Spore formation by Bacillus cereus in broth as affected by temperature, nutrient availability, and manganese

A study was done to determine the effect of interacting factors on sporulation of Bacillus cereus in broth. Vegetative cells (1.4 to 2.2 log CFU/ml) of B. cereus strain 038-2 (capable of growing at 12 degrees C) and strain F3812/84 (capable of growing at 8 degrees C) were inoculated into 30 ml of tryptic soy broth (TSB), TSB supplemented with manganese (50 microg/ml), diluted (10%) TSB (dTSB), and dTSB supplemented with manganese (50 microg/ml) and incubated at 8, 12, or 22 degrees C for up to 30, 30, or 10 days, respectively. Unheated and heated (80 degrees C for 10 min) cultures were plated on brain heart infusion agar to determine total cell counts (vegetative cells plus spores) and the number of spores produced, respectively. Both strains of B. cereus survived in TSB and dTSB for 30 days at 8 degrees C but did not sporulate. At 12 degrees C, cells grew in TSB to a population of 6.0 +/- 0.8 log CFU/ml, which was maintained for 30 days. Neither strain grew in dTSB at 12 degrees C and survived for at least 30 days. Spores were not produced in any of the test broths at 12 degrees C. At 22 degrees C, cells reached a stationary growth phase between 12 and 24 h in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese, and approximately 1% of the CFU were spores. In dTSB, cell growth and spore formation were retarded at 22 degrees C and a significantly lower number of spores was produced compared with the number of spores produced in TSB, TSB supplemented with manganese, and dTSB supplemented with manganese. The addition of manganese to TSB did not affect cell growth or spore formation, but manganese did enhance sporulation in dTSB. This study provides useful information on spore formation by B. cereus as affected by conditions that may be imposed in liquid milieus on the surface of foods and on food contact surfaces in processing environments.     

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times (D-values) were calculated by linear regression analysis (D = -1/slope of a plot of log surviving cells versus time). The log(10) of the resulting D-values were plotted against their corresponding temperatures to calculate (-1/slope of the curve) the thermal resistance (z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 degrees C) to 33.2 min (50 degrees C) for B. cereus and from 0.9 min (65 degrees C) to 16.3 min (55 degrees C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 degrees C) to 32.1 min (85 degrees C) and from 2.2 min (100 degrees C) to 34.2 min (90 degrees C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 degrees C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 degrees C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 degrees C for 12s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 degrees C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.     

Effective chemical control of psychrotrophic Bacillus cereus EPSO-35AS and INRA TZ415 spore outgrowth in carrot broth

The growth kinetic parameters of germinated cells from heat-activated spores of the psychrotrophic Bacillus cereus EPSO-35AS strain in nutrient broth (NB) and in tyndallized carrot broth (TCB) were evaluated at different temperatures (8, 12, and 16 degrees C) for control samples and for samples acidified with citric acid or lemon juice at pH values between 4.7 and 5.5. Lowering the pH from 7.4 or 6.2 to 5.2 inhibited bacterial growth in both tested media after 60 days at 12 degrees C and lower temperatures, confirming the effectiveness of acidification in association with refrigeration to control B. cereus proliferation in minimally processed foods (MPFs) based on carrot. The activities of selected concentrations of cinnamon essential oil, cinnamaldehyde, carvacrol, and eugenol against B. cereus EPSO-35AS and INRA TZ415 strains in both media over the same temperature range were also studied. Addition of either cinnamon essential oil or cinnamaldehyde at concentrations of 5 and 2 microL 100mL(-1), respectively, caused complete inhibition of the growth of both psychrotrophic strains even if mild temperature abuse occurred (12 degrees C). Hence, a combination of one of these compounds and refrigerated storage may be useful for preservation of MPFs in which major ingredient was carrot. On the contrary, carvacrol and eugenol were not able to prevent B. cereus growth in TCB during storage at 8 degrees C. Their effects on the organoleptic characteristics of TCB are discussed.     

The effect of 100% CO2 on the growth of nonproteolytic Clostridium botulinum at chill temperatures

The growth of a cocktail of spores from six nonproteolytic Clostridium botulinum type B and E isolates at 5 and 10 degrees C was used to assess the combined effect of NaCl (0.5-4.5% w/v), pH (5.5-6.5) and atmosphere (10% H2:90% N2, 5% CO2:10% H2:85% N2, or 100% CO2) in buffered peptone, yeast, glucose, starch broth with an Eh of approximately -350 mV. Under all atmospheres growth tended to be slower as the concentration of NaCl increased and with NaCl combined with pH levels below 6.0. Of the atmospheres tested, growth occurred at a slower rate and over a narrower range of conditions when C. botulinum was exposed to 100% CO2. This effect was enhanced when the incubation temperature was 5 degrees C. The results indicate that while CO2 decreased C. botulinum growth at chill temperatures, prevention of growth also depended on the NaCl concentration and the pH of the medium.     

Influence of pH and temperature on growth of Bacillus cereus in vegetable substrates

Bacillus cereus is a food-borne pathogen which most often contaminates foods of plant origin. Spores of psychrotrophic strains have the ability to germinate and grow at refrigeration temperatures in different vegetable substrates, such as carrot broth, zucchini broth, and cooked carrot purée. In some circumstances, factors such as pH, heat treatment, and storage temperature play a fundamental role in controlling the growth of these psychrotrophic strains and in extending the shelf life of refrigerated, minimally processed vegetable-based products in relation to pathogenic spore-forming bacteria. The combination of mild acidification (pH 5.0) and refrigeration (相似文献   

Effect of temperature on growth characteristics of Bacillus cereus TZ415

The effect of temperature on the maximal specific growth rate was studied in Bacillus cereus between 5 and 40 degrees C cultivated in courgette broth and rich medium (J broth). B. cereus grown from 5 to 38 degrees C in rich medium. No growth was observed in courgette broth below 10 degrees C. The Arrhenius plot was fitted from experimental data of B. cereus grown in rich medium and at regulated pH, oxygen and temperature. Two domains which are separated by a critical temperature around 13 degrees C can be distinguished with regard to temperature dependence of maximal specific growth rate. Over the cold domain from 5 to 13 degrees C, the temperature characteristic was 2.6 fold higher than over the sub-optimal domain from 13 to 38 degrees C suggesting that the growth temperature regulates several metabolic pathways.     

Survival, isolation and characterization of a psychrotrophic Bacillus cereus strain from a mayonnaise-based ready-to-eat vegetable salad

Incidence and population levels of Bacillus cereus in American salad, an industrially manufactured, packaged and refrigerated deli salad containing vegetables and mustard, were determined. Of 12 ready-to-eat samples examined, one (8.3%) was positive for B. cereus at less than 5 x 10(3)cfu g(-1). According to the ISO confirmation procedure, a strain was isolated and further characterized and identified as B. cereus EPSO-35AS by API 50CH/20E phenotypic system, combined with additional tests of motility, oxidase activity and anaerobic growth. This strain produced diarrhoeal enterotoxin in tryptic soy broth culture as detected by BCET-RPLA test, hydrolysed starch and had a low D(90)-value (2.1 min), with an estimated z-value of 6.79 degrees C. After a lengthy lag phase (9-12 days of incubation), the strain was able to grow at 8 degrees C in both nutrient broth and tyndallized carrot broth with specific growth rates from 0.009 to 0.037 h(-1), respectively. In the vegetable substrate, lag time was approximately 3 days (66 h) shorter than in laboratory medium. The effect of temperature abuses on the safety of the product during the time of use or consumption is discussed.     

An assessment of pasteurization treatment of water, media, and milk with respect to Bacillus spores

This study evaluated the ability of spore-forming Bacillus spp. to resist milk pasteurization conditions from 72 to 150 degrees C. Spores from the avirulent surrogate Sterne strain of Bacillus anthracis, as well as a representative strain of a common milk contaminant that is also a pathogen, Bacillus cereus ATCC 9818, were heated at test temperatures for up to 90 min in dH2O, brain heart infusion broth, or skim milk. In skim milk, characteristic log reductions (log CFU per milliliter) for B. anthracis spores were 0.45 after 90 min at 72 degrees C, 0.39 after 90 min at 78 degrees C, 8.10 after 60 min at 100 degrees C, 7.74 after 2 min at 130 degrees C, and 7.43 after 0.5 min at 150 degrees C. Likewise, log reductions (log CFU per milliliter) for viable spores of B. cereus ATCC 9818 in skim milk were 0.39 after 90 min at 72 degrees C, 0.21 after 60 min at 78 degrees C, 7.62 after 60 min at 100 degrees C, 7.37 after 2 min at 130 degrees C, and 7.53 after 0.5 min at 150 degrees C. No significant differences (P < 0.05) in thermal resistance were observed for comparisons of spores heated in dH2O or brain heart infusion broth compared with results observed in skim milk for either strain tested. However, spores from both strains were highly resistant (P < 0.05) to the pasteurization temperatures tested. As such, pasteurization alone would not ensure complete inactivation of these spore-forming pathogens in dH2O, synthetic media, or skim milk.     

Synergistic bactericidal effect of carvacrol, cinnamaldehyde or thymol and refrigeration to inhibit Bacillus cereus in carrot broth

Possible use of three different essential oil components as natural food preservatives was studied by examining their influence in the kinetics of growth from activated spores of four Bacillus cereus strains in tyndallized carrot broth over the temperature range 5-16 degrees C. Selected low concentrations of carvacrol, cinnamaldehyde, or thymol showed a clear antibacterial activity against B. cereus in the vegetable substrate. The addition of 2 microl cinnamaldehyde or 20 mg thymol to 100 ml of broth in combination with refrigeration temperatures (相似文献   

Modeling the germination kinetics of clostridium botulinum 56A spores as affected by temperature, pH, and sodium chloride

The germination kinetics of proteolytic Clostridium botulinum 56A spores were modeled as a function of temperature (15, 22, 30 degrees C), pH (5.5, 6.0, 6.5), and sodium chloride (0.5, 2.0, 4.0%). Germination in brain heart infusion (BHI) broth was followed with phase-contrast microscopy. Data collected were used to develop the mathematical models. The germination kinetics expressed as cumulated fraction of germinated spores over time at each environmental condition were best described by an exponential distribution. Quadratic polynomial models were developed by regression analysis to describe the exponential parameter (time to 63% germination) (r2 = 0.982) and the germination extent (r2 = 0.867) as a function of temperature, pH, and sodium chloride. Validation experiments in BHI broth (pH: 5.75, 6.25; NaCl: 1.0, 3.0%; temperature: 18, 26 degrees C) confirmed that the model's predictions were within an acceptable range compared to the experimental results and were fail-safe in most cases.     

Antibacterial activity of 11 essential oils against Bacillus cereus in tyndallized carrot broth

The antibacterial activity of 11 essential oils from aromatic plants against the strain INRA L2104 of the foodborne pathogen Bacillus cereus grown in carrot broth at 16 degrees C was studied. The quantity needed by the essential oils of nutmeg, mint, clove, oregano, cinnamon, sassafras, sage, thyme or rosemary to produce 14-1110% relative extension of the lag phase was determined. Total growth inhibition of bacterial spores was observed for some of the antimicrobial agents assayed. The addition of 5 microl cinnamon essential oil per 100 ml of broth in combination with refrigeration temperatures of 相似文献   

Inactivation of Bacillus cereus spores by high hydrostatic pressure at different temperatures

The effect of pH on the initiation of germination and on the inactivation of Bacillus cereus (KCTC 1012) spores during high hydrostatic pressure processing (HPP) with pressures of 0.1 to 600 MPa at different temperatures was investigated. Two different high-pressure treatments were adopted to evaluate the effect of pH on the inactivation of B. cereus on sporulation medium and in suspension medium. Inactivation of B. cereus spores with HPP treatment was affected more by sporulation medium pH than by suspension medium pH. B. cereus spores obtained through sporulation at pH 6.0 showed more resistance to inactivation by HPP at 20, 40, and 60 degrees C than did those obtained through sporulation at pHs of 7.0 and 8.0. Constituents of B. cereus spores obtained through sporulation at pH 6.0 may undergo electrochemical charge changes comparable to those for spores obtained through sporulation at pH 7.0. The initiation of B. cereus spore germination was more sensitive to pressure around 300 MPa at 20 degrees C. Increasing processing temperatures during HPP enhanced the effect of sporulation medium pH (i.e., environmental pH) on the inactivation of B. cereus spores.     

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.     

The potential of vancomycin-resistant enterococci to persist in fermented and pasteurised meat products

Experiments with 148 isolates of vancomycin-resistant enterococci (VRE) were performed to assess their potential to persist and grow in fermented sausages and pasteurised meat products. All strains were meat isolates and Van-type A, except a single VanC1 strain. In total, 143 strains of Enterococcus faecium were involved. Eight selected strains were examined for their potential to grow at high salt and nitrite levels and at reduced pH. The same isolates were used in experiments with fermented sausages. All available strains were subjected to heating tests in meat suspensions with added curing ingredients. All but one of the eight tested isolates grew at pH 4.0 in tryptone soya broth (TSB). With the combination of 8% w/w NaCl, 400 ppm NaNO2 and 0.5% w/w glucose in the meat suspension, all isolates grew at 37 degrees C, whereas none grew at 7 degrees C even after 56 days. With the addition of 10% w/w NaCl, 200 ppm NaNO2 and 0.5% w/w glucose, still one E. faecium isolate grew at 37 degrees C, although very slowly. Overall, the strains tolerated high salt and nitrite concentrations and reduced pH very well, even beyond levels applied in the regular production of fermented and/or pasteurised meat products. The tested strains could be isolated after the fermentation and further ripening of "boerenmetworst" and "snijworst". Overall, their colony counts decreased on average about 1 log-unit over a period of 60 days after batter manufacture. All 148 isolates demonstrated a relatively weak thermal resistance compared to results for selected vancomycin-sensitive enterococci strains reported in the literature and to results collected under identical experimental conditions in this laboratory. None of the strains (log inoculation level about 5-6 ml(-1) for each isolate) could be cultured after heating at 70 degrees C for 10 min.     

Effect of stress induced by suboptimal growth factors on survival of Escherichia coli O157:H7.

This study investigated the growth and survival of E. coli O157:H7 exposed to a combination of suboptimal factors (22 degrees C, 7 degrees C, -18 degrees C/0.5% NaCl, 5.0% NaCl/pH 7.0, pH 5.4, pH 4.5/addition of lactic acid) in a simulation medium for red meat (beef gravy). Prolonged survival was noted as the imposed stress was more severe, and as multiple growth factors became suboptimal. At a defined temperature (7 degrees C or -18 degrees C), survival was prolonged at the more acid, more suboptimal pH (pH 4.5 > pH 5.4 > pH 7.0) while at a defined pH (pH 4.5), better survival was observed at 7 degrees C than at 22 degrees C. This suggests that application of the hurdle concept for preservation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in minimal processed foods. At both 22 degrees C and 7 degrees C, the addition of lactic acid instead of HCl to reduce pH (to pH 4.5) resulted in a more rapid decrease of E. coli O157:H7. High survival was observed in beef gravy, pH 5.4 at -18 degrees C (simulation of frozen meat)-reduction of log 3.0 to log 1.9 after 43 days--and in beef gravy, pH 4.5 and 5% NaCl at 7 degrees C (simulation of a fermented dried meat product kept in refrigeration)--less than 1 log reduction in 43 days. In these circumstances, however, a high degree of sublethal damage of the bacterial cells was noted. The degree of sublethal damage can be estimated from the difference in recovery of the pathogen on the non-selective TSA medium and the selective SMAC medium.     

The effect of calcium and sodium lactates on growth from spores of Bacillus cereus and Clostridium perfringens in a 'sous-vide' beef goulash under temperature abuse

The effect of calcium and sodium lactates on growth from spores of Bacillus cereus and Clostridium perfringens at three different concentrations (0, 1.5 and 3% w/w) and at different temperatures (10, 15 and 20 degrees C for B. cereus and 15, 20 and 25 degrees C for C. perfringens) was investigated, using beef goulash as a model system for pasteurised vacuum-packaged convenience foods. Calcium lactate at a level of 3% reduced the pH values of the samples from 6.0 to 5.5. No B. cereus growth was observed at 10 degrees C, but after 7 days at an incubation temperature of 15 degrees C, cell number increased by 1 log cfu/g in the control samples. At this temperature, lactates were seen to be effective at inhibiting growth. Calcium lactate was more inhibitory than sodium lactate as the growth of B. cereus was inhibited at 1.5 and 3% concentrations at 20 degrees C, respectively. Growth of C. perfringens was arrested in the presence of 1.5% calcium lactate at all storage temperatures, whereas growth was inhibited by 3% sodium lactate only at 15 degrees C.     

Thermal resistance of spores from virulent strains of Bacillus anthracis and potential surrogates

The objective of this study was to determine the thermal resistance of spores of Bacillus anthracis and potential surrogates. The heat resistance of spores suspended in buffer (pH 7.0 or 4.5), milk, or orange juice was determined at 70, 80, and 90 degrees C. D-values for B. anthracis strains Sterne, Vollum, and Pasteur ranged from < 1 min at 90 degrees C to approximately 200 min at 70 degrees C and were lower under acidic than under neutral conditions. The D-values for B. anthracis spores fell within the range obtained for spores from eight strains of Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis. However, there were significant differences (P < 0.001) among the D-values of the strains. The z-values in pH 7.0 buffer and milk averaged approximately 10.5 degrees C and were not significantly different among strains (P < 0.05). The z-values in pH 4.5 buffer and orange juice averaged 12.9 and 13.9 degrees C, respectively, significantly (P < 0.05) higher than those obtained in milk or in pH 7.0 buffer. The significance of this difference was driven by large differences among a few strains. The z-values for B. anthracis strain Pasteur were twice as high in the acid media than in the neutral media. This study confirms that B. anthracis spores are not unusually heat resistant and that spores from validated Bacillus species are appropriate surrogates for thermal resistance studies.     

Influence of the sporulation temperature on the impact of the nutrients inosine and l-alanine on Bacillus cereus spore germination

The effect of sporulation temperature on Bacillus cereus spore germination triggered by the nutrient germinants inosine and l-alanine was investigated. The germination (expressed as % A(600nm) fall) of heat-activated spores of B. cereus strain ATCC14579 produced at 20 and 37 degrees C, and of psychrotrophic strains LM9 and D15 produced at 15 and 37 degrees C was followed in a germination buffer containing inosine at concentrations between 0.01 and 10mmoll(-1), or l-alanine between 1 and 100mmoll(-1). Spores wet-heat resistance at 90 degrees C was also determined. Spores of the three strains produced at the lowest temperatures generally showed a higher germination capacity in response to both inosine and in l-alanine than those produced at 37 degrees C, and were also more susceptible to heat. Low sporulation temperature is confirmed as a detrimental factor to B. cereus spore wet-heat resistance, and conversely gave spores with a sensitivity to the nutrient germinants inosine and l-alanine higher than that of spores formed close to the maximal sporulation temperature.     

Presence and growth of Bacillus cereus in dehydrated potato flakes and hot-held, ready-to-eat potato products purchased in New Zealand

Potato products prepared from dehydrated potato flakes have been implicated in foodborne illness incidents involving Bacillus cereus intoxications. B. cereus can survive as spores in potato flakes and can germinate and multiply in the rehydrated product. This study assessed the frequency and concentration of B. cereus in dehydrated potato flakes and hot-held, ready-to-eat mashed potato products. Of 50 packets of potato flakes tested, eight contained greater than 100 CFU/g B. cereus (maximum 370 CFU/g). The temperature of the potato portion of 44 hot-held food products was measured immediately after purchase, and 86% were below the safe hot-holding temperature of 60 degrees C. The potato portions were subsequently tested for B. cereus. Only two of the potato portions contained B. cereus at greater than 100 CFU/g, a potato-topped pastry (1000 CFU/g) and a container of potato and gravy (120 CFU/g). To assess multiplication of B. cereus in this food, we held rehydrated potato flakes with naturally occurring B. cereus at 37, 42, and 50 degrees C and tested them over 6 h. By 6 h, the number of B. cereus in potato stored at 37 degrees C had exceeded 10(3) CFU/g, was greater than 10(4) CFU/g at 50 degrees C, and was close to 10(6) CFU/g at 42 degrees C. Growth data were compared to predictions from the U.S. Department of Agriculture Pathogen Modeling Program (PMP 7.0). The PMP predictions were found to simulate the measured growth better at 42 degrees C than at 37 degrees C. Hot-held potato products should be safe for consumption if held at 60 degrees C or above or discarded within 2 h.     

Thermal inactivation of Bacillus cereus spores affected by the solutes used to control water activity of the heating medium

The heat resistance of B. cereus spores (ATCC 7004, 4342 and 9818) over a wide temperature range (92-125 degrees C) in aqueous solutions of NaCl, LiCl, sucrose and glycerol at different water activities (1.00-0.71) was investigated. Sodium chloride in the heating medium tended to protect the spores of B. cereus against heat. The z-values increased significantly (P < 0.05) at and above a concentration of 4.0 M. The effects of LiCl were lower than those caused by the NaCl at the same a(w) values. An increase in z-values was observed. but the differences were only statistically significant (P<0.05) at the highest concentration tested (5.0 M). A concentration of sucrose 0.87 M caused in all cases a reduction in D-values, which was most pronounced for strains 4342 and 9818. With increasing concentration of sucrose ( > 0.87 M), the D-values showed an increase, although only those obtained for strain 4342 in sucrose solutions 2.22 M were higher than those found in pure water. The z-values were significantly higher (P < 0.05) when sucrose was added at concentrations above 1.42 M, except for strain 4342. When a(w) was lowered from 0.96 to 0.71 with glycerol, D-values obtained gradually increased, about 30, 50 and 60 fold for 4342, 7004 and 9818 strains, respectively. No significant effect on z-values were detected.