Effect of pH-dependent,stationary phase acid resistance on the thermal tolerance of Escherichia coli O157:H7

The ability of pH-dependent, stationary phase acid resistance to cross-protect Escherichia coli O157:H7 against a subsequent lethal thermal stress was evaluated using microbiological media and three liquid foods. Three strains were grown for 18 h at 37°C in acidogenic (TSB+G, final pH 4·6–4·7) and non-acidogenic (TSB-G, final pH 7·0–7·2) media to provide stationary phase cells with and without induction of pH-dependent acid resistance. The cells were then heated in BHI broth (pH 6·0) at 58°C, using a submerged coil apparatus. The TSB+G grown strains had greatly increased heat resistance, with the heating time needed to achieve a five-log inactivation, being increased two- to four-fold. The z -values of TSB+G and TSB-G grown cells were 4·7°C and 4·3°C, respectively. Increases in heat resistance with TSB+G-grown E. coli O157:H7 were also observed using milk and chicken broth, but not with apple juice. However, cross-protection was restored if the pH of the apple juice was increased from 3·5 to 4·5. The data indicate that pH-dependent acid resistance provides E. coli O157:H7 with cross-protection against heat treatments, and that this factor must be considered to estimate this pathogen's thermal tolerance accurately.     

Influence of Temperature, NaCI, and pH on the Growth of Aeromonas Hydrophila

Growth of clinical isolates of Aeromonas hydrophila at various temperatures, pH values, and salt levels was studied in BHI broth. A majority of the isolates grew at 4-5°C and 42°C, and all grew equally well over the range 20-35°C. At 28°C, most isolates could tolerate 4% NaCl, while at 4°C only a limited number grew in 3% NaCl. Similarly, isolates could better tolerate acidic conditions when cultured at 28°C as compared to 4°C. These data suggest that it is likely that A. hydrophila strains associated with human gastroenteritis are capable of growing in foods at refrigeration temperatures currently considered adequate for preventing the growth of foodborne pathogens.     

Influence of Temperature on Germination and Growth of Spores of Emetic and Diarrheal Strains of Bacillus cereus in a Broth Medium and in Rice

Germination and growth of Bacillus cereus spores from emetic and diarrheal strains were measured in Trypticase soy broth (TSB) and in autoclaved rice/beef extract from 5°– 55°C. Growth for some strains occurred from 15°– 50°C, and little difference was noted between responses of diarrheal and emetic types or between media, except a higher maximum population was achieved in rice. Germination was more extensive in rice than in TSB at <15°C and was generally more extensive for diarrheal strains in either medium.     

Growth and Survival of Salmonella typhimurium at Low Temperature in Nutrient Deficient Media

The growth and survival of two strains of Salmonella typhimutium in nutrient-deficient media at 7°C were studied. Cultures were propagated at 37°C, 24°C or 7°C in tryptic soy broth (TSB), inoculated into optimal, minimal and nutrient-deficient media (TSB, 1% TSB; 1.0%, 0.1% and 0.01% peptone water; 0.1% and 0.01% nonfat dry milk; and 30% glycol with and without 0.1% added milk solids) and incubated at 7°C. Numbers at 7°C declined initially when first propagated at 37°C. No decline was seen when propagated at the lower temperatures (24°C and 7°C). S. typhimurium survived in all media for extended periods; one strain survived for more than 1 year at 7°C in 0.1% peptone water and TSB.     

Growth and survival of Yersinia enterocolitica at acidic pH

The ability of three strains of Yersinia enterocolitica to survive and grow in tryptic soy broth (TSB) at pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 was determined. In addition, experiments were done to assess survival of Y. enterocolitica in mayonnaise, spoonable salad dressing, and tartar sauce. Two of the strains were able to grow in TSB at pH 4.5 and above when incubated at 25°C. Cell concentrations of all strains decreased at lower pH values at this temperature. No growth of Yersinia was observed when TSB was incubated at 5°C for any pH tested, although survival was prolonged. Viable cells were not recovered from artificially contaminated tartar sauce (pH 3.2) or spoonable salad dressing (pH 3.4) at any time. Cells survived for at least 48 h in artificially contaminated mayonnaise (pH 3.8) held at 5°C. No viable cells were recovered after 24 h from mayonnaise held at 21°C.     

Development of twoListeria monocytogenesgrowth models in a meat broth and their application to beef meat

Growth variation ofListeriastrains was taken into account by building two growth models with strains previously characterized, respectively, by their slow (L. monocytogenes CLIP 19532) and fast (L. monocytogenes 14) growth in different conditions of pH, a_wand temperature. Strains of intermediate growth were studied in a meat broth and strains used for the models were grown on the surface of beef meat. Ten growth repetitions at 14°C– a_w0.98–pH 6.2 showed that generation times were similar [ratio value (R=standard deviation/average): 3.2%] but that range of lag times was wide (R=27.4%). In broth, calculated lag and generation times were not significantly different between strains from 30 to 14°C, but variations became larger as temperatures came close to 4°C. Model values corresponded well to experimental generation times and to a lesser extent to lag times. On meat at 4°C and 14°C both strains had experimental lag times three-fold longer than predicted lag times. Experimental generation times were shorter than predicted values at 14°C and longer at 4°C: differences between growth in broth and on meat could be due to the characteristics of the meat, the experimental conditions of growth, the mode of inoculation and the way of adjustment of a_w. Growth variations were found between available predictive models.     

Comparison of growth and disinfectant resistance of Bacillus cereus isolated from fresh-cut produce and organic vegetables

Bacillus cereus strains were inoculated into tryptic soy broth (TSB) and incubated (7–35°C). Exponential and stationary phase cells were subjected to TSB plus sodium hypochlorite (0–3.6%), hydrogen peroxide (0–0.28%), and acetic acid (0–1.5%). Populations were counted on tryptic soy agar. Linear regression was fitted to survival data to calculate death rates. Cell counts decreased at 7°C. Differences in growth patterns were observed at 10°C and 15°C. Exponential and stationary phase cells showed significant (p<0.05) differences in disinfectant resistance. Strains treated with different disinfectants showed differences in growth and disinfectant resistance during different growth phases.     

Short communication: Comparison of growth kinetics at different temperatures of Streptococcus macedonicus and Streptococcus thermophilus strains of dairy origin

Within the genus Streptococcus, S. thermophilus and S. macedonicus are the 2 known species related to foods. Streptococci are widely used as starter cultures to rapidly lower milk pH. As S. macedonicus has been introduced quite recently, much less information is available on its technological potential. Because temperature is an important factor in fermented food production, we compared the growth kinetics over 24 h of 8 S. thermophilus and 7 S. macedonicus strains isolated from various dairy environments in Italy, at 4 temperatures, 30°C, 34°C, 37°C and 42°C. We used the Gompertz model to estimate the 3 main growth parameters; namely, lag phase duration (λ), maximum growth rate (µ_max), and maximum cell number at the stationary phase (N_max). Our results showed significant differences in average growth kinetics between the 2 species. Among the strains tested, 37°C appeared to be the optimal temperature for the growth of both species, particularly for S. macedonicus strains, which showed mean shorter lag phases and higher cell numbers compared with S. thermophilus. Overall, the growth curves of S. macedonicus strains were more similar to each other whereas S. thermophilus strains grew very differently. These results help to better define and compare technological characteristics of the 2 species, in view of the potential use of S. macedonicus in place of S. thermophilus in selected technological applications.     

Microbiological safety of UHT milk treated at 120 °C for 2 s,as estimated from the distribution of high-heat-resistant Bacillus cereus in dairy environments

Unlike common ultra-high-temperature (UHT) milk, so-called “UHT milk” in Japan is typically pasteurised at 120–130 °C for 2 s and distributed at 10 °C or less, and there is a potential risk of Bacillus cereus. To estimate the microbiological safety of UHT milk, we surveyed the distribution of high-heat-resistant B. cereus strains (defined as showing <∼3 log reduction after treatment at 120 °C for 2 s) among 200 isolates from dairy environments. Only four strains, which were isolated from the milk plant environment, showed high-heat resistance. All of them were unable to grow at 10 °C but grew at 12 °C. In contrast, heat-labile strains grew well at 10 °C. Therefore, UHT milk pasteurised at 120 °C for 2 s can be microbiologically safe, provided it is kept at 10 °C or less, within a rational shelf-life and avoiding contamination with B. cereus, especially of milk-plant-environment origin.     

Synthesis of extracellular lipase by a strain ofPseudomonas fluorescensisolated from raw camel milk

Ten strains of psychrotrophic bacteria were isolated from raw camel milk and were arranged according to their lipase production. Lipolytic activities ranged between 0.26 to 3.43 meq of palmetic acid 100 g^-;1of bovine milk fat h^-;1.Pseudomonas fluorescensRM₄was the most active strain. This bacterium could grow and secrete lipase over a wide range of temperatures. The optimum temperature for growth was 37°C, and the maximum lipase production was at 25°C. Growth was maximal after 96 h of incubation at 25°C, and lipase activity was maximal at 72 h post-inoculation at 25°C (during the late logarithmic phase). Shaking of the cultures (100 rpm) led to an increase in both growth and enzyme activities. Pseudomonas fluorescensRM₄was able to grow and secrete lipase over a pH range of 5.5-;8.50. Synthesis of the enzyme appeared to be inducible because no enzyme was detected in the absence of organic nitrogen. Supplementation of the basal medium with milk proteins enhanced lipase production. Tryptophan and lysine induced enzyme synthesis most effectively. Addition of 4 g l^-;1of glucose to broth stimulated both growth and production of lipase. Beyond this level of supplementation, lipase activity was considerably depressed.     

The effect of pH,sodium chloride,sodium nitrite and storage temperature on the growth of Clostridium perfringens and faecal streptococci in laboratory media

The effects of salt concentration (1–12% w/v) in combination with unheated sodium nitrite (0–400 μg/ml) on growth of mixed strains of Clostridium perfringens and of faecal streptococci at three pH values (5.6, 6.2, 6.8) and storage temperatures ranging from 10°C to 35°C is reported. At pH 6.2, following storage at 15°C, 1% salt and 50 μg/ml nitrite inhibited growth of C. perfringens. At 20°C and pH 6.2, 200 μg/ml nitrite plus 3% salt, or 50 μg/ml plus 4% salt were required to inhibit growth. Growth of C. perfringens was prevented by levels of curing salts used commercially providing the pH was 6.2 or below. At pH 6.8 or above at least 4% salt and 50 μg/ml nitrite was required to prevent growth at 20°C. The faecal streptococci grew in medium containing 6% salt and 400 μg/ml nitrite irrespective of pH or storage temperature. In 8% salt growth was prevented by storing at or below 17.5°C or, if pH was 6.2 or lower, by adding 200 μg/ml nitrite irrespective of storage temperature. Growth of faecal streptococci was not controlled by concentrations of curing salts which would be acceptable in meat products.     

The effects of salt and pH stress on the growth rates of persistent strains of Listeria monocytogenes collected from specific ecological niches

This study aimed to investigate the growth responses of persistent strains of Listeria monocytogenes to NaCl (5–10%) and pH (4–8.5) stress. Seven isolates of two persistent strains, collected from cheeses with 7 and 42 days of ripening, and from the shelf of one maturation room, and two reference strains were analysed. The experiments were carried out, in Tryptic Soy Broth (TSB), at 37 °C. Response surface methodology (RSM) was used to compare the effects of the selected variables on the growth rate of the isolates. Persistent strains showed, in general, higher growth rates than reference strains. The factor NaCl was highly significant to the isolate collected from the shelf. There was a different growth response between isolates from cheese with 7 and 42 days of ripening. These results suggest the adaptation of L. monocytogenes to specific environments within the dairies.     

Growth and histamine production by Morganella morganii under various temperature conditions

The generation times (G) for Morganella morganii were determined using absorbance measurements (at 0–10°C) or conductance measurements (at 15–25°C). Growth was rapid at 15–25°C (G = 2.6 at 15°C and G = 1.08 h at 25°C) but at the lower temperatures growth was greatly reduced. Growth and histamine production by M. morganii in histidine-containing broth (HDB) and in mackerel (packed in air-permeable plastic bags) was studied. It was found that large amounts of histamine are formed at low temperatures (0–5°C), where no growth takes place, following storage at higher temperatures (10–25°C). Measurements of conductance can be used to separate between samples with no or only insignificant content of histamine (< 50 ppm) which had detection times (DT) above 10 h and those with elevated content of histamine (> 50 ppm) which had shorter DT.     

Antimicrobial Activity of Vanillin and Mixtures with Cinnamon and Clove Essential Oils in Controlling <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 in Milk

The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of vanillin against Listeria monocytogenes Scott A and Escherichia coli O157:H7 was determined in tripticase soy broth (TSB), pH 7 and 6, incubated at 35 °C/24 h and in semi-skim milk incubated at 35 °C/24 h and 7 °C/14 days. The influence of the fat content of milk on the antimicrobial activity of vanillin was tested in whole and skim milk incubated at 7 °C/14 days. Mixtures of clove and cinnamon with vanillin were also evaluated in semi skim milk incubated at 7 °C. The MICs for L. monocytogenes were 3,000 ppm in TSB (pH 7) and 2,800 ppm in TSB (pH 6). The MICs for E. coli O157:H7 were 2,800 ppm in TSB (pH 7) and 2,400 ppm in TSB (pH 6). The MBCs in TSB were 8,000 ppm for L. monocytogenes and 6,000 ppm for E. coli O157:H7. The pH values assayed did not influence significantly the MIC or MBC in TSB. The MICs in semi-skim milk for L. monocytogenes and E. coli O157:H7 were 4,000 and 3,000 ppm at 35 °C/24 h, and 2,500 and 1,000 ppm at 7 °C/7 days, respectively. The MBCs were 20,000 ppm for L. monocytogenes and 11,000 ppm for E. coli O157:H7. High incubation temperatures did not affect the MBC but increased the MIC of the vanillin in milk. This effect could be attributed to the increased membrane fluidity and to the membrane perturbing activity of vanillin at low temperatures. The fat in milk reduced significantly the antimicrobial activity of vanillin, probably due to effect protective of the fat molecules. Mixtures of clove and cinnamon leaves inhibited the growth of L. monocytogenes in a similar way that vanillin alone but had a synergistic effect on the E. coli O157:H7. Mixtures of cinnamon bark and vanillin had always a synergistic effect and some of the combination assayed showed bactericidal activity on the population of L. monocytogenes and E. coli O 157:H7.     

Effect of Fish Attributes and Handling Stress on Quality of Smoked Arctic Char Fillets

ABSTRACT: Arctic char hatched at 6.5 and 9.5 °C and later divided into large, medium, and small size groups were assigned to processing (1) without handling stress, (2) immediately after handling stress, (3) at 24 h after handling stress, and (4) at 48 h after handling stress. Stress reduced gut weight and muscle a* value, increased muscle L* for the 9.5 °C group, decreased muscle L* for the 6.5 °C group, and increased fillet weight loss after brining. Fillets from the stressed, 9.5 °C group absorbed more brine, increasing salt and ash content of smoked products. A 24‐h stress recovery decreased total‐ and water‐phase salt. Fish hatched at 9.5 °C appeared to be more sensitive to handling stress than fish hatched at 6.5 °C. A 48‐h stress recovery increased gut weight and enhanced muscle L*.     

Suitability ofLactococcus lactissubsp.lactisATCC 11454 as a protective culture for lightly preserved fish products

This study is part of strategy to control the human pathogenListeria monocytogenesin lightly preserved fish products by using food-grade lactic acid bacteria. When the nisin- producingLactococcus lactissubsp.lactisATCC 11454 was cultured in the same vessel asL. monocytogenesScott A in brain–heart infusion broth (BHI) at 30°C, the pathogen declined from 5×10⁵to fewer than 5 cfu ml⁻¹within 31 h. The effect was not due to lactic acid inhibition. Growth and nisin production byL. lactisATCC 11454 were investigated under the conditions of temperature and salt used for light preservation. At 5°C in M17 broth, the organism grew well and produced nisin. In an infusion of cold-smoked salmon the organism did not grow at 5°C, although it did at 10°C. NaCl up to 4% allowed for efficient growth and nisin production, while 5% NaCl resulted in very slow growth and no detectable nisin. On slices of commercial cold-smoked salmon at 10°C, no net propagation ofL. lactisATCC 11454 could be detected within 21 days. However, when salmon slices were inoculated withL. monocytogenesat 10⁴cfu g⁻¹and a 300-fold excess of washed lactococcus cells, the pathogen's population declined a half log the first 1.5 days, then increased at a rate slightly lower than that of the control not inoculated with the lactococcus.     

Effects of cold storage and heat–acid shocks on growth and verotoxin 2 production ofEscherichia coliO157:H7

Escherichia coliO157:H7 was cold-stored (4°C) either in nutritious menstruum [buffered Brain Heart Infusion (BHI) broth] or with starvation (buffered saline) at pH 7.0 or 5.5. Cultures grown in BHI broth at 37°C for 24h served as non-cold-stored controls. After 4-weeks cold storage, bacterial cells were shocked by heat (45°C for 5min) and acid (pH 2.5 for 30min at 37°C) and subsequently moved to optimal conditions (BHI broth of pH 7.4 incubated at 37°C). The results showed: (a) both lag-phase duration and growth rate of this pathogen at 37°C significantly increased after cold-storage with starvation, but not after cold storage in the nutritious menstruum; (b) combined heat–acid shocks increased growth rates at 37°C of both previously cold-stored and non-cold-stored bacterial cells; (c) final concentrations of verotoxin produced by bacterial cells at 37°C were not affected by previous cold storage in the nutritious menstruum; (d) verotoxin production by bacterial cells at 37°C increased after cold storage with starvation, and heat–acid shocks further enhanced that production. Further research is needed to evaluate the food safety implications of these results, i.e. whether cells ofE. coliO157:H7 originating from nutrient-poor/lower-pH environments may be more harmful to humans than those from nutrient-rich/higher-pH foods.     

A Study of Factors Affecting Growth and Recovery of Listeria monocytogenes Scott A

The growth parameters for Listeria monocytogenes Scott A at various temperatures, pH values, water activity values, and in different carbohydrates were assessed. Growth was seen from 4°C through 45°C and at pH values from 4.7 through 9.2. Generation times were calculated for each parameter at which growth was observed. Optimum growth was defined as shortest generation time. L. monocytogenes grew optimally over the temperature range 30–37°C. The optimum pH was 7.0. Growth was demonstrated in solutions of up to 39.4% sucrose (a_w of 0.92). Carbohydrate fermentation was variable. Further studies involved examination of growth at optimal water activities (0.97) with suboptimal temperatures and pH values, and at 7.0, with suboptimal temperatures and water activities. Growth was seen when these parameters were combined.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Heat Resistance of Some Selected Toxigenic Enterococci in Milk and Other Suspending Media

Streptococcus faecium IF-100, S. faecalis var. zymogenes RCM-29 (both toxigenic) and S. faecalis var. liquefaciens N.P. were subjected to 50–70°C in reconstituted nonfat dry cow's milk (NFDCM), spray-dried infant food (RIF), full cream buffaloe's milk (FCBM), normal saline and brain heart infusion (BHI) broth for different periods of time. S. faecium IF-100 was the most heat resistant in all the heating media as evidenced by high D values, i.e., 8.5, 8.5, and 6.5 min in RIF, FCBM and NFDCM, respectively, even at 70°C. The corresponding D values in respect to S. faecalis var. liquefaciens were 3.5 min each in the three menstrua. However, the heat resistance of the test cultures was the least in normal saline (D values ranging from 1 min (RCM-39) to 2.5 min (IF-100). The variations in D values of the 3 enterococci were statistically significant at 1%.