B. de las Rivas

Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration

The occurrence of in vitro amino acid activity in bacterial strains associated with fresh pork sausages packaged in different atmospheres and kept in refrigeration was studied. The presence of biogenic amines in decarboxylase broth was confirmed by ion-exchange chromatography and by the presence of the corresponding decarboxylase genes by PCR. From the 93 lactic acid bacteria and 100 enterobacteria strains analysed, the decarboxylase medium underestimates the number of biogenic amine-producer strains. 28% of the lactic acid bacteria produced tyramine and presented the tdc gene. All the tyramine-producer strains were molecularly identified as Carnobacterium divergens. Differences on the relative abundance of C. divergens were observed among the different packaging atmospheres assayed. After 28 days of storage, the presence of argon seems to inhibit C. divergens growth, while packing under vacuum seems to favour it. Among enterobacteria, putrescine was the amine more frequently produced (87%), followed by cadaverine (85%); agmatine and tyramine were only produced by 13 and 1%, respectively, of the strains analysed. Packing under vacuum or in an atmosphere containing nitrogen seems to inhibit the growth of enterobacteria which produce simultaneously putrescine, cadaverine, and agmatine. Contrarily, over-wrapping or packing in an atmosphere containing argon seems to favour the growth of agmatine producer-enterobacteria. The production of putrescine and cadaverine was associated with the presence of the corresponding amino acid decarboxylase genes. The biogenic amine-producer strains were included in a wide range of enterobacterial species, including Kluyvera intermedia, Enterobacter aerogenes, Yersinia kristensenii, Serratia grimesii, Serratia ficaria, Yersinia rodhei, Providencia vermicola and Obesumbacterium proteus.

Methods for the Detection of Bacteria Producing Biogenic Amines on Foods: A Survey

Abstract.The presence of biogenic amines in foods is of considerable public concern for the food industry and the regulatory agencies, since given the potential health hazard, there is a growing demand from consumers and control authorities to reduce the allowable limits of biogenic amines in foods and beverages. Rapid and simple methods are needed for the analysis of the ability to form biogenic amines by bacteria in order to evaluate the potential risk of bacterial occurring in some food products. Analytical chromatographic methods used for routine biogenic amine analysis of food substrates have been applied to bacterial cultures. Specific differential culture media for the presumptive identification of biogenic amine-producer bacteria have been developed. Recently, several PCR based methods targeting amino acid decarboxylases have been described. These latter molecular methods, in addition to its rapidity and specificity, offer the advantage of the identification of producer bacteria before the amine is synthesized.Zusammenfassung (Redaktion).Es wird darüber berichtet, unter welchen Umständen biogene Amine in Lebensmitteln vorkommen können und dass – aufgrund der damit verbundenen, möglichen gesundheitlichen Gefährdung – von den Verbrauchern und der Lebensmittelaufsicht angemahnt wird, die festgesetzten Grenzwerte für biogene Amine in Lebensmitteln und Getränken abzusenken. Im Sinne der Vorsorge stellt sich aber die Frage nach schnellen und praktikablen Nachweismethoden, um Bakterien, die biogene Amine produzieren können, frühzeitig zu identifizieren. Analytische chromatographische Methoden, die für die Routine-Kontrolle von Lebensmitteln auf biogene Amine eingesetzt werden, sind für die Untersuchung von Bakterienkulturen modifiziert worden. Spezielle Kulturmedien sind für die Identifizierung von biogene Amine produzierenden Bakterien entwickelt worden. Außerdem sind mehrere PCR-Methoden beschrieben worden, die nicht nur schnell und sehr spezifisch sind, sondern auch den Nachweis relevanter Bakterien ermöglichen, bevor diese mit der Synthese von biogenen Aminen begonnen haben.

Technological and safety properties of lactic acid bacteria isolated from Spanish dry-cured sausages

Technological and safety-related properties were analyzed in lactic acid bacteria isolated from Spanish dry-cured sausages in order to select them as starter cultures. In relation to technological properties, all the strains showed significative nitrate reductase activity; Lactobacillus plantarum, Lactobacillus paracasei and 52% of the Enterococcus faecium strains showed lipolytic activity and only Lactobacillus sakei strains (43%) were able to form biofilms. Related to safety aspects, E. faecium strains were the most resistant to antibiotics, whereas, L. sakei strains were the most sensitive. In relation to virulence factors, in the E. faecium strains analyzed, only the presence of efaA gene was detected. The analysis of biogenic amine production showed that most E. faecium strains and L. sakei Al-142 produced tyramine. In conclusion, L. paracasei Al-128 and L. sakei Al-143 strains possess the best properties to be selected as adequate and safe meat starter cultures.

Molecular cloning and functional characterization of a histidine decarboxylase from Staphylococcus capitis

Aims: Histamine intoxication is probably the best known toxicological problem of food‐borne disease. A histamine‐producing Staphylococcus capitis strain has been isolated from a cured meat product. The aim of this study was to gain deeper insights into the genetic determinants for histamine production in Staph. capitis.

Evaluation of Exopolysaccharide Production by Leuconostoc mesenteroides Strains Isolated from Wine

Exopolysaccharide (EPS)-producing lactic acid bacteria are responsible for the alteration of wine and other fermented beverages. The potential to produce EPS was investigated for Leuconostoc mesenteroides strains isolated from Spanish grape must and wine. Most strains were able to produce EPS from sucrose containing media. Based on their EPS-producing phenotype and on their EPS monosaccharide composition, the L. mesenteroides strains analyzed could be arranged in 2 groups. One group comprises mucoid strains producing a glucan polymer, and the other group includes strains producing a fructan polymer. The presence of a glucosyltransferase encoding gene in the glucan producing L. mesenteroides strains was assayed by PCR. Two primer sets, PF1-PF8 and GTFF-GTFR, were used to amplify internal fragment of known glucosyltransferase genes. None of the glucan-producing strains gave a positive amplicon by the primer sets used. Therefore, new tools need to be developed to broaden the range of potentially spoiling agents detected by PCR in fermented beverages.

An amperometric affinity penicillin-binding protein magnetosensor for the detection of β-lactam antibiotics in milk.

The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of β-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of β-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min.

Characterization of coagulase-negative staphylococci isolated from Spanish dry cured meat products.

Technological and safety-related properties were analyzed in a coagulase-negative staphylococci (CNS) collection isolated from Spanish dry-cured meat products in order to use them as starter cultures. The highest nitrate reductase and proteolytic activity was showed by Staphylococcus carnosus and Staphylococcus equorum. Only a few strains were able to form biofilms and the presence of the ica gene was analyzed on them. In relation to antibiotic resistance, all S. carnosus and most of the S. equorum strains were sensitive to the antibiotics tested and the presence of the blaZ gene in the β-lactamic resistant strains was studied. Biogenic amines were produced by 25% of the strains analyzed being all the S. carnosus strains tyramine producers. Taking into account the studied properties, two S. equorum strains could be selected as adequate and safe potential starter cultures for the elaboration of meat products.

Production and characterization of a tributyrin esterase from Lactobacillus plantarum suitable for cheese lipolysis

Lactobacillus plantarum is a lactic acid bacterium that can be found during cheese ripening. Lipolysis of milk triacylglycerols to free fatty acids during cheese ripening has fundamental consequences on cheese flavor. In the present study, the gene lp_1760, encoding a putative esterase or lipase, was cloned and expressed in Escherichia coli BL21 (DE3) and the overproduced Lp_1760 protein was biochemically characterized. Lp_1760 hydrolyzed p-nitrophenyl esters of fatty acids from C2 to C16, with a preference for p-nitrophenyl butyrate. On triglycerides, Lp_1760 showed higher activity on tributyrin than on triacetin. Although optimal conditions for activity were 45°C and pH 7, Lp_1760 retains activity under conditions commonly found during cheese making and ripening. The Lp_1760 showed more than 50% activity at 5°C and exhibited thermal stability at high temperatures. Enzymatic activity was strongly inhibited by sodium dodecyl sulfate and phenylmethylsulfonyl fluoride. The Lp_1760 tributyrin esterase showed high activity in the presence of NaCl, lactic acid, and calcium chloride. The results suggest that Lp_1760 might be a useful tributyrin esterase to be used in cheese manufacturing.

Gene organization of the ornithine decarboxylase‐encoding region in Morganella morganii

Aims:  The production of putrescine is a relevant property related to food quality and safety. Morganella morganii is responsible for putrescine production in fresh fish decomposition. The aim of this study was to gain deeper insights into the genetic determinants for putrescine production in M. morganii.

Biotransformation of Phenolics by Lactobacillus plantarum in Fermented Foods

Abstract Food phenolics exhibit a wide range of beneficial health effects due to their antioxidant properties. Natural phenolic compounds often occur as glycosides, esters, or polymers on which their bioactivity is reduced. Some food phenolics are transformed by the microbiota present during fermentation. This conversion is often essential for absorption and modulates the biological activity of these dietary compounds. Lactobacillus plantarum is the species most frequently used to ferment plant food products where phenolic compounds are abundant. The knowledge of the metabolism of phenolic compounds in L. plantarum is of interest as this bacterium possesses relevant enzymatic activities to obtain bioactive compounds. Complex phenolics are hydrolyzed by L. plantarum to simpler and more biologically active compounds. The complete enzymatic pathways for the biodegradation of polymeric hydroxybenzoic- and hydoxycinnamic-derived compounds are described. The adaptative behavior of L. plantarum under stress induced by phenolics also modulates traits beneficial for its gastrointestinal survival.