Effects of heat, cold, acid and bile salt adaptations on the stress tolerance and protein expression of kefir-isolated probiotic Lactobacillus kefiranofaciens M1.

Lactobacillus kefiranofaciens M1 is a probiotic strain isolated from Taiwanese kefir grains. The present study evaluated the effects of heat, cold, acid and bile salt adaptations on the stress tolerance of L. kefiranofaciens M1. The regulation of protein expression of L. kefiranofaciens M1 under these adaptation conditions was also investigated. The results showed that adaptation of L. kefiranofaciens M1 to heat, cold, acid and bile salts induced homologous tolerance and cross-protection against heterologous challenge. The extent of induced tolerance varied depending on the type and condition of stress. Proteomic analysis revealed that 27 proteins exhibited differences in expression between non-adapted and stress-adapted L. kefiranofaciens M1 cells. Among these proteins, three proteins involved in carbohydrate metabolism (triosephosphate isomerase, enolase and NAD-dependent glycerol-3-phosphate dehydrogenase), two proteins involved in pH homeostasis (ATP synthase subunits AtpA and AtpB), two stress response proteins (chaperones DnaK and GroEL) and one translation-related protein (30S ribosomal protein S2) were up-regulated by three of the four adaptation treatments examined. The increased synthesis of these stress proteins might play a critical protective role in the cellular defense against heat, cold, acid and bile salt stresses.

Optimizing Production of Two Potential Probiotic Lactobacilli Strains Isolated from Piglet Feces as Feed Additives for Weaned Piglets.

Two probiotic strains, Lactobacillus johnsonii x-1d-2 and Lactobacillus mucosae x-4w-1, originally isolated from piglet feces, have been demonstrated to possess antimicrobial activities, antibiotic resistances and interleukin-6 induction ability in RAW 267.4 macrophages in our previous study. These characteristics make L. johnsonii x-1d-2 and L. mucosae x-4w-1 good candidates for application in feed probiotics. In this study, soybeal meal, molasses and sodium acetate were selected to optimize the growth medium for cultivation of L. johnsonii x-1d-2 and L. mucosae x-4w-1. These two strains were then freeze-dried and mixed into the basal diet to feed the weaned piglets. The effects of L. johnsonii x-1d-2 and L. mucosae x-4w-1 on the growth performance and fecal microflora of weaned piglets were investigated. The results showed that the bacterial numbers of L. johnsonii x-1d-2 and L. mucosae x-4w-1 reached a maximum of 8.90 and 9.30 log CFU/mL, respectively, when growing in optimal medium consisting of 5.5% (wt/vol) soybean meal, 1.0% (wt/vol) molasses and 1.0% (wt/vol) sodium acetate. The medium cost was 96% lower than the commercial de Man, Rogosa and Sharpe medium. In a further feeding study, the weaned piglets fed basal diet supplemented with freeze-dried probiotic cultures exhibited higher (p<0.05) body weight gain, feed intake, and gain/feed ratio than weaned piglets fed basal diet. Probiotic feeding also increased the numbers of lactobacilli and decreased the numbers of E. coli in the feces of weaned piglets. This study demonstrates that L. johnsonii x-1d-2 and L. mucosae x-4w-1 have high potential to be used as feed additives in the pig industry.

Effect of acid adaptation on the environmental stress tolerance of three strains of Vibrio parahaemolyticus.

Three strains of Vibrio parahaemolyticus (690, BCRC 13023, and BCRC 13025), involved in foodborne outbreaks in Taiwan, were subjected to acid adaptation at pH 5.5 for 90 min. The effects of acid adaptation on the tolerance of V. parahaemolyticus to various environmental stresses, including heat (47°C), cold (4°C and -20°C), ethanol (8%), high salt (20% NaCl), and hydrogen peroxide (20 ppm) were examined. Results showed that acid adaptation increased the thermal tolerance of the three test strains of V. parahaemolyticus, while it did not affect their cold tolerance. Acid adaptation also increased the ethanol tolerance in V. parahaemolyticus 690 and BCRC 13025, but not in BCRC 13023. Differences in the tolerance to high salts were noted among the three strains after prior acid adaptation. However, these acid-adapted V. parahaemolyticus strains were more susceptible to hydrogen peroxide than their nonadapted controls. These findings demonstrated that acid adaption responses of V. parahaemolyticus varied among strains and types of stress challenge.

Enhancing the antitumor cell proliferation and Cu(2+)-chelating effects of black soybeans through fermentation with Aspergillus awamori.

In the present study, black soybeans were fermented with Aspergillus awamori at 30°C for 3 days. The effect of fermentation on the antiproliferative effect against human colon cancer cells, Caco-2 and HT-29 as well as Cu(2+)-chelating effect of black soybeans was investigated. It was found that the water, 80% methanol or 80% ethanol extract of fermented black soybeans showed a significantly higher (P < 0.05) antiproliferative and Cu(2+)-chelating effect than did the respective extract of non-fermented black soybeans. Generally, the methanol extract and the ethanol extract of fermented black soybeans exerted higher antiproliferative effect on both Caco-2 and HT-29 cells. While water extract of fermented black soybeans showed the highest Cu(2+)-chelating effect among the various extracts examined. Taking into account of extraction yields further revealed that bioactive principles that exhibit Cu(2+)-chelating effect could be extracted to the largest extent with water as the extraction solvent. With same amount of sample, water extract obtained from fermented black soybeans possesses the highest Cu(2+)-chelating abilities.

Investigation of microorganisms involved in biosynthesis of the kefir grain.

The purpose of this study was to understand the significance of each microorganism in grain formation by evaluating their microbial aggregation and cell surface properties during co-aggregation of LAB and yeasts together with an investigation of biofilm formation. Non-grain forming strains from viili were also evaluated as a comparison. Results indicated that the kefir grain strains, Lactobacillus kefiranofaciens and Saccharomyces turicensis possess strong auto-aggregation ability and that Lactobacillus kefiri shows significant biofilm formation properties. Significant co-aggregation was noted when S. turicensis and kefir LAB strains (Lb. kefiranofaciens and Lb. kefiri) were co-cultured. Most of the tested LAB strains are hydrophilic and had a negative charge on their cell surface. Only the kefir LAB strains, Lb. kefiranofaciens HL1 and Lb. kefiri HL2, possessed very high hydrophobicity and had a positive cell surface charge at pH 4.2. In contrast, the LAB and yeasts in viili did not show any significant self-aggregation or biofilm formation. Based on the above results, we propose that grain formation begins with the self-aggregation of Lb. kefiranofaciens and S. turicensis to form small granules. At this point, the biofilm producer, Lb. kefiri, then begins to attach to the surface of granules and co-aggregates with other organisms and components in the milk to form the grains. On sub-culturing, more organisms attach to the grains resulting in grain growth. When investigated by scanning electron microscopy, it was found that short-chain lactobacilli such as Lb. kefiri occupy the surface, while long-chain lactobacilli such as Lb. kefiranofaciens have aggregated towards the center of the kefir grains. These findings agree with the above hypothesis on the formation of grains. Taken together, this study demonstrates the importance of cell surface properties together with fermentation conditions to the formation of grains in kefir.

Adaptive acid tolerance response of Vibrio parahaemolyticus as affected by acid adaptation conditions, growth phase, and bacterial strains.

Vibrio parahaemolyticus strain 690 was isolated from gastroenteritis patients. Its thermal and ethanol stress responses have been reported in our previous studies. In this study, we further investigated the effects of various acid adaptation conditions including pH (5.0-6.0) and time (30-90 min) on the acid tolerance in different growth phases of V. parahaemolyticus 690. Additionally, the adaptive acid tolerance among different V. parahaemolyticus strains was compared. Results indicated that the acid tolerance of V. parahaemolyticus 690 was significantly increased after acid adaptation at pH 5.5 and 6.0 for 30-90 min. Among the various acid adaptation conditions examined, V. parahaemolyticus 690 acid-adapted at pH 5.5 for 90 min exhibited the highest acid tolerance. The acid adaptation also influenced the acid tolerance of V. parahaemolyticus 690 in different growth phases with late-exponential phase demonstrating the greatest acid tolerance response (ATR) than other phases. Additionally, the results also showed that the induction of adaptive ATR varied with different strains of V. parahaemolyticus. An increase in acid tolerance of V. parahaemolyticus was observed after prior acid adaptation in five strains (556, 690, BCRC 13023, BCRC 13025, and BCRC 12864), but not in strains 405 and BCRC 12863.

DDA3 stabilizes microtubules and suppresses neurite formation.

We have previously shown that DDA3 - also known as proline/serine-rich coiled-coil protein 1 (PSRC1) - is a microtubule-associated protein that promotes cell growth by stimulating the ß-catenin pathway. Here, we report that DDA3 can bundle and stabilize microtubules in vivo and in vitro. We found that overexpression of DDA3 increased the abundance of acetylated and tyrosinated microtubules. We employed PC12 and N2a cell lines, as well as cultured hippocampal neurons, and demonstrated that overexpression of DDA3 suppressed neurite/axon outgrowth, whereas its depletion accelerated neurite/axon formation and elongation. Knockdown of DDA3 reduced ß3-tubulin levels in N2a cells, which contributed to the spontaneous neurite formation caused by DDA3 depletion. Consistent with its role in suppressing neuritogenesis, DDA3 was downregulated during induced neuronal differentiation. Moreover, expression of DDA3 was detected in the rat brain at embryonic (E) day E15 and in the cortical region at E17, the period of active neurogenesis. Levels of cortical DDA3 decreased at the beginning of E19, when active neuritogenesis is completed. Overall our results demonstrate that DDA3 is a so-far-unknown microtubule-stabilizing protein that is involved in regulating neurite formation and elongation.

Heat shock and cold shock treatments affect the survival of Listeria monocytogenes and Salmonella Typhimurium exposed to disinfectants.

The foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium were subjected to heat shock at 48°C for 10 and 30 min, respectively, and then cold shocked at 15°C for 3 h. The effect of these shocks on the viability of test organisms exposed to chlorine dioxide and quaternary ammonium compounds was then determined. After exposure to the disinfectants, the viable population of each test organism, regardless of heat shock or cold shock treatment, decreased as the exposure period was extended. Both heat shock and cold shock treatments reduced the susceptibility of L. monocytogenes to both disinfectants at 25°C. However, for Salmonella Typhimurium, exposure to the chlorine dioxide disinfectant or quaternary ammonium compounds at 25°C significantly reduced (P < 0.05) survival of heat-shocked cells but significantly increased (P < 0.05) survival of cold-shocked cells compared with control cells. Survival of both L. monocytogenes and Salmonella Typhimurium generally was reduced after exposure to disinfectants at 40°C compared with 25°C.

Use of Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris isolated from TRFM in manufacturing of functional low-fat cheeses.

UNLABELLED: The purpose of this study was to manufacture new functional low-fat cheeses using Taiwanese ropy fermented milk (TRFM) and Lactococcus lactis subsp. cremoris strains isolated from TRFM. After 28 d of ripening and storage, the viable populations of lactic acid bacteria (LAB) in the low-fat cheeses made with L. lactis subsp. cremoris TL1 (TL1), L. lactis subsp. cremoris TL4 (TL4), and TRFM still maintained above 10(8) CFU/g. The low-fat cheeses made with TL1 and TRFM showed higher moisture contents than the cheeses made with TL4, full-fat, and low-fat cheese controls. The low-fat cheeses made with TL1 and TL4 had higher customer preferential scores similar to full-fat cheese control in the sensory evaluation. Additionally, the low-fat cheeses fermented with TL1, TL4, and TRFM for 4 h had higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging and ferrous ion-chelating abilities than the cheeses fermented with the starters for 8 h, full-fat, and low-fat cheese controls. A better angiotensin-converting enzyme (ACE) inhibition activity was also observed in the low-fat cheeses made with TL1, TL4, and TRFM than that in the full-fat and low-fat cheese controls during ripening and storage period. PRACTICAL APPLICATION: As health-conscious consumers continue to seek low-fat alternatives in their diets, there remain strong interests for the dairy industry to develop low-fat cheeses to meet the demands. This study clearly demonstrated that the low-fat cheeses fermented with TL1 for 4 h showed a better overall acceptability and possessed antioxidative abilities and ACE inhibitory activities than other cheeses tested in this study. By improving its flavor and investigating the possible mechanisms of its functionalities in the future, this low-fat cheese might possibly be commercialized and give a positive impact on cheese consumption in the future.

The effect of heat shock on the response of Cronobacter sakazakii to subsequent lethal stresses.

Cronobacter sakazakii, formerly Enterobacter sakazakii, has been implicated in a severe form of neonatal meningitis. In this study, C. sakazakii BCRC 13988 was first exposed to heat-shock treatment at 47 degrees C for 15 min. The heat-shocked C. sakazakii was subjected to several lethal challenges including low temperature (3 degrees C and -20 degrees C), pH 3.3, 15% ethanol, high osmotic pressure (tryptic soy broth + 75% sorbitol, a(w) 0.81), and drying. It was found that heat shock significantly (p < 0.05) enhanced the resistance of C. sakazakii to all the lethal stresses examined. After 60 min of exposure to 15% ethanol, the survival of the heat-shocked cells was approximately 752 times that of the nonshocked cells. Compared with the nonshocked C. sakazakii, the heat-shocked cells exhibited a 322- and 1.6-fold increase in survival after 7 days of exposure to -20 degrees C and 3 degrees C, respectively. A 48-fold increase in the survival was noted with the heat-shocked cells after 6 h of exposure to dry air (relative humidity 37%) at 25 degrees C, showing a survival of 0.00107% which is approximately 50-fold that of the control. After 36 h of exposure to the high osmotic stress environment, the survival of the heat-shocked C. sakazakii was found to be approximately 119 times that of the control cells. Finally, an increased survival of approximately 72 times that of the control cells was observed with the heat-shocked C. sakazakii after 60 min of challenge at pH 3.3.

The effect of temperature and length of heat shock treatment on the thermal tolerance and cell leakage of Cronobacter sakazakii BCRC 13988.

Enterobacter sakazakii is an emerging opportunistic pathogen associated with life-threatening illnesses in infants, with infant formula serving as the principal mode of transmission. In the present study, C. sakazakii (formely E. sakazakii) BCRC 13988 was subjected to various heat shock treatments (42-48 degrees C for 5-15 min). Its subsequent survival at 51 degrees C and the leakage of intracellular materials was investigated. It was found that 47 degrees C was the maximum growth temperature of the test organism. In addition, heat shock enhanced the thermal tolerance of C. sakazakii BCRC 13988. Within heat shock temperatures between 42 and 47 degrees C, the thermal tolerance enhancing effect increased as the length or temperature of the heat shock treatment was increased. However, increasing the heat shock temperature to 48 degrees C reduced the thermal tolerance enhancing effect. Among the various heat shocked cells examined, the 47 degrees C-15 min-heat shocked C. sakazakii exhibited the highest thermal tolerance. Moreover, electron micrograph analysis showed that heat shock treatment caused damage and disruption in C. sakazakii cells. There was a significant increase (P<0.05) in the leakage of nucleic acid and protein in the supernatant of the heat shocked cell suspension that increased as the temperature and duration of heat shock increased.

Ethanol and NaCl susceptibility and protein expression of acid-adapted B. cereus 1-4-1 as well as its growth patterns in the presence of various carbon and nitrogen sources.

In the present study, the foodborne pathogen Bacillus cereus 1-4-1 was subjected to acid adaptation treatment by suspending the test organism in tryptic soy broth (pH 5.5) for 2 hours. The susceptibility of the acid-adapted and nonadapted cells of B. cereus 1-4-1 to high concentrations of ethanol (20%) and NaCl (20%) was then examined. In addition, the effect of acid adaptation on the protein expression profile of B. cereus 1-4-1 as well as the growth patterns of the acid-adapted and nonadapted cells of the test organism in the presence of various carbon and nitrogen sources were compared. Results revealed that acid-adapted B. cereus 1-4-1 was more susceptible to ethanol (20%) and NaCl (20%) than its nonadapted counterpart. Analysis with one-dimensional SDS-PAGE showed no distinct difference in the expression of the 16 proteins bands noted in the nonadapted cells compared with those of acid-adapted cells. Two-dimensional electrophoresis revealed that the acid adaptation treatment affected the expression of 26 species of protein, with the levels of 12 proteins increasing and 14 proteins decreasing in the cells of acid-adapted B. cereus 1-4-1 compared with those of the control cells. Furthermore, immunoblotting detected GroEL-like protein with a similar level in the acid-adapted and nonadapted cells of B. cereus 1-4-1 while failing to find the presence of a DnaK-like protein. B. cereus 1-4-1, regardless of acid adaptation, exhibited the highest maximum growth with sucrose as the carbon source while the maximum growth was found in the presence of either peptone, soytone, tryptone, or yeast extract as the nitrogen source, with these showing no significant difference. Finally, the growth patterns of the acid-adapted and nonadapted cells were similar.

Survival of Vibrio parahaemolyticus under environmental stresses as influenced by growth phase and pre-adaptation treatment.

In this study, the susceptibility of Vibrio parahaemolyticus in different growth phases after exposure to lethal stresses including 47 degrees C and 8% ethanol was first investigated. The effect of a culture's growth phase on both the heat and ethanol shock response of V. parahaemolyticus was then examined. It was found that cells of V. parahaemolyticus in the mid-exponential phase, regardless of adaptation, were most susceptible to environmental stresses, while cells in the stationary phase were least susceptible to the lethal stresses examined. Adaptation with heat shock at 42 degrees C for 45 min or ethanol shock with 5% ethanol for 60 min induced an increased resistance of V. parahaemolyticus to subsequent lethal stresses at 47 degrees C and 8% ethanol. While the adaptation treatments resulted in a reduced resistance of the test organism to pH 4.4 and 20% NaCl. Generally, the extent of changes in the resistance of V. parahaemolyticus to lethal stresses between the adapted and control cells was found to be growth phase dependent. Compared with the respective control cells, the adapted late-exponential phase cells exhibited the greatest extent of change, while the adapted stationary phase cells showed the least change in their resistance to the lethal stresses examined.

The effect of acid adaptation on the susceptibility of Bacillus cereus to the stresses of temperature and H2O2 as well as enterotoxin production.

In the present study, Bacillus cereus 1-4-1, which is capable of causing diarrheal syndrome, was subjected to acid adaptation at pH 5.5 for 2 hours. The effect of acid adaptation on the survival of B. cereus subjected to subsequent lethal challenges at both low (4 degrees and -18 degrees C) and high temperatures (49 degrees C) as well as in the presence of 5 mM H(2)O(2) was investigated. Additionally, enterotoxin production by B. cereus as influenced by acid adaptation was examined. Results revealed that acid adaptation increased the survival of B. cereus during storage at -18 degrees C while a decreased survival was noted for the acid-adapted cells during storage at 4 degrees C. In addition, the acid-adapted cells were less susceptible to high temperature than the nonadapted cells. On the other hand, acid adaptation did not change the susceptibility of test organism to H(2)O(2). It was also found that acid adaptation time affected the enterotoxin production of B. cereus cells. The 1- to 2-hour acid-adapted cells exhibited a reduced level of enterotoxin production while cells acid adapted for 4 hours and the nonadapted cells showed no difference in the level of enterotoxin production. Besides, the acid-adapted cells showed a reduced lag period for growth and enterotoxin production when they were grown in tryptic soy broth.

Survival of the acid-adapted Bacillus cereus in acidic environments.

In this study, the acid tolerance of Bacillus cereus 1-4-1 after adaptation at pH 5.5 for 1, 2 and 4 h was first determined. The survival of acid-adapted and non-adapted cells of B. cereus in phosphate buffer solution (PBS pH 4.0) containing various organic acids such as acetic, propionic, citric, lactic or tartaric acid as well as in a commercial acidic beverage of mixed fruits and vegetables (pH 3.7) was then examined. Results revealed that acid adaptation time influenced the increased tolerance of B. cereus in PBS (pH 4.0). The 2 h-adapted cells exhibited the highest acid tolerance in PBS. The presence of chloramphenicol during the acid adaptation reduced the extent of increased acid tolerance. Acid adaptation was also found to enhance the tolerance of the test organism in the presence of the various organic acids tested. While the extent of increased acid tolerance varied with the organic acid examined. Acid-adapted B. cereus cells exhibited the largest extent of increased tolerance, showing an increased survival of ca. 1000 folds, in the propionic acid-containing PBS. Additionally, a higher survival percentage was noted with the acid-adapted than the non-adapted cells of B. cereus in the acidic beverage stored at 4 or 25 degrees C.

Protein expression in Vibrio parahaemolyticus 690 subjected to sublethal heat and ethanol shock treatments.

Cells of Vibrio parahaemolyticus 690 were subjected either to heat shock at 42 degrees C for 45 min or to ethanol shock in the presence of 5% ethanol for 60 min. The protein profiles of the unstressed and stressed V. parahaemolyticus cells were compared. Additionally, the induction of DnaK- and GroEL-like proteins in the unstressed and stressed cells of V. parahaemolyticus was also examined. Analysis with one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that three proteins with molecular masses of 93, 77, and 58 kDa were induced by both heat shock and ethanol shock. The protein patterns revealed by two-dimensional electrophoresis were more detailed than those revealed by one-dimensional SDS-PAGE. It was found that heat shock and ethanol shock affected the expression of a total of 28 proteins. Among them, four proteins with molecular masses of 94, 32.1, 26.7, and 25.7 kDa were enhanced by both heat shock and ethanol shock. Furthermore, immunoblot analysis showed the presence of a GroEL-like protein with a molecular mass of 61 kDa in the test organism, with the heat-shocked and ethanol-shocked cells producing a GroEL-like protein in a larger quantity than the unstressed cells. However, DnaK-like protein was not detectable in either the unstressed or the stressed cells.

p53 target DDA3 binds ASPP2 and inhibits its stimulation on p53-mediated BAX activation.

The p53 tumor suppressor functions in maintaining the integrity of the genome. We have previously reported that DDA3 is an oncoprotein transcriptionally regulated by p53. To explore mechanisms underlying DDA3 action, we searched for its interacting proteins by yeast two-hybrid screening, and identified ASPP2, a p53 binding protein, as its binding partner. The DDA3/ASPP2 binding was confirmed in vitro by GST pull-down and in vivo by immunofluorescence assay, which indicated colocalization of DDA3 and ASPP2. Interacting domain of DDA3 was mapped to amino acids 118-241, whereas both the N- and C-terminal regions of ASPP2 were capable of binding to DDA3. DDA3 dose-dependently inhibited ASPP2 in stimulating the p53-mediated BAX promoter activation without interfering the binding of ASPP2 to p53. Together these results identify ASPP2 as a bona fide DDA3 interacting protein, and suggest that the ASPP2/DDA3 interaction may inhibit ASPP2 in stimulating the apoptotic signaling of p53.

Ethanol shock changes the fatty acid profile and survival behavior of Vibrio parahaemolyticus in various stress conditions.

Vibrio parahaemolyticus 690, a clinical strain, was subjected to ethanol shock in the presence of 5% ethanol for a period of 30 and 60 min. Survival behaviors of the ethanol shocked and control cells of V. parahaemolyticus in the presence of H(2)O(2) (20 ppm), crystal violet (3 ppm), NaCl (20%), and low pH solution (pH 4.4) containing various organic acids including lactic acid, acetic acid, citric acid and tartaric acid (25 mM) were compared. In addition, the effects of ethanol shock on the fatty acid profile and recovery of V. parahaemolyticus on tryptic soy agar (TSA) plus various amounts of NaCl were also investigated. After ethanol shock, it was found that the proportion of vaccenic acid (18:1) increased, while the proportion of palmitic acid (16:0) and ratio of saturated fatty acid to unsaturated fatty acid decreased in cells of V. parahaemolyticus. The recovery of the ethanol-shocked cells on TSA plus 6.0% or 7.5% NaCl was significantly less than the control cells. Furthermore, ethanol shock increased the survival of V. parahaemolyticus in the presence of H(2)O(2), while made the test organism less resistant to crystal violet, high NaCl and organic acids. The degree of decreased acid tolerance observed on the ethanol-shocked cells of test organism varied with the organic acid examined. Finally, ethanol shock for 60 min exhibited either a higher or similar degree of effect on the test organism (depending on the type of stress encountered) than did ethanol shock for 30 min. Data obtained from the present study does provide useful information that is indispensable when control measure of V. parahaemolyticus is to be performed efficiently and adequately.

Expression of superoxide dismutase, catalase and thermostable direct hemolysin by, and growth in the presence of various nitrogen and carbon sources of heat-shocked and ethanol-shocked Vibrio parahaemolyticus.

Vibrio parahaemolyticus 690 was subjected either to heat shock at 42 degrees C or ethanol shock in the presence of 5% ethanol. The effects of those shocks on superoxide dismutase (SOD) and catalase (CAT) activities, and thermostable direct hemolysin (TDH) production were examined. In addition, the growth behaviors of the stressed and unstressed cells of V. parahaemolyticus in the presence of various nitrogen and carbon sources were compared. Both heat shock and ethanol shock reduced the levels of SOD and CAT activities in V. parahaemolyticus. Gel activity staining assay failed to detect the expression of CAT, while one SOD enzyme with an electrophoretic mobility greater than the [Mn]SOD and [Fe]SOD of Escherichia coli was detected in the unstressed, heat-shocked and ethanol-shocked cells of V. parahaemolyticus. Heat shock for 15-60 min and ethanol shock for 45-60 min were found to enhance the synthesis of TDH. Ethanol-shocked and unstressed cells of V. parahaemolyticus grew similarly and produced similar amounts of TDH when they were grown in TSB-3% NaCl, but slower growth and less production of TDH occurred with heat-shocked cells until after 200 min of cultivation. The growth rate and maximum growth of the unstressed, heat-shocked and ethanol-shocked cells varied with the nitrogen and carbon sources used. With the same nitrogen or carbon source, the growth patterns of the ethanol-shocked and unstressed cells were similar while the heat-shocked cells exhibited an extended lag period.