Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media.

Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days. The spore count was determined at different intervals over the 180-day storage period. There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C. The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C.     

Thermal destruction of Clostridium botulinum spores suspended in tomato juice in aluminum thermal death time tubes.

The heat destruction characteristics of Clostridium botulinum spores suspended in tomato juice and phosphate buffer were determined by the survivor curve method with aluminum thermal death time tubes. Two type A strains of C. botulinum and a type B strain were evaluated. Strains A16037 and B15580 were implicated in outbreaks of botulism involving home-canned tomato products. Strain A16037 had a higher heat resistance than either 62A or B15580. The mean thermal resistance (D-values) for A16037 in tomato juice (pH 4.2) were: 115.6 degrees C, 0.4 min; 110.0 degrees C, 1.6 min; and 104.4 degrees C, 6.0 min. The mean D-values for A16037 in Sorensen 0.067 M phosphate buffer (pH 7) were: 115.6 degrees C, 1.3 min; 110.0 degrees C, 4.4 min; and 104.4 degrees C, 17.6 min. At each test temperature, the D-values were approximately three times higher in buffer than in tomato juice. The z-value for C. botulinum A16037 spores in tomato juice was 9.4 degrees C, and in buffer the z-value was 9.9 degrees C. The use of aluminum thermal death time tubes in a miniature retort system makes it possible to determine survivor curves for C. botulinum spores at 121.1 degrees C. This is possible because the lag correction factor for the aluminum tubes is only about 0.2 min, making possible heating times as short as 0.5 min.     

Acid tolerance of Shigella sonnei and Shigella flexneri

AIMS: Determination of the behaviour of Shigella sonnei and Sh. flexneri under acid conditions. METHODS AND RESULTS: The growth and survival of Shigella spp. (9 isolates) in acidified Brain Heart Infusion (BHI) (pH 5.0-3.25 with pH intervals of 0.25) was determined after 6, 24 and 30 h incubation at 37 degrees C. Subsequently, survival of shigellae was studied in apple juice and tomato juice stored at 7 degrees C and 22 degrees C for up to 14 days and in strawberries and a fresh fruit salad, kept at 4 degrees C for 4 and 48 h. CONCLUSIONS: The minimum pH for growth in acidified BHI for Sh. flexneri and Sh. sonnei was, respectively, pH 4.75 and pH 4.50. Survival in fruit juices and fresh fruits depended upon their pH, the type of strain and the incubation temperature. Shigella spp. Survived for up to 14 days in tomato juice and apple juice stored at 7 degrees C. The shortest survival time (2-8 d) was observed in apple juice at 22 degrees C. Sh. sonnei but not Sh. flexneri was recovered after 48 h from strawberries and fruit salad kept at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid foods, especially if kept at refrigeration temperatures, support survival of Shigella spp. and may cause Shigella food poisoning.     

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.     

Clostridium botulinum growth and toxin production in tomato juice containing Aspergillus gracilis.

The ability of spores of one type A and one type B strain of Clostridium botulinum to grow and produce toxin in tomato juice was investigated. The type A strain grew at pH 4.9, but not at pH 4.8; the type B strain grew at pH 5.1, but not at pH 5.0. Aspergillus gracilis was inoculated along with C. botulinum spores into pH 4.2 tomato juice; in a nonhermetic unit, a pH gradient developed under the mycelial mat, resulting in C. botulinum growth and toxin production. In a hermetic unit, mold growth was reduced, and no pH gradient was detected; however, C. botulinum growth and low levels of toxin production (less than 10 50% lethal doses per ml) still occurred and were associated with the mycelial mat. The results of tests to find filterable or dialyzable growth factors were negative. It was demonstrated that for toxin production C. botulinum and the mold had to occupy the same environment.     

Effect of storage time and temperature and the variation among replicate tests (on different days) on the performance of spore disks and strips.

Laboratory-prepared spore disks were stored for 96 weeks at 22 degrees C with 50% relative humidity (RH) and at 4 degrees C with less than 1% RH. At the same time commercial spore strips were stored for 64 weeks at 22 degrees C with 50% RH. The spore count per unit and the heat resistance were measured at the beginning of the experiment and after 16, 32, 48, 64, 80, and 96 weeks of storage. The laboratory-prepared spore disks stored at 4 degrees C with less than 1% RH showed less change in numbers of spores per disks and decrease in the survival time than did the disks stored at 22 degrees C with 50% RH. Both the laboratory-prepared spore disks and the commercial spore strips stored at 22 degrees C with 50% RH decreased in survival times with increased storage time. The relative change in the survival times with storage was less for the commercial spore strips than for the laboratory-prepared spore disks.     

Behavior of Listeria monocytogenes inoculated into raw tomatoes and processed tomato products

Rates of death and growth of Listeria monocytogenes inoculated onto raw whole and into chopped tomatoes stored at 10 and 21 degrees C were not influenced by prior treatment of tomatoes with chlorine or packaging under an atmosphere of 3% O2 and 97% N2. Growth of the pathogen occurred in whole tomatoes held at 21 degrees C but not at 10 degrees C, while death occurred in chopped tomatoes stored at these temperatures. Likewise, growth patterns of mesophilic aerobic microorganisms, psychrotrophic microorganisms, and yeasts and molds on whole and chopped tomatoes were essentially unaffected by chlorine and modified atmosphere packaging treatments. Populations of L. monocytogenes inoculated into commercially processed tomato juice and sauce and held at 5 degrees C remained constant for 14 days. A gradual decrease in the number of viable L. monocytogenes cells was observed in juice and sauce held at 21 degrees C. In contrast, the organism died rapidly when suspended in commercial tomato ketchup at 5 and 21 degrees C. Unlike low-acid raw salad vegetables such as lettuce, broccoli, asparagus, and cauliflower on which we have observed L. monocytogenes grow at refrigeration temperatures, tomatoes are not a good growth substrate for the organism. Nevertheless, L. monocytogens can remain viable on raw whole and chopped tomatoes and in commercial tomato juice and sauce for periods extending beyond their normal shelf-life expectancy.     

Alterations in the major heterotrophic bacterial populations isolated from a still bottled mineral water

The heterotrophic bacterial population of a bottled mineral water stored in returnable glass bottles and in polyvinyl chloride (PVC) bottles at room temperature was studied over 9-12 months. The plate counts in R2A medium incubated at 22 degrees and 37 degrees C were low initially, increasing to 10(4)-10(5) cfu/ml within a few days of bottling. The number of bacteria recovered at 22 degrees C from PVC bottles was fairly constant during the storage period, but the population isolated at 37 degrees C decreased markedly after storage for 1 year. The major components of the population were Pseudomonas strains, one of which was identified as Pseudomonas vesicularis. Major changes took place during storage; two groups of bacteria (B and C) were dominant initially, but during the latter period of storage other groups (F, G and H) increased in number.     

Behavior of Listeria monocytogenes inoculated into raw tomatoes and processed tomato products.

Rates of death and growth of Listeria monocytogenes inoculated onto raw whole and into chopped tomatoes stored at 10 and 21 degrees C were not influenced by prior treatment of tomatoes with chlorine or packaging under an atmosphere of 3% O2 and 97% N2. Growth of the pathogen occurred in whole tomatoes held at 21 degrees C but not at 10 degrees C, while death occurred in chopped tomatoes stored at these temperatures. Likewise, growth patterns of mesophilic aerobic microorganisms, psychrotrophic microorganisms, and yeasts and molds on whole and chopped tomatoes were essentially unaffected by chlorine and modified atmosphere packaging treatments. Populations of L. monocytogenes inoculated into commercially processed tomato juice and sauce and held at 5 degrees C remained constant for 14 days. A gradual decrease in the number of viable L. monocytogenes cells was observed in juice and sauce held at 21 degrees C. In contrast, the organism died rapidly when suspended in commercial tomato ketchup at 5 and 21 degrees C. Unlike low-acid raw salad vegetables such as lettuce, broccoli, asparagus, and cauliflower on which we have observed L. monocytogenes grow at refrigeration temperatures, tomatoes are not a good growth substrate for the organism. Nevertheless, L. monocytogens can remain viable on raw whole and chopped tomatoes and in commercial tomato juice and sauce for periods extending beyond their normal shelf-life expectancy.     

The Effect of Oidium lactis on the Sporulation of Bacillus coagulans in Tomato Juice

Oidium lactis can raise the pH of tomato juice in thin layers to a pH range optimum for sporulation of Bacillus coagulans in 24 hr at 25 C and in 48 hr at 35 C. The percentage of sporulation of B. coagulans was greater in tomato juice where O. lactis had grown than in the thermoacidurans broth. As high as 61.7% sporulation occurs within 24 hr in the tomato juice in which Oidium lactis had grown when this juice was incubated at 52 C. When tomato juice was adjusted to pH 5.0 and B. coagulans or a mixture of O. lactis and the vegetative cells of B. coagulans were added as an inoculum, only in the juice in which O. lactis was growing were spores produced within 72 hr at 35 C.     

Probiotication of tomato juice by lactic acid bacteria

This study was undertaken to determine the suitability of tomato juice as a raw material for production of probiotic juice by four lactic acid bacteria (Latobacillus acidophilus LA39, Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Tomato juice was inoculated with a 24-h-old culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were measured. The lactic acid cultures reduced the pH to 4.1 or below and increased the acidity to 0.65% or higher, and the viable cell counts (CFU) reached nearly 1.0 to 9.0 x 10(9)/ml after 72 h fermentation. The viable cell counts of the four lactic acid bacteria in the fermented tomato juice ranged from 10(6) to 10(8) CFU/ml after 4 weeks of cold storage at 4 degrees C. Probiotic tomato juice could serve as a health beverage for vegetarians or consumers who are allergic to dairy products.     

Comparative study of the inactivation kinetics of pectinmethylesterase in tomato juice and purified form

Pectinmethylesterase (PME) extracted from tomato fruit was purified by affinity chromatography. A single peak of PME activity was observed, presenting a molar mass of 33.6 kDa, an isoelectric point higher than 9.3, and an optimal temperature and pH for activity of 55 degrees C and 8.0, respectively. The processing stability of purified tomato PME in buffer solution was compared to PME stability in tomato juice. In both media, thermal inactivation of PME presented first-order inactivation kinetics, PME in tomato juice being more heat-labile than purified PME. Regarding high-pressure treatment, tomato PME showed to be very pressure-resistant, revealing an outspoken antagonistic effect of temperature and pressure. To avoid cloud loss in tomato juice, a time-temperature treatment of 1 min at 76.5 degrees C was calculated in order to have a residual PME activity of 1 x 10(-)(4) U/mL.     

Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa

The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.     

The influence of pH and temperature on the behaviour of Salmonella enteritidis phage type 4 in home-made mayonnaise

The behaviour of Salmonella enteritidis phage type 4 in home-made mayonnaise was studied. Samples of mayonnaise were prepared with different pH values using wine vinegar or lemon juice in order to bring down the pH to 5,4.5,4 and 3.6, inoculate and incubated at 4, 24 and 35C for 5 days. The results showed a better bactericidal activity of the vinegar (acetic acid) than the lemon juice (citric acid), both of these acids being more active at higher temperatures. For preventing salmonellosis transmission by home-made mayonnaise the use of vinegar as an acidulant in order to achieve a pH between 3.6 and 4 and storage in a warm place is recommended.     

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.     

Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes

The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology. Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production. Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960. No toxin was detected when the aw was 0.955. The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time. The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear. Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes.

The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology. Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production. Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960. No toxin was detected when the aw was 0.955. The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time. The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear. Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.     

The fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid

The fate of Salmonella enteritidis PT4 in home-made mayonnaise prepared with citric acid solution (citric acid concentration of > or = 4.98% (w/v)) was investigated. It was found that pH of mayonnaise is closely related to the ratio of egg yolk to citric acid, and the inactivation rate of the micro-organisms increases as the ratio decreases and/or incubation temperature increases. To achieve Salm. enteritidis PT4-free home-made mayonnaise prepared with pure lemon juice (citric acid concentration > or = 5% (w/v)), it is recommended that the pH should be 3.30 or below, or, in practice, at least 20 ml pure lemon juice per fresh egg yolk should be used. For the use of 20-35 ml pure lemon juice per egg yolk, the product should be held at 22 degrees C or over for at least 72 h and for the use of over 35 ml pure lemon juice per egg yolk, for at least 48 h before consumption or refrigeration.     

Effect of temperature, sodium chloride, and pH on growth of Listeria monocytogenes in cabbage juice.

Human illness and death have resulted from the consumption of milk, cheese, and cole slaw contaminated with Listeria monocytogenes. Since the effects of temperature, NaCl, and pH on the growth of the organism in cabbage were unknown, a series of experiments was designed to investigate these factors. Two strains (LCDC 81-861 and Scott A, both serotype 4b) were examined. At 30 degrees C, the viable population of the LCDC 81-861 strain increased in sterile unclarified cabbage juice (CJ) containing 0 to 1.5% NaCl; a decrease in the population of both strains occurred in juice containing greater than or equal to 2% NaCl. At 5 degrees C, the population of the Scott A strain in CJ containing up to 5% NaCl was reduced by about 90% over a 70-day period; the LCDC 81-861 strain was more sensitive to refrigeration but remained viable in CJ containing less than or equal to 3.5% NaCl for 70 days. Growth in CJ at 30 degrees C resulted in a decrease in pH from 5.6 to 4.1 within 8 days. Death of L. monocytogenes occurred at 30 degrees C when the organism was inoculated into sterile CJ adjusted to pH less than or equal to 4.6 with lactic acid. No viable cells were detected after 3 days at pH less than or equal to 4.2. At 5 degrees C, the rate of death at pH less than or equal to 4.8 was slower than at 30 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)