The ability of riboflavin-overproducing lactiplantibacillus plantarum strains to survive under gastrointestinal conditions

Riboflavin, vitamin B2, is essential for humans and has to be obtained from the diet. Some lactic acid bacteria (LAB) produce this vitamin, and they can be used for in-situ fortification of foods. This could be an alternative to supplementation with chemically synthesized vitamin, to palliate riboflavin deficiencies in specific groups of people. Moreover, if the producing LAB could survive in the gastrointestinal stress (GIT) they could be added as probiotics in this environment. In the present study we tested two riboflavin-overproducing Lactiplantibacillus plantarum strains (M5MA1-B2 and M9MG6-B2), spontaneous mutants of LAB isolated from chicha, a traditional Andean beverage. These two LAB, and also their isogenic strains M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12], expressing the mCherry protein from the pRCR12 plasmid, were evaluated in vitro under simulated GIT conditions. Among other, specifically developed protein fluorescence assays were used. The four LAB showed similar levels of adhesion (>6.0%) to Caco-2 cells, higher than that of the probiotic Lacticaseibacillus rhamnosus GG strain (4.51%). Thus, LAB biofilm formation was assessed in the labeled cells by intracellular mCherry fluorescence and in the unlabeled parental strains by crystal violet staining. Both methods detected the formation of consistent biofilms by the L. plantarum strains. The quantification of mCherry fluorescence was also used to analyze LAB auto-aggregation properties. High levels of auto-aggregation were detected for both M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12]. Survival of LAB included in a commercial cereal-based food matrix (Incaparina) under GIT conditions was also evaluated. The four LAB were resistant in vitro to the stomach and intestinal stresses, and proliferated in this environment, indicating a protective and nutritional effect of the Incaparina on the bacteria. Also, M9MG6-B2 survival in the presence or absence of Incaparina was evaluated in vivo in a BALB/c mouse model. The administration of the M9MG6-B2 strain alone or together with Incaparina had no adverse effect on the health, growth and/or well-being of the rodents. In addition, an increment in the villus length/crypt depth ratio was observed. The overall results obtained indicate that the LAB studied have probiotic characteristics of interest for the development of functional foods.Fil: Hernández Alcántara, Annel M.. Consejo Superior de Investigaciones Científicas; EspañaFil: Pardo, Sandra. Consejo Superior de Investigaciones Científicas; EspañaFil: Mohedano, Mari Luz. Consejo Superior de Investigaciones Científicas; EspañaFil: Vignolo, Graciela Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Aznar, Rosa. Consejo Superior de Investigaciones Científicas. Instituto de Agroquímica y Tecnología de Alimentos; España. Universidad de Valencia; EspañaFil: López, Paloma. Consejo Superior de Investigaciones Científicas; Españ

Lactic Acid Bacteria Isolated from Fermented Doughs in Spain Produce Dextrans and Riboflavin

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B2). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species (Weissella cibaria, Leuconostoc citreum, Leuconostoc falkenbergense and Leuconostoc mesenteroides). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B2. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585–685 μg/and 6.5–7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense.This research was funded by the Spanish Ministry of Science, Innovation and Universities, (grants RTI2018-097114-B-I00 and PCIN-2017-075), by the Basque Government Industry and Education Department (grant PIBA_2020_1_0032) and by the University of the Basque Country (General Grant to Research Groups (GIU 19/014))

Lactic Acid Bacteria Isolated from Fermented Doughs in Spain Produce Dextrans and Riboflavin

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B2). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species (Weissella cibaria, Leuconostoc citreum, Leuconostoc falkenbergense and Leuconostoc mesenteroides). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B2. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585–685 μg/and 6.5–7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense.This research was funded by the Spanish Ministry of Science, Innovation and Universities, (grants RTI2018-097114-B-I00 and PCIN-2017-075), by the Basque Government Industry and Education Department (grant PIBA_2020_1_0032) and by the University of the Basque Country (General Grant to Research Groups (GIU 19/014))

Probiotic properties and stress response of lactic acid bacteria isolated from cooked meat products

3 p.This work was supported by the Universidad Autónoma Metropolitana and the Spanish Ministry of Economy and Competitiveness (grant AGL2015-65010-C3-1-R).Peer reviewe

Evaluation of Agro-Industrial Co-Products as Source of Bioactive Compounds: Fiber, Antioxidants and Prebiotic

Agro-industrial co-products derived of fruit processing represents an important source of bioactive compounds as fiber, antioxidants and prebiotics. The objective of this work was to determine the content of fiber, antioxidant capacity and prebiotic activity of three flours obtained from commonly co-products (banana peel, apple peel, and carrot bagasse). The results showed a higher total fiber content in carrot bagasse, and lower in apple peel. Significantly differences were found in antioxidant activity. Fruit co-products flours were a suitable carbon source increasing specific growth rate with a reduction in duplication time as compared to glucose. The prebiotic activity was positive in the three co-products, all flours survived at pH 1.0 and showed resistance to simulated gastric acid for about 60 min. Banana peel, apple peel and carrot bagasse showed to be a good source of bioactive compounds as fiber and antioxidants and can be used as prebiotics for lactic acid bacteria

Probiotic properties and stress response of thermotolerant lactic acid bacteria isolated from cooked meat products

29 p.-5 fig.-1 tabThe aim of this study was to evaluate the probiotic properties of six thermotolerant lactic acid bacteria isolated from cooked meat products. The bacteria were typed, by determination of the DNA sequence of their 16S rRNA coding genes, as one Enterococcus faecium (UAM1 strain) and five Pediococcus pentosaceus (UAM2-UAM6 strains). Under gastric stress conditions the viability of the Pediococci decreased more than five-fold, whereas E. faecium showed a high resistance (61% survival). Exposure to small intestine stress did not drastically affect the survival of any of the strains (less than one-fold decrease), which were able to grow in the presence of 0.3% bile. A hydrophilic surface profile was observed, with higher affinity for chloroform than for xylene. Strains showed high levels of auto-aggregation as well as co-aggregation with Gram-positive and Gram-negative bacterial pathogens. The adherence of E faecium UAM1 to human Caco-2 cells (around 20%) was significantly higher than that obtained with the P. pentosaceus strains (2%–5%) and Lactobacillus acidophilus LA-5 (6%). The overall results indicate that E. faecium UAM1, has probiotic properties that predict its capability to colonize in competition with pathogens in the intestinal tract. This bacterium deserves further investigation for its potential as a component of functional food.A. M. Hernández-Alcántara was supported by graduate grants from the Consejo Nacional de Ciencia y Tecnología (CONACyT) Mexico (National Grant 290817 and Mixed Grant 291062). This work was also supported by the Spanish Ministry of Economy and Competitiveness (grant AGL2015-65010-C3-1-R).Peer reviewe

Biological functions of exopolysaccharides from lactic acid bacteria and their potential benefits for humans and farmed animals

35 p.-1 tab.Lactic acid bacteria (LAB) synthesize exopolysaccharides (EPS), which are structurally diverse biopolymers with a broad range of technological properties and bioactivities. There is scientific evidence that these polymers have health-promoting properties. Most commercialized probiotic microorganisms for consumption by humans and farmed animals are LAB and some of them are EPS-producers indicating that some of their beneficial properties could be due to these polymers. Probiotic LAB are currently used to improve human health and for the prevention and treatment of specific pathologic conditions. They are also used in food-producing animal husbandry, mainly due to their abilities to promote growth and inhibit pathogens via different mechanisms, among which the production of EPS could be involved. Thus, the aim of this review is to discuss the current knowledge of the characteristics, usage and biological role of EPS from LAB, as well as their postbiotic action in humans and animals, and to predict the future contribution that they could have on the diet of food animals to improve productivity, animal health status and impact on public healthThe present work was supported by the Spanish Ministry of Science, Innovation and Universities (grant RTI2018-097114-B-I00), by the University of the Basque Country (GIU19/014) and by CONICET (Proyecto PUE 058), National Agency for Scientific and Technological Promotion (Proyecto PICT 2016-3495) and Universidad Nacional del Litoral (Proyecto CAI+D 50120150100152LI, CAI+D 50120150100151LI, CAI+D 50620190100152LI and CAI+D Orientado 2016 2-14).Peer reviewe

Proteomic and in silico analyses of dextran synthesis influence on Leuconostoc lactis AV1n adaptation to temperature change

21 p.-9 fig.-1 tab.Leuconostoc lactis is found in vegetables, fruits, and meat and is used by the food industry in the preparation of dairy products, wines, and sugars. We have previously demonstrated that the dextransucrase of Lc. lactis (DsrLL) AV1n produces a high-molecular-weight dextran from sucrose, indicating its potential use as a dextran-forming starter culture. We have also shown that this bacterium was able to produce 10-fold higher levels of dextran at 20°C than at 37°C, at the former temperature accompanied by an increase in dsrLL gene expression. However, the general physiological response of Lc. lactis AV1n to cold temperature in the presence of sucrose, leading to increased production of dextran, has not been yet investigated. Therefore, we have used a quantitative proteomics approach to investigate the cold temperature-induced changes in the proteomic profile of this strain in comparison to its proteomic response at 37°C. In total, 337 proteins were found to be differentially expressed at the applied significance criteria (adjusted p-value ≤ 0.05, FDR 5%, and with a fold-change ≥ 1.5 or ≤ 0.67) with 204 proteins overexpressed, among which 13% were involved in protein as well as cell wall, and envelope component biosynthesis including DsrLL. Proteins implicated in cold stress were expressed at a high level at 20°C and possibly play a role in the upregulation of DsrLL, allowing the efficient synthesis of the protein essential for its adaptation to cold. Post-transcriptional regulation of DsrLL expression also seems to take place through the interplay of exonucleases and endonucleases overexpressed at 20°C, which would influence the half-life of the dsrLL transcript. Furthermore, the mechanism of cold resistance of Lc. lactis AV1n seems to be also based on energy saving through a decrease in growth rate mediated by a decrease in carbohydrate metabolism and its orientation toward the production pathways for storage molecules. Thus, this better understanding of the responses to low temperature and mechanisms for environmental adaptation of Lc. lactis could be exploited for industrial use of strains belonging to this species.This study was supported by the Spanish Ministry of Science, Innovation and Universities (grant no. RTI2018-097114-B-I00) and the Tunisian Ministry of Higher Education and Scientific Research.Peer reviewe

The ability of Riboflavin-overproducing Lactiplantibacillus plantarum strains to survive under gastrointestinal conditions

17 p.-5 fig.-4 tab.+mat. supl.:5 p.-3 fig.-1 tab.Riboflavin, vitamin B2, is essential for humans and has to be obtained from the diet. Some lactic acid bacteria (LAB) produce this vitamin, and they can be used for in-situ fortification of foods. This could be an alternative to supplementation with chemically synthesized vitamin, to palliate riboflavin deficiencies in specific groups of people. Moreover, if the producing LAB could survive in the gastrointestinal stress (GIT) they could be added as probiotics in this environment. In the present study we tested two riboflavin-overproducing Lactiplantibacillus plantarum strains (M5MA1-B2 and M9MG6-B2), spontaneous mutants of LAB isolated from chicha, a traditional Andean beverage. These two LAB, and also their isogenic strains M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12], expressing the mCherry protein from the pRCR12 plasmid, were evaluated in vitro under simulated GIT conditions. Among other, specifically developed protein fluorescence assays were used. The four LAB showed similar levels of adhesion (>6.0%) to Caco-2 cells, higher than that of the probiotic Lacticaseibacillus rhamnosus GG strain (4.51%). Thus, LAB biofilm formation was assessed in the labeled cells by intracellular mCherry fluorescence and in the unlabeled parental strains by crystal violet staining. Both methods detected the formation of consistent biofilms by the L. plantarum strains. The quantification of mCherry fluorescence was also used to analyze LAB auto-aggregation properties. High levels of auto-aggregation were detected for both M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12]. Survival of LAB included in a commercial cereal-based food matrix (Incaparina) under GIT conditions was also evaluated. The four LAB were resistant in vitro to the stomach and intestinal stresses, and proliferated in this environment, indicating a protective and nutritional effect of the Incaparina on the bacteria. Also, M9MG6-B2 survival in the presence or absence of Incaparina was evaluated in vivo in a BALB/c mouse model. The administration of the M9MG6-B2 strain alone or together with Incaparina had no adverse effect on the health, growth and/or well-being of the rodents. In addition, an increment in the villus length/crypt depth ratio was observed. The overall results obtained indicate that the LAB studied have probiotic characteristics of interest for the development of functional foods.This work was supported by the Spanish Ministry of Science, Innovation and Universities (grants RTI2018-097114-B-I00, PCIN-2017-075, and PCIN-2017-003), the Ibero-American Program of Science and Technology for the Development (CYTED, Strategic Project Ref: 917PTE0537), and the European Cooperation in Science and Technology (SOURDOMICS Cost Action CA1801). JL, GV, and AM acknowledge the CONICET (Argentina) for its financial contributions.Peer reviewe

Análisis de las propiedades probióticas de cepas sobreproductoras de riboflavina derivadas de bacterias aisladas de chicha Argentina

Trabajo presentado al XI Workshop Sociedad Española de Microbiota, Probióticos y Prebióticos, celebrado en Granada del 12 al 14 de febrero de 2020.[Introducción/Objetivos]: Algunas bacterias ácido lácticas (BAL) producen riboflavina, vitamina B2 esencial para los seres humanos. Por ello pueden ser utilizadas para la elaboración de alimentos funcionales. En este trabajo se pretendió caracterizar in vitro e in vivo tres cepas de Lactobacillus plantarum sobreproductoras de riboflavina, derivadas de BAL aisladas de chicha argentina.[Metodología]: Las BAL fueron marcadas por transferencia del plásmido pRCR12, que codifica la proteína fluorescente mCherry. Los niveles de riboflavina y de mCherry se determinaron fluorimetricamente. Se analizó la adhesión de las BAL a células epiteliales Caco-2. La formación de biopelículas se evaluó por tinción con cristal violeta y por la fluorescencia de mCherry, que también se utilizó para analizar la autoagregación de las BAL. Se determinó la supervivencia de las BAL bajo estrés gastrointestinal en presencia de una matriz alimentaria comercial (Incaparina). La supervivencia in vivo de las BAL libres o en conjunción con Incaparina se evaluó en un modelo murino con ratones convencionales BALB/c.[Resultados]: El marcaje de las BAL con pRCR12 no afectó de forma significativa su crecimiento o su producción de riboflavina. Las tres cepas mostraron niveles semejantes de adhesión a los enterocitos (4-6%) y similares a los del probiótico Lactobacillus rhamnosus LGG (4%). Las tres cepas formaron biopelículas consistentes. M9MG6B2 y M5MA1B2 mostraron una agregación superior (30-40%) a la de M9MM1B2 (18%). Se detectó in vitro un efecto protector de la Incaparina y una resistencia satisfactoria de las tres cepas al estrés gastrointestinal. Únicamente M9MM1B2 mostró cierta susceptibilidad a pH 2,0 (supervivencia del 63%). La administración de las BAL solas o junto con Incaparina a roedores, no tuvo efecto adverso sobre la salud, el crecimiento y/o el bienestar de los roedores.[Conclusiones]: Los resultados obtenidos indican que las tres BAL estudiadasposeen características probióticas de interés para el desarrollo de alimentos funcionales.Peer reviewe