Nucleotide sequencing,purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA

An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.     

Accumulation of short n-chain ethyl esters by esterases of lactic acid bacteria under conditions simulating ripening Parmesan cheese

EstA from Lactobacillus helveticus CNRZ32 (Lbh-EstA), EstB, and EstC from Lactobacillus casei LILA, and EstA from Lactococcus lactis MG1363 (Lcl-EstA) were evaluated for their ability to accumulate esters in a model system simulating Parmesan cheese ripening conditions (10 degrees C, 2 to 3% NaCl, pH 5.4 to 5.5, aw = 0.850 to 0.925) using Capalase K from kid goat as a positive control. All of the LAB esterases and Capalase K mediated the accumulation of esters in the model system in an enzyme specific manner, which was influenced by a, and selectivity for fatty acid chain-length. In general, enzyme mediated accumulation of ethyl esters was higher at aw values of 0.850 and 0.900 than at aw of 0.925, demonstrating that aw is a critical parameter influencing ester accumulation. The substrate selectivity of esterases, aw, and enzyme type may be important factors in the development of fruity flavors, as evidenced by results in this model system simulating Parmesan cheese ripening conditions.     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

A NaCl-stable serine proteinase from Virgibacillus sp. SK33 isolated from Thai fish sauce

An extracellular proteinase from Virgibacillus sp. SK33, isolated from 1 month-old fish sauce, was purified to electrophoretic homogeneity, using hydrophobic interaction chromatography and hydroxyapatite with purification fold of 2.5 and 7% yield. The anomalous molecular weight (MW) of 19 kDa was obtained from SDS–PAGE, whereas a MW of 33.7 kDa was determined by MALDI-TOF. Optimum conditions for catalytic activity were 55 °C and pH 7.5. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferentially hydrolysed Suc-Ala-Ala-Pro-Phe-AMC, indicating a serine proteinase with subtilisin-like characteristics. K_m and k_cat of the purified proteinase were 27 μM and 12 s⁻¹, respectively. Proteinase activity, toward both synthetic and anchovy substrates, increased with NaCl up to 25%. The proteinase exhibited high stability in both the absence and presence of NaCl up to 25%. Approximately 2.5-fold increase in activity was observed in the presence of divalent cations, including Ca²⁺, Mg²⁺ and Sr²⁺ at 100 mM. MALDI-TOF MS and LC–ESI-MS/MS analyses, as well as N-terminal sequences, revealed that the purified enzyme did not match microbial proteinases in the database, indicating it to be a novel proteinase.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

The processing of high-molecular-weight xylanase (XynE, 110 kDa) from Aeromonas caviae ME-1 to 60-kDa xylanase (XynE60) in Escherichia coli and purification and characterization of XynE60

A xylanase gene (xynE) encoding XynE (110 kDa) was cloned from a lambda phage genomic library of Aeromonas caviae ME-1 which is a multiple-xylanase-producing bacterium. Upon nucleotide sequence analysis, we found that xynE comprises 2823 by and encodes a protein of 941 amino acid residues (104,153 Da), which was similar to endo-beta-1,4-xylanases which are categorized to glycosyl hydrolase family 10. An Escherichia coli transformant that harbored pXED30 carrying xynE produced 110-, 84-, 72-, and 66-kDa xylanases in the cell-free extract, and 72- and 66-kDa xylanases in the culture supernatant. We purified the 66-kDa xylanase to electrophoretic homogeneity from a culture supernatant by a series of column chromatographies. The calculated molecular mass of the purified xylanase determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was 60,154.50 Da, and the xylanase was designated XynE60. Analysis of the N-terminal 10 amino acid residues and the determined molecular mass of XynE60 revealed that XynE60 is a product processed at the Gly26-Gly27, and Thr565-Ala566 sites of XynE by proteolytic cleavage. XynE60 showed optimal activity at 55 degrees C and pH 8.0, and was stable below 45 degrees C and at pH 7.0-8.5. The K(m) and V(max) of XynE60 were calculated to be 8.1 mg/ml and 6897 nkat/mg, respectively.     

Characterization of a thermostable family 10 endo-xylanase (XynB) from Thermotoga maritima that cleaves p-nitrophenyl-beta-D-xyloside

Thermotoga maritima MSB8 possesses two xylanase genes, xynA and xynB. The xynB gene was isolated from the genomic DNA of T. maritima, cloned, and expressed in Escherichia coli. XynB was purified to homogeneity by heat treatment, affinity chromatography and ion-exchange column chromatography. The purified enzyme produced a single band upon SDS-PAGE corresponding to a molecular mass of 42 kDa. At 70 degrees C, the enzyme was stable between pH 5.0 and pH 11.4, and it was stable at temperatures of up to 100 degrees C from pH 7.0 to pH 8.5. At 50 degrees C, XynB displayed an optimum pH of 6.14 and at this pH the temperature for optimal enzyme activity was 90 degrees C. XynB exhibited broad substrate specificity and was highly active towards p-nitrophenyl-beta-D-xylobioside with K(m) and k(cat) values of 0.0077 mM and 5.5 s(-1), respectively, at 30 degrees C. It was also active towards p-nitrophenyl-beta-D-xyloside. The initial product of the cleavage of p-nitrophenyl-beta-D-xyloside was xylobiose, indicating that the major reaction in the cleavage was transglycosylation, not hydrolysis.     

Effect of environmental parameters on growth kinetics of Bacillus cereus (ATCC 7004) after mild heat treatment

The influence of temperature (10 to 50 degrees C), initial pH (4.0 to 6.0) and sodium chloride concentration (0.5 to 3.0%) on the growth in nutrient broth and in meat extract of Bacillus cereus after mild heat treatment (90 degrees C--10 min) was determined. B. cereus spores survived after heating and they were able to germinate and grow in both media when post-treatment conditions were favourable. Heated B. cereus did not grow at 10 and 50 degrees C or in a medium with pH 4.0. Decreasing pH values and increasing levels of sodium chloride decreased growth rate and increased the lag phase of B. cereus. pH 4.5 was unable to prevent the growth of heated spores in a meat substrate with 0.5% NaCl at 12 degrees C. The combination of pH /=1.0% and temperatures /=60 degrees C could control heated B. cereus ATCC 7004 growth.     

Purification and characterization of a novel cyclohexylamine oxidase from the cyclohexylamine-degrading Brevibacterium oxydans IH-35A

Cyclohexylamine oxidase (CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to electrophoretic homogeneity, by serial chromatographies. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by gel filtration and SDS-PAGE. The optimum pH was 7.4 and the stable pH range was 6.0 to 7.0. The enzyme was thermostable up to 30 degrees C. The enzyme was found to be highly specific for the deamination of alicyclic monoamines such as cyclopentylamine, cycloheptylamine, and N-methylcyclohexylamine and aliphatic monoamines, such as sec-butylamine. The apparent K(m) value for cyclohexylamine was 1.23 mM. The enzyme was inhibited by flavin enzyme inhibitors such as quinine and quinacrine. The N-terminal 27 amino acid residues were determined as Gly-Ser-Val-Thr-Pro-Asp-Pro-Asp-Val-Asp-Val-Ile-Ile-His-Gly-Ala-Gly-Ile-Ser-Gly-Ser-Ala-Ala-Ala-Lys-Ala-Leu-, revealing homology to conventional flavin-containing amine oxidases (EC 1.4.3.4).     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.     

Thermal injury and recovery of Salmonella enterica serovar enteritidis in ground chicken with temperature, pH, and sodium chloride as controlling factors

Cells of Salmonella enterica serovar Enteritidis were grown at 25 and 35 degrees C, heat injured (55, 60, and 62.5 degrees C), and recovered in tryptic soy broth (TSB) at various NaCl concentrations (2.0 and 3.5%) and pH levels (5.5 and 6.5). To assess the interactions of growth temperature, heating temperature, NaCl concentration and pH on the thermal injury and recovery of Salmonella Enteritidis in ground chicken, a randomized design with each experimental combination was used. When a logistic equation for nonlinear survival curves was used, D-values of cells of Salmonella Enteritidis grown at 25 degrees C were 7.60, 5.73, and 4.81 min at 55, 60, and 62.5 degrees C, respectively. For cells grown at 35 degrees C, the D-values were 12.38, 7.45, and 5.70 min at 55, 60, and 62.5 degrees C. The influence of tryptic soy agar and double modified lysine agar (DMLIA) on the recovery of heat-injured cells was determined. Recovery was significantly reduced on DMLIA at increased pH levels and NaCl concentrations. Higher numbers of cells were recovered in TSB with 2.0% NaCl than in TSB with 3.5% NaCl. It was observed that the rate of recovery of heat-injured cells was similar at each pH. Therefore, a pH range of 5.5 to 6.5 does not have a major inhibitory effect on the recovery of Salmonella Enteritidis.     

Effect of pH, NaCl content, and temperature on growth and survival of Arcobacter spp

Growth and survival of six human isolates of the pathogenic Arcobacter spp. in the presence of selected environmental factors were studied. Four strains of Arcobacter butzleri and two strains of Arcobacter cryaerophilus were exposed to pH levels of 3.5 to 8.0. Most strains grew between pH 5.5 and 8.0, with optimal growth of most A. butzleri and A. cryaerophilus strains at pH 6.0 to 7.0 and 7.0 to 7.5, respectively. The 24-h optimal growth range in the presence of NaCl was 0.5 to 1.0% for A. cryaerophilus. However, after 96 h, the optimum was between 0.5 and 2.0% NaCl. The optimum range for growth of A. butzleri strains was 0.09 to 0.5% NaCl after 96 h. The upper growth limits were 3.5 and 3.0% NaCl for A. butzleri and A. cryaerophilus, respectively. Survival at 25 degrees C in up to 5% NaCl was noted for A. butzleri 3556 and 3539 and A. cryaerophilus 3256. Decimal reduction times (D-values) at pH 7.3 in phosphate-buffered saline for three A. butzleri strains were 0.07 to 0.12 min at 60 degrees C, 0.38 to 0.76 min at 55 degrees C, and 5.12 to 5.81 min at 50 degrees C. At pH 5.5, decreased thermotolerance was observed, with D-values of 0.03 to 0.11 min at 60 degrees C, 0.30 to 0.42 min at 55 degrees C, and 1.97 to 4.42 min at 50 degrees C. Calculated z-values ranged from 5.20 to 6.28 degrees C. D-values of a three-strain mixture of A. butzleri in raw ground pork were 18.51 min at 50 degrees C and 2.18 min at 55 degrees C. Mild heat (50 degress C) followed by cold shock (4 or 8 degrees C exposure) had a synergistic lethal effect, reducing more cells than with an individual 50 degrees C treatment or with cold shock temperatures of 12 or 16 degrees C.     

Porcine blood cell concentrates for food products: hygiene, composition, and preservation

The objective of this study was to determine whether porcine blood cell concentrates (BCC) can be produced and stored using hygienic measures independent of the temperature acting upon the substrate. A number of additives widely accepted by the consumer (NaCl, sugars, food-grade acids) were used to form so-called hurdles (water activity [a(w)], pH) to spoilage, and their impact was tested on microbiological and sensory parameters of the BCC. BCC, whole blood, plasma, and the anticoagulant were collected on 23 days in a slaughterhouse. The BCC with the additives were stored for 27 days at + 3 degrees C and at +20 degrees C. Microbiological and chemical tests were carried out on the raw materials, and a(w) and the pH values of the stored BCC combinations were determined; the combinations were also submitted to sensory testing. The amounts of protein (33.4%) and hemoglobin (29.5 g/dl) in the BCC were significantly higher than in whole blood (19.4%; 13.8 g/dl). The mean total aerobic plate count was similar in all three substrates. However, the highest count (4.83 log CFU/g) was found in BCC; the count was lower in whole blood (4.62 log CFU/g) and lowest in plasma (4.22 log CFU/g). Storability (defined as a count of <5 log CFU/g) for 27 days at +20 degrees C was achieved only with two additive types: 15% NaCl and 10% NaCl plus 10% glucose plus 1% of a food-grade acid. Spoilage of the BCC was inhibited by an a(w) of 0.824 (with 15% NaCl) and by the combination of a(w) 0.87 and a pH of 5 (with 10% NaCl, 10% sugar, 1% acid). Both substrates retained their red color and fresh odor over the entire storage time.     

Purification and characterization of 3-isopropylmalate dehydrogenase from Thiobacillus thiooxidans

3-Isopropylmalate dehydrogenase was purified to homogeneity from the acidophilic autotroph Thiobacillus thiooxidans. The native enzyme was a dimer of molecular weight 40,000. The apparent K(m) values for 3-isopropylmalate and NAD+ were estimated to be 0.13 mM and 8.7 mM, respectively. The optimum pH for activity was 9.0 and the optimum temperature was 65 degrees C. The properties of the enzyme were similar to those of the Thiobacillus ferrooxidans enzyme, expect for substrate specificity. T. thiooxidans 3-isopropylmalate dehydrogenase could not utilize malate as a substrate.     

Novel substrate specificity of D-arabinose isomerase from Klebsiella pneumoniae and its application to production of D-altrose from D-psicose

d-Arabinose isomerase from Klebsiella pneumoniae 40bXX was purified 12-fold with a 62.5% yield indicated by its electrophoretic homogeneity. The purified enzyme showed the highest activities toward d-arabinose and l-fucose as substrates at optimum conditions (50 mM glycine-NaOH, pH 9.0, 40 degrees C). The enzyme had a broad range of substrate specificities toward various d/l-aldoses, i.e., d-arabinose, l-fucose, d/l-xylose, d-mannose, d/l-lyxose, l-glucose, d-altrose and d/l-galactose. The equilibrium ratios between d-arabinose and d-ribulose, l-fucose and l-fuculose, d-altrose and d-psicose, and l-galactose and l-tagatose were 90:10, 90:10, 13:87 and 25:75, respectively. Using a combination of the immobilized d-tagatose 3-epimerase and d-arabinose isomerase, we achieved the production of d-altrose from d-fructose in a batch reactor. We successfully produced approximately 12 g of d-altrose from 200 g of d-fructose in a reaction series with an overall yield of 6%. The product obtained was confirmed to be d-altrose by HPLC and (13)C-NMR. To the best of our knowledge, this is the first report on the production of d-altrose from a cheap sugar, d-fructose, using an enzymatic method.     

Phytase activity in sourdough lactic acid bacteria: purification and characterization of a phytase from Lactobacillus sanfranciscensis CB1

The phytase activity of 12 species of sourdough lactic acid bacteria was screened. It was intracellular only, largely distributed among the species and strains of Lactobacillus sanfranciscensis possessed the highest levels of activity. A monomeric ca. 50-kDa phytase was purified to homogeneity from L. sanfranciscensis CB1 by three chromatographic steps. L. sanfranciscensis CB1 exhibited the highest hydrolysing activity on Na-phytate after reaching the stationary phase of growth (ca. 12 h). Cells cultivated in the presence of maltose and fructose showed an increase of the phytase activity of ca. 35% with respect to the other carbon sources used. The phytase was optimally active at pH 4.0 and 45 degrees C. The enzyme was strongly inhibited by 2 mM of phenylmethylsulfonyl fluoride (PMSF), and 2 mM Hg(2+) and Fe(2+). It had a pI of ca. 5.0. The substrate specificity was dependent on the type of phosphate ester; a very low activity was detected on alpha-D-glucose-1-phosphate and D-fructose-6- and 1,6-phosphate, while the highest hydrolysis was found towards adenosine-5'-tri-, di- and mono-phosphate. Compared to these substrates, the activity on Na-phytate was also relevant. The enzyme was thermo-stable after exposure to 70 degrees C for 30 min; the D value calculated at 80 degrees C was ca. 10 min. As shown by the Central Composite Design (CCD) applied to study the individual and interactive effects of pH, temperature and NaCl, acidic conditions and elevated temperatures were indispensable for the enzyme adaptation to high NaCl concentrations. L. sanfranciscensis CB1 cells or the correspondent cytoplasmic extract were used to ferment a sourdough for 8 h at 37 degrees C; a marked decreased (64-74%) of the Na-phytate concentration was found compared with the unstarted dough. The sourdough started with L. sanfranciscensis CB1 cells was re-used for several times and the phytase activity was maintained to a considerable level.     

Purification and some characteristics of a monomeric alanine racemase from an extreme thermophile, Thermus thermophilus

We purified to homogeneity an alanine racemase (EC 5.1.1.1) from Thermus thermophilus HB8, an extreme thermophile. Interestingly, the enzyme possessed a monomeric structure with a molecular weight of about 38,000. The enzyme was most active at pH 8 and 75 degrees C, and remained active after incubation at 80 degrees C for 30 min.     

Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S: cloning and characterization

The carboxypeptidase gene from Geobacillus SBS-4S was cloned and sequenced. The sequence analysis displayed the gene consists of an open reading frame of 1503 nucleotides encoding a protein of 500 amino acids (CBP(SBS)). The amino acid sequence comparison revealed that CBP(SBS) exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBP(SBS) contained an active site motif (265)HEXXH(269) which is conserved in family-M32 of carboxypeptidases. The gene was expressed with His-Tag utilizing Escherichia coli expression system and purified to apparent homogeneity. The purified CBP(SBS) showed highest activity at pH 7.5 and 70°C. The enzyme activity was metal ion dependent. Among metal ions highest activity was found in the presence of Co(2+). Thermostability studies of CBP(SBS) by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77°C. The enzyme exhibited K(m) and V(max) values of 14 mM and 10526 μmol min(-1) mg(-1) when carbobenzoxy-alanine-arginine was used as substrate. k(cat) and k(cat)/K(m) valves were 10175 s(-1) and 726 mM(-1) s(-1). To our knowledge this is the highest ever reported enzyme activity of a metallocarboxypeptidase and the first characterization of a metallocarboxypeptidase from genus Geobacillus.     

Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

Growth kinetics of Vibrio parahaemolyticus O3:K6 under varying conditions of pH, NaCl concentration and temperature

The growth responses of Vibrio parahamolyticus to pH, NaCl concentration and temperature changes were studied using serotype O3:K6 and other strains. Growth curves were obtained for 27 different sets of conditions, comprised of three levels of NaCl concentration, pH and temperature. The temperature, pH and NaCl concentrations most favorable for growth were in the order of 25 degrees C, 20 degrees C and 15 degrees C, pH 8, 7 and 5.8, and 1%, 3% and 7%, respectively. The bacteria grew most rapidly at 25 degrees C, at a pH of 7 or 8 in the presence of 1% or 3% NaCl, with the population (initial, ca. 2.5 log CFU/mL) reaching a level log 7 CFU/mL at 12 h. A growth predictive model using the Gompertz equation was generated from the experimental data for any combination of NaCl concentration, pH and temperature within the range used in this study.