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Cheese is a food in which toxic concentrations of biogenic amines (BA) may be reached, mainly as a consequence of the decarboxylation of determined amino acids by certain lactic acid bacteria (LAB). To maintain the food safety of cheese, environmentally friendly strategies are needed that specifically prevent the growth of BA-producing LAB and the accumulation of BA. The bacteriocins produced by LAB are natural compounds with great potential as food biopreservatives. This work examines the antimicrobial potential of 7 bacteriocin-containing, cell-free supernatants (CFS: coagulin A-CFS, enterocin A-CFS, enterocin P-CFS, lacticin 481-CFS, nisin A-CFS, nisin Z-CFS and plantaricin A-CFS) produced by LAB against 48 strains of the LAB species largely responsible for the accumulation of the most important BA in cheese, i.e., histamine, tyramine and putrescine. Susceptibility to the different CFS was strain-dependent. The histamine-producing species with the broadest sensitivity spectrum were Lentilactobacillus parabuchneri (the species mainly responsible for the accumulation of histamine in cheese) and Pediococcus parvulus. The tyramine-producing species with the broadest sensitivity spectrum was Enterococcus faecium, while Enterococcus faecalis and Enterococcus hirae were among the most sensitive putrescine producers. Nisin A-CFS was active against 31 of the 48 BA-producing strains (the broadest antimicrobial spectrum recorded). Moreover, commercial nisin A prevented biofilm formation by 67% of the BA-producing, biofilm-forming LAB strains. These findings underscore the potential of bacteriocins in the control of BA-producing LAB, and support the use of nisin A as a food grade biopreservative for keeping BA-producing LAB in check and reducing BA accumulation in cheese.

Cheese is a fermented food in which toxic concentrations of biogenic amines (BAs) may accumulate, mainly as a result of the metabolism of certain lactic acid bacteria (LAB); the formation of these compounds needs to be limited. This work reports the antimicrobial potential of bacteriocin AS-48 against the LAB largely responsible for the accumulation of the BA histamine, tyramine and putrescine in cheese. Most of the 50 analysed BA-producing LAB strains were sensitive to AS-48. The bacteriocin also prevented biofilm formation of 92% of the BA-producing strains tested. This result is of great technological importance since, in cheese production, biofilms are an important source of contamination by BA-producing LAB. This study supports the potential use of AS-48 as a biopreservative for controlling BA-producing LAB in fermented food.

Tryptamine is a neuroactive compound that derives from the decarboxylation of L-tryptophan. Tryptamine may act as a neurotransmitter or neuromodulator of the human central nervous system, where it is found in low concentrations. Tryptamine is considered to be a SRA (serotonin releasing agent), a type of drug that induces the release of serotonin into the neuronal synaptic cleft. SRA have been used clinically as appetite suppressants, and they have also been proposed as novel antidepressants and anxiolytics. Therefore, we considered the screening of tryptamine-producing lactic acid bacteria (LAB). Tryptamine has been detected in fermented food such as cheese, although data about the type of cheese where it accumulates and the concentrations that it can reach are very scarce. Supposedly, microbial tryptophan decarboxylases are the responsible enzymes for tryptamine biosynthesis in cheese. However, neither the microbial species nor the enzyme have been yet identified. Our hypothesis is that cheese accumulating tryptamine could be used as source of potential LAB able to decarboxylate L-tryptophan and produce tryptamine. In this work, we have quantified the tryptamine content of hundreds of different cheeses. Moreover, following a knowledge discovery in databases approach we identified those technological/environmental/metabolic profiles of cheeses accumulating the highest amounts of tryptamine. Furthermore, from those cheeses we have isolated LAB with the ability to convert tryptophan into tryptamine. These results open the possibility of the future use of such LAB as therapeutic psychobiotics for conferring a mental health benefit to the host.

Biogenic amines (BA) are nitrogenous compounds with biological activity that can accumulate in cheese in toxic concentrations due to the metabolism of some lactic acid bacteria (LAB). Putrescine, one of the BA most frequently found in cheese, is synthesized from arginine through the agmatine deiminase (AGDI) route. Arginine is first decarboxylated to produce agmatine, which is subsequently deaminated by the agmatine deiminase pathway (AgdI) to produce putrescine. Our group has identified Enterococcus faecalis and some strains of Lactococcus lactis as those responsible for the conversion of agmatine to putrescine in cheese. However, little is known about which microorganisms perform the decarboxylation of arginine to produce agmatine. Moreover, these microorganisms would be of additional interest since there is evidence of the usefulness of agmatine in the treatment of neuropathies and mood disorders. The identification of BA-producing microorganisms in food samples is usually based on the pH rise due to the decarboxylation of the amino-acid substrate, revealed by a pH indicator. However, for the detection of agmatine-producing microorganisms in cheese, this would lead to a large number of false positives, since many LAB possess the arginine deiminase activity which, from arginine, produces ornithine and ammonium, which causes a pH increase even greater. To solve this problem, we have designed a new biosensor for the detection of agmatine-producing microorganisms based on the agmatine-inducible transcription system (ACE) designed in our group. Here we describe the design and optimization of a protocol that has allowed us to isolate and identify agmatine-producing microorganisms from dairy samples.

The rise of antimicrobial resistant (AMR) bacteria is a major health concern, especially with regard to members of the ESKAPE group, to which vancomycin-resistant (VRE) Enterococcus faecium belongs. Phage therapy has emerged as a novel alternative for the treatment of AMR infections. This, however, relies on the isolation and characterisation of a large collection of phages. This work describes the exploration of human faeces as a source of new E. faecium-infecting phages. Phage vB_EfaH_163 was isolated and characterised at the microbiological, genomic, and functional levels. vB_EfaH_163 phage, a new member of Herelleviridae, subfamily Brockvirinae, has a dsDNA genome of 150,836 bp that does not harbour any virulence factors or antibiotic resistance genes. It infects a wide range of E. faecium strains of different origins, including VRE strains. Interestingly, it can also infect Enterococcus faecalis strains, even some that are linezolid-resistant. Its capacity to control the growth of a clinical VRE isolate was shown in broth culture and in a Galleria mellonella animal model. The discovery and characterisation of vB_EfaH_163 increases the number of phages that might be used therapeutically against AMR bacteria.

Bacterial biofilms on the surfaces of food industry equipment act as reservoirs of spoilage and pathogenic bacteria that contaminate food matrix constituting a health threat. Lentilactobacillus parabuchneri is a heterofermentative lactic acid bacteria present in the microbiota of fermented dairy products that was identified as the main responsible for the production and accumulation of elevated concentrations of histamine -a toxic biogenic amine- in cheese [1]. Some L. parabuchneri strains have been shown to produce biofilms on the surfaces of dairy industrial equipment [2]. Such biofilms are a reservoir of histamine producers that contaminate the cheese during the elaboration processes and results in the accumulation of histamine in the final product. The ability to form biofilms in 25 L. parabuchneri strains isolated from cheese was evaluated. The strains were categorized as strong, moderate and weak biofilms formers and two representatives from each category were selected for further work. The influence of temperature and pH -relevant factors during cheese elaboration- in the capacity of the selected strains to form biofilm, was studied. The adherence to different materials -plastic, rubber, stainless steel, and wood- commonly used in the manufacture of cheese, was also evaluated. In addition, the composition of the extracellular matrix of L. parabuchneri biofilms was also investigated using different approaches, namely confocal microscopy, real-time infrared spectroscopy and enzymatic treatments. Genome sequencing and analysis of the selected strains made it possible to identify a cluster of 4 genes -present only in the strong biofilm-forming strains- which showed high similarity with genes involved in pili formation, following the sortase-mediated pilus model. Therefore, such genes could be related to the ability to adhere to surfaces, the first step of biofim formation. The heterologous expression of the cluster in Lactococcus lactis demonstrated its relevant

Lentilactobacillus parabuchneri, a lactic acid bacterium, is largely responsible for the production and accumulation of histamine, a toxic biogenic amine, in cheese. L. parabuchneri strains can form biofilms on the surface of industry equipment. Since they are resistant to cleaning and disinfection, they may act as reservoirs of histamine-producing contaminants in cheese. The aim of this study was to investigate the biofilm-producing capacity of L. parabuchneri strains. Using the crystal violet technique, the strains were first categorized as weak, moderate or strong biofilm producers. Analysis of their biofilm matrices revealed them to be mainly composed of proteins. Two strains of each category were then selected to analyze the influence on the biofilm-forming capacity of temperature, pH, carbon source, NaCl concentration and surface material (i.e., focusing on those used in the dairy industry). In general, low temperature (8 °C), high NaCl concentrations (2–3% w/v) and neutral pH (pH 6) prevented biofilm formation. All strains were found to adhere easily to beech wood. These findings increase knowledge of the biofilm-forming capacity of histamine-producing L. parabuchneri strains and how their formation may be prevented for improving food safety.

La triptamina es una amina biógena (AB) que se sintetiza en animales, plantas y microorganismos mediante la descarboxilación del aminoácido triptófano. La triptamina puede actuar como neurotransmisor o neuromodulador del sistema nervioso central, donde se encuentra en bajas concentraciones. Como otras AB, se puede acumular en los alimentos y ha sido reconocida por la Agencia Europea de Seguridad Alimentaria (EFSA) como potencial agente tóxico. Hasta el momento ningún organismo regulador ha establecido oficialmente un umbral de seguridad para su ingesta, aunque en nuestro grupo de investigación hemos demostrado su citotoxicidad in vitro [1]. Sin embargo, la triptamina se considera un SRA (agente liberador de serotonina) [2], un tipo de fármaco que induce la liberación de serotonina en la hendidura sináptica neuronal. Los SRA se han utilizado clínicamente como supresores del apetito y también se han propuesto como nuevos antidepresivos y ansiolíticos. Además, se ha propuesto que la triptamina es un potenciador de la actividad serotoninérgica [3]. También hay estudios que muestran los efectos beneficiosos de la triptamina producida por la microbiota intestinal sobre el sistema gastrointestinal, ya que aumenta la secreción de fluidos dependientes de aniones y acelera el tránsito gastrointestinal [4]. Durante la maduración del queso se produce la proteólisis de las caseínas y, al igual que sucede con otros aminoácidos, tiene lugar la liberación de triptófano a partir del cual podría sintetizarse la triptamina. Dado que los datos disponibles actualmente son muy escasos, nos propusimos como objetivo estudiar el contenido de triptófano y triptamina en distintos tipos de quesos e identificar los microorganismos productores. En este trabajo, se ha analizado mediante un método de cromatografía líquida (UHPLC) desarrollado en nuestro grupo, el contenido de triptófano y triptamina en más de 250 variedades de queso. Aplicando técnicas de análisis multivariante se han identificado

The quality and commercial value of a cheese depends on its organoleptic qualities and nutritional properties. Moreover, consumers and industry interest in a cheese may be increased if it also contains bioactive compounds with demonstrated health-promoting effects, such as gamma amino butyric acid (GABA), the main inhibitory neurotransmitter of the human central nervous system. Indeed, the ingestion of a GABA-enriched cheese has proven an anti-hypertensive effect on human [1]. In our group, we have demonstrated the presence of large quantities of GABA in several cheese samples from Europe [2]. Gy¿r-Moson-Sopron megyei Csemege is one of the most traditional cow¿s milk cheese specialities in Hungary. It has been registered as a protected geographical indication (PGI) in the European Union, based on tradition, a unique production method, the specific skills required for production, and the reputation of the cheese. To date, no published studies concerning to this cheese variety are available in the scientific literature. The aim of the present study was to initiate the chemical and microbiological characterization of Gy¿r-Moson-Sopron megyei Csemege PGI cheese. Thus, we determined the content of free proteinogenic aminoacids, and GABA by ultra-high performance liquid chromatography (UHPLC). Also, for safety considerations, we examined the content of biogenic amines. Finally, we determined the volatile organic composition of the cheese by gas chromatography coupled to mass spectrometry. The microbial diversity of Gy¿r-Moson-Sopron megyei Csemege PGI cheese was explored by means of viable counting on selective culture media. Moreover, individual colonies grown on MRS and M17 culture media whose morphology resembled that of lactic acid bacteria were cultured in acidic conditions in the presence of the precursor monosodium glutamate and screened for its potential ability to change the colour of the medium, due to the presumptive production of GABA. The confirmation of thos

Las aminas biógenas (AB) son compuestos nitrogenados derivados de aminoácidos y que tienen actividad biológica. Las AB se pueden acumular en algunos tipos de quesos a concentraciones tóxicas, debido al metabolismo de algunas bacterias del ácido láctico (BAL). La putrescina es una de las AB que se encuentra más frecuentemente en quesos y se sintetiza a partir del aminoácido arginina mediante una descarboxilación y una desaminación [1]. Dependiendo del orden de estas dos reacciones, se diferencian dos rutas diferentes, siendo la ruta de la agmatina deiminasa (AGDI) la que prevalece en alimentos fermentados, incluido el queso. En esta ruta, la arginina es descarboxilada por la arginina decarboxilasa (Adc) para dar lugar a agmatina, la cual es posteriormente desaminada por la actividad de la agmatina deiminasa (AgdI) generándose como producto final la putrescina. En nuestro grupo hemos identificado Enterococcus faecalis y algunas cepas de Lactococcus lactis como los principales microorganismos responsables de la conversión de agmatina a putrescina en quesos [2]. Sin embargo, se desconoce que microorganismos realizan el primer paso de la ruta, es decir, la descarboxilación de la arginina para producir agmatina. Nuestro objetivo es aislar e identificar los microorganismos responsables de este paso imprescindible para la acumulación de putrescina. Además, estos microorganismos tendrían un doble interés, ya que existen evidencias clínicas de la utilidad de la agmatina en el tratamiento de enfermedades relacionadas con neuropatías y con trastornos del estado de ánimo como la ansiedad y la depresión [4]. También está aprobada como suplemento alimenticio para el aumento de la masa muscular. La identificación de microorganismos productores de AB en muestras de alimentos suele basarse en el aumento de pH debido a la descarboxilación del aminoácido sustrato, revelado por un indicador de pH añadido al medio de cultivo suplementado con el aminoácido correspondiente, arginina en est

Currently, consumption of spontaneously fermented milks is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. In this context, to select eligible indigenous strains which could be applied as bioprotective and/or starter cultures, the present study aimed to characterize the genomic variability, biotechnological potential, and safety of thirty-eight LAB isolated from Algerian dairy and farm sources of western Algeria. The isolates were unequivocally identified by 16S rRNA gene and fingerprint-based methods. The following species were identified: Enterococcus faecium (n = 15), Enterococcus durans (n = 2), Enterococcus hirae (n = 2), Enterococcus lactis (n = 1), Lactiplantibacillus plantarum (n = 6), Lactococcus lactis (n = 4), Levilactobacillus brevis (n = 3), Lacticaseibacillus paracasei (n = 3), Lacticaseibacillus rhamnosus (n = 1), and Pediococcus acidilactici (n = 1). Among the strains, three of them, L. lactis LGMY8, Lb. plantarum LGMY30 and Lb. paracasei LGMY31 were safe and showed some valuable biotechnological properties, such as high acidification, proteolytic activity, EPS production, and inhibition of undesirable bacteria that made them powerful candidates to be used as starter.

La histamina es una amina biógena que se sintetiza mediante descarboxilación enzimática del aminoácido histidina y que debido al metabolismo de algunas bacterias se puede acumular en concentraciones elevadas en los alimentos, provocando reacciones adversas en los consumidores. El queso es uno de los alimentos en los que más frecuentemente se acumula histamina a concentraciones tóxicas, siendo Lentilactobacillus parabuchneri el principal responsable. Trabajos previos revelaron la capacidad de algunas de sus cepas de formar biofilms en distintas superficies, lo que constituye un riesgo de contaminación de los quesos durante su producción, especialmente de los quesos procesados, como los rallados y cortados. Los biofilms son comunidades de microorganismos que permanecen adheridos a una superficie y embebidos en una matriz extracelular. Su presencia en la industria alimentaria es muy habitual, pudiendo adherirse a las superficies de los equipamientos utilizados, provocando la contaminación de los alimentos. Dependiendo de las especies pueden causar su deterioro, con la consecuente pérdida económica, e incluso pueden ser responsables de intoxicaciones alimentarias. El objetivo del presente trabajo fue identificar los genes de L. parabuchneri involucrados en la formación del biofilm y determinar su funcionalidad. Con este fin, se secuenció y analizó el genoma de 6 cepas con diferentes capacidades de producción de biofilm (fuertes, moderadas y débiles). En ellas, se confirmó la presencia del cluster HDC de producción de histamina. Además, se identificó un cluster de 4 genes, presente únicamente en las cepas formadoras de biofilm fuerte, que mostró similitud con genes implicados en la formación de pili, siguiendo el modelo sortase-mediated pilus, y que por tanto podría estar relacionado con la capacidad de adhesión a superficies. Se realizó la clonación de las 8kb del cluster, y su expresión heteróloga en Lactococcus lactis NZ9000 permitió comprobar su funcionalidad e impli

La depresión es una enfermedad creciente en todo el mundo, que se estima afectan a más de 300 millones de personas. En la actualidad existen evidencias de que los microorganismos en el intestino podrían estar involucrados en los trastornos mentales a través del eje cerebro-intestino. De hecho, se ha propuesto el uso de probióticos como terapia adjunta en el manejo de la depresión. Recientemente, se ha visto que los efectos beneficiosos de estos microorganismos estarían mediados por su capacidad para producir y liberar compuestos neuroactivos. Entre estos microorganismos, destacan las bacterias del ácido láctico (BAL) ya que son especies seguras, que juegan un papel fundamental en la elaboración de alimentos fermentados, como el queso, y que podrían utilizarse para la elaboración de alimentos fermentados funcionales enriquecidos en compuestos neuroactivos para ayudar en el tratamiento de la depresión. Dentro de estos compuestos destaca la ¿-feniletilamina (PEA), la cual se utiliza en la actualidad como suplemento nutricional para mejorar el estado de ánimo y el bienestar, entre otros beneficios. En este trabajo se llevó a cabo una búsqueda de BAL capaces de producir PEA y que tuviese potencial biotecnológico para ser utilizadas como cultivos, ya sea iniciadores o adjuntos, para la elaboración de alimentos lácteos fermentados funcionales. Para ello se partió de muestras de queso con alto contenido en PEA y se llevó a cabo una búsqueda, identificación y tipificación de potenciales productores de PEA. Las cepas identificadas como Enterococcus durans, fueron caracterizadas a nivel tecnológico para su inclusión en productos lácteos fermentados. Se seleccionó la cepa con mejores aptitudes tecnológicas y se realizó un estudio de la influencia de factores tecnológicos en su capacidad de producir PEA, comprobando que la temperatura y la sal afectan positiva y negativamente a su producción.

Some bacteria decarboxylate amino acids as a means of resisting acid stress. It is a mechanism commonly used by bacteria in fermented dairy products. The decarboxylation of histidine to histamine occurs frequently in some types of cheese, but high concentrations of histamine can be harmful to consumers. Not all amino acid decarboxylation products are, however, undesirable. GABA, the decarboxylation product of glutamate, has positive effects on human health. Although histamine and GABA accumulate in large quantities in many types of cheese, there is little information regarding their relationship, and that which is available is restricted to a few cheese varieties. The present work examines whether cheeses exist with high concentrations of GABA but safe concentrations of histamine. The GABA and histamine contents of 250 cheeses representing 143 European varieties were examined, and the technological, environmental and metabolic (TEM) traits governing their accumulation identified. Two TEM profiles were associated with high GABA contents and safe histamine concentrations. These findings could help the dairy industry select the best technological conditions for making GABA-rich, histamine-safe cheeses.

Lactic acid bacteria (LAB) belonging to the genus classically known as Lactobacillus, recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor Escherichia coli carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested Lactocaseibacillus casei strains, including many natural isolates, and to several other genera, including Lentilactobacillus parabuchneri, for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the oriT and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant Staphylococcus epidermidis. Conjugative DNA transfer from E. coli to S. aureus was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into S. epidermidis. This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.

Research background. Consumption of spontaneously fermented camel’s milk is common in Algeria, making it a feasible source of diverse lactic acid bacteria (LAB) with the potential to be used as adjunct cultures to improve quality and safety of fermented dairy products. Experimental approach. Twelve raw camel´s milk samples were used as a source of indigenous LAB, which were further characterised by examining 39 phenotypic traits with technological relevance. Results and conclusions. Thirty-five non-starter LAB (NSLAB) were isolated from 12 Algerian raw camel's milk samples and they were microbiologically, biochemically and genetically characterised. Some isolates showed proteolytic activity, acidifying capacity, the ability to use citrate, and to produce dextran and acetoin. Ethanol, acetaldehyde, methyl acetate, acetoin and acetic acid were the major volatile compounds detected. Cluster analysis performed using the unweighted group with arithmetic average (UPGMA) method, and based on the thirty-nine phenotypic characteristics investigated, reflected the microbial diversity that can be found in raw camel´s milk. Novelty and scientific contribution. The isolated strains, from a non-typical source, showed interesting technological traits to be considered as potential adjunct cultures. Cluster analysis based on the examined phenotypic characteristics proved to be a useful tool for the typification of isolates when no genetic information is available. These findings may be of use towards an industrialised production of camel's milk dairy products

Histamine, one of the most toxic and commonly encountered biogenic amines (BA) in food, is produced by the microbial decarboxylation of histidine. It may accumulate at high concentrations in fish and fermented food. Cheese has some of the highest histamine concentrations, the result of the histidine-decarboxylase activity of certain lactic acid bacteria (LAB). The present work describes the nucleotide sequence and transcriptional organization of the gene cluster responsible for histamine biosynthesis (the HDC cluster) in Lactobacillus vaginalis IPLA 11064 isolated from cheese. The influence of histidine availability and pH on histamine production and the expression of the HDC cluster genes is also examined. As expected, the results suggest that the production of histamine under acidic conditions improves cell survival by maintaining the cytosol at an appropriate pH. However, the transcriptional regulation of the HDC cluster is quite different from that described in other dairy histamine-producing LAB, probably due to the lack of a termination-antitermination system in the histidyl-tRNA synthetase gene (hisS).

Consumers' demand for functional foods that contains health-promoting substances is currently increasing. Gamma amino butyric acid (GABA) is a beneficial amino compound highly valued for its anti-hypertensive, anti-diabetic and relaxing effects. A multifactorial analysis has been performed to classify cheese according to their GABA content. We have analysed thirty-seven cheese manufactures (one-hundred samples) including dominant climate, milk treatment, milk origin, moulds presence, proteolysis, ripening time, final texture and fat content as categorical variables. We also quantified free amino acids, glutamic acid, glutamine and ammonium ions as chemical signatures of GABA metabolism. Cheese samples were classified into four distinct clusters. The spatial correlation between samples and variables revealed the existence of two profiles of high-GABA-content cheeses. These findings may help the food industry design functional cheeses with high GABA contents.

La tiramina es una amina biógena traza que los seres vivos sintetizan a partir de la descarboxilación enzimática de la tirosina. A pesar de su importancia fisiológica, se sabe que la ingesta de alimentos que contienen altas concentraciones de tiramina puede ocasionar distintos problemas de salud en el consumidor, como hipertensión, dolores de cabeza, migrañas, etc. En alimentos fermentados como productos cárnicos, derivados del pescado y productos lácteos se pueden acumular concentraciones altas de tiramina. El queso es el alimento fermentado en el que se han registrado concentraciones más elevadas de tiramina (hasta 2130 mg/kg). De hecho, la primera vez que se describieron los efectos tóxicos hipertensivos de la tiramina fue en pacientes con depresión que estaban recibiendo tratamiento con inhibidores de MAO, después de comer queso. Debido a esto, se conoce como >reacción del queso> al conjunto de efectos tóxicos que la ingesta de alimentos ricos en tiramina causa en el consumidor. Los principales responsables de la acumulación de tiramina en quesos son bacterias del ácido láctico pertenecientes a la especie Enterococcus faecalis; nuestros estudios indican que en estos microorganismos la producción de tiramina es una característica de especie. A pesar de que la tiramina se considera una de las AB más tóxicas, no existen límites máximos legales para la concentración de esta AB en alimentos. En el último informe que emitió sobre aminas biógenas (2011), la Autoridad Europea de Seguridad Alimentaria (EFSA) consideraba necesario realizar estudios científicos que cuantificasen la toxicidad de la tiramina, para poder determinar cuáles son las concentraciones máximas de esta AB en alimentos, cuya ingesta no afecte la salud de los consumidores. Motivados por este informe, desarrollamos un modelo in vitro con el que hemos cuantificado la citotoxicidad de la tiramina en líneas intestinales humanas (HT29), así como la citotoxicidad de otras AB alimentarias que se encuentran fr

Biogenic amines (BA) - nitrogenous compounds of low molecular weight - are the result of metabolism of certain amino acids. They are biologically present in all living organisms and play essential physiological roles. However, their accumulation in foodstuffs due to the metabolic activity of certain microorganisms represents a toxicological risk. Containing such microorganisms, and with an abundance of precursor substrate amino acids, fermented foods in general, and cheeses in particular, provide an ideal matrix for the accumulation of these toxic compounds. Unfortunately, the main microorganisms responsible for BA accumulation are members of the lactic acid bacteria (LAB) group, which are also essential for the development of the organoleptic characteristics of the final product. The methods used to reduce the BA content of cheese, such as milk pasteurization, commonly fail to do so, and affect desirable non-BA-producing LAB as well. Bacteriophages have been proposed as biotechnological tools for diminishing the presence of undesirable microorganisms in dairy products. Given their specificity, they could be used to target the population of BA-producing bacteria. In this work, we aimed to explore the use of Enterococcus faecalis infecting phages as a tool to reduce the content of BA in dairy products. For this, we proceeded to the isolation and characterization of E. faecalis bacteriophage 156, a member of the family Myoviridae. Its genome was sequenced and compared with that of E. faecalis family Myoviridae phages available in public databases. Its capacity to decrease the accumulation of the BA tyramine and putrescine in an experimental laboratory-scale cheese model was proven.

Histamine, a biogenic amine, is abundant in fermented foods and beverages, notably wine. A high intake of this monoamine may produce adverse reactions in humans, which may be severe in individuals with a reduced capacity to catabolise extrinsic histamine. Thus, control of histamine concentration during wine production and before distribution is advisable. Simple, rapid, point-of-use bioanalytical platforms are needed because traditional methods for the detection and quantification of histamine are expensive and time-consuming. This work applies the lateral flow immunoassay technique to histamine detection. Superparamagnetic particle labels, and an inductive sensor designed to read the test line in the immunoassay, enable magnetic quantification of the molecule. The system is calibrated with histamine standards in the interval of interest for wine production. A commercial optical strip reader is used for comparison measurements. The lateral flow system has a limit of detection of 1.2 and 1.5 mg/L for the inductive and optical readers, respectively. The capability of the inductive system for histamine quantification is demonstrated for wine samples at different processing points (at the end of alcoholic fermentation, at the end of malolactic fermentation, in freshly bottled wine, and in reserve wine). The results are validated by ultra-high-performance liquid chromatography. [Figure not available: see fulltext.].

Enterococcus faecalis is a lactic acid bacterium characterized by its tolerance of very diverse environmental conditions, a property that allows it to colonize many different habitats. This species can be found in food products, especially in fermented foods where it plays an important role as a biopreservative and influences the development of organoleptic characteristics. However, E. faecalis also produces the biogenic amines tyramine and putrescine. The consumption of food with high concentrations of these compounds can cause health problems. The present work reports the construction, via homologous recombination, of a double mutant of E. faecalis in which the clusters involved in tyramine and putrescine synthesis (which are located in different regions of the chromosome) are no longer present. Analyses showed the double mutant to grow and adhere to intestinal cells normally, and that the elimination of genes involved in the production of tyramine and putrescine has no effect on the expression of other genes.

Putrescine and cadaverine are among the most common biogenic amines (BA) in foods, but it is advisable that their accumulation be avoided. Present knowledge about their toxicity is, however, limited; further research is needed if qualitative and quantitative risk assessments for foods are to be conducted. The present work describes a real-time analysis of the cytotoxicity of putrescine and cadaverine on intestinal cell cultures. Both BA were cytotoxic at concentrations found in BA-rich foods, although the cytotoxicity threshold for cadaverine was twice that of putrescine. Their mode of cytotoxic action was similar, with both BA causing cell necrosis; they did not induce apoptosis. The present results may help in establishing legal limits for both putrescine and cadaverine in food

Lactic acid bacteria (LAB) are among the most widely used microorganisms in food fermentation. However, some LAB species can also be used as live vehicles for the in situ delivery of therapeutic molecules to the mucosa of the human gastrointestinal tract (GIT). Many LAB species have ‘qualified presumption of safety’ status and survive passage through the GIT. Indeed, some are part of the usual GIT microbiota. These are appropriate candidates for the in situ production of recombinant prophylactic and therapeutic proteins. Live recombinant LAB that produce microbial antigens have been shown to elicit an immune response that confers protection against the corresponding pathogens; these LAB could therefore be used as oral vaccines. In addition, some LAB have been genetically engineered to produce therapeutic, neutralizing antibodies. The variable domain of heavy-chain-only antibodies from camelids – known as VHH antibodies or nanobodies – has peculiar properties (nanoscale size, robust structure, acid resistance, high affinity and specificity, easily produced in bacteria, etc.) that make them ideal choices as LAB-produced immunotherapeutic agents. The present review examines the advantages offered by LAB for the in situ production of therapeutic proteins in the human GIT, discusses the use of in situ produced VHH antibody fragments, and assesses the usefulness of this strategy in the treatment of infectious and non-infectious gastrointestinal diseases.

Se denomina biofilms o biopelículas a comunidades de microorganismos que permanecen adheridos a una superficie y embebidos en una matriz extracelular formada por polisacáridos, proteínas, ADN de las células lisadas y/o ácidos teicoicos. La formación y el desarrollo de los biofilms dependen de múltiples factores, incluyendo la especie y cepa microbiana, las propiedades de los materiales de la superficie a la que se adhieren y las condiciones ambientales (pH, temperatura¿). La presencia de biofilms es habitual en la industria alimentaria, ya que las bacterias pueden adherirse a la superficie de algunos de los materiales empleados: plástico, metal, goma y madera. Estos biofilms son muy resistentes a los métodos de lavado y desinfección de las instalaciones y actúan como un reservorio de microorganismos que contaminan los alimentos, deteriorándolos y provocando pérdidas económicas significativas. Además, dependiendo de las especies presentes en el biofilm, pueden incluso ser responsables de intoxicaciones alimentarias y de la transmisión de enfermedades. Las bacterias del ácido láctico (BAL) cumplen un papel fundamental en la industria alimentaria. Sin embargo, algunas BAL son responsables de la acumulación de aminas biógenas (AB), como la histamina, en altas concentraciones en alimentos fermentados. El consumo de alimentos con concentraciones elevadas de histamina provoca reacciones adversas en los consumidores y por este motivo, las autoridades sanitarias recomiendan evitar su presencia en los alimentos. Lentilactobacillus parabuchneri ha sido descrito como el principal responsable de la acumulación de histamina en quesos. Trabajos previos realizados en nuestro grupo de investigación revelaron la capacidad de algunas cepas de L. parabuchneri de formar biofilms, incluso sobre el acero empleado en la maquinaria industrial, lo que constituye un riesgo de contaminación de los quesos durante el proceso de elaboración que tendrá como consecuencia la posterior acumulación de

Lactic acid bacteria (LAB) belonging to the genus classically known as Lactobacillus, recently split into 25 different genera, include many relevant species for the food industry. The well-known properties of lactobacilli as probiotics make them an attractive model also for vaccines and therapeutic proteins delivery in humans. However, scarce tools are available to accomplish genetic modification of these organisms, and most are only suitable for laboratory strains. Here, we test bacterial conjugation as a new tool to introduce genetic modifications into many biotechnologically relevant laboratory and wild type lactobacilli. Using mobilizable shuttle plasmids from a donor Escherichia coli carrying either RP4 or R388 conjugative systems, we were able to get transconjugants to all tested Lactocaseibacillus casei strains, including many natural isolates, and to several other genera, including Lentilactobacillus parabuchneri, for which no transformation protocol has been reported. Transconjugants were confirmed by the presence of the oriT and 16S rRNA gene sequencing. Serendipitously, we also found transconjugants into researcher-contaminant Staphylococcus epidermidis. Conjugative DNA transfer from E. coli to S. aureus was previously described, but at very low frequencies. We have purified this recipient strain and used it in standard conjugation assays, confirming that both R388 and RP4 conjugative systems mediate mobilization of plasmids into S. epidermidis. This protocol could be assayed to introduce DNA into other Gram-positive microorganisms which are resistant to transformation.