Stress response of Listeria monocytogenes isolated from cheese and other foods

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.     

Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses

The majority of published studies on the adaptive heat or acid tolerance response of Listeria monocytogenes have been performed with a single strain exposed to a single adaptation treatment; however, in food ecosystems, microorganisms commonly exist as multi-species communities and encounter multiple stresses, which may result in "stress hardening". Therefore, the present study evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (T; 46 degrees C) stresses, sequentially or simultaneously within 1.5h, in tryptic soy broth with 0.6% yeast extract (TSBYE). All treatments induced adaptive responses on L. monocytogenes at 57 degrees C, while no such cross-protection was observed at 52 and 63 degrees C. Survivor curves at 57 degrees C appeared convex with profound shoulders determined by a Weibull model. The highest thermotolerance was observed after combined exposure to acid and heat shock (pH-T), followed by exposure to osmotic shock, and by the combination of osmotic with heat shock (NaCl-T). Regarding acid tolerance, prior exposure to low pH, pH-T, or a combination of NaCl, pH and T resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log(10)CFU ml(-1)) than the control cultures. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses (e.g., NaCl-pH-T, NaCl-T and NaCl-pH) resulted in higher survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. The results indicate that combinations and sequences of sublethal hurdles may affect L. monocytogenes acid and heat tolerance, especially in acidic environments with mild heating or in low moisture environments.     

酸适应海德尔堡沙门氏菌对环境胁迫耐受性分析

目的：研究海德尔堡沙门氏菌（Salmonella heidelberg）低pH值酸适应性及酸适应对细胞其他胁迫环境耐受性的影响。方法：海德尔堡沙门氏菌在pH 5.5酸性培养基中生长2 h诱导酸适应性,以pH 3.0酸胁迫培养基及不同温度、NaCl、胆盐、消毒剂环境的存活率评价其耐受性。结果：pH 5.5酸适应2 h后,海德尔堡沙门氏菌在pH 3.0酸胁迫环境的存活率大幅提高。低pH值酸适应也使海德尔堡沙门氏菌在45、50 ℃高温条件下的存活率显著提高,对4 ℃冷藏环境的耐受性显著低于非酸处理菌株,但对－20 ℃冷冻环境的生存无影响。酸适应海德尔堡沙门氏菌对高渗、胆盐胁迫也表现良好的耐受性,5～10 g/100 mL NaCl溶液处理1 h,酸适应海德尔堡沙门氏菌的存活率大于60%,在1～10 g/100 mL胆盐溶液的存活率比对照组高3.8 倍。海德尔堡沙门氏菌酸适应性也使其对消毒剂的敏感性发生变化,酸适应性海德尔堡沙门氏菌在10～25 mmol/L H2O2溶液的存活率显著高于对照菌株,最高达3.1 倍;但对NaClO、乙醇消毒剂的耐受性显著降低,高质量浓度下二者无显著差异。结论：海德尔堡沙门氏菌在低pH值酸性条件生长一段时间会诱导产生酸适应性,还会协同提高其对高温、高渗、胆盐、H2O2胁迫环境的交叉抗性;但对4、－20 ℃低温、NaClO、乙醇等杀菌、保藏措施敏感性提高。     

PROTEIN PROFILE CHANGES IN ACID ADAPTED LISTERIA MONOCYTOGENES EXHIBITING CROSS-PROTECTION AGAINST AN ACTIVATED LACTOPEROXIDASE SYSTEM IN TRYPTIC SOY BROTH

Foodborne pathogens often tolerate and survive environmental stress conditions including extreme acidity to varying degrees. One possible reason for this survival may be the production of protective stress proteins during acid shock (ASR) and/or tolerance (ATR) responses. The ASR and ATR of Listeria monocytogenes strains V7, V37 and CA in tryptic soy broth without dextrose acidified with lactic acid were studied. Possible cross-protection of acid adapted cells against an activated lactoperoxidase system was also determined. The strains were either directly challenged at pH 4.0 and 3.5 to study their ASR or initially adapted at pH 5.5 for the equivalent of 1 generation before challenging at pH 4.0 and 3.5 to study their ATR. Adapted and nonadapted cells were challenged at pH 4.5 with or without an activated lactoperoxidase system. In all cases viability was determined by enumeration over a period of 24 or 48 h after challenge and the production of stress proteins analyzed by 2-dimensional gel electrophoresis. While there were some differences in the survival responses for each strain, the acid adapted cells of each strain survived to a greater degree than nonadapted cells at both pH 4.0 (at least 10 fold at 24 h) and pH 3.5 (at least 1000 fold at 6 h) but not at pH 4.5. The acid adapted cells exposed to the lactoperoxidase system survived better (at least 5-fold) than their nonadapted counterparts for all 3 strains at 24 and 48 h. The 2-dimensional gel analysis for all 3 strains showed that the adapted and nonadapted cells underwent a change in their physiology, (at pH 4.0 compared to the control at pH 7.0; at pH 4.5 with the addition of lactoperoxidase system components) in that there was induction as well as repression of several proteins.     

Effect of acid tolerance response (ATR) on attachment of Listeria monocytogenes Scott A to stainless steel under extended exposure to acid or/and salt stress and resistance of sessile cells to subsequent strong acid challenge

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate (ca. 10? CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly (p<0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment.     

Tolerance to high osmolality of the lactic acid bacterium Oenococcus oeni and identification of potential osmoprotectants

Growth of the lactic acid bacterium Oenococcus oeni under hyperosmotic constraint was investigated in a chemically defined medium. The bacterium could grow on media with an elevated osmolality, preferably below 1.5 Osm kg(-)(1) H(2)O. At osmolalities comprised between 0.6 and 1.5 Osm kg(-)(1) H(2)O, the growth deficit elicited by the sugars glucose and fructose was slightly more severe than with salts (NaCl or KCl). In contrast to what was observed in other lactic acid bacteria, proline, glycine betaine and related molecules were unable to relieve inhibition of growth of O. oeni under osmotic constraint. This was correlated to the absence of sequences homologous to the genes coding for glycine betaine and/or proline transporters described in Lactococcus lactis and Lactobacillus plantarum. The amino acid aspartate proved to be osmoprotective under electrolyte and non-electrolyte stress. Examination of the role of peptides during osmoregulation showed that proline- and glutamate-containing peptides were protective under salt-induced stress, and not under sugar-induced stress. Under high salt, PepQ a cytoplasmic prolidase that specifically liberated proline from di-peptides increased activity, while PepX (X-prolyl-dipeptidyl aminopeptidase) and PepI (iminopeptidase) activities were unaffected. Our data suggest that proline- and glutamate-containing peptides may contribute to the adaptation of O. oeni to high salt through their intracellular hydrolysis and/or direct accumulation.     

A study on the implications of NaCl reduction in the fermentation profile of Conservolea natural black olives

This study examined the impact of different mixtures of NaCl, KCl, and CaCl₂ on the fermentation profiles of Conservolea natural black olives. Five different combinations of chloride salts were investigated, namely (i) 8% NaCl (control treatment), (ii) 4% NaCl and 4% KCl, (iii) 4% NaCl and 4% CaCl₂, (iv) 4% KCl and 4% CaCl₂, and (v) 2.6% NaCl-2.6% KCl-2.6% CaCl₂. The changes in the microbial association (lactic acid bacteria, yeasts, Enterobacteriaceae), pH, titratable acidity, organic acids, volatile compounds, and mineral content in olive flesh were analyzed. Results demonstrated that all salt combinations led to vigorous lactic acid processes based on the obtained values of pH (3.9-4.2) and titratable acidity (0.70-0.86 g lactic acid per 100 ml brine). Organoleptic evaluation was a critical factor in the acceptability of the final product. Increasing concentrations of CaCl₂ or a combination of KCl and CaCl₂ rendered the product bitter with low acceptability by the taste panel. Only one combination of chloride salts (4% NaCl and 4% KCl) could finally produce olives with lower sodium content and good organoleptic attributes. The results of this study could be employed by the Greek table olive industry in an attempt to produce natural black olives with less sodium without affecting the traditional taste of fermented olives in order to meet consumers’ demand for low sodium dietary intake.     

Heat stress adaptation induces cross-protection against lethal acid stress conditions in Arcobacter butzleri but not in Campylobacter jejuni

The ability of many bacteria to adapt to stressful conditions may later protect them against the same type of stress (specific adaptive response) or different types of stresses (multiple adaptive response, also termed cross-protection). Arcobacter butzleri and Campylobacter jejuni are close phylogenetic relatives that occur in many foods of animal origin and have been linked with human illness (mainly diarrhoea). In the present study, sublethal stress adaptation temperatures (48 °C and 10 °C) and mild and lethal acid conditions (pH 5.0 and pH 4.0) were determined for A. butzleri and C. jejuni. In addition, it was evaluated whether these sublethal stress adaptations cause specific adaptive responses or cross-protection against subsequent mild or lethal acid stresses in these bacteria. The studies were conducted in broth adjusted to the different conditions and the results were determined by the dilution series plating method. It was shown that heat stress adapted A. butzleri (incubated for 2 h at 48 °C) were significantly more resistant to subsequent lethal acid stress (pH 4.0) than non-adapted cells at the 1 h time-point (p < 0.01 in Wilcoxon rank sum test). No specific adaptive responses against the stresses in A. butzleri or C. jejuni and no cross-protection in C. jejuni were found. The ability of heat stressed A. butzleri to tolerate later lethal acid conditions should be taken into account when designing new food decontamination and processing strategies.     

Acid adaptation affects the viability of Salmonella typhimurium during the lactic fermentation of skim milk and product storage

In this study, Salmonella typhimurium was acid adapted at pH 5.5 for 4 h. The viability of the acid-adapted and non-adapted cells of S. typhimurium was investigated both during the lactic fermentation of skim milk with Streptococcus thermophilus or Lactobacillus bulgaricus, and during the storage of lactic fermented milk products at 5 degrees C. It was found that the viable population of S. typhimurium, regardless of acid adaptation, increased in skim milk during the initial 24 h of lactic fermentation and then declined. However, the viable population of acid-adapted S. typhimurium was significantly higher (P<.05) than that of non-adapted cell at the end of 48 h of fermentation. Acid-adapted cells of S. typhimurium were also found to have survived better than non-adapted cells in the S. thermophilus-prepared fermented milk and two commercial lactic fermented milk products. The viability of the acid-adapted and non-adapted S. typhimurium at 5 and 37 degrees C in cell-free fermented milks that had their pHs adjusted to 6.4 and skim milk (pH 6.4) was further investigated. Results revealed that acid adaptation, in addition to enhancing acid tolerance, reduced the susceptibility of S. typhimurium to refrigerated temperature and other detrimental factors which might be present in lactic fermented milk products. These responses all contribute to the enhanced survival of acid-adapted S. typhimurium in the lactic fermented milk products observed in the present study.     

Characterization of low salinity stress in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine foodborne pathogenic bacterium commonly found in seawater or seafood. This bacterium often encounters low salinity stress when the contaminated seafood is washed with fresh water during food processing. This study was conducted to investigate the response of exponential- and stationary-phase cells of V. parahaemolyticus ST550 to lethal or sublethal low salinity. Tolerance to lethal low salinity (0.25% NaCl) was enhanced in V. parahaemolyticus cells in the exponential phase by previous adaptation in sublethal low salinity (0.6% NaCl). Low salinity-adapted cells in the exponential phase were also cross-protected against the challenge of lethal low pH, indifferent to heat, and sensitized to bile, acetic acid, and lactic acid stress. The adapted cells in the stationary phase were significantly protected against heat treatment at 44°C for 10 and 15 min, sensitized to bile and acetic acid treatment, and indifferent to low pH and lactic acid.     

Effect of Partial Substitution of NaCl with KCl on Halloumi Cheese during Storage: Chemical Composition,Lactic Bacterial Count,and Organic Acids Production

Abstract: The effect of partial substitution of NaCl with KCl on chemical composition, lactic bacterial count, and organic acids profile of Halloumi cheese was investigated. Halloumi cheeses were made and kept in 4 different brine solutions at 18% including NaCl only (HA), 3NaCl : 1KCl (HB), 1NaCl : 1KCl (HC), and 1NaCl : 3KCl (HD) and then stored at 4 °C for 56 d. No significant effect was observed between control and experimental cheeses in terms of moisture, fat, protein, lactic bacterial count, and pH values at the same storage period. There was a significant difference in ash, sodium, and potassium contents among experimental cheeses at the same storage period. Ash, sodium, and potassium contents increased significantly during storage at same salt treatment. There was no significant difference in lactic and citric acid contents among experimental cheeses and that of the control. In contrary, there was a significant difference in acetic acid among experimental cheeses. A strong positive correlation was observed between ash, Na, and K contents. An inverse correlation between organic acids and both Na and K contents was also observed.     

牛源性大肠杆菌O26热休克-酸应激响应

为探讨热休克-酸应激对大肠杆菌O26存活及相关基因表达的影响，以本实验收集的22 株牛源性O26为对象，首先进行乳酸和盐酸耐受实验，进而选取乳酸耐受性能不同的菌株混合进行热休克-酸应激存活实验，最后选取1 株代表菌株采用实时聚合酶链式反应分析应激2 h和4 h时一般应激基因rpoS、酸应激基因（asr、ycfR、gadA）、热应激基因（rpoH、dnaK、clpB和groEL）的表达差异。结果表明，乳酸或盐酸处理2 h后22 株O26存活菌数均显著下降（P＜0.05），耐受程度呈现菌株差异，且同一菌株对乳酸、盐酸的耐受有差异。与正常胰蛋白胨大豆肉汤对照组相比，5 株O26混菌酸应激和热休克-酸应激均导致存活菌数逐渐下降，而热休克-酸应激组存活菌数高于酸应激组，表明热休克发挥交叉保护效应，增强了O26抗酸能力。酸应激导致菌株G10Z1应激相关基因表达基本下调，而热休克-酸应激组与酸应激组相比上述基因的表达基本上调，表明热休克的交叉保护作用与上述基因表达水平的增加有关。提示食品实际生产加工中，当非致死性热处理与酸化联合使用时应注意交叉保护可能导致的食品安全风险增加的现象。     

Chloride salt mixtures affect Gordal cv. green Spanish-style table olive fermentation

This work studies the effects of different sodium (in the range of 4-10%), potassium (0-4%) and calcium (0-6%) chloride salt mixtures on the fermentation profile of Gordal olives processed according to the Spanish style. For this purpose, response surface methodology based on a simplex centroid mixture design with constrain (sum of salt percentages = 10%) was used. All treatments reached appropriate titratable acidity levels, but this parameter could not be related to the initial chloride salt concentration. The presence of CaCl₂ led to lower initial and after-fermentation pHs, delayed sugar diffusion into the brine, its maximum concentration and titratable acidity formation. CaCl₂ also delayed Enterobacteriaceae and yeast sprang, decreasing their overall growth. This chloride salt also showed a tendency to reduce overall lactic acid bacteria growth. KCl had a similar behaviour to NaCl but, in general, increased overall microbial growth. Thus, a partial substitution of NaCl in Spanish-style green olives with KCl and CaCl₂ does not substantially modify the fermentation profile but does produce some changes, which, when properly managed, could help to improve product processing.     

Individual effects of sodium, potassium, calcium, and magnesium chloride salts on Lactobacillus pentosus and Saccharomyces cerevisiae growth

A quantitative investigation on the individual effects of sodium (NaCl), potassium (KCl), calcium (CaCl2), and magnesium (MgCl2) chloride salts against Lactobacillus pentosus and Saccharomyces cerevisiae, two representative microorganisms of table olives and other fermented vegetables, was carried out. In order to assess their potential activities, both the kinetic growth parameters and dose-response profiles in synthetic media (deMan Rogosa Sharpe broth medium and yeast-malt-peptone-glucose broth medium, respectively) were obtained and analyzed. Microbial growth was monitored via optical density measurements as a function of contact time in the presence of progressive chloride salt concentrations. Relative maximum specific growth rate and lag-phase period were modeled as a function of the chloride salt concentrations. Moreover, for each salt and microorganism tested, the noninhibitory concentrations and the MICs were estimated and compared. All chloride salts exerted a significant antimicrobial effect on the growth cycle; particularly, CaCl2 showed a similar effect to NaCl, while KCl and MgCl2 were progressively less inhibitory. Microbial susceptibility and resistance were found to be nonlinearly dose related.     

Proteolysis of low-moisture Mozzarella cheese as affected by substitution of NaCl with KCl

The proteolytic and ACE inhibitory activities of low-moisture Mozzarella cheese (LMMC) as affected by partial substitution of NaCl with KCl were investigated. Experimental LMMC were made and salted with 4 salt mixtures: NaCl only (control), 3NaCl:1KCl, 1NaCl:1KCl, and 1NaCl:3KCl, and then proteolytic activity and angiotensin-converting enzyme inhibitory activity were determined. Salt treatment significantly affected angiotensin-converting enzyme inhibitory activity and phosphotungstic acid-soluble N of LMMC during storage. Water-soluble N, trichloroacetic acid-soluble N, lactic acid bacteria population, and total free amino acids were unaffected during storage. Nonetheless, water-soluble N and trichloroacetic acid-soluble N increased significantly during storage within a salt treatment. Peptide profiles and urea-PAGE gels did not differ between experimental cheeses at the same storage time.     

EFFECT OF ACID ADAPTATION AND DIFFERENT SALT CONCENTRATIONS ON SURVIVAL OF LISTERIA MONOCYTOGENES IN TURKISH WHITE CHEESE

ABSTRACT

Survival of acid‐adapted and nonacid adapted Listeria monocytogenes in Turkish white cheese with different salt concentrations (3.8 and 6.7% NaCl) was investigated. Cheese blocks were inoculated with L. monocytogenes and stored at 4C for 15 days. Samples were taken on different days and analyzed for the numbers of the pathogen, and the samples were challenged with simulated gastric fluid (SGF) for determining acid tolerance. There was no significant differences between the numbers of acid‐adapted and nonadapted L. monocytogenes cells regardless of the salt concentration on sampling days (P > 0.05). Challenge in SGF revealed that there was no significant difference between acid‐adapted and nonadapted bacteria in cheese with 3.8% NaCl throughout the storage period, while significant (P < 0.05) differences between those cells in cheese with 6.7% NaCl were found after day 5. These results suggest that acid‐adapted L. monocytogenes exposed to high salt concentration such as 6.7% may have an advantage in SGF.

PRACTICAL APPLICATIONS

Evaluation of potential stress responses of pathogens in food is important to understand the behavior of pathogens that can survive under stresses. As an environmental contaminant, Listeria monocytogenes may acquire adaptation to low acid conditions in cheese plants. Our results suggested that although there was no significant difference between acid‐adapted and nonadapted cells both in industrial (3.8% salt) and traditional (6.7% salt) white cheese, salt concentration significantly affected the survival of acid‐adapted cells in simulated gastric fluid (pH 2.3). These findings may indicate that L. monocytogenes, which is adapted to acid before contaminating high salt cheeses, may survive in stomach and cause infection. It can be said that increased salt concentration in cheese may not provide a barrier against acid‐adapted L. monocytogenes.

     

利用溶剂分离氯化钾和氯化钠的新工艺探索

文章考察了乙醇加入量、温度等因素对氯化钾和氯化钠单独存在及二者共存时在水溶液中的溶解度的影响,研究结果表明:在不同温度下加入乙醇可使氯化钾和氯化钠在水中的溶解度均降低,但其降低的幅度不同;两种盐类在水中溶解度的差异将随温度的升高而减小。在此基础上,文章提出了利用溶剂分离氯化钾和氯化钠的新工艺初步方案。     

Relationship between the effects of stress induced by human bile juice and acid treatment in Vibrio cholerae

The effects of low pH and human bile juice on Vibrio cholerae were investigated. A mild stress condition (exposure to acid shock at pH 5.5 or exposure to 3 mg of bile per ml for 20 min) slightly decreased (by < or = 1 log unit) V. cholerae cell viability. However, these treatments induced tolerance to subsequent exposures to more severe stress. In the O1 strain, four proteins were induced in response to acid shock (ca. 101, 94, 90, and 75 kDa), whereas only one protein (ca. 101 kDa) was induced in response to acid shock in the O139 strain. Eleven proteins were induced in response to bile shock in the O1 strain (ca. 106, 103, 101, 96, 88, 86, 84, 80, 66, 56, and 46 kDa), whereas only one protein was induced in response to bile shock in the O139 strain (ca. 88 kDa). V. cholerae O1 and O139 cells that had been preexposed to mild acid shock were twofold more resistant to pH 4.5 (with times required to inactivate 90% of the cell population [D-values] of 59 to 73 min) than were control cells (with D-values of 24 to 27 min). Likewise, cells that were preexposed to mild bile shock (3 mg/ml) were almost twofold more tolerant of severe bile shock (30 mg/ml; D-values, 68 to 87 min) than were control cells (with D-values of 37 to 43 min). These protective effects persisted for at least 1 h after the initial shock but were abolished when chloramphenicol was added to the culture during the shock. Cells preexposed to acid shock exhibited cross-protection against subsequent bile shock. However, cells preexposed to bile shock exhibited no changes in acid tolerance. Bile shock induced a modest reduction (0 to 20%) in enterotoxin production in V. cholerae, whereas acid shock had no effect on enterotoxin levels. Adaptation to acid and bile juice and protection against bile shock in response to preexposure to acid shock would be predicted to enhance the survival of V. cholerae in hosts and in foods. Thus, these adaptations may play an important role in the development of cholera disease.     

Fermentation profiles of Manzanilla-Aloreña cracked green table olives in different chloride salt mixtures

NaCl plays an important role in table olive processing affecting the flavour and microbiological stability of the final product. However, consumers demand foods low in sodium, which makes necessary to decrease levels of this mineral in fruits. In this work, the effects of diverse mixtures of NaCl, CaCl₂ and KCl on the fermentation profiles of cracked directly brined Manzanilla-Aloreña olives, were studied by means of response surface methodology based in a simplex lattice mixture design with constrains. All salt combinations led to lactic acid processes. The growth of Enterobacteriaceae populations was always limited and partially inhibited by the presence of CaCl₂. Only time to reach half maximum populations and decline rates of yeasts, which were higher as concentrations of NaCl or KCl increased, were affected, and correspondingly modelled, as a function of salt mixtures. However, lactic acid bacteria growth parameters could not be related to initial environmental conditions. They had a longer lag phase, slower growth and higher population levels than yeasts. Overall, the presence of CaCl₂ led to a slower Enterobacteriaceae and lactic acid bacteria growth than the traditional NaCl brine but to higher yeast activity. The presence of CaCl₂ in the fermentation brines also led to higher water activity, lower pH and combined acidity as well as a faster acidification while NaCl and KCl had fairly similar behaviours. Apparently, NaCl may be substituted in diverse proportions with KCl or CaCl₂ without substantially disturbing water activity or the usual fermentation profiles while producing olives with lower salt content.