The effect of pH, salt concentration and temperature on the survival and growth of Listeria monocytogenes

Factorially designed experiments have been used to study the growth and survival of Listeria monocytogenes in different combinations of pH and salt concentrations at ambient and chill temperatures. Survival at low pH and high salt concentration was strongly temperature dependent. The minimum pH values that allowed survival after 4 weeks from an initial 10⁴ cells were 4·66 at 30†C, 4·36 at 10†C and 4·19 at 5†C. These limits were salt dependent, low (4–6%) salt concentrations improved and higher concentrations reduced survival at limiting pH values. The lowest pH that allowed a 100-fold increase in cell numbers within 60 d was 4·66 at 30†C but this was increased to 4·83 at 10†C. At 5†C growth occurred at pH 7·0 but not at pH 5·13. Simple predictive models describing the effect of hydrogen-ion and salt concentration on the time for at least a 100-fold increase in numbers at 10†C and 30†C were constructed after analysis of the results for a least squares fit to a quadratic model. The interactions between salt and hydrogen-ion concentration on growth were found to be purely additive.     

Survival and growth of Cronobacter species (Enterobacter sakazakii) in wheat-based infant follow-on formulas

Aims:  To determine the survival and growth characteristics of Cronobacter species ( Enterobacter sakazakii ) in infant wheat-based formulas reconstituted with water, milk, grape juice or apple juice during storage.
Methods and Results:  Infant wheat-based formulas were reconstituted with water, ultra high temperature milk, pasteurized grape or apple juices. The reconstituted formulas were inoculated with Cronobacter sakazakii and Cronobacter muytjensii and stored at 4, 25 or 37°C for up to 24 h. At 25 and 37°C, Cronobacter grew more (>5 log₁₀) in formulas reconstituted with water or milk than those prepared with grape or apple juices ( c. 2–3 log₁₀). The organism persisted, but did not grow in any formulas stored at 4°C. Formulas reconstituted with water and milk decreased from pH 6·0 to 4·8–5·0 after 24 h, whereas the pH of the formulas reconstituted with fruit juices remained at their initial pH values, c. pH 4·8–5·0.
Conclusions:  Cronobacter sakazakii and C. muytjensii can grow in reconstituted wheat-based formulas. If not immediately consumed, these formulas should be stored at refrigeration temperatures to reduce the risk of infant infection.
Significance and Impact of the Study:  The results of this study will be of use to regulatory agencies and infant formula producers to recommend storage conditions that reduce the growth of Cronobacter in infant wheat-based formulas.     

Modelling the effectiveness of a natural antimicrobial on Salmonella enteritidis as a function of concentration, temperature and pH, using conductance measurements

The growth of Salmonella enteritidis in a brain heart infusion medium was monitored using the traditional viable count method and by conductance measurements using a Rabit impedance instrument. Growth curves (log₁₀ cfu ml⁻¹ vs time) at three different concentrations of oleuropein (0, 0·2 and 0·8%), pH values in the range of 5–8 and incubation temperatures from 22 to 42 °C were modelled using the Gompertz equation. A good correlation between the maximum growth rate from the viable count method and the maximum slope of the conductance curve from the impedance instrument was established. Based on this correlation, the maximum specific growth rate of Salm. enteritidis was modelled as a function of the oleuropein concentration, initial pH values and the incubation temperature with a quadratic equation, using a new, larger dataset of growth measurements by conductance. The developed model was validated by statistical comparison of predicted growth rates with growth rates determined by the viable count method, within the limits of the antimicrobial, pH and temperature domain.     

The fate of Bacillus anthracis in unpasteurized and pasteurized milk

Growth of Listeria monocytogenes at pH 5·0 did not increase growth of the organism at pH 7·0 after exposure to low pH (3·0, 3·5), compared with cells initially grown at pH 7·0. However, growth at pH 5·0 significantly increased the survival of cells at low pH as determined by plate counts compared with cells grown at neutral pH. Thus, pH adaptation not only occurs in enteric bacteria but also in this Gram-positive organism. Alterations in the cytoplasmic membrane could be responsible.     

Inactivation effect of X-ray treatments on Cronobacter species (Enterobacter sakazakii) in tryptic soy broth, skim milk, low-fat milk and whole-fat milk*

Aims:  To determine the inactivation effect of X-ray treatments on Cronobacter ( E. sakazakii ) in tryptic soy broth (TSB), skim milk (0% fat), low-fat milk (1% and 2%) and whole-fat milk (3·5%).
Methods and Results:  X-rays were produced using the RS 2400 generator system (Rad Source Technologies Inc.). Cronobacter (in TSB), inoculated skim milk (0% fat), low-fat milk (1% and 2% fat) and whole-fat milk (3·5% fat) were treated with 0·0, 0·1, 0·5, 0·75, 1·0, 2·0, 3·0, 4·0, 5·0 and 6·0 kGy X-ray doses. Surviving bacteria in the TSB and inoculated milk, before and after treatment, were enumerated using plating method onto trypticase soy agar. Greater than 7·0-log CFU reduction in Cronobacter population was observed with 4·0, 5·0, 6·0, 6·0 and 6·0 kGy X-ray in the TSB, skim milk, 1% fat milk, 2% fat milk and 3·5% fat milk, respectively.
Conclusions:  Treatment with X-rays significantly ( P  <   0·05) reduced Cronobacter to less than detectable limits (<1 log CFU ml⁻¹) in skim milk at 5·0 kGy and milk with 1% fat content and greater at 6·0 kGy dose levels. The D-value for Cronobacter in TSB was significantly ( P  <   0·05) lower than those in milk samples.
Significance and Impact of the Study:  Treatment with X-rays could be an effective and safe alternative technology to control pathogenic bacteria ( Cronobacter ) in the dairy industry.     

Use of the molecular typing methods to evaluate the control ofListeria monocytogenes contamination in a raw milk and dairy products

Nineteen serogroup 1/2a Listeria monocytogenes strains isolated from raw milk, dairy products and salt water in one dairy were analyzed. Pulsed field gel electrophoresis (PFGE) and ribotyping were used to determine whether these strains isolated over a 8-month period are epidemiologically related. The samples of raw milk were contaminated by different L. monocytogenes clones. The clones isolated from dairy products (with the exception of one sample) and salt water were identical. Comparative genetic analysis of the clones isolated from raw milk, salt water and dairy products revealed the source of contamination and identified the L. monocytogenes strain involved in this process.     

The association of prepartum urine pH,plasma total calcium concentration at calving and postpartum diseases in Holstein dairy cattle

The use of anionic salts to prevent milk fever in dairy cattle has been an effective nutritional strategy; however, the degree of acidification that determines the most acceptable productive responses and well-being of the cow is still a controversial topic. The objective of this study was to assess urine pH in prepartum Holstein cows fed anionic diets and determine its association with plasma total Ca, Mg, P, β-hydroxyl-butyrate (BHB) concentrations at parturition and the occurrence of peripartum disorders. This investigation consisted of 2 studies. Study 1 was conducted on a grazing dairy. Between February and May 2019, 60 prepartum multiparous cows were tested for urine pH and plasma metabolite concentration at parturition. Total Ca, P, Mg and BHB at day 1 in milk (DIM) were assessed and statistically analyzed by ANOVA (models for polynomial regression). Study 2 was conducted on a drylot dairy farm. Between July 2018 and January 2019, 203 cows were evaluated for urine pH and followed-up for 30 DIM to obtain the incidence of dystocia, stillbirths, milk fever, retained fetal membranes, metritis, clinical mastitis and ketosis. Cows were categorized based on their last urine pH as group 1: pH > 7.0 (n = 135); group 2: pH between 6.0 and 7.0 (n = 46) and group 3: pH < 6.0 (n = 22). A logistic regression model for each health event was conducted considering urine pH group as the main effect. Urine sample was collected at 2.71 ± 2.84 days before parturition. In study 1, there was a quadratic effect of urine pH on total Ca. Total Ca concentration was higher between urine pH 6.0 and 7.0, while decreasing below pH 6.0 and above pH 7.0. There was a trend (P = 0.11) for a quadratic effect of urine pH on the concentration of plasma BHB at parturition. β-Hydroxyl-butyrate was lower approximately between urine pH 6.5 and 7.5. In study 2, the odds for a stillborn in cows with urine pH < 6.0 was 2.39 (95% CI = 1.06–5.40) times the odds for a stillborn in cows with urine pH ≥ 7.0. There was no association between urine pH and the other diseases. In conclusion, cows with prepartum urine pH < 6.0 and > 7.0 had lower concentration of plasma total Ca and tended to have a higher concentration of BHB. Cows with urine pH < 6.0 had a higher incidence of stillbirths than cows with urine pH > 7.0.     

Growth and reducing capacity of Listeria monocytogenes under different initial redox potential

Aims:  To determine the reducing capacity of Listeria monocytogenes and to highlight the effect of redox potential on its growth parameters.
Methods and Results:  The reducing capacity of L. monocytogenes was monitored in Brain Heart Infusion Broth media at different initial redox potential (Eh) and pH at 37°C. The effect of Eh obtained by gas flushing (air, N₂ and N₂-H₂) or by adding potassium ferricyanide and dithiotreitol in concentration from 1 to 10 mmol l⁻¹on L. monocytogenes growth parameters at pH 6·0, 7·0 and 8·0 was investigated. A total change of 539 mV (±44 mV) from an initial redox value of +330 ± 8 mV to a more negative potential in redox curves was observed resulting from L. monocytogenes growth at pH 7·0 at 37°C. A significant influence of pH and redox potential on L. monocytogenes lag phase of growth was shown ( P  < 0·05).
Conclusions:  Listeria monocytogenes exhibited longer lag phase in reducing conditions and at pH 6·0. The method used to modify the redox potential was shown to have no effect on growth parameters at pH 7·0.
Significance and Impact of the Study:  The provided information on the extending lag time and the possible delayed growth of this major pathogen in reducing conditions might be useful for its control in foods.     

Changes in the acidity and lactic acid content of 'Nono', a Nigerian cultured milk product

Changes in the pH value and lactic acid content of the uninoculated, cultured Nigerian whole milk product, 'Nono', during incubation at room temperature (27°± 2°C) for 120 h were monitored. The pH decreased from 6·78 ± 0·19 to 4·30 ± 0·11 while the lactic acid content increased from 0·32 ± 0·04% to 2·50 ± 0·02% (w/v). This was accompanied by souring and curdling of the milk particles.     

Validation of a polynomial regression model: the thermal inactivation of Bacillus subtilis spores in milk

AIMS: The predicted survival of Bacillus subtilis 168 spores from a polynomial regression equation was validated in milk. METHODS AND RESULTS: Bias factor suggested as an index of model performance was used to validate the polynomial model predictions in ultrahigh temperature (UHT) treated and sterilized whole and skim milk. Model predictions were fail safe, predicting higher D-values (decimal reduction times) in buffer than actually noted in milk. CONCLUSIONS: The D-values for spores were lower in milk as compared with those predicted in potassium phosphate buffer contrary to the popular expectation of better spore survival in complex food systems. The Bias factor, a quantitative measure of the model performance, indicated that on average the model predictions exceed the observations by 40% in the case of whole milk and by 60% in the case of skim milk. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work is an attempt to ascertain the extent of reliability that one can safely place in polynomial model predictions, without compromising on the safety or palatability of foods where it is eventually intended to be applied. The work has also highlighted the differences in the thermal inactivation pattern of spores in buffer and in milk with a possible influence of the various constituents of milk. The work will assist the dairy industry to better design thermal processes to ensure longer shelf life of dairy foods.     

Use of a Coulter Counter to Detect Discrete Changes in Cell Numbers and Volume During Growth of Escherichia coli

Escherichia coli could be detected at a concentration of 10⁵ cells/ml. Growth curves of the organism grown in filtered tryptone soya broth were made by taking viable and Coulter counts. Both curves were similar in shape and varied little during 10 h growth. Cell volume and volume distribution were seen to vary considerably however, volume reaching a stable minimum value of 0·61 μm³ after 8 h. The Coulter Counter was considered to be potentially useful for detecting significant bacteriuria.     

'BITTY' CREAM: THE OCCURRENCE AND SIGNIFICANCE OF BACILLUS CEREUS SPORES IN RAW MILK SUPPLIES

SUMMARY: Methods for determining the Bacillus cereus content of milks and of rinses of dairy equipment are described and their limitations discussed. Milk samples from various sources were examined throughout the year for 'bittiness' as well as for B. cereus. The organism was not detected in all samples showing bittiness. There was a marked seasonal variation in the B. cereus index of raw milk supplies; maximum numbers were obtained from July to September and minimum numbers in April and May. Rinses of farm dairy equipment yielded few B. cereus spores but milk cans not uncommonly contained large numbers, especially in the summer months, when 10·5% of cans showed more than 5 × 10³/can. Preliminary observations on other sources of B. cereus are described and some of the problems of the control of this organism in raw milk supplies are discussed.     

Thermal resistance of Enterobacter sakazakii in reconstituted dried-infant formula

Enterobacter sakazakii , designated a unique species in 1980, has been implicated in a rare but severe form of neonatal meningitis, with dried-infant formula being implicated as the mode of transmission. The high mortality rate (40–80%) and the lack of information about this organism led to a study of the heat resistance of Ent. sakazakii in reconstituted dried-infant formula. Ten Canadian Ent. sakazakii strains (5 clinical and 5 food isolates) were used to determine the heat resistance of this organism at 52, 54, 56, 58 and 60°C in reconstituted dried-infant formula. D - values of 54·8, 23·7, 10·3, 4·2 and 2·5 min were obtained for each temperature, respectively. The overall calculated z -value was 5·82°C. In a comparison of the D -values of several members of the Enterobacteriaceae in dairy products, Ent. sakazakii appeared to be one of the most thermotolerant organisms. The importance of process control during manufacture and the use of aseptic procedures during the preparation, use and storage of dried-infant formula is discussed.     

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram-negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log₁₀ cfu g⁻¹ than counts in curd made in January, and were lower by about 2·5 log₁₀ cfu g⁻¹ than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Factors affecting the survival of Salmonella and Escherichia coli in anaerobically fermented pig waste

The survival of Salmonella typhimurium was investigated in acidogenic, anaerobically fermented pig wastes and in synthetic media, each containing volatile fatty acids (VFA). Salm. typhimurium survived at pH 6·8, but not at pH 4·0, when incubated at 37°C for 24 h in either fermented or synthetic medium containing VFA. The minimum inhibiting concentration of VFA for Salm. typhimurium after 48 h incubation at 30°C at pH 4·0 was 0·03 mol/l and for Escherichia coli it was 0·09 mol/l. Fermented pig wastes in a digester, maintained at pH 5·9, were inoculated with Salm. typhimurium and then incubated at 37°C for 24 h. The pH was adjusted to either 4·0 or 5·0 and after a further 48 h at 30°C, Salm. typhimurium survived at pH 5·0 but not at pH 4·0. It was concluded that pH is critical in determining the survival of this organism in acidogenic anaerobically fermented pig waste.     

Synbiotics growth optimization of Bifidobacterium pseudocatenulatum G4 with prebiotics using a statistical methodology

Aims:  This study demonstrated the optimum growth of Bifidobacterium pseudocatenulatum G4 with prebiotics via statistical model.
Methods and Results:  Commercial prebiotics [inulin and fructooligosaccharide (FOS)], together with sorbitol, arabinan and inoculum rate, were tested by fractional factorial design to determine their impact on growth of Bif. pseudocatenulatum G4 in skim milk. At 48 h incubation, bacterial growth was mainly influenced by FOS and inoculum rate. Growth reduction was observed in all samples incubated for 72 h. Central composite design (CCD) was adopted using FOS and inoculum rate at 48 h incubation to develop the statistical model for optimization. The model predicted that 2·461 log CFU ml⁻¹ produced the optimum growth increase of Bif. pseudocatenulatum G4. The combination that produced the optimum point was 2·86% FOS (g/v) and 0·67% inoculum rate (v/v).
Conclusion:  At optimum combination of inoculum rate and FOS, validation experiments recorded 2·40 ± 10·02 log CFU ml⁻¹. The application in 1-l bioreactor for 24 h showed higher growth increase of 2·95 log CFU ml⁻¹.
Significant and Impact of the Study:  Response surface methodology approach is useful to develop optimum synbiotics combination for strain G4 with FOS.     

Prediction of toxigenic fungal growth in buildings by using a novel modelling system.

There is growing concern about the adverse effects of fungal bioaerosols on the occupants of damp dwellings. Based on an extensive analysis of previously published data and on experiments carried out within this study, critical limits for the growth of the indoor fungi Eurotium herbariorum, Aspergillus versicolor, and Stachybotrys chartarum were mathematically described in terms of growth limit curves (isopleths) which define the minimum combination of temperature (T) and relative humidity (RH) at which growth will occur. Each growth limit curve was generated from a series of data points on a T-RH plot and mathematically fitted by using a third-order polynomial equation of the form RH = a(3)T(3) + a(2)T(2) + a(1)T + a(0). This fungal growth prediction model was incorporated within the ESP-r (Environmental Systems Performance [r stands for "research"]) computer-based program for transient simulation of the energy and environmental performance of buildings. For any specified location, the ESP-r system is able to predict the time series evolution of local surface temperature and relative humidity, taking explicit account of constructional moisture flow, moisture generation sources, and air movement. This allows the predicted local conditions to be superimposed directly onto fungal growth curves. The concentration of plotted points relative to the curves allows an assessment of the risk of fungal growth. The system's predictive capability was tested via laboratory experiments and by comparison with monitored data from a fungus-contaminated house.     

Comparison of a quadratic response surface model and a square root model for predicting the growth rate of Yersinia enterocolitic

Polynomial equations, relating the growth rate of Yersinia enterocolitica to temperature (0–25°C) and pH (4.5–6-5) in a liquid medium were constructed for four different acidulants. The logarithm of the time for a 100-fold increase in bacterial numbers could be represented by a quadratic response surface function of pH and temperature. The interactions between pH and temperature on growth rate were found to be additive. Values for a 2 log cycle increase in growth derived from the model were in good agreement with experimental values. Predictions from the quadratic model and from a square root model were compared with experimental values in laboratory media and UHT milk. The mean square error (MSE) for the quadratic response surface model was smaller than that for the square root model in 81% of cases. In UHT milk the square root model increasingly underestimated growth rate, as the temperature decreased and would 'fail dangerous' if used for predictive purposes. This indicated that the response surface model is more reliable for predicting the growth of Y. enterocolitica under conditions of sub-optimal temperature and pH.