Probiotic Characteristics of Lactiplantibacillus plantarum CECT 9435 and Its Survival and Competitive Properties Under Simulated Conditions of the Child Gut Microbiota.

Probiotics are valuable microorganisms effective in reducing malnutrition-related infections in children. In this work, a collection of lactobacilli strains representative of traditional Andean fermented beverages was in vitro screened for their capability to survive the gastrointestinal transit, to adhere to the intestinal epithelium and to compete under simulated conditions of the child gut microbiota. The results allowed the selection of the riboflavin overproducing strain Lactiplantibacillus plantarum CECT 9435 based on its good rate of survival under in vitro gastrointestinal conditions when included in a food matrix representing the fortified food supplement Incaparina. The strain also showed good adhesion to HT29 cells producing mucus and outstanding performance in E. coli competition for the adhesion to this epithelial cell line. L. plantarum CECT 9435 gut performance was also evaluated in the child intestinal microbiota simulated in a dynamic gut model (BFBL simulator). The viability of the probiotic candidate in the gut conditions was high during the 7-day intervention period, reaching over 1 × 107 counts in each of the reactors simulating the three colonic regions. The transient viability of L. plantarum CECT 9435 within the child gut microbiota and its adhesion capacity to intestinal cells could facilitate the strain potential benefits as probiotic added to fortified supplementary foods destined to malnourished children.

Human intestinal enteroids and predictive models validate the operational limits of sanitizers used for viral disinfection of vegetable process wash water.

Vegetables are globally associated with a considerable number of foodborne outbreaks caused by viral infections, specifically human norovirus. In fresh produce industry, washing represents a critical step for food safety as process wash water (PWW) needs to be maintained at appropriate microbial quality to prevent water-mediated cross-contamination. This study aimed to explore the disinfection efficacy of chlorine (free chlorine, FC), chlorine dioxide (ClO2) and peracetic acid (PAA) in PWW against infectious human norovirus and Tulane virus (TV). First, we tested the extent of TV inactivation in baby leaf, bell pepper, and vegetables mix PWW and monitored the viral decay by cell culture. Then, inactivation kinetics were defined for infectious human norovirus exposed to FC, ClO2 and PAA in baby leaves PWW using the human intestinal enteroids (HIE) system. Finally, kinetic inactivation models were fitted to TV reduction and decay of sanitizers to aid the implementation of disinfection strategies. Results showed that >8 log10 human norovirus and 3.9 log10 TV were inactivated by 20 ppm FC within 1 min; and by 3 ppm ClO2 in 1 min (TV) or 5 min (norovirus). PAA treatment at 80 ppm reduced ca. 2 log10 TV but not completely inactivated the virus even after 20 min exposure, while 5 min treatment prevented norovirus replication in HIE. TV inactivation in PWWs was described using an exponential decay model. Taking these data together, we demonstrated the value of applying the HIE model to validate current operational limits for the most commonly used sanitizers. The inactivation kinetics for human norovirus and TV, along with the predictive model described in this study expand the current knowledge to implement post-harvest produce safety procedures in industry settings.

Exploring the impact of lactic acid bacteria on the biocontrol of toxigenic Fusarium spp. and their main mycotoxins.

The occurrence of fungi and mycotoxins in foods is a serious global problem. Most of the regulated mycotoxins in food are produced by Fusarium spp. This work aimed to assess the antifungal activity of selected lactic acid bacteria (LAB) strains against the main toxigenic Fusarium spp. isolated from cereals. Various machine learning (ML) algorithms such as neural networks (NN), random forest (RF), extreme gradient boosted trees (XGBoost), and multiple linear regression (MLR), were applied to develop models able to predict the percentage of fungal growth inhibition caused by the LAB strains tested. In addition, the ability of the assayed LAB strains to reduce/inhibit the production of the main mycotoxins associated with these fungi was studied by UPLC-MS/MS. All assays were performed at 20, 25, and 30 °C in dual culture (LAB plus fungus) on MRS agar-cereal-based media. All factors and their interactions very significantly influenced the percentage of growth inhibition compared to controls. The efficacy of LAB strains was higher at 20 °C followed by 30 °C and 25 °C. Overall, the order of susceptibility of the fungi to LAB was F. oxysporum > F. poae = F. culmorum ≥ F. sporotrichioides > F. langsethiae > F. graminearum > F. subglutinans > F. verticillioides. In general, the most effective LAB was Leuconostoc mesenteroides ssp. mesenteroides (T3Y6b), and the least effective were Latilactobacillus sakei ssp. carnosus (T3MM1 and T3Y2). XGBoost and RF were the algorithms that produced the most accurate predicting models of fungal growth inhibition. Mycotoxin levels were usually lower when fungal growth decreased. In the cultures of F. langsethiae treated with LAB, T-2 and HT-2 toxins were not detected except in the treatments with Pediococcus pentosaceus (M9MM5b, S11sMM1, and S1M4). These three strains of P. pentosaceus, L. mesenteroides ssp. mesenteroides (T3Y6b) and L. mesenteroides ssp. dextranicum (T2MM3) inhibited fumonisin production in cultures of F. proliferatum and F. verticillioides. In F. culmorum cultures, zearalenone production was inhibited by all LAB strains, except L. sakei ssp. carnosus (T3MM1) and Companilactobacillus farciminis (T3Y6c), whereas deoxynivalenol and 3-acetyldeoxynivalenol were only detected in cultures of L. sakei ssp. carnosus (T3MM1). The results show that an appropriate selection and use of LAB strains can be one of the most impacting tools in the control of toxigenic Fusarium spp. and their mycotoxins in food and therefore one of the most promising strategies in terms of efficiency, positive impact on the environment, food safety, food security, and international economy.

Complete genome sequences of Lacticaseibacillus paracasei INIA P272 (CECT 8315) and Lacticaseibacillus rhamnosus INIA P344 (CECT 8316) isolated from breast-fed infants reveal probiotic determinants.

Lacticaseibacillus paracasei INIA P272 and Lacticaseibacillus rhamnosus INIA P344, isolated from breast-fed infants, are two promising bacterial strains for their use in functional foods according to their demonstrated probiotic and technological characteristics. To better understand their probiotic characteristics and evaluate their safety, here we report the draft genome sequences of both strains as well as the analysis of their genetical content. The draft genomes of L. paracasei INIA P272 and L. rhamnosus INIA P344 comprise 3.01 and 3.26 Mb, a total of 2994 and 3166 genes and a GC content of 46.27 % and 46.56 %, respectively. Genomic safety was assessed following the EFSA guidelines: the identification of both strains was confirmed through Average Nucleotide Identity, and the absence of virulence, pathogenic and antibiotic resistance genes was demonstrated. The genome stability analysis revealed the presence of plasmids and phage regions in both genomes, however, CRISPR sequences and other mechanisms to fight against phage infections were encoded. The probiotic abilities of both strains were supported by the presence of genes for the synthesis of SCFA, genes involved in resistance to acid and bile salts or a thiamine production cluster. Moreover, the encoded exopolysaccharide biosynthesis genes could provide additional protection against the deleterious gastrointestinal conditions, besides which, playing a key role in adherence and coaggregation of pathogenic bacteria together with the high number of adhesion proteins and domains encoded by both genomes. Additionally, the bacteriocin cluster genes found in both strains, could provide an advantageous ability to compete against pathogenic bacteria. This genomic study supports the probiotic characteristics described previously for these two strains and satisfies the safety requirements to be used in food products.

Discrimination of non-infectious SARS-CoV-2 particles from fomites by viability RT-qPCR.

The ongoing coronavirus 2019 (COVID-19) pandemic constitutes a concerning global threat to public health and economy. In the midst of this pandemic scenario, the role of environment-to-human COVID-19 spread is still a matter of debate because mixed results have been reported concerning SARS-CoV-2 stability on high-touch surfaces in real-life scenarios. Up to now, no alternative and accessible procedures for cell culture have been applied to evaluate SARS-CoV-2 infectivity on fomites. Several strategies based on viral capsid integrity have latterly been developed using viability markers to selectively remove false-positive qPCR signals resulting from free nucleic acids and damaged viruses. These have finally allowed an estimation of viral infectivity. The present study aims to provide a rapid molecular-based protocol for detection and quantification of viable SARS-CoV-2 from fomites based on the discrimination of non-infectious SARS-CoV-2 particles by platinum chloride (IV) (PtCl4) viability RT-qPCR. An initial assessment compared two different swabbing procedures to recover inactivated SARS-CoV-2 particles from fomites coupled with two RNA extraction methods. Procedures were validated with human (E229) and porcine (PEDV) coronavirus surrogates, and compared with inactivated SARS-CoV-2 suspensions on glass, steel and plastic surfaces. The viability RT-qPCR efficiently removed the PCR amplification signals from heat and gamma-irradiated inactivated SARS-CoV-2 suspensions that had been collected from specified surfaces. This study proposes a rapid viability RT-qPCR that discriminates non-infectious SARS-CoV-2 particles on surfaces thus helping researchers to better understand the risk of contracting COVID-19 through contact with fomites and to develop more efficient epidemiological measures.

Chryseobacterium potabilaquae sp. nov., Chryseobacterium aquaeductus sp. nov. and Chryseobacterium fistulae sp. nov., from drinking water systems.

A polyphasic taxonomic study was conducted on three strains isolated from drinking water systems that had previously been deposited as Chryseobacterium species at the Spanish Type Culture Collection in order to complete their classification. Strains CECT 9293T, CECT 9390T and CECT 9393T were isolated from sites in Barcelona, Spain, in the framework of a project aimed at generating the first MALDI-TOF database specific for bacteria present in water for human consumption. Their partial 16S rRNA sequences showed that their closest relatives among the type strains of Chryseobacterium exhibited 98 % similarity or less, supporting their taxonomic novelty. At the same time, comparison between them revealed that strains CECT 9293T and CECT 9393T could perhaps be related at the species level as they shared 99.5 % similarity. However, whole genome sequencing was performed and the subsequent calculation of relatedness indices, average nucleotide identity and estimated DNA-DNA hybridization, ruled out that possibility and confirmed instead that each of the strains should be considered a separate species in the genus Chryseobacterium. Having clarified their status, we also performed phylogenomic analyses and searched for possible environmental or non-type material sequences that could be related to any of them at the species level. In parallel, the strains were characterized phenotypically and compared to their closest relatives to determine diagnostic traits to support their formal proposal. The proposed species are Chryseobacterium potabilaquae sp. nov. with the type strain CECT 9293T (=LMG 32084T), Chryseobacterium aquaeductus sp. nov. with the type strain CECT 9390T (=LMG 32085T) and Chryseobacterium fistulae sp. nov. with the type strain CECT 9393T (=LMG 32086T).

Rapid and improved identification of drinking water bacteria using the Drinking Water Library, a dedicated MALDI-TOF MS database.

According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment facilities. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is potentially such a method, although most of the currently available mass spectral libraries have been developed in a clinical setting and have limited environmental applicability. In this work, a MALDI-TOF MS drinking water library (DWL) was defined and developed by targeting bacteria present in water intended for human consumption. This database, made up of 319 different bacterial strains, can contribute to the routine microbiological control of either treated drinking water or mineral bottled water carried out by water treatment and distribution operators, offering a faster identification rate compared to a clinical sample-based library. The DWL, made up of 96 bacterial genera, 44 of which are not represented in the MALDI-TOF MS bacterial Bruker Daltonics (BDAL) database, was found to significantly improve the identification of bacteria present in drinking water.

Genome Sequence of Bifidobacterium breve INIA P734 (CECT 8178), a Strain Isolated from Human Breast Milk.

The draft genome sequence of Bifidobacterium breve INIA P734, a strain shared by mother and child, is reported. It consists of 50 contigs, with 2,391,925 bp, 2,099 genes, and a G+C content of 58.8%. The genome analysis revealed the absence of antibiotic resistance and pathogenicity-related genes.

Pseudidiomarina piscicola sp. nov., isolated from cultured European seabass, Dicenthrarchus labrax.

Strain CECT 9734 T, a Gram-negative, aerobic, chemoorganotrophic bacterium, motile by polar flagella, was isolated from cultured European seabass, Dicenthrarchus labrax, in Spain. It grows from 5 to 42 ºC, 6-9 pH and 1-12% total salinity. Major cellular fatty acids are C15:0 iso, summed feature 9 (C17:1 iso w9c/C16:0 10-methyl) and C17:0 iso. The genome size is 2.5 Mbp and G + C content is 49.5 mol%. Comparative analysis of the 16S rRNA gene sequence shows that the strain is a member of Pseudidiomarina, with highest similarities with Pseudidiomarina halophila (97.0%) and Pseudidiomarina salinarum (96.9%). Phylogenomic tree based on UBCG program shows P. halophila as its closest relative. ANI and in-silico DDH with other Pseudidiomarina spp. are lower than 87 and 20%, respectively, suggesting that strain CECT 9734 T represents a new species, for which we propose the name Pseudidiomarina piscicola sp. nov. and CECT 9734 T (= LUBLD50 7aT = LMG 31044 T) as type strain.

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.

Effectiveness of a comprehensive care protocol in patients with new diagnoses of type 2 diabetes mellitus and associated comorbidities in primary care: study protocol of a quasi-experimental trial.

INTRODUCTION: Type 2 diabetes mellitus (T2DM) is a highly prevalent chronic disease in the Spanish population. Typically, T2DM is associated with other chronic conditions. Intensive medication at the time of diagnosis has proven effective in reducing cardiovascular risk, improving glycaemic control and preventing T2DM complications. However, it has not yet been demonstrated that a comprehensive and intensive health education protocol at the time of diagnosis has the benefits described previously. Currently, there is great variability in the practices of primary care nurses regarding health education at the time of disease diagnosis.We aimed to evaluate the effectiveness of a systematic protocol with a comprehensive care programme in people with newly diagnosed T2DM with associated comorbidities. METHODS AND ANALYSIS: A multicentre quasi-experimental design comparing a group of individuals taking part in the intervention (intervention group (IG)) with a similar group receiving standard diabetes care (comparison group (CG)) is planned. The intervention will take place during the 3 months after study enrolment. Data will be collected at baseline and at 3, 6 and 12 months. Ten primary care centres in Barcelona city will be selected for participation: 5 for the IG and 5 for the CG. The IG will include five structured individual visits postdiagnosis with the primary care nurse, during which aspects of diabetes education will be discussed with the patient and his/her family. The results will be measured in terms of health-related quality of life and the change in main outcomes (glycated haemoglobin and weight). ETHICS AND DISSEMINATION: The study fully met the requirements of the Ethical Committee of Clinical Investigation of the IDIAP Jordi Gol (approval code: P13/118). Patients will be informed that their data are confidential, and they have the right to withdraw at any time without penalty. Dissemination will include publishing the findings in peer-reviewed journals and sharing our findings at scientific conferences. TRIAL REGISTRATION NUMBER: NCT03990857; Pre-results.

Methylobacterium symbioticum sp. nov., a new species isolated from spores of Glomus iranicum var. tenuihypharum.

Strain SB0023/3 T, isolated from spores of the arbuscular mycorrhizal fungus Glomus iranicum var. tenuihypharum, was analysed to determine whether it represents a new species. It was studied for its applicability in the field of agriculture to reduce the input of nitrogen fertilizers. Comparative analysis of the 16S rRNA gene sequence shows the strain to be affiliated to the genus Methylobacterium, the closest similarities (98.7%) being shared with Methylobacterium dankookense. Further phylogenomic analysis through Up-to-date Bacterial Core Gene (UBCG) confirmed Methylobacterium dankookense as its closest relative. Average Nucleotide Identity (ANI) and in silico DNA-DNA hybridization (DDH) were lower than 92% and 44%, respectively, of the values shown by its phylogenetic relatives. Its genome had an approximate length of 6.05 Mb and the G + C content of the genome was 70.1 mol%. The main cellular fatty acid was Summed Feature 8 (C18:1ω7c and/or C18:1ω6c). It is a Gram-staining-negative, pink-pigmented, strictly aerobic and facultative methylotroph; it grows at 28 ºC and can grow at up to 3% salinity in the presence of sodium chloride. All the data collected support the naming of a novel species to accommodate the strain SB0023/3 T, for which the name Methylobacterium symbioticum sp. nov. is proposed. The type strain is SB0023/3 T (=CECT 9862 T =PYCC 8351 T).

Mesonia oceanica sp. nov., isolated from oceans during the Tara oceans expedition, with a preference for mesopelagic waters.

Strain ISS653T, isolated from Atlantic seawater, is a yellow pigmented, non-motile, Gram-reaction-negative rod-shaped bacterium, strictly aerobic and chemoorganotrophic, slightly halophilic (1-15 % NaCl) and mesophilic (4-37 °C), oxidase- and catalase-positive and proteolytic. Its major cellular fatty acids are iso-C15 : 0, iso-C15 : 0 2-OH, and iso-C17 : 0 3-OH; the major identified phospholipid is phosphatidylethanolamine and the major respiratory quinone is MK6. Genome size is 4.28 Mbp and DNA G+C content is 34.9 mol%. 16S rRNA gene sequence similarity places the strain among members of the family Flavobacteriaceae, with the type strains of Mesonia phycicola (93.2 %), Salegentibacter mishustinae (93.1 %) and Mesonia mobilis (92.9 %) as closest relatives. Average amino acid identity (AAI) and average nucleotide identity (ANI) indices show highest values with M. mobilis (81 % AAI; 78.9 % ANI), M. phycicola (76 % AAI; 76.3 % ANI), Mesonia maritima (72 % AAI, 74.9 % ANI), Mesonia hippocampi (64 % AAI, 70.8 % ANI) and Mesonia algae (68 % AAI; 72.2 % ANI). Phylogenomic analysis using the Up-to-date-Bacterial Core Gene set (UBCG) merges strain ISS653T in a clade with species of the genus Mesonia. We conclude that strain ISS653T represents a novel species of the genus Mesonia for which we propose the name Mesonia oceanica sp. nov., and strain ISS653T (=CECT 9532T=LMG 31236T) as the type strain. A second strain of the species, ISS1889 (=CECT 30008) was isolated from Pacific Ocean seawater. Data obtained throughout the Tara oceans expedition indicate that the species is more abundant in the mesopelagic dark ocean than in the photic layer and it is more frequent in the South Pacific, Indian and North Atlantic oceans.

Thalassocella blandensis gen. nov., sp. nov., a novel member of the family Cellvibrionaceae.

Strain ISS155T, isolated from surface Mediterranean seawater, has cells that are Gram-reaction-negative, motile, strictly aerobic chemoorganotrophic, oxidase-positive, unable to reduce nitrate to nitrite, and able to grow with cellulose as the sole carbon and energy source. It is mesophilic, neutrophilic, slightly halophilic and has a requirement for sodium and magnesium ions. Its 16S rRNA gene sequence places the strain among members of Cellvibrionaceae, in the Gammaproteobacteria, with Agarilytica rhodophyticola 017T as closest relative (94.3 % similarity). Its major cellular fatty acids are C18 : 1, C16 : 0 and C16 : 1; major phospholipids are phosphatidyl glycerol, phosphatidyl ethanolamine and an unidentified lipid, and the major respiratory quinone is Q8. The genome size is 6.09 Mbp and G+C content is 45.2 mol%. A phylogenomic analysis using UBCG merges strain ISS155T in a clade with A. rhodophyticola, Teredinibacter turnerae, Saccharophagus degradans and Agaribacterium haliotis type strain genomes, all of them possessing a varied array of carbohydrate-active enzymes and the potential for polysaccharide degradation. Average amino acid identity indexes determined against available Cellvibrionaceae type strain genomes show that strain ISS155T is related to them by values lower than 60 %, with a maximum of 58 % to A. rhodophyticola 017T and 57 % to T. turnerae T7902T and S. degradans 2-40T. These results, together with the low 16S rRNA gene sequence similarities and differences in phenotypic profiles, indicate that strain ISS155T represents a new genus and species in Cellvibrionaceae, for which we propose the name Thalassocella blandensis gen. nov., sp. nov., and strain ISS155T (=CECT 9533T=LMG 31237T) as the type strain.

Genomic Insights Into Five Strains of Lactobacillus plantarum With Biotechnological Potential Isolated From chicha, a Traditional Maize-Based Fermented Beverage From Northwestern Argentina.

Lactic acid bacteria (LAB) are indigenous microorganisms that have been involved in food fermentations throughout history to preserve food and also to give special characteristics to them. The traditional fermented foods that are still being elaborated in indigenous populations around the world are a potential source of LAB with important biotechnological properties and/or beneficial to health. In a previous work, LAB biodiversity associated with chicha, a traditional maize-based fermented beverage from Northwestern Argentina, was studied, both by culture dependent and independent methods. From that study, 392 isolates were recovered, mostly members of Lactobacillus and Leuconostoc. Biotechnological characterization of representative isolates led to the selection of five strains belonging to the species Lactobacillus plantarum for their ability to produce vitamin B2 (riboflavin) and vitamin B9 (folates), their antimicrobial properties and antibiotics susceptibility. In this work, we present the Whole Genome Sequences (WGS) of these five strains that have been deposited in the Spanish Type Culture Collection: M5MA1 (= CECT 8962), M9MM1 (= CECT 8963), M9MM4 (= CECT 8964), M9MG6 (= CECT 8965), and M9Y2 (= CECT 8966), and a detailed description of their characterization, through a genomic approach, analyzing the genes responsible for these biotechnological properties, making a comparative study of the five genomes and reporting the aspects related to food safety, in accordance with the recommendations of the European Food Safety Authority (EFSA FEEDAP Panel, 2018) aiming at their use in the design of functional foods. The analysis unveiled, for the five strains, the complete set of genes for folate and riboflavin biosynthesis, the absence of pathogenic factors, the presence of CRISPR and cas genes, phage sequences, insertion elements and an aminoglycosides resistance gene, aadA, whose resistance could not be proved phenotypically in any strain. Genomic comparisons showed that strain CECT 8962 was significantly different in terms of genetic content and allowed the identification of carbohydrates metabolism and membrane transport related genes as the main components of the unique and accessory genome.

Shimia thalassica sp. nov., and reclassification of Pseudopelagicola gijangensis as Shimia gijangensis comb. nov., and Thalassobius activus as Cognatishimia activa comb. nov.

Strain CECT 7735T, a marine Gram-reaction negative, aerobic, non-motile bacterium, was isolated from coastal seawater in Valencia, Spain. Strain CECT 7735T is chemoorganotrophic, mesophilic, slightly halophilic, grows at 15-28 °C but not at 4 or 37 °C, requires seawater for growth and grows up to 6 % salinity. The major cellular fatty acid is summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genome is 55.7 mol%. Comparative analysis of the 16S rRNA gene sequence shows the strain is affiliated to the family Rhodobacteraceae, in the class Alphaproteobacteria, with highest similarities to Phaeobacter species (97.0-97.5 %), Shimia species (96.5-97.3 %) and Pseudopelagicola gijangensis (96.5 %). Further phylogenomic analysis through the up-to-date-bacterial core gene (UBCG) set showed P. gijangensis to be its closest relative. Average nucleotide identity and in silico DNA-DNA hybridization values are lower than 85 and 21 %, respectively, with its phylogenetic relatives, suggesting that strain CECT 7735T represents a new species. The average amino acid identity value was over 70 % with the genome of the type strain of P. gijangensis and with all those of Shimia species. These values, together with UBCG set trees, suggest that the new species and P. gijangensisbelong to the same genus and that Pseudopelagicola should be reclassified as a Shimia species. We conclude that strain CECT 7735T represents a new species in the genus Shimia, for which we propose the name Shimiathalassica sp. nov. In addition, Pseudopelagicola gijangensis is reclassified as Shimiagijangensis comb. nov. From the same phylogenomic study, it can be concluded that Thalassobius activus should be reclassified in the genus Cognatishimia as Cognatishimiaactiva comb. nov.

Real-Time Detection of Riboflavin Production by Lactobacillus plantarum Strains and Tracking of Their Gastrointestinal Survival and Functionality in vitro and in vivo Using mCherry Labeling.

Some strains of lactic acid bacteria (LAB) produce riboflavin, a water-soluble vitamin of the B complex, essential for human beings. Here, we have evaluated riboflavin (B2 vitamin) production by five Lactobacillus plantarum strains isolated from chicha, a traditional maize-based fermented alcoholic beverage from north-western Argentina and their isogenic riboflavin-overproducing derivatives previously selected using roseoflavin. A direct fluorescence spectroscopic detection method to quantify riboflavin production in bacterial culture supernatants has been tested. Comparison of the efficiency for riboflavin fluorescence quantification with and without prior HPLC fractionation showed that the developed method is a rapid and easy test for selection of B2 vitamin-producing strains. In addition, it can be used for quantitative detection of the vitamin production in real time during bacterial growth. On the basis of this and previous analyses, the L. plantarum M5MA1-B2 riboflavin overproducer was selected for in vitro and in vivo studies after being fluorescently labeled by transfer of the pRCR12 plasmid, which encodes the mCherry protein. The labeling did not affect negatively the growth, the riboflavin production nor the adhesion of the strain to Caco-2 cells. Thus, L. plantarum M5MA1-B2[pRCR12] was evaluated for its survival under digestive tract stresses in the presence of microbiota in the dynamic multistage BFBL gut model and in a murine model. After exposure to both models, M5MA1-B2[pRCR12] could be recovered and detected by the pink color of the colonies. The results indicated a satisfactory resistance of the strain to gastric and intestinal stress conditions but a low colonization capability observed both in vitro and in vivo. Overall, L. plantarum M5MA1-B2 could be proposed as a probiotic strain for the development of functional foods.

Different Modes of Regulation of the Expression of Dextransucrase in Leuconostoc lactis AV1n and Lactobacillus sakei MN1.

Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter P dsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the P dsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the P dsrLL -mrfp fusion or the P dsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from P dsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from P dsrLL . A different behavior was detected, when expression from P dsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.

In situ riboflavin fortification of different kefir-like cereal-based beverages using selected Andean LAB strains.

Cereal-based functional beverages represent social, economic, and environmental sustainable opportunities to cope with emerging trends in food consumption and global nutrition. Here we report, for the first time, the polyphasic characterization of three cereal-based kefir-like riboflavin-enriched beverages, obtained from oat, maize and barley flours, and their comparison with classical milk-based kefir. The four matrices were successfully fermented with commercial starters: i) milk-kefir and ii) water-kefir, proving the potential of cereal ingredients in the formulation of dairy-like fermented beverages with milk-kefir starter behavior better in these matrices. In the light of their potentiality, seven riboflavin-producing Andean Lactic Acid Bacteria (LAB) were tested for tolerance to food stresses commonly encountered during food fermentation. Moreover, the LAB strains investigated were screened for spontaneous riboflavin overproducing derivatives. Lactobacillus plantarum M5MA1-B2 with outstanding response to stress, was selected to improve riboflavin content in an in situ fortification approach. The combination of L. plantarum M5MA1-B2 riboflavin overproducing strain with milk kefir starter in oat, lead to cover, for one serving of 100 g, 11.4% of Recommended Dietary Allowance (RDA). Besides, addition of L. plantarum M5MA1-B2 improved performance of water kefir in oat and maize matrices. Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) analysis provided the on-line Volatile Organic Compounds profiles supporting the best combination of starter, LAB and cereal matrix for novel functional foods development.

Antiviral activity of aged green tea extract in model food systems and under gastric conditions.

Aged-green tea extract (GTE) is known to reduce the infectivity of hepatitis A virus (HAV) and murine norovirus (MNV), a human norovirus surrogate, in vitro and in washing solutions. Initially, the effect of aged-GTE was evaluated on virus like particles (VLPs) of human norovirus (HuNoV) genogroup I (GI) by a porcine gastric mucine (PGM)-enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM), and on HuNoV GI suspensions by an in situ capture-RT-qPCR method, suggesting that HuNoVs are very sensitive to aged-GTE treatment at 37 °C. Moreover, the potential application of aged-GTE was evaluated using model foods and simulated gastric conditions. Then, aged-GTE samples prepared in orange juice, apple juice, horchata, and milk, respectively, were individually mixed with each virus and incubated overnight at 37 °C. Aged-GTE at 5 mg/ml in apple juice reduced MNV infectivity to undetectable levels and from 1.0 to 1.8 log in milk, horchata and orange juice. Aged-GTE at 5 mg/ml in orange juice, apple juice, horchata and milk reduced HAV infectivity by 1.2, 2.1, 1.5, and 1.7 log, respectively. Additionally, aged-GTE at 5 mg/ml in simulated intestinal fluid reduced MNV titers to undetectable levels and reduced HAV infectivity by ca. 2.0 log. The results show a potential for aged-GTE as a suitable natural option for preventive strategies for foodborne viral diseases.