Optimal temperature input design for estimation of the square root model parameters: parameter accuracy and model validity restrictions

As part of the model building process, parameter estimation is of great importance in view of accurate prediction making. Confidence limits on the predicted model output are largely determined by the parameter estimation accuracy that is reflected by its parameter estimation covariance matrix. In view of the accurate estimation of the Square Root model parameters, Bernaerts et al. have successfully applied the techniques of optimal experiment design for parameter estimation [Int. J. Food Microbiol. 54 (1-2) (2000) 27]. Simulation-based results have proved that dynamic (i.e., time-varying) temperature conditions characterised by a large abrupt temperature increase yield highly informative cell density data enabling precise estimation of the Square Root model parameters. In this study, it is shown by bioreactor experiments with detailed and precise sampling that extreme temperature shifts disturb the exponential growth of Escherichia coli K12. A too large shift results in an intermediate lag phase. Because common growth models lack the ability to model this intermediate lag phase, temperature conditions should be designed such that exponential growth persist even though the temperature may be changing. The current publication presents (i) the design of an optimal temperature input guaranteeing model validity yet yielding accurate Square Root model parameters, and (ii) the experimental implementation of the optimal input in a computer-controlled bioreactor. Starting values for the experiment design are generated by a traditional two-step procedure based on static experiments. Opposed to the single step temperature profile, the novel temperature input comprises a sequence of smaller temperature increments. The structural development of the temperature input is extensively explained. High quality data of E. coli K12 under optimally varying temperature conditions realised in a computer-controlled bioreactor yield accurate estimates for the Square Root model parameters. The latter is illustrated by means of the individual confidence intervals and the joint confidence region.     

Misidentification of Vibrio cholerae O155 isolated from imported shrimp as O serogroup O139 due to cross-agglutination with commercial O139 antisera

Fish and shellfish products imported into Denmark are routinely analyzed for pathogenic Vibrio spp., particularly Vibrio cholerae, if products originate from subtropical or tropical areas. A V. cholerae strain that agglutinated commercial O139 antiserum but not the O1, Inaba, or Ogawa antisera was isolated from imported raw frozen shrimp. The toxigenicity of the strain was analyzed, and the results of a polymerase chain reaction showed that the V. cholerae strain did not contain the virulence genes ctx, tcpA, and zot, which are normally found in V. cholerae O1 and O139. The strain was resistant to colistin and spectinomycin. The high susceptibility of the strain to antimicrobial agents was confirmed by the lack of an SXT element, a self-transmissible, chromosomal genetic element that is normally present in 0139 strains and encodes resistance to sulfonamides, trimethoprim, and streptomycin. The strain contained two plasmids, in contrast to other O139 strains, which normally do not contain plasmids. The characteristics of the strain led to further agglutination testing with other antisera that are not commercially available, and the strain was found to agglutinate O155 antiserum in repeated testing. Manufacturers of 0139 antiserum should be aware of the closely related O antigens of the O139, O22, and O155 serogroups and should be aware that their commercial diagnostic O139 antiserum must be absorbed to remove cross-reacting agglutinins of O22 and O155 strains.     

Enzymatic modification of selected functional properties of myofibril preparation obtained from mechanically recovered poultry meat

The effect of addition of 3 g/L of commercially available transglutaminase preparation to protein extracts obtained from mechanically recovered poultry meat was studied. The content of free thiol groups (–SH), thermal drip and gel texture were determined. After pre-incubation at 7–8 °C for 1, 3, 5 and 24 h, the samples were subjected to one-step heating at 50, 60, 70 and 80 °C and two-step heating at 50/80, 55/80 and 60/80 °C. The addition of preparation and the extension of pre-incubation time led to decrease of free -SH groups content. After heating, the number of thiol groups decreased, the texture was improved, but thermal drip from gels increased. The amount of –SH groups in gel extracts subjected to one-step heating decreased with simultaneous increase of mechanical strength of gels. Protein gels subjected to two-step heating exhibited higher firmness than gels subjected to one-step heating. Thus, the 3 g/L addition of transglutaminase preparation in combination with one-step thermal processing at 70 °C and pre-incubation for 3 h contributed to improvement of texture properties of model gels and low thermal drip.     

Estrogen-induced alterations in amh and dmrt1 expression signal for disruption in male sexual development in the zebrafish

Dmrt1 and amh are genes involved in vertebrate sex differentiation. In this study, we cloned dmrt1 and amh cDNAs in zebrafish (Danio rerio) and investigated the effects of exposure to 17a-ethinylestradiol (EE2), during early life on their patterns of expression and impact on the subsequent gonadal phenotype. Expression of both amh and dmrt1 in embryos was detected as early as at 1 day post fertilization (dpf) and enhanced expression of amh from 25 dpf was associated with the period of early gonadal differentiation. Sex-dependent differences in enhanced green fluorescent protein transgene expression driven by the promoter of the germ cell-specific vas gene were exploited to show that at 28dpf and 56dpf both amh and dmrt1 mRNA were overexpressed in males compared with females. Exposure during early life to environmentally relevant concentrations of EE2 had a suppressive effect on the expression of both amh and dmrt1 mRNAs and this was associated with a cessation/retardation in male gonadal sex development. Our findings indicate that estrogen-induced suppression in expression of dmrt1 and amh during early life correlate with subsequent disruptive effects on the sexual phenotype in males.     

Purification and characterization of natural Bet v 1 from birch pollen and related allergens from carrot and celery

Birch pollen allergy is predominantly caused by the major allergen Bet v 1 and can lead to crossreactions with homologous proteins in food. Two major cross-reactive food allergens are Dau c 1 from carrot and Api g 1 from celery, which have never been purified from their natural source. Here, we describe a non-denaturing purification method for obtaining natural Bet v 1, Dau c 1 and Api g 1, comprising of ammonium sulfate precipitation, hydrophobic interaction chromatography and size exclusion chromatography. This method resulted in 98-99% pure isoform mixtures for each allergen. Characterization of these isoform mixtures with Q-TOF MS/MS clearly showed earlier reported isoforms of Bet v 1, Dau c 1 and Api g 1, but also new isoforms. The presence of secondary structure in the three purified allergens was demonstrated via circular dichroism and showed high similarity. The immune reactivity of the natural allergens was compared with recombinant proteins by Western blot and ELISA and showed similar reactivity.     

Isolation and characterization of a psychrotolerant toxin producer, Bacillus weihenstephanensis, in liquid egg products

A psychrotolerant bacteria of the Bacillus cereus group was found responsible for the spoilage of whole liquid egg products. By sequencing a 16S rRNA region and performing a PCR amplification of specific 16S rRNA and cspA signatures, a Bacillus weihenstephanensis was identified. Characterization of this strain shows its ability to grow in defined medium as well as in whole liquid egg at refrigerated temperatures. The strain isolated possesses genes encoding for hemolysin BL, nonhemolytic enterotoxin, and B. cereus enterotoxins and produces enterotoxins with cytotoxic activity in whole liquid egg, even at refrigerated temperatures. The isolate exhibits a clear ability to stick and form biofilms on stainless steel, the most common material used in egg breaking factories, as well as on model hydrophilic (glass) and hydrophobic (polytetrafluoroethylene) materials. These findings show the necessity to monitor for Bacillus contamination in egg products that are often used in the composition of particularly susceptible finished products such as cream, dessert, dairy, meat, and seafood.     

Solubilisation and Degradation of Wheat Gluten Proteins by Barley Malt Proteolytic Enzymes

Solubilisation and degradation of wheat gluten proteins by barley malt proteolytic enzymes (BMPE) was investigated with a model buffer system at pH 4.0 and pH 5.6, representing optimal pH for proteolysis and a pH value typical for beer brewing conditions respectively. Under the experimental conditions, incubation of commercial wheat gluten with BMPE solubilised 70% and 20% of the gluten proteins at pH 4.0 and pH 5.6 respectively. Gel permeation chromatography profiles and SDS‐PAGE showed that wheat gluten proteins were more degraded by BMPE at pH 4.0 than at pH 5.6. In a laboratory scale barley malt brewing experiment, proteins of worts, prepared with and without wheat gluten, were characterised. Results comparable to those in the model buffer system at pH 5.6 were obtained, which indicated that BMPE indeed solubilise wheat gluten during mashing, but that further degradation is rather limited under these conditions.     

Dehalogenation of chlorinated ethenes and immobilization of nickel in anaerobic sediment columns under sulfidogenic conditions

A sediment column study was carried out to demonstrate the bioremediation of chloroethene- and nickel-contaminated sediment in a single anaerobic step under sulfate-reducing conditions. Four columns (one untreated control column and three experimental columns) with sediment from a chloroethene- and nickel-contaminated site were investigated for 1 year applying different treatments. By stimulating the activity of sulfate-reducing bacteria by the addition of sulfate as supplementary electron acceptor, complex anaerobic communities were maintained with lactate as electron donor (with or without methanol), which achieved complete dehalogenation of tetra- and trichloroethenes (PCE and TCE) to ethene and ethane. A few weeks after sulfate addition, production of sulfide increased, indicating an increasing activity of sulfate-reducing bacteria. The nickel concentration in the effluent of one nickel-spiked column was greatly reduced, likely due to the enhanced sulfide production, causing precipitation of nickel sulfide. At the end of the study, 94% of the initial amount of nickel added to that column was recovered in the sediment As compared to the untreated (nonspiked) control column, all chloroethene-spiked columns ladditions of PCE and TCE) showed a permanent release of small chloride ion quantities (approximately 0.5-0.7 mM chloride), which were detected in the effluents a few weeks after sulfide production was observed for the first time. The formation of ethene and ethane as final products after dechlorination of PCE and TCE was detected in some effluents and in some gas phases of the columns. Other metabolites or intermediates (such as DCE isomers) were only detected sporadically in negligible quantities. The results of this study demonstrated thatmicrobial activity stimulated under sulfate-reducing conditions can have a beneficial effect on both the precipitation of heavy metals and the complete dechlorination of organochlorines. The strongly negative redox potential created by the activity of sulfate-reducing bacteria may be one factor responsible for stimulating the activity of the dehalogenating bacteria in the test columns.