Potencial probiótico de "Lactobacillus reuteri" y aplicación de la reuterina como bioconservante alimentario

Actualmente, el consumidor demanda alimentos menos procesados, más naturales y con una mayor vida útil. Esta tendencia ha aumentado el consumo de productos RTE (del inglés ready-to-eat), alimentos que se consumen directamente sin tratamiento higienizante o con un procesado mínimo, siendo susceptibles de contaminación con microorganismos patógenos durante el procesado y manipulación. Listeria monocytogenes, Salmonella spp. y Escherichia coli O157:H7 pueden entrar en contacto con los RTE en las operaciones anteriores, entrañando un riesgo para el consumidor. En el presente trabajo se ha propuesto la reuterina (REU), un compuesto antimicrobiano producido por algunas cepas de Lactobacillus reuteri durante el metabolismo anaerobio del glicerol, como bioconservante en alimentos RTE. Para ello, la reuterina se ha producido y purificado, añadiéndose a los alimentos de forma individual y en combinación con las altas presiones hidrostáticas (APH). Además, se han empleado otros bioconservantes como el sistema lactoperoxidasa (SLP) o la lactoferrina (LF) junto con las APH. La citotoxicidad de la reuterina se ha investigado mediante el empleo de células Hep G2. Asimismo, se ha comprobado el efecto antimicrobiano de la reuterina producida in situ mediante la adición de Lb. reuteri INIA P572 como cultivo bioprotector y glicerol en la elaboración de productos lácteos, así como su impacto en las características del queso. Finalmente, se ha evaluado el potencial probiótico de Lb. reuteri INIA P572 en un modelo de colon, en cultivos celulares y en un modelo murino de colitis ulcerosa inducida por sulfato de dextrano sódico. Se estudió el efecto antimicrobiano de los tres bioconservantes (REU, SLP y LF) junto con las APH (450 MPa/ 5 min) frente a L. monocytogenes, Salmonella spp. y E. coli O157:H7 en salmón ahumado y jamón cocido y su influencia sobre las características de los productos. La reuterina ejerció un efecto bacteriostático frente a L. monocytogenes y bactericida frente a E. coli O157:H7 y S. Enteritidis en jamón cocido y en salmón ahumado. En combinación con APH se consiguieron reducciones de aproximadamente 4 unidades logarítmicas para L. monocytogenes y superiores para el resto de patógenos. En salmón ahumado, se consiguió además reducir la formación de aminas biógenas tanto con la reuterina aplicada individualmente como combinada con las altas presiones hidrostáticas..

Probiotic and Functional Properties of Limosilactobacillus reuteri INIA P572

Limosilactobacillus reuteri INIA P572 is a strain able to produce the antimicrobial compound reuterin in dairy products, exhibiting a protective effect against some food-borne pathogens. In this study, we investigated some probiotic properties of this strain such as resistance to gastrointestinal passage or to colonic conditions, reuterin production in a colonic environment, and immunomodulatory activity, using different in vitro and in vivo models. The results showed a high resistance of this strain to gastrointestinal conditions, as well as capacity to grow and produce reuterin in a human colonic model. Although the in vitro assays using the RAW 264.7 macrophage cell line did not demonstrate direct immunomodulatory properties, the in vivo assays using a Dextran Sulphate Sodium (DSS)-induced colitic mice model showed clear immunomodulatory and protective effects of this strain.This work was supported by project no. RTA2017-00002-00-00 from the Spanish Ministry of Science and Innovation, by the Junta de Andalucía (CTS 164) and Instituto de Salud Carlos III (PI19/01058) with funds from the European Union.Ye

Reuterin, lactoperoxidase, lactoferrin and high hydrostatic pressure on the inactivation of food-borne pathogens in cooked ham

The antimicrobial effect of high hydrostatic pressure (HHP) processing combined with reuterin, lactoperoxydase system (LPS) and lactoferrin (LF) on the survival of Listeria monocytogenes, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli O157:H7 in sliced cooked ham stored under strict refrigeration temperature (4 °C) and mild temperature abuse conditions (10 °C) was investigated. One day after treatment, L. monocytogenes counts in HHP at 450 MPa for 5 min were 0.8 log units lower, but a recovery was observed with counts not significantly different to those observed in control after 35 d. S. Enteritidis and E. coli O157:H7 levels were reduced around 5 log cfu/g by the pressure treatment (450 MPa/5 min) and the numbers of these pathogens did not increase significantly during the 35 d of storage at 4 °C. The individual application of reuterin and LPS influenced the survival of the three pathogens studied, extending the lag phase of L. monocytogenes and diminishing S. Enteritidis and E. coli levels throughout storage, whereas no effect was recorded when LF was added. When reuterin or LPS were applied in combination with HHP there was a synergistic antimicrobial effect against L. monocytogenes, avoiding at 4 °C the recovery observed with individual treatments. These combined treatments also kept the levels of S. Enteritidis and E. coli O157:H7 below the detection limit (<1 log unit) in cooked ham stored at 4 and 10 °C during 35 d. The results obtained in the present work suggest that HHP at 450 MPa for 5 min in combination with LPS or reuterin would be useful as a hurdle technology approach against L. monocytogenes, S. Enteritidis and E. coli O157:H7 in cooked ham.MINECO (AGL2010- 16600 y CSD2007-00016)Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEpu

Natural antimicrobials and high‐pressure treatments on the inactivation of <i>Salmonella</i> Enteritidis and <i>Escherichia coli</i><scp>O157</scp>:<scp>H7</scp> in cold‐smoked salmon

BACKGROUND High hydrostatic pressure (HHP) combined with reuterin and lactoperoxidase system (LPS) has exerted antimicrobial activity against Listeria monocytogenes in cold-smoked salmon at chilled temperatures. Therefore the purpose of this work was to evaluate the effect of HHP combined with reuterin, LPS and lactoferrin (LF) on the survival of Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli O157:H7 in cold-smoked salmon stored at 4 and 10 °C. RESULTS Salmonella Enteritidis and E. coli O157:H7 were reduced more than 3 log colony-forming units (CFU) g−1 by the pressure treatment (450 MPa/5 min). LPS slightly diminished pathogen levels throughout storage, whereas no effect was recorded when reuterin or LF was added. The Salmonella population was below the detection limit (<1 log CFU g−1) during the storage of HHP-treated smoked salmon at 4 and 10 °C. The antimicrobial activity of HHP against E. coli O157:H7 was increased when 450 MPa was applied in combination with LPS in cold-smoked salmon at 4 and 10 °C. CONCLUSION HHP at 450 MPa/5 min inactivated S. Enteritidis in cold-smoked salmon and in combination with LPS would be useful as a hurdle technology approach against E. coli O157:H7, even under mild temperature abuse conditions. © 2015 Society of Chemical IndustryMINECO (AGL2010-16600 and RTA2013-0070-C03-01)Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEpu

Reuterin and High Hydrostatic Pressure Treatments on the Inactivation of Listeria monocytogenes and Effect on the Characteristics of Cold-Smoked Salmon

t The effect of reuterin and high hydrostatic pressure (HHP) processing at 450 MPa for 5 min on the inactivation of Listeria monocytogenes and the characteristics of coldsmoked salmon during 35 days at 4 and 10 °C were investigated. The growth rate of the pathogen was reduced by reuterin addition and a synergistic antimicrobial effect against L. monocytogenes was recorded when the biopreservative was applied in combination with HHP at 450 MPa for 5 min. This combined treatment prevented the pathogen recovery observed with individual treatments and delayed the spoilage of smoked salmon maintaining total viable counts under 3.5 log units during 35 days of storage at 4 °C. All treatments assayed induced changes in lightness (L*) and redness (a*), resulting in a brighter appearance of smoked salmon, whereas no modifications were recorded in shear strength values immediately after treatments. Moreover, reuterin and HHP treatments, individually or in combination, avoided the formation of biogenic amines during the 35 days of storage at 4 and 10 °C. The addition of reuterin in combination with HHP at 450 MPa for 5 min might be applied as a hurdle technology to improve the safety and extend the shelf life of lightly pMINECO (AGL2010-16600)Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEpu

In situ reuterin production by Lactobacillus reuteri in dairy products

In situ reuterin production during the manufacture and storage of two dairy model systems elaborated from milk supplemented with an optimized concentration of glycerol (50 mM) and inoculated with a commercial starter and with different reuterin-producing Lactobacillus reuteri strains was investigated. L. reuteri was able to survive and to produce reuterin in cheese and yogurt models. The highest reuterin production was achieved by L. reuteri INIA P572 and INIA P579, which displayed reuterin concentrations up to 5.5 mM in cheese and up to 1.5 mM in yogurt. The addition of reuterin-producing L. reuteri and glycerol to milk reduced the viable counts of the cheese starter from day 10 onwards, while did not influence the counts of the yogurt starter, compared to control dairy models without L. reuteri. Strains L. reuteri INIA P572 and INIA P579 could be promising candidates in the development of bioprotective cultures to control pathogenic microorganisms in dairy products due to the potentially inhibitory concentrations of reuterin achieved in situ.MINECO (RTA 2010-00116-00-00 y AGL2010-16600)Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEpu

Optimización de la producción de reuterina en quesos por Lactobacillus reuteri

Cheeses manufactured from pasteurized milk supplemented with glycerol and reuterin-producing Lactobacillus reuteri INIA P572 as adjunct to the commercial starter culture were analysed in order to optimize a biopreservation strategy. The highest reuterin concentration determined by a colorimetric assay was detected on day 1 in cheeses with 100–500 mM glycerol. The presence of reuterin was confirmed by a direct detection technique as HPLC. Cheeses made with L. reuteri and 200 or 500 mM glycerol showed a red tendency in color in comparison with control. The results with purified reuterin suggested that the development of slightly rosy colour in cheese was related to some compound produced/overproduced when higher levels of glycerol were present in cheese, but not due to reuterin. Application of L. reuteri INIA P572 as adjunct to the commercial starter with 100 mM glycerol led to such a reuterin concentration in cheese that could control undesirable microorganisms, avoiding the presence of color-changing compounds.Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Fac. de VeterinariaTRUEpu

Bee products as an alternative for the preservation of nitrate and nitrite-reduced dry fermented sausages

Authorship contribution statement: Xavier F. Hospital: Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Eva Hierro: Writing – review & editing, Writing – original draft, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization. Izaskun Martín-Cabrejas: Writing – original draft, Methodology, Investigation. Natalia Caballero: Investigation. Begona Jimenez: Investigation. Vanesa Sanchez-Martín: Investigation. Paloma Morales: Funding acquisition, Formal analysis. Ana I. Haza: Funding acquisition, Formal analysis. Manuela Fernandez: Writing – review & editing, Writing – original draft, Supervision, Methodology, Funding acquisition, Formal analysis, Conceptualization.Food regulations are becoming increasingly restrictive on the use of nitrate and nitrite as additives in meat products, so different alternatives are being investigated to replace their functions. In this study, we tested the antioxidant activity of mixtures of bee products to partially replace nitrate and nitrite in dry fermented sausages. Two combinations of chestnut honey, propolis and royal jelly were added to sausages at 2% concentration, together with 0 and 75 mg/kg of nitrate and nitrite. TBARs and volatile profile analysis revealed a lower intensity of oxidation phenomena in sausages prepared with bee products, but these differences were not perceived by the tasters in the sensory analysis. Only instrumental differences in the a* colour parameter were observed when nitrate and nitrite were not used in the formulation. The addition of bee products did not affect the typical microbiota (lactic acid bacteria and gram-positive catalase-positive cocci) of sausages. The combination of 2% bee products and 75 mg/kg of nitrate and nitrite may be useful to obtain dry fermented sausages with a sensory quality and oxidation stability similar to a standard product containing the maximum amounts of these additives allowed in the European Union (150 mg/kg each).Ministerio de Ciencia e Innovación (España)Sección Deptal. de Farmacia Galénica y Tecnología Alimentaria (Veterinaria)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

A Highly Sensitive Method for the Detection of Hydrolyzed Gluten in Beer Samples Using LFIA

Most gluten analysis methods have been developed to detect intact gluten, but they have shown limitations in certain foods and beverages in which gluten proteins are hydrolyzed. Methods based on G12/A1 moAbs detect the sequences of gluten immunogenic peptides (GIP), which are the main contributors to the immune response of celiac disease (CD). Immunogenic sequences with tandem epitopes for G12/A1 have been found in beers with <20 mg/kg gluten, which could be consumed by CD patients according to the Codex Alimentarius. Therefore, an accurate method for the estimation of the immunogenicity of a beer is to use two moAbs that can recognize celiac T cell epitopes comprising most of the immunogenic response. Here, a specific and sensitive method based on G12/A1 LFIA was developed to detect GIP in beers labeled gluten-free or with low gluten content, with an LOD of 0.5 mg/kg. A total of 107 beers were analyzed, of those 6.5% showed levels higher than 20 mg/kg gluten and 29% showed levels above the LOD. In addition, G12/A1 LFIA detected gluten in 15 more beer samples than competitive ELISA with another antibody. Despite their labeling, these beers contained GIP which may cause symptoms and/or intestinal damage in CD patients

A Highly Sensitive Method for the Detection of Hydrolyzed Gluten in Beer Samples Using LFIA

Most gluten analysis methods have been developed to detect intact gluten, but they have shown limitations in certain foods and beverages in which gluten proteins are hydrolyzed. Methods based on G12/A1 moAbs detect the sequences of gluten immunogenic peptides (GIP), which are the main contributors to the immune response of celiac disease (CD). Immunogenic sequences with tandem epitopes for G12/A1 have been found in beers with <20 mg/kg gluten, which could be consumed by CD patients according to the Codex Alimentarius. Therefore, an accurate method for the estimation of the immunogenicity of a beer is to use two moAbs that can recognize celiac T cell epitopes comprising most of the immunogenic response. Here, a specific and sensitive method based on G12/A1 LFIA was developed to detect GIP in beers labeled gluten-free or with low gluten content, with an LOD of 0.5 mg/kg. A total of 107 beers were analyzed, of those 6.5% showed levels higher than 20 mg/kg gluten and 29% showed levels above the LOD. In addition, G12/A1 LFIA detected gluten in 15 more beer samples than competitive ELISA with another antibody. Despite their labeling, these beers contained GIP which may cause symptoms and/or intestinal damage in CD patients.Corporación Tecnológica de Andalucía PRJ20220443