Survival,growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature

A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acid-shocked Shigella flexneri 2a cells as affected by pH and temperature. The pathogen was grown at 37 degrees C for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5+/-0.05 with lactic acid. All three cell types were separately inoculated into tryptic soy broth (6.6-7.0 log(10) cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5-5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 degrees C for up to 144 h. Overall, inactivation of S. flexneri cells at low pH was enhanced with an increase in incubation temperature. All three types of cells survived for 144 h at 4 degrees C in TSB acidified to pH 3.5, compared to < 24 h at 30 degrees C and 2 h at 48 degrees C. The population of all three cell types increased significantly (alpha = 0.05) within 24 h when cells were incubated at 12, 21, or 30 degrees C in TSB at pH 5.0, 5.5, or 7.3. Prior exposure of the S. flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions. Acid-adapted cells decreased by approximately 2.5 log(10) cfu/ml when incubated at 4 degrees C for 144 h, compared to a 6-log(10) reduction in control (unadapted) cells. When cells were exposed to low pH (3.5-4.5) and high temperature (48 degrees C), significantly higher (alpha = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S. flexneri cells were injured. Results show that the ability of S. flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature.     

Growth and viability of commercial Bifidobacterium spp. in honey-sweetened skim milk.

Two commercial Bifidobacterium bifidum (Bf-1 and Bf-6) were cultured in 12% (wt/vol) reconstituted nonfat dry milk (NDM) containing 5% (wt/wt) honey, sucrose, fructose, or glucose. Inoculated samples were incubated anaerobically at 37 degrees C for 48 h. Samples were collected at 12-h intervals and examined for (i) specific growth rate, (ii) pH, and (iii) levels of fermentation end products (lactic and acetic acids) as measured by high-performance liquid chromatography (HPLC). Viability of the organisms during 28 days of refrigerated storage at 4 degrees C was also assessed at 7-day intervals. Growth promotion and acid production were greatest when Bf-1 and Bf-6 were grown in the presence of honey. For both Bf-1 and Bf-6, retention of viability was greatest up to 14 days of refrigerated storageat 4 degrees C when they were grown and stored in the presence of honey compared to other sweeteners.     

SUGAR UPTAKE AND SUBSEQUENT ESTER AND HIGHER ALCOHOL PRODUCTION BY SACCHAROMYCES CEREVISIAE

The production of a number of esters and higher alcohols by brewing strains of Saccharomyces cerevisiae, in synthetic media containing only glucose, fructose or maltose as sole carbohydrate source, has been investigated. Results indicated that production of most volatiles was generally lower when maltose was the sugar being fermented, despite maltose-grown cells having higher viabilities and vitalities than glucose or fructose-grown cells. There was no significant difference in the levels of esters and higher alcohols produced during fermentation when glucose or fructose was metabolised, although strain variation was observed. Similar results were obtained when wort was supplemented with either glucose, fructose or maltose. Wort supplemented with maltose produced fewer volatiles, especially higher alcohols, than that which had a hexose sugar added. The activity of estersynthesising enzymes present in glucose or maltose PYN grown cells was also examined. Similar levels of ethyl acetate and isoamyl acetate were obtained when cells grown in either glucose or maltose PYN were disrupted and ester production monitored. The implications of these results for the fermentation of high-gravity worts are discussed.     

Changes in the proteome of Escherichia coli during growth at 15 degrees C after incubation at 2, 6 or 8 degrees C for 4 days

For better understanding of the complex behaviour of Escherichia coli at chiller temperatures, log phase E. coli grown at 15 degrees C were incubated at 8, 6, or 2 degrees C for 4 days, and were then incubated at 15 degrees C for 12 h. Cultures were sampled after incubation at the lower temperatures, and during subsequent incubation at 15 degrees C. Proteins extracted from the samples were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Spots of 45 previously identified proteins that were differentially expressed at 15 or < or =8 degrees C were quantified by image analysis. After incubation at 8 or 6 degrees C for 4 days cells were growing with or without formation of elongated cells (filaments), respectively, but growth did not occur at 2 degrees C. In cells incubated at 8 or 6 degrees C proteins associated with the stress response and energy generation were upregulated and proteins associated with protein synthesis were downregulated, while protein levels in cells incubated at 2 degrees C were little changed. When cells were then incubated at 15 degrees C, the levels of differentially expressed proteins in cells that had been incubated at 8 or 6 degrees C decreased or increased towards the levels found in cells growing at 15 degrees C, but some proteins were still under or over expressed after 12 h. In cells incubated at 15 degrees C after incubation at 2 degrees C, the levels of many of the proteins declined but the levels of proteins associated with protein synthesis increased. The findings indicate that the physiological states of log phase E. coli incubated at < or =2 degrees C or at higher chiller temperature are different, but that for both states incubation at an above chiller temperature for >3 generations is required before protein levels adjusted to those usual for the higher temperature. Cells in these different physiological states may respond differently to other stresses encountered during warming of chilled foods.     

Isolation of a novel mutant strain of Saccharomyces cerevisiae by an ethyl methane sulfonate-induced mutagenesis approach as a high producer of bioethanol

In order to obtain mutant strains showing higher bioethanol production than wild-type strains, a commercial Saccharomyces cerevisiae type was subjected to mutagenesis using ethyl methane sulfonate (EMS). After adding EMS to a shaken yeast suspension, the viability of yeast cells was assessed by diluted sample inoculation to solid yeast-extract peptone glucose (YEPG) medium at 15-min intervals. At 45 min, the viability of yeast cells was estimated to be about 40%. Mutagenized cells were recovered from YEPG broth after incubation at 30 degrees C for 18 h. After this period, EMS-treated yeast cells were grown on solid aerobic low-peptone (ALP) medium containing 2-12% (v/v) ethanol. All plates were incubated at 30 degrees C for 2-6 d in order to form colonies. The mutant strains that tolerated high concentrations of ethanol were selected for bioethanol production in microfuge tubes containing fermentation medium. Formation of bioethanol in small tubes was detected by the distillation-colorimetric method. In addition, trehalose content and invertase activity were determined in each mutant strain. Among many isolated mutant strains, there were six isolated colonies that grew on ALP medium supplemented with 10% (v/v) ethanol and one of them produced bioethanol 17.3% more than the wild type.     

Differential expression of proteins in cold-adapted log-phase cultures of Escherichia coli incubated at 8, 6 or 2 degrees C

Temperature is used to control the growth of microorganisms in foods. The minimum temperature for sustained growth of Escherichia coli is 7 degrees C. E. coli cells in the logarithmic phase of growth at 15 degrees C were incubated at 8, 6 or 2 degrees C. The cells grew with the formation of filaments at the two higher temperatures, but did not grow at 2 degrees C. In order to investigate more thoroughly the nature of filament formation in E. coli at temperatures near the minimum temperature for sustained growth, cells were harvested after 1 day at 2 degrees C or at times up to 4 or 8 days at 8 or 6 degrees C, respectively. Proteins extracted from the cells were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and spots containing differentially expressed proteins were identified by quadropole time-of-flight tandem (Q-ToF-2) mass spectrometry. For most of the identified proteins, the amounts were not substantially different in cells grown at 15 degrees C or incubated at 2 degrees C. In cells incubated at 8 or 6 degrees C, proteins associated with stress responses, the tricarboxylic acid cycle and electron transport were present in substantially greater amounts, and proteins associated with protein synthesis were present in substantially smaller amounts than in cells grown at 15 degrees C. These findings suggest that the stringent response is induced in E. coli incubated at temperatures near the minimum for growth, so the formation of filaments at those temperatures may be a result of the stringent response.     

Improving recovery of Salmonella enterica serovar typhimurium DT104 cells injured by heating at different water activity values

This study describes the evaluation of potentially more sensitive methods for the recovery of Salmonella cells injured by heating (54 to 60 degrees C) at different water activity values (0.65 to 0.90, reduced using equal portions of glucose and fructose). These methods included gradual rehydration, the use of diluting media with added solutes or blood, the addition of blood to plating agar, and the use of different incubation temperatures and times. Gradual rehydration of cells that had been challenged at low water activity (0.65 and 0.70) and high temperature markedly improved recovery, measured as a >50% increase in the time to obtain a 3-log10 reduction in cell numbers, compared to dilution into media with a high water activity. Adding sucrose, glycerol, or blood to the diluting media (maximal recovery diluent) did not improve recovery, but a plating agar containing blood recovered approximately 38% more cells than nutrient agar. Prolonged incubation of agar plates allowed recovery of injured Salmonella cells that presumably had extended lag periods, with significantly higher recovery rates after 48 h incubation at 37 degrees C than after 24 h (P = 0.05). This work highlights that by recovering Salmonella using a method specific to the nature of the injury, a better prediction of food safety and the success of food processing can be made.     

Influence of temperature,water activity and pH on growth of some xerophilic fungi

The combined effects of water activity (aw), pH and temperature on the germination and growth of seven xerophilic fungi important in the spoilage of baked goods and confectionery were examined. Eurotium rubrum, E. repens, Wallemia sebi, Aspergillus penicillioides, Penicillium roqueforti, Chrysosporium xerophilum and Xeromyces bisporus were grown at 25, 30 and 37 degrees C on media with pH values of 4.5, 5.5, 6.5 and 7.5 and a range of water activities (aw) from 0.92 to 0.70. The aw of the media was controlled with a mixture of equal parts of glucose and fructose. Temperature affected the minimum aw for germination for most species. For example, P. roqueforti germinated at 0.82 aw at 25 degrees C, 0.86 aw at 30 degrees C and was unable to germinate at 37 degrees C. E. repens germinated at 0.70 aw at 30 degrees C, but at 25 and 37 degrees C, its minimum aw for germination was 0.74. C. xerophilum and X. bisporus germinated at 0.70 aw at all three temperatures. The optimum growth occurred at 25 degrees C for P. roqueforti and W. sebi, at 30 degrees C for Eurotium species, A. penicillioides and X. bisporus and at 37 degrees C for C. xerophilum. These fungi all grew faster under acidic than neutral pH conditions. The data presented here provide a matrix that will be used in the development of a mathematical model for the prediction of the shelf life of baked goods and confectionery.     

Heat resistance of Yersinia enterocolitica grown at different temperatures and heated in different media.

In the range of 4-20 degrees C, growth temperature did not influence the heat resistance at 54-66 degrees C for Yersinia enterocolitica at pH 7 in citrate phosphate buffer. However, when cells were grown at 37 degrees C. the D62 increased from 0.044 to 0.17 min. This increase was constant at all heating temperatures tested (z = 5.7-5.8). Growth temperature did not influence the proportion of heat-damaged cells after a heat treatment, as measured by their response to a 2% of sodium chloride added to the recovery medium. The sensitivity of heat treated cells to nisin or lysozyme depended on growth temperature: Whereas the number of cells grown at 4 degrees C surviving heat treatment was the same regardless of the presence of 100 IU/ml of nisin or 100 microg/ml of lysozyme in the recovery medium, that of cells grown at 37 degrees C was, in these media, lower. The pH of maximum heat resistance in citrate phosphate buffer was pH 7 for cells grown at 37 degrees C, but pH 5 for those grown at 4 degrees C. In both suspensions the magnitude of the effect of pH on heat resistance was constant at all heating temperatures. For cells grown at 4 degrees C the heat resistance at 54-66 degrees C, in skimmed milk or pH 7 buffer, was the same. For cells grown at 37 degrees C this also applied for heat treatment at 66 degrees C but at 56 degrees C the heat resistance in skimmed milk was higher.     

Effect of incubation temperature on aerobic plate counts of beef and sheep carcasses

Australian regulations for microbiological testing of carcasses specify a number of incubation temperatures and media for meat processed at both domestic and export establishments. Accordingly, the effect of incubation temperature and media on aerobic plate counts of samples from beef and sheep carcasses was investigated. For both species, aerobic plate counts on Petrifilm incubated at 35 degrees C were significantly lower than those counts on Petrifilm and pour plates incubated at 25 and 30 degrees C, reflecting the inability of many psychrotrophs to grow at 35 degrees C. When samples were taken from carcasses that had been stored in abattoir chillers for periods between 16 h and 5 days, difference between counts at 35 degrees C versus those incubated at 25 and 30 degrees C became greater as the period of refrigerated storage increased. For export beef carcasses, the effect of this difference is minimal, since the vast majority of counts incubated at 35 degrees C are done on carcasses that have been chilled for less than 24 h and will not have a large proportion of psychrotrophs.     

High-level production of erythritol by strain 618A-01 isolated from pollen

We have isolated a microorganism (strain 618A-01) from pollen which has the ability to produce erythritol when grown in the presence of glucose as the carbon source. When cultivated in a medium consisting of 20% glucose and 1% dried bouillon in a shake flask, 75 g/l erythritol was produced after 950 h, corresponding to a 37.5% yield against glucose consumption. No other polyols, including glycerol, were detected in the medium. Positive-ion fast atom bombardment mass spectrometry and 1H- and 13C-NMR analyses confirmed that the fermentation product was erythritol. Scanning electron microscopic analysis clearly demonstrated that the cells grown on YPD medium at 30 degrees C showed yeast-like morphology, while they appeared like hyphae at 37 degrees C. The complete 18S rRNA sequence of the isolate was determined, which showed high identity (99.5%) with the genus Ustilago of the phylum Basidiomycota. The data strongly suggest that strain 618A-01 belongs to the class Ustilaginomycetes. The culture conditions for the production of erythritol by the isolate were examined. The use of medium containing 1% tryptone, 0.5% yeast extract and 0.5% NaCl yielded the highest cell growth and erythritol productivity among the media tested. Continuous glucose feeding at 6-7% to the fermentor further increased the production of erythritol, and we obtained a maximal 100 g/l erythritol after 530 h, with a 39.3% yield.     

Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake

The effects of freezing method, freeze drying process, and the use of protective agents on the viability of the biocontrol yeast Candida sake were studied. Freezing at -20 degrees C was the best method to preserve the viability of C. sake cells after freeze drying using 10% skim milk as a protectant (28.9% survival). Liquid nitrogen freezing caused the highest level of damage to the cells with viability < 10%. Different concentrations of exogenous substances including sugars, polyols, polymers and nitrogen compounds were tested either alone or in combination with skim milk. There was little or no effect when additives were used at 1% concentration. Galactose, raffinose and sodium glutamate at 10% were the best protective agents tested alone but the viability of freeze-dried C. sake cells was always < 20%. Survival of yeast cells was increased from 0.2% to 30-40% by using appropriate protective media containing combinations of skim milk and other protectants such as 5% or 10% lactose or glucose, and 10% fructose or sucrose.     

Influence of temperature on growth of Legionella pneumophila biofilm determined by precise temperature gradient incubator

Bacterial growth is influenced by several different culture conditions. Temperature is one of an essential component which regulates bacterial growth and their morphology. The influence of temperature on the length of bacteria was investigated in broth and on agar in a temperature range from 30.0 degrees C to 47.0 degrees C in 0.5 degrees C steps using a newly developed temperature gradient incubator. The incubator is able to reach a set temperature within 2 h and maintain temperature as accurate as +/-0.1 degrees C of the set temperature. Three Legionella pneumophila serotype 1 strains were incubated for 48 h in BCYE-alpha agar at various temperatures ranging from 30.0 degrees C to 48.0 degrees C and length of bacteria grown at each temperature was microscopically measured. Ability of bacteria to multiply at a given temperature was also determined. L. pneumophila serotype 1 strains ATCC 33152, a clinical isolate Okinawa 02-001 were going to elongate to longer than 100 mum when cultured higher than at 39.5 degrees C and at 41.5 degrees C, respectively. Each strain was unable to multiply when cultured higher than at 44.2 degrees C (ATCC 33152) or at 44.0 degrees C (Okinawa 02-001). Those data would provide insights for establishing regulations in terms of maintaining hot water temperature in a facility where a circulating hot water supply-system is available and contamination with Legionella spp. is likely to happen.     

The effect of pH on the initiation of growth of cottage cheese spoilage bacteria

The influence of pH on strains of Pseudomonas spp. and Enterobacter agglomerans that cause spoilage of cottage cheese varieties during storage at 7 degrees C has been investigated. In a culture medium adjusted to the required pH with HCl, 57 of 64 strains of Pseudomonas spp. grew at pH 4.8 when incubated at 7 degrees C but a very low proportion of strains grew at pH 4.7 or pH 4.6 and none at pH 4.5. At 20 degrees C some of the pseudomonads grew at pH 4.4. Three out of nine strains of E. agglomerans grew at pH 3.8 when incubated at 7 degrees C and at pH 3.6 when incubated at 20 degrees C. In cultures of E. agglomerans at controlled pH and 20 degrees C, after a lag phase the doubling time at pH 4.1 was 2 h, and at pH 7.0 was 1.4 h.     

Exopolysaccharide production by Pediococcus damnosus 2.6 in a semidefined medium under different growth conditions

Ropy Pediococcus damnosus (strain 2.6) was used for production of exopolysaccharide (EPS) in a semidefined medium. From a kinetic point of view, an experiment conducted in SMD medium containing 30 g l(-1) glucose and 5 g l(-1) Bacto casamino acids (Difco Laboratories, Detroit, MI), without pH control, showed that EPS production took place mainly during the growth phase. The viscosity of the cultures developed in parallel to the EPS synthesis until 94 h of incubation; after 200 h of fermentation, viscosity decreased. The effect of glucose, Bacto casamino acid concentrations and temperature on growth and EPS production was determined by using a full factorial design. Within the domain of experimental conditions considered, the concentration of glucose and Bacto casamino acids has a significant effect on the production of exopolysaccharide. The incubation at 12 degrees C produced a prolonged lag phase and due to the lower growth yield, higher specific EPS production was found at this temperature. At 25 degrees C the EPS production was mainly enhanced by the increase in glucose concentration. The increase in nitrogen concentration from 5 to 15 g l(-1) did not yield greater EPS production. However, at 12 degrees C optimal EPS production was obtained when both higher glucose and nitrogen concentrations were used.     

Interaction of water activity and temperature on aflatoxin production by Aspergillus flavus and A. parasiticus in irradiated maize seeds.

The effects of aw (0.90, 0.95, 0.98) and temperature (25 degrees C, 30 degrees C, 35 degrees C) on aflatoxin production by Aspergillus flavus and Aspergillus parasiticus growing on irradiated maize seeds, were examined. Highest levels of aflatoxin were produced by A. parasitious at 25 degrees C and 0.98 aw and by A. flavus at 30 degrees C at 0.95 and 0.98 aw. At 0.90 aw toxin production was consistently low for both species at all temperatures. Temperature cycling of A. flavus between 25 degrees C and 35 degrees C each for 12 h resulted in higher aflatoxin synthesis than when incubated either at 25 degrees C or 35 degrees C.     

Influence of selected sugars and temperature on fatty acids composition in Candida tropicalis

Temperature, mono- and disaccharides affected lipids, biomass production as well as odd-chain and unsaturated fatty acids contents of Candida tropicalis. With various sugars as carbon sources at 30 degrees C, the order for biomass production was, galactose greater than glucose greater than sucrose greater than fructose greater than lactose, while lipids production/g biomass decreased as follows: galactose, glucose, sucrose, fructose and lactose. On the other hand, the odd-chain fatty acids contents decreased in the following order: fructose, sucrose, glucose and lactose. Lowering the temperature of cultivation to 15 degrees C, decreased biomass and lipids production. However, a notable decrease in odd-chain fatty acids contents was detected.     

HEAT RESISTANCE IN DIFFERENT HEATING MEDIA OF LISTERIA MONOCYTOGENES ATCC 15313 GROWN AT DIFFERENT TEMPERATURES

This paper examines the influence of growth temperature on thermal resistance of Listeria monocytogenes ATCC 15313. Regardless of the incubation temperature, the heat resistance of Listeria monocytogenes increased during incubation until the stationary phase of growth was reached. The maximum heat resistance of cells grown at 4C, 20C or 37C was attained after 14 days, 36 h and 18 h of incubation, respectively. After longer incubation times the heat resistance of cells grown at 4C and 20C did not change but that of cells grown at 37C decreased. The maximum heat resistance was usually greater at higher incubation temperatures. However, the magnitude of these differences depended on the pH and composition of heating media. By raising the incubation temperature from 4C to 37C, the D₆₂ value in pH 7 citrate-phosphate buffer increased from 0.13 to 0.34 min. However, when skimmed milk was used as menstruum this difference was not observed. Cells grown at 37C attained maximum heat resistance at pH 7 but those grown at 4C, at pH 6. The magnitude of the effect of the incubation temperature on heat resistance was constant at all heating temperatures tested (z = 6 ± 0.8C). The higher heat resistance of cells grown at higher temperatures was not due to a greater capacity of heat damage repair     

The effects of growth temperature and growth phase on the inactivation of Listeria monocytogenes in whole milk subject to high pressure processing

The aim of this study was to explore the effect of a wide range of growth temperatures, growth phases and plating media on the inactivation of Listeria monocytogenes by high pressure processing (HPP). In part one, L. monocytogenes was grown to mid-stationary phase at 4, 15, 25, 35 or 43 degrees C, inoculated into whole UHT milk at approximately 10(7) CFU/ml and high pressure processed at 400 MPa at room temperature (20-25 degrees C). Afterward, the HPP milk was plated on Tryptic Soy Yeast Extract Agar (TSYEA) and Modified Oxford Agar (MOX) to determine the degree of injury. For part two, cells were grown to mid-exponential, late-exponential or mid-stationary phase at 15 or 43 degrees C and processed in the same way. Time to reach a 5-log reduction was determined and data were analysed by ANOVA. The results from part one showed that both growth temperature and plating medium had a significant effect (P < 0.001) on the inactivation of stationary phase L. monocytogenes by HPP. Tukey's pairwise comparisons revealed that the effects of all temperatures, except 35 and 43 degrees C, were significantly different (P < 0.05). Cells grown at 15 degrees C were most sensitive to HPP, followed by cells grown at 4, 25 or 35 degrees C, with cells grown at 43 degrees C appearing to be the most resistant. Inactivation of cells grown at 4, 15 or 25 degrees C followed first order kinetics, whereas cells grown at 35 or 43 degrees C displayed non-linear inactivation kinetics due to tailing. In part two, both growth phase and plating medium had significant effects on the inactivation (P < or = 0.001) of L. monocytogenes by HPP. Cells grown at 15 degrees C to mid-stationary phase were the most pressure-resistant when tested on both media, and were significantly more resistant (P < 0.05) than cells grown at the same temperature to the other two phases of growth. There was no significant difference between mid- and late-exponential phase cells grown at 15 degrees C. When cells were grown at 43 degrees C, mid-exponential phase cells were significantly more sensitive (P < 0.05) than either late-exponential or mid-stationary phase cells, with no difference between late-exponential or mid-stationary phase cells. It was postulated that membrane composition, stationary phase proteins and/or stress proteins may affect pressure resistance.