decreased lag times. The ability to initiate growth under severe salt stress appears to depend on the presence of a resistant sub-fraction of the population, although high cell densities assist adaptation of those resistant cells to the unfavourable growth conditions by some unspecified medium conditioning effect. These results are relevant to the prediction of lag times and probability of growth from low numbers of stressed cells in food.     

The effect of inoculum size on the growth of Penicillium expansum in apples

Penicillium expansum is the most important cause of blue mould rot, a major post-harvest disease of apples worldwide. The objective of this study was to evaluate the effect of the inoculum size on the germination and growth parameters of P. expansum under different storage conditions in apples. Growth of P. expansum was observed in more than 90% of the inoculations, when the inoculum was equal or higher than 2x10(4) spores. The use of a low inoculum level resulted in longer lag phases and a larger variability of the estimated lag phase. This indicates that more replications are necessary to estimate the lag phase of the mould in the specified circumstances. At lower temperature, more inoculum was necessary to reduce the variability of the estimated lag phase, showing that this effect is temperature dependent. Moreover, the effect of the inoculum level on the lag phase is even more pronounced for a slower growing strain. These results imply that the inoculum size influences the estimated growth parameters and should be considered in quantitative risk assessments and for the design of challenge tests and experiments to gather data for predictive growth models.     

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。     

Fumonisin production on irradiated corn kernels: effect of inoculum size.

Production of fumonisins B1, B2, and B3 by Fusarium moniliforme was evaluated on irradiated corn kernels inoculated with different spore concentrations (10, 10(2), 10(3), 10(5), and 10(6)), a water activity of 0.97, and a temperature of 25 degrees C. There was a direct relationship between the level of toxin produced and inoculum size. The highest levels of total fumonisin produced after 35 days of incubation were 5,028 and 9,063 ng/g at 10(5) and 10(6) spores per ml, respectively. The pattern of fumonisin production (FB1 > FB2 > FB3) in cultures growing from different inocula was not affected during the 35 days of incubation. The ratio between FB2 and FB1 varied from 0.15 to 0.42, whereas the ratio between FB3 and FB1 varied from 0.34 to 0.87.     

Application of artificial neural networks to describe the combined effect of pH and NaCl on the heat resistance of Bacillus stearothermophilus

A model for prediction of bacterial spore inactivation was developed. The influence of temperature, pH and NaCl on the heat resistance of Bacillus stearothermophilus spores was described using low-complexity, black box models based on artificial neural networks. Literature data were used to build and train the neural network, and new experimental data were used to evaluate it. The neural network models gave better predictions than the classical quadratic response surface model in all the experiments tried. When the neural networks were evaluated using new experimental data, also good predictions were obtained, providing fail-safe predictions of D values in all cases. The weights and biases values of neurons of the neural network that gave the best results are presented, so the reader can use the model for their own purposes. The use of this non-linear modelling technique makes it possible to describe more accurately interacting effects of environmental factors when compared with classical predictive microbial models.     

热压协同对嗜热脂肪芽孢杆菌芽孢灭活动力学的研究

研究了热压协同(500～600MPa,80-90℃)时嗜热脂肪芽孢杆菌芽孢在磷酸缓冲液和牛奶(经超高温灭菌)中灭活的动力学规律,并对超高静压的升压过程及相应的灭活效果进行了研究.结果表明,升压过程对嗜热脂肪芽孢杆菌芽孢灭活的影响不能忽略,且随压力增加这种效果越强,最高使其下降1.23个数量级;嗜热脂肪芽孢杆菌在牛奶中比在磷酸缓冲液中有更高的抗性;在3种拟合模型(线性、Weibull和Log-logistic模型)中,线性模型不适合模拟这些存活曲线,而Log-logistic模型能更好地模拟这些存活曲线,其次是Weibull模型.     

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R² values of 0.9969 and 0.9958 respectively. Linear correlations between the log₁₀ input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10¹ CFU/mL to 1.65 × 10⁶ CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.     

Bacillus stearothermophilus Spore Recovery Altered by Media Concentration and Formulation

Bacillus stearothermophilus spores, subjected to a range of heating times and temperatures, were recovered using dextrose tryptone agar and soybean casein digest agar, with and without additives, at normal and reduced concentrations. For very mild heat treatments, for all media tested, there was essentially no difference in number of spores recovered. For severely heat-stressed B. stearothermophilus spores, reduction in the media manufacturer's recommended concentration to one-tenth significantly increased recovery; the 0.1 concentration media produced a 2- to 60-fold increase in numbers recovered. Recovery of heat-stressed spores using the 0.1 concentration media increased with increasing heating time and temperature.     

An explanation for the effect of inoculum size on MIC and the growth/no growth interface

The inoculum effect (IE) is the phenomenon observed where changes in the inoculum size used in an experiment alters the outcome with respect to, for example, the minimum inhibitory concentration of an antimicrobial or the growth/no growth boundary for a given set of environmental conditions. Various hypotheses exist as to the cause of the IE such as population heterogeneity and quorum sensing, as well as the null hypothesis - that it is artefactual. Time to detection experiments (TTD) were carried out on different initial inoculum sizes of several bacterial species (Aeromonas hydrophila, Enterobacter sakazakii, Salmonella Poona, Escherichia coli and Listeria innocua) when challenged with different pH and with combined pH and sodium acetate. Data were modelled using a modification to a Gamma model (Lambert and Bidlas 2007, Int. J. Food Microbiology 115, 204-213), taking into account the inoculum size dependency on the TTD obtained under ideal conditions. The model suggests that changes in minimum inhibitory concentration (MIC) or in the growth-no growth boundary with respect to inoculum size are due to using a smaller or larger inoculum (i.e. is directly related to microbial number) and is not due to other, suggested, phenomena. The model used further suggests that the effect of a changing inoculum size can be modelled independently of any other factor, which implies that a simple 1 to 2-day experiment measuring the TTD of various initial inocula can be used as an adjunct to currently available models.     

The effect of incubation temperature and inoculum size on growth of salmonellae in minced beef

Growth rates and lag times of Salmonella organisms were determined in minced beef at temperatures between 10 and 35 degrees C. A mixture of serotypes containing antibiotic-resistance markers was used to allow small numbers of salmonellae to be counted in the presence of an excess of competing organisms. Maximum growth rates and lag times of salmonellae were unaffected by inoculum size. The effect of temperature on growth rate could be described by the simple square root relationship of Ratkowsky et al. (J. Bacteriol. 149, 1-5, 1982).     

碳源诱导嗜热脂肪芽孢杆菌生产淀粉分支酶的研究

利用嗜热脂肪芽孢杆菌（Bacillus stearothermophilus）生产淀粉分支酶,研究不同碳源种类及浓度对嗜热脂肪芽孢杆菌产酶性能的影响。结果表明,以0.20%麦芽糊精为碳源时,淀粉分支酶的酶活最高,达20.02 U/mL,是其他碳源的2～10倍。采用最优培养基在小试试验的基础上进行了扩大培养中试试验,淀粉分支酶酶活稳定,为19.87 U/mL,是小试试验的1.1倍。     

Bioproduction of Perillyl Alcohol and Related Monoterpenes by Isolates of Bacillus stearothermophilus

Bacillus stearothermophilus BR388 was isolated from orange peel by an enrichment culture using (+)-limonene. The thermophilic isolate exhibited growth between 45 and 68°C., with optimum growth near 55°C. BR388 could grow on limonene as a sole carbon source, but grew and degraded limonene more effectively when supplemented with small amounts of yeast extract. Perillyl alcohol was the major conversion product, with α-terpineol and perillyl aldehyde as minor products. Strains of B. stearothermophilus previously isolated from aromatic enrichments also grew on limonene, but had higher sensitivity to limonene toxicity than did BR388.     

Factors affecting the heat resistance of Bacillus stearothermophilus spores

Summary. Maximum colony counts of unheated and heated spores of Bacillus stearothermophilus were obtained after incubation at 50-65°C and 45-50°C, respectively. the composition of the plating medium was found to have a marked effect upon recovery of unheated and heated spores. the effects of diluent and incubation time on recovery have also been investigated.     

嗜热脂肪芽孢杆菌试管法检测牛乳中β-内酰胺类抗生素残留

采用嗜热脂肪芽孢杆菌试管法对牛乳中6种最为常见的β-内酰胺类抗生素残留进行检测,指出嗜热脂肪芽孢杆菌试管法为半定量检测法,对牛乳中β-内酰胺类抗生素残留具有较好的灵敏性.牛乳中青霉素G、氨苄青霉素、羟氨苄青霉素的检测限为6μg/kg,头孢氨苄的检测限为150μg/kg,苯唑青霉素的检测限为15μg/kg、头孢匹林的检测限为30μg/kg.在检测时间、操作难易程度上较BSDA法具有一定的优越性,可应用于牛乳中β-内酰胺类抗生素残留的检测.     

Reduction of Bacillus subtilis, Bacillus stearothermophilus and Streptococcus faecalis in meat batters by temperature-high hydrostatic pressure pasteurization

People have a growing preference for fresh, healthy, palatable and nutritious meals and drinks. However, as food deterioration is a constant threat along the entire food chain, food preservation remains as necessary now as in the past. High pressure processing is one of the emerging technologies being studied as an alternative to the classical pasteurization and sterilization treatments of food. Samples of fried minced pork meat were inoculated with strains of Streptococcus faecalis and with sporulating microorganisms like Bacillus subtilis and stearothermophilus. The samples were subjected to several combined temperature-high pressure treatments predicted by the mathematical model applied in Response Surface Methodology. Using the "Box-Behnken" concept, the number of tests for a whole area of pressure-temperature-time-combinations (pressure variation: 50-400 MPa, temperature variation 20-80°C, time variation 1-60 min) could be limited to 15. In the center point of the model, the experimental combination was performed in triple to estimate the experimental variance. All the tests were executed in a randomized order to exclude the disturbing effect of environmental factors. Microbial analysis revealed for each microorganism an important reduction in total plate count, demonstrating a superior pressure resistance of the sporulating microorganisms in comparison with the most pressure resistant vegetative species Streptococcus faecalis. The effect of the medium composition could be neglected, showing little protective effect of, e.g. the fat fraction as seen in heat preservation techniques.