Survival and growth of Escherichia coli O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature.

A study was undertaken to determine the fate of Escherichia coli O157:H7 in ground, roasted beef as influenced by the combined effects of pH, acidulants, temperature, and time. There was essentially no change in the viable population of E. coli O157:H7 when beef salads (pH 5.40 to 6.07) containing up to 40% mayonnaise were incubated at 5 degrees C for up to 72 h. At 21 and 30 degrees C, significant (P < or = 0.05) increases in populations of the organism occurred in salads containing 16 to 32% mayonnaise (pH 5.94 to 5.55) between 10 and 24 h of incubation. Death was more rapid as the pH of acidified beef slurries incubated at 5 degrees C was decreased from 5.98 to 4.70. E. coli O157:H7 grew in control slurries (pH 5.98) and in slurries containing citric and lactic acids (pHs 5.00 and 5.40) incubated at 21 degrees C for 24 h; decreases occurred in slurries acidified to pHs 4.70, 5.00, and 5.40 with acetic acid or pH 4.70 with citric or lactic acid. At 30 degrees C, populations decreased in slurries acidified to pHs 4.70 and 5.00 with acetic acid. Citric and lactic acids failed to prevent significant increases in populations in slurries at pH 4.70 to 5.40 between 10 and 24 h of incubation. The order of effectiveness of acidulants in inhibiting growth was acetic acid > lactic acid > or = citric acid. The same order was observed for inactivation of E. coli O157:H7 in acidified (pH 5.00) beef slurry heated at 54 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of pH, acidulant and temperature on the survival of Yersinia enterocolitica

The survival of Yersinia enterocolitica at sub-optimal temperatures (0–23°C) and growth inhibitory pH values, achieved using a range of acidulants, was investigated. At a given pH, survival was greater the lower the temperature. Sulphuric and citric acids had lower bactericidal activity than acetic and lactic acids and in nearly all cases where the four acids could be compared at the same pH the order of bactericidal activity was acetic > lactic > citric > sulphuric. Attempts to model this behaviour by a negative square root relationship gave good correlation coefficients for plots of the square root of death rate against temperature at different combinations of pH and acidulant but so too did several other functions of death rate. The high coefficient of variation for T ₀ determined from square root plots prevented construction of a combined temperature/pH model similar to that already described for growth.     

Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.     

Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media.

Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid. All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter). Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C. botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6. The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration. A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks. At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C. botulinum spores. Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C. botulinum spores under these conditions. Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6.     

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.     

The effect of various acidulants on the growth of Listeria monocytogenes

The ability of four Listeria monocytogenes strains to initiate growth in brain heart infusion broth adjusted to various pH values with either acetic, lactic, citric or hydrochloric acid was investigated. Acetic acid was the most effective inhibitor tested, since in broth adjusted with this acid a higher minimum pH was required for growth of the various strains at both 4 and 30°C, as compared with broth adjusted with the other acidulants. The minimum pH value required for the initiation of growth of L. monocytogenes ranged from 5·0 to 5·7 at 4°C, and from 4·3 to 5·2 at 30°C, depending upon the acidulant used.     

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°C, inocula containing < 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give > 10⁸ 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 10⁶. 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.     

Citric acid production by a novel Aspergillus niger isolate: II. Optimization of process parameters through statistical experimental designs

In this work, sequential optimization strategy, based on statistical designs, was employed to enhance the production of citric acid in submerged culture. For screening of fermentation medium composition significantly influencing citric acid production, the two-level Plackett-Burman design was used. Under our experimental conditions, beet molasses and corn steep liquor were found to be the major factors of the acid production. A near optimum medium formulation was obtained using this method with increased citric acid yield by five-folds. Response surface methodology (RSM) was adopted to acquire the best process conditions. In this respect, the three-level Box-Behnken design was applied. A polynomial model was created to correlate the relationship between the three variables (beet molasses, corn steep liquor and inoculum concentration) and citric acid yield. Estimated optimum composition for the production of citric acid is as follows pretreated beet molasses, 240.1g/l; corn steep liquor, 10.5g/l; and spores concentration, 10(8)spores/ml. The optimum citric acid yield was 87.81% which is 14 times than the basal medium. The five level central composite design was used for outlining the optimum values of the fermentation factors initial pH, aeration rate and temperature on citric acid production. Estimated optimum values for the production of citric acid are as follows initial pH 4.0; aeration rate, 6500ml/min and fermentation temperature, 31.5 degrees C.     

不同酸性条件对双歧杆菌存活的影响

以胡萝卜复合汁为保存基质,试验了不同的基质起始PH值、常用的酸味剂和盐酸、不同比例的乳酸乙酸混合酸对两歧双歧杆菌（Bb）和长双歧杆菌（Bl）存活的影响。结果表明,适于菌体存活的pH值,Bb为6．2,Bl为4．5～5．0;常用的酸味剂和盐酸对Bb存活的影响次序为柠檬酸＞盐酸＞乳酸＞乙酸,对Bl则为乳酸＞盐酸＞柠檬酸＞乙酸;对Bb和Bl存活最为不利的乳酸：乙酸（摩尔）比例分别为肝1:1和1:1．5.总体上,Bl的保存性优于Bb.     

Activity of p-aminobenzoic acid compared with other organic acids against selected bacteria

The antibacterial activity of p -aminobenzoic acid against Listeria monocytogenes, Salmonella enteritidis and Escherichia coli was compared with the activity of commonly used acidulants: formic, propionic, acetic, lactic and citric acids. Viable count evaluations and MIC determinations indicated that p -aminobenzoic acid caused greater inhibitory effects than the other organic acids. The activity of p -aminobenzoic acid on the growth of the test organisms at selected pH values indicated that p -aminobenzoic acid was more active at low pH than at high pH. Uptake studies showed that the uptake of p -aminobenzoic acid by E. coli was markedly decreased as the pH values increased. Electron micrographs of E. coli cells grown in the presence of p -aminobenzoic acid indicate that p -aminobenzoic acid caused marked damage to the cell envelope. It is suggested that p -aminobenzoic acid has at least two mechanisms of action: one mechanism in common with other organic acids and the other mechanism by interfering with the synthesis of the peptidoglycan layer by an action on the dihydrofolate reductase enzyme.     

Short communication: Influence of inoculum preparation on citric acid production by Aspergillus niger

The type of sporulation medium and time of incubation had an effect on spore viability and citric acid production by mycelia grown from Aspergillus niger spores. Shu & Johnson agar (SJA) and potato dextrose agar gave higher citric acid titres than malt-extract agar. SJA also gave better germinability than the other media. Viability increased with time of incubation, but higher production of citric acid was achieved with spores incubated for less than 7 days.     

Heat resistance of Paenibacillus polymyxa in relation to pH and acidulants

The efficacy of different organic acids in decreasing the heat resistance of Paenibacillus polymyxa spores was assessed. The relationship between concentration of the undissociated form of different organic acids and decrease in heat resistance was also investigated. The heat resistance of P. polymyxa spores was tested in distilled water at 85, 90 and 95 degrees C, at pH4 and in the presence of 50, 100 and 200 mmol l(-1) of the undissociated form of lactic, citric or acetic acid and sodium citrate or acetate. The undissociated form of organic acids was responsible for increasing the heat sensitivity of spores. The most effective acid was lactic acid. The D values of the spores decreased rapidly (between 74 and 43%) in the presence of 50 mmol l(-1) of the undissociated form of organic acid, and increasing concentrations of these forms affected the heat resistance of spores less than proportionally. The heat resistance of the spores in milk was approximately threefold lower than in distilled water. This work has shown that the undissociated fraction of organic acids increases, albeit non-linearly, the sensitivity of spores to heat, even in complex substrates such as milk. By knowing the amount of organic acids added to a given substrate, their dissociation constants and the final pH, it could be possible to estimate the concentration of undissociated forms and the corresponding increase in lethality of heat treatments. This would help the food industry to maximize the lethality achieved by heat processes and/or safely reduce the heat treatments already in use.     

The influence of intracellular pH of spores on citric acid production by Aspergillus niger

The spores with different intracellular pH values were produced by cultivating Aspergillus niger on potato-dextrose agar at different pH_e values. High citric acid production is obtained using spores with the highest initial pH values (pH_i) around 7.5. After a drop of intracellular pH during germination of spores of about 0.7 units, the conditions for highest rate of metabolic flow through the glycolytic pathway were achieved only by the mycelium grown out of spores with the highest pH_i, since a very narrow pH optimum of 6-phosphofructokinase activity has been found around 7.5.     

Toxicity of citric and succinic acids for the pycnidiospores ofBotryodiplodia theobromae

The toxic effect of citric and succinic acids on the germination of the pycnidiospores ofBotryodiplodia theobromae, mycelial growth and the killing rate of theB. theobromae spores was investigated. The percentage inhibition of germination of viable fungal spores by 0.01% succinic or citric acid ranged between 51.6 and 58.1%, respectively.B. theobromae was found to grow in 2% malt extract broth at 28°C at the rate of 0.13 CFU/h. Citric acid exhibited a higher killing rate of 0.26 CFU/h and was more effective against the germination of the fungal spores. At concentrations of 0.3% and above, citric acid could be used as pre- and post-infectional fungicide.     

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.     

Effect of acetic and trans-aconitic acids on citric acid production by Aspergillus niger.

The effect of acetic and trans-aconitic acids on citric acid production by A. niger at different pH values was studied. The presence of acetic acid at pH 2 prevented spore germination, while it decreased the fungal growth and citric acid production at other pH values. In the presence of trans-aconitic acid the inhibition was less marked at lower than at higher pH values.     

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.     

黑曲霉菌株柠檬酸合成酶基因的敲除及功能分析

【背景】柠檬酸合成酶是碳代谢途径的中心酶,其在三羧酸循环(tricarboxylic acid cycle,TCA)、氨基酸合成和乙醛酸循环中发挥着重要作用,是柠檬酸合成的关键酶。本论文所选用的是一株高产柠檬酸的黑曲霉菌株CGMCC10142。【目的】克隆柠檬酸合成酶关键基因,构建柠檬酸合成酶的敲除菌株并鉴定其在黑曲霉菌株高产柠檬酸过程中的功能及影响。【方法】采用根癌农杆菌转化方法并利用同源重组原理,采用抗性筛选和致死型反向筛选的双重筛选方法获得正确敲除株。对转化子在不同碳源下的生长情况进行观察并对柠檬酸发酵过程中菌丝球变化和产酸量进行分析,最后通过荧光定量PCR分析柠檬酸合成酶基因对黑曲霉积累柠檬酸的影响,及其对主要代谢途径中重要酶相关基因和其他的表达量的影响。【结果】以柠檬酸高产菌株黑曲霉CGMCC10142为出发菌,构建一株遗传稳定的柠檬酸合成酶敲除的菌株T1-2。结果发现该菌株在以葡萄糖为碳源的培养基上生长缓慢并且产生孢子量减少。通过摇瓶发酵产酸实验,结果表明敲除菌在84 h产酸量为64.3 g/L,相对于出发菌的98.7g/L降低了34.85%。通过荧光定量PCR发现柠檬酸合成酶的表达量是下降的,同时重要酶的表达量都下降。【结论】该菌株的柠檬酸合成酶基因对柠檬酸积累具有重要作用,但存在其他同工酶基因,该基因敲除仅使产酸合成降低34.85%,同时发现该柠檬酸合成酶的顺畅表达有助于主代谢途径中各关键酶的高效表达,本研究可为研究黑曲霉高产柠檬酸机理奠定基础。     

Effects of humoral factors on ventilation kinetics during recovery after impulse-like exercise

To clarify the ventilatory kinetics during recovery after impulse-like exercise, subjects performed one impulse-like exercise test (one-impulse) and a five-times repeated impulse-like exercises test (five-impulse). Duration and intensity of the impulse-like exercise were 20 sec and 400 watts (80 rpm), respectively. Although blood pH during recovery (until 10 min) was significantly lower in the five-impulse test than in the one-impulse test, ventilation (.VE) in the two tests was similar except during the first 30 sec of recovery, in which it was higher in the five-impulse test. In one-impulse, blood CO2 pressure (PCO2) was significantly increased at 1 min during recovery and then returned to the pre-exercise level at 5 min during recovery. In the five-impulse test, PCO2 at 1 min during recovery was similar to the pre-exercise level, and then it decreased to a level lower than the pre-exercise level at 5 min during recovery. Accordingly, PCO2 during recovery (until 30 min) was significantly lower in the five-impulse than in one-impulse test..VE and pH during recovery showed a curvilinear relationship, and at the same pH, ventilation was higher in the one-impulse test. These results suggest that ventilatory kinetics during recovery after impulse-like exercise is attributed partly to pH, but the stimulatory effect of lower pH is diminished by the inhibitory effect of lower PCO2.     

The effect of acid shock on sporulating Bacillus subtilis cells

AIMS: To study the effect of acid shock in sporulation on the production of acid-shock proteins, and on the heat resistance and germination characteristics of the spores formed subsequently. METHODS AND RESULTS: Bacillus subtilis wild-type (SASP-alpha+beta+) and mutant (SASP-alpha-beta-) cells in 2 x SG medium at 30 degrees C were acid-shocked with HCl (pH 4, 4.3, 5 and 6 against a control pH of 6.2) for 30 min, 1 h into sporulation. The D85-value of B. subtilis wild-type (but not mutant) spores formed from sporulating cells acid-shocked at pH 5 increased from 46.5 min to 78.8 min, and there was also an increase in the resistance of wild-type acid-shocked spores at both 90 degrees C and 95 degrees C. ALA- or AGFK-initiated germination of pH 5-shocked spores was the same as that of non-acid-shocked spores. Two-dimensional gel electrophoresis showed only one novel acid-shock protein, identified as a vegetative catalase 1 (KatA), which appeared 30 min after acid shock but was lost later in sporulation. CONCLUSIONS: Acid shock at pH 5 increased the heat resistance of spores subsequently formed in B. subtilis wild type. The catalase, KatA, was induced by acid shock early in sporulation, but since it was degraded later in sporulation, it appears to act to increase heat resistance by altering spore structure. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first proteomic study of acid shock in sporulating B. subtilis cells. The increasing spore heat resistance produced by acid shock may have significance for the heat resistance of spores formed in the food industry.