Tropomyosin from tilapia (Oreochromis mossambicus) as an allergen.

BACKGROUND: Tilapia is among the most common fresh water fish species raised by fish farms and can cause allergic reactions upon ingestion. OBJECTIVE: To investigate important allergens in Tilapia (Oreochromis mossambicus). METHODS: Allergens were detected using immunoblotting. An important allergen was purified to homogeneity by reversed-phase High Pressure Liquid Chromatography and characterized by enzyme linked immunosorbent assay (ELISA), competitive ELISA, Mass spectrometry (MS), circular dichroism measurements and differential scanning calorimetry. RESULTS: By immunoblotting using sera from 10 patients with confirmed tilapia allergy, we identified a number of allergens with apparent molecular weights 114 to 17 kD. All patients produced IgE against a 32 kD allergen, Ore m 4, which was identified by MS as tropomyosin (TM). IgE binding of the pure protein was confirmed by immunoblotting, ELISA and ELISA inhibition. cDNA from tilapia tropomyosin (TM) was sequenced and compared with TMs from other species. The tilapia TM showed 53.5% homology to TM from shrimp. Homology was much higher to human TM isoform 5 (87.7%). CONCLUSION AND CLINICAL RELEVANCE: TMs are the major allergens in allergy to crustaceans. Auto-antibodies against human TM isoform 5 have been implicated as a causative agent in inflammatory bowel disease (IBD). Intriguingly, six of the 10 tilapia allergic patients had also been diagnosed with IBD, corroborating a connection between allergy and IBD. To our knowledge, this is the first report of tropomyosin from vertebrates as an allergen.

Effects of post-processing treatments on sensory quality and Shiga toxigenic Escherichia coli reductions in dry-fermented sausages.

The effects of post-processing treatments on sensory quality and reduction of Shiga toxigenic Escherichia coli (STEC) in three formulations of two types of dry-fermented sausage (DFS; salami and morr) were evaluated. Tested interventions provided only marginal changes in sensory preference and characteristics. Total STEC reductions in heat treated DFS (32°C, 6days or 43°C, 24h) were from 3.5 to >5.5 log from production start. Storing of sausages (20°C, 1month) gave >1 log additional STEC reduction. Freezing and thawing of sausages in combination with storage (4°C, 1month) gave an additional 0.7 to 3.0 log reduction in STEC. Overall >5.5 log STEC reductions were obtained after storage and freezing/thawing of DFS with increased levels of glucose and salt. This study suggests that combined formulation optimisation and post-process strategies should be applicable for implementation in DFS production to obtain DFS with enhanced microbial safety and high sensory acceptance and quality.

Reduction of verotoxigenic Escherichia coli by process and recipe optimisation in dry-fermented sausages.

Outbreaks of verotoxigenic Escherichia coli (VTEC) linked to dry-fermented sausages (DFSs) have emphasized the need for DFS manufacturers to introduce measures to obtain enhanced safety and still maintain the sensory qualities of their products. To our knowledge no data have yet been reported on non-O157:H7 VTEC survival in DFS. Here, the importance of recipe and process variables on VTEC (O157:H7 and O103:H25) reductions in two types of DFS, morr and salami, was determined through three statistically designed experiments. Linear regression and ANOVA analyses showed that no single variable had a dominant effect on VTEC reductions. High levels of NaCl, NaNO(2), glucose (low pH) and fermentation temperature gave enhanced VTEC reduction, while high fat and large casing diameter (a(w)) gave the opposite effect. Interaction effects were small. The process and recipe variables showed similar effects in morr and salami. In general, recipes combining high batter levels of salt (NaCl and NaNO(2)) and glucose along with high fermentation temperature that gave DFS with low final pH and a(w), provided approximately 3 log(10) reductions compared to approximately 1.5 log(10) reductions obtained for standard recipe DFS. Storage at 4°C for 2months provided log(10) 0.33-0.95 additional VTEC reductions and were only marginally affected by recipe type. Sensory tests revealed only small differences between the various recipes of morr and salami. By optimisation of recipe and process parameters, it is possible to obtain increased microbial safety of DFS while maintaining the sensory qualities of the sausages.

Reduction of verotoxigenic Escherichia coli by process and recipe optimisation in dry-fermented sausages.

Outbreaks of verotoxigenic Escherichia coli (VTEC) linked to dry-fermented sausages (DFSs) have emphasized the need for DFS manufacturers to introduce measures to obtain enhanced safety and still maintain the sensory qualities of their products. To our knowledge no data have yet been reported on non-O157:H7 VTEC survival in DFS. Here, the importance of recipe and process variables on VTEC (O157:H7 and O103:H25) reductions in two types of DFS, morr and salami, was determined through three statistically designed experiments. Linear regression and ANOVA analyses showed that no single variable had a dominant effect on VTEC reductions. High levels of NaCl, NaNO(2), glucose (low pH) and fermentation temperature gave enhanced VTEC reduction, while high fat and large casing diameter (a(w)) gave the opposite effect. Interaction effects were small. The process and recipe variables showed similar effects in morr and salami. In general, recipes combining high batter levels of salt (NaCl and NaNO(2)) and glucose along with high fermentation temperature that gave DFS with low final pH and a(w), provided approximately 3 log(10) reductions compared to approximately 1.5 log(10) reductions obtained for standard recipe DFS. Storage at 4 degrees C for 2 months provided log(10) 0.33-0.95 additional VTEC reductions and were only marginally affected by recipe type. Sensory tests revealed only small differences between the various recipes of morr and salami. By optimisation of recipe and process parameters, it is possible to obtain increased microbial safety of DFS while maintaining the sensory qualities of the sausages.

Cross-resistance to antibiotics of Escherichia coli adapted to benzalkonium chloride or exposed to stress-inducers.

AIMS: To study the effects of adaptation and stress on the resistance to benzalkonium chloride (BC) and cross-resistance to antibiotics in Escherichia coli. METHODS AND RESULTS: Precultivation of E. coli ATCC 11775 and E. coli DSM 682 in the presence of subinhibitory concentrations of BC or stress inducers (salicylate, chenodeoxycholate and methyl viologen) resulted in higher minimum inhibitory concentration (MIC) of BC and chloramphenicol (CHL). Adaptation to growth in sixfold of the initial MIC of BC resulted in stable BC resistance and enhanced tolerance to several antibiotics and ethidium bromide (EtBr). The MIC of CHL increased more than 10-fold for both strains. Enhanced efflux of EtBr in adapted E. coli ATCC 11775 indicated that the observed resistance was due to efflux. Changes in outer membrane protein profiles were detected in the BC-adapted cells. There were no indications of lower membrane permeability to BC. CONCLUSIONS: Induction of stress response or gradual adaptation to BC or CHL results in acquired cross-tolerance between BC and antibiotics in E. coli. Enhanced efflux was one of the observed differences in adapted cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Provided not taking due precautions, extensive use of disinfectants could lead to emergence of antibiotic-resistant isolates.

Meat starters have individual requirements for Mn(2+).

The effect of different Mn(2+) concentrations on sausage fermentation was evaluated. A screening experiment was carried out with six lactobacilli starters in a sausage model. To further investigate the effects found, two selected lactobacilli strains were tested in pilot-scale sausage production. For all starters an increased fermentation rate was observed after Mn(2+) addition. Differences in the development of microbial, textural and sensory parameters were observed in the sausages. For one of the cultures these differences levelled out during sausage production yielding identical end products with and without Mn(2+), for the other strain the differences due to Mn(2+) addition in the sausages remained throughout the production process yielding sausages with different properties. Knowing a starter culture's requirements for Mn(2+) will allow optimisation of dry fermented sausage production in order to increase reliability and reproducibility of production decrease fermentation time and ensure microbial safety of the final product.

Identification and characterization of quaternary ammonium compound resistant staphylococci from the food industry.

The distribution of known genes conferring resistance to quaternary ammonium compounds (QACs) among different species of staphylococci isolated from the food industry was investigated. Twenty-four isolates hosting one of the genes qacA/qacB, smr, qacG or qacH, were subjected to species identification. Species determination was performed by biochemical analyses (API STAPH), comparative 16S rDNA sequence analysis and tDNA intergenic spacer length polymorphism analysis. Good correlation was obtained between the different methods. The isolates belonged to six different species of coagulase-negative Staphylococcus. The most commonly found species were Staphylococcus epidermidis and Staphylococcus saprophyticus. The results also indicated the possible spread of specific isolates of staphylococci which may reflect the dominance of certain strains in environments were QACs are used on a regular basis. The isolates were further characterized by the resistance phenotype to antimicrobial agents including antibiotics and disinfectants. Resistance to ampicillin, penicillin G and dyes was prevalent in strains harbouring the qacA or qacB genes, features also common among clinical staphylococci containing qacA/qacB. One QAC resistant strain harbouring the smr gene showed resistance to ampicillin, penicillin, tetracycline, erythromycin and trimethoprim. No enterotoxin production was detected among the QAC resistant strains.

The qacG gene on plasmid pST94 confers resistance to quaternary ammonium compounds in staphylococci isolated from the food industry.

The 2.3 kb resistance plasmid pST94 revealed a new gene (qacG) encoding resistance to benzalkonium chloride (BC), a commonly used quaternary ammonium disinfectant, and the intercalating dye ethidium bromide (Eb) in staphylococci isolated from the food industry. The 107 amino acid QacG protein showing 69.2% identity to the staphylococcal multi-drug resistance protein Smr is a new member of the small multi-drug resistance (SMR) protein family. QacG conferred resistance via proton dependent efflux. An additional ORF on pST94 encoded a protein with extensive similarity to replication proteins of other Gram-positive bacteria. Gene constructs containing the qacG and smr gene region combined with the smr or qacG promoter, respectively, indicated that QacG is more efficient than Smr and that qacG has a weaker promoter. Resistant qacG-containing cells could be adapted to withstand higher concentrations of BC. Adapted qacG-containing cells showed increased resistance mainly to BC. In contrast, adaptation of sensitive cells showed cross-resistance development to a range of compounds. Induction of proton-dependent efflux was observed for BC-adapted staphylococci cells not containing qacG. The ability of sublethal concentrations of BC to develop cross-resistance and induce efflux mechanisms could be of practical significance; it should be considered before use of any new disinfectant and in the design of better disinfection procedures.

The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance.

The prevalence of disinfectant-resistant food-related microorganisms is of concern to the food industry. The Staphylococcus saprophyticus strain ST2H6 isolated from a poultry processing plant contained a 2.4-kb plasmid (p2H6) harbouring qacH, which encodes resistance to disinfectants based on quaternary ammonium compounds. The complete p2H6 nucleotide sequence revealed an open reading frame encoding a putative protein of 107 amino acid residues with strong similarity to members of the small multidrug resistance protein family. QacH also conferred high-level ethidium bromide resistance and low-level proflavine resistance and thus differed phenotypically from the similar proteins Smr and QacG. Fluorimetry indicated that the high-level ethidium bromide resistance was due to improved efflux energised by the proton motive force. Site-directed mutagenesis substituting the Asp-24 residue with Glu-24 had no effect on resistance characteristics. An additional open reading frame on p2H6 encoded a putative protein with similarity to rolling circle replication proteins.

Accelerated production of dry fermented sausage.

The scope of this paper is to review work connected with accelerated ripening of dry fermented sausages by addition of proteolytic enzymes. An overview of the following topics is given: practical sausage experiments with addition of various proteinases of bacterial origin, including data from sensory, biochemical and gc/ms analyses; biochemical and genetic characterization of the enzyme shown to be most useful in these experiments, the serine proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151; experiments to transform starter cultures with the genes for production of this proteinase and proposals for future work in this field.

Resistance to quaternary ammonium compounds in Staphylococcus spp. isolated from the food industry and nucleotide sequence of the resistance plasmid pST827.

The complete nucleotide sequence of the 2.8 kb plasmid pST827 involved in resistance to the quaternary ammonium compound (QAC) benzalkonium chloride in meat-associated staphylococci was determined. An open reading frame (ORF) similar to the QAC resistance genes qacC, ebr and smr previously reported from clinical staphylococcal strains was identified (qacC'). In addition an ORF coding for a protein (Rep827) showing extensive homology to reported replication proteins of Gram-positive organisms was found. The occurrence of known QAC resistance gene (qacA-C) among staphylococcal strains isolated from food processing plants was studied by hybridization analysis. Of 191 isolates, 25 were resistant to benzalkonium chloride. Five of these gave no hybridization signals to probes specific for qacA-C. Further hybridization analysis indicated that pST827 or closely related plasmids are widespread among QAC-resistant staphylococcal strains. The finding of resistant staphylococci in different areas of the food processing industry indicates that QAC resistance is a potential problem in the food processing industry.

Purification and cloning of piscicolin 61, a bacteriocin from Carnobacterium piscicola LV61.

Piscicolin 61, a bacteriocin produced by Carnobacterium piscicola LV61, inhibits the growth of strains of Carnobacterium, Lactobacillus, and Enterococcus. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential hydrophobic interaction and reversed-phase chromatography. The N-terminal amino acid sequence of piscicolin 61 was determined by Edman degradation. The plasmid-located structural gene encoding piscicolin (psc61) was cloned and sequenced. It encoded a primary translation product of 71 amino acid residues, which is cleaved between amino acid residues 18 and 19 to yield the active bacteriocin. The calculated M(r) from the deduced protein sequence, 5052.6, agreed with that obtained by mass spectrometry. Piscicolin 61 did not show any sequence similarities to other known bacteriocins. However, the leader sequence resembled those of the pediocin-like bacteriocins. Piscicolin 61 may be able to form amphiphilic helices and may thus act on the membrane of sensitive cells.

Purification and cloning of sakacin 674, a bacteriocin from Lactobacillus sake Lb674.

Sakacin 674, a bacteriocin produced by Lactobacillus sake Lb764 and which inhibits the growth of Listeria monocytogenes, was purified to homogeneity by ammonium sulphate precipitation and sequential ion exchange, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence of sakacin 674 was determined by Edman degradation. The bacteriocin consisted of 43 amino acid residues and had a calculated molecular mass of 4436.6 Da, which is in good agreement with the molecular mass of 4437.2 as determined by mass spectrometry. The structural gene encoding sakacin 674 (sakR) was located on the chromosome. This gene was cloned and sequenced. It encoded a primary translation product of 61 amino acid residues which was cleaved between amino acids 18 and 19 to yield the active sakacin 674. Sakacin 674 resembled other known bacteriocins and was very similar to sakacin P.

The nucleotide sequence of a putative membrane transport gene from Clostridium perfringens.

A gene from Clostridium perfringens encoding a highly hydrophobic polypeptide of M(r) = 30,698 was cloned and sequenced. The polypeptide showed similarities with the inner membrane proteins of binding protein-dependent transport systems of the gram negative enterobacteria. The sequence homology to the UgpE and MalG proteins of Escherichia coli was 27.6%, or 60.4 and 52.2%, respectively, taking into account the conservative amino acid changes. The polypeptide contained the EAA---G---------(I/V)-LP motif of the gram negative transport proteins. This motif may thus be very old, as clostridia and E. coli separated some 1.5 x 10(9) years ago. The similarities suggest a common evolutionary origin of these genes.

Identity of the 17-kilodalton protein, a DNA-binding protein from Escherichia coli, and the firA gene product.

The 17-kilodalton protein, a DNA-binding protein encoded by the skp gene of Escherichia coli, was found to be identical to histonelike protein I, the product of the firA gene. This conclusion was reached after chromosomal localization, using the recently constructed high- and low-resolution E. coli restriction maps, and by direct comparison of the N-terminal amino acid sequence of histonelike protein I and the 17-kilodalton protein.