Biochemical, Biophysical and Functional Characterization of an Insoluble Iron Containing Hepcidin-Ferritin Chimeric Monomer Assembled Together with Human Ferritin H/L Chains at Different Molar Ratios

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin-ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery

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 PdsrLL 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 PdsrLL-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 PdsrLL-mrfp fusion or the PdsrLS 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 PdsrLL 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 PdsrLL. A different behavior was detected, when expression from PdsrLS 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

Microbiological analysis and assessment of biotechnological potential of lactic acid bacteria isolated from Tunisian flours

International audienceThis work was carried out to identify lactic acid bacteria (LAB) from baking wheat flours and to evaluate their technological capabilities for potential incorporation in sourdough process. Six samples of wheat flours obtained from different geographical regions of Tunisia were microbiologically analyzed. Several technological features including acidification, antimicrobial, amylolytic, proteolytic, and antioxidant activities of six selected LAB strains were investigated for future in situ applications. Moreover, LAB were investigated for their ability to produce exopolysaccharides. A total of 45 autochthonous LAB were isolated and identified by genetic analysis of 16S–23S rRNA intergenic transcribed spacer (ITS)-generated patterns ITS-PCR. One of each ITS-PCR pattern was subjected to partial 16S rRNA gene sequencing, and strains were identified as Weissella cibaria, Lactobacillus plantarum, Lactobacillus brevis, Pediococcus pentosaceus, Pediococcus pentoseus, Pediococcus acidilactici, Enterococcus faecium, Enterococcus casseliflavus, and Enterococcus faecalis. All tested LAB showed good acidifying ability by decreasing significantly (p < 0.05) the pH of flour extract below 4.0 after 24 h and below 3.0 after 72 h. Pediococcus pentoseus and P. acidilactici presented fermentation quotient (FQ, ratio of lactic and acetic acids) close to the optimal range. All LAB isolates demonstrated extracellular proteolytic activity. Weissella cibaria S25 had the highest radical-scavenging activity with 25.57%. Lactobacillus plantarum S28 demonstrated the highest amylolytic activity (1386 U/mL) followed by P. acidilactici S16 (1086 U/mL). Although, L. plantarum S28 showed the highest production of exopolysaccharides (0.97 g/L). Moreover, varying halo of inhibition was detected against Escherichia coli, Staphylococcus aureus, Aspergillus niger, and Penicillium expansum. This study revealed that autochthonous flour LAB had interesting technological features and thus could be used in sourdough production

Diversity of vaginal lactic acid bacterial microbiota in 15 Algerian pregnant women with and without bacterial vaginosis by using culture independent method

International audienceBacterial Vaginosis (BV) is the most common lower genital tract disorder among women of reproductive age (pregnant and non-pregnant) and a better knowledge of Lactobacillus species richness in healthy and infected vaginal microbiota is needed to efficiently design better probiotic products to promote the maintenance of normal flora which will help prevent bacterial vaginosis

Functional Probiotic Assessment and In Vivo Cholesterol-Lowering Efficacy of Weissella sp. Associated with Arid Lands Living-Hosts

The research and the selection of novel probiotic strains from novel niches are receiving increased attention on their proclaimed health benefits to both humans and animals. This study aimed to evaluate the functional properties of Weissella strains from arid land living-hosts and to select strains with cholesterol-lowering property in vitro and in vivo, for use as probiotics. They were assessed for acid and bile tolerance, antibiotic susceptibility, membrane properties, antibacterial activity, antiadhesive effect against pathogens to host cell lines, and cholesterol assimilation in vitro. Our results showed that the majority of strains revealed resistance to gastrointestinal conditions. All the strains were nonhemolytic and sensitive to most of the tested antibiotics. They also exhibited high rates of autoaggregation and some of them showed high coaggregation with selected pathogens and high adhesion ability to two different cell lines (Caco-2 and MIM/PPk). Particularly, W. halotolerans F99, from camel feces, presented a broad antibacterial spectrum against pathogens, reduced Enterococcus faecalis and Escherichia coli adhesion to Caco-2 cells, and was found to reduce, in vitro, the cholesterol level by 49 %. Moreover, W. halotolerans F99 was evaluated for the carbohydrate utilization as well as the serum lipid metabolism effect in Wistar rats fed a high-cholesterol diet. W. halotolerans F99 showed an interesting growth on different plant-derivative oligosaccharides as sole carbon sources. Compared with rats fed a high-fat (HF) diet without Weissella administration, total serum cholesterol, low-density lipoprotein cholesterol, and triglycerides levels were significantly (p<0.001) reduced in W. halotolerans F99-treated HF rats, with no significant change in high-density lipoprotein cholesterol HDL-C levels. On the basis of these results, this is the first study to report that W. halotolerans F99, from camel feces, can be developed as cholesterol-reducing probiotic strain. Further studies may reveal their potential and possible biotechnological and probiotic applications

In-vitro characterization of potentially probiotic Lactobacillus strains isolated from human microbiota: interaction with pathogenic bacteria and the enteric cell line HT29

Among the various tests commonly used for selecting probiotic microorganisms, the tolerance to gastrointestinal transit conditions remains being commonly used to evaluate the probiotic potential of the strains. Besides, the adhesion to epithelial cells and the competition with pathogens constitute significant traits for evaluating the colonization ability and functional performance of candidate strains. In this study, a total of 13 lactic acid bacteria strains isolated from human feces were first identified by biochemical tests and 16S rRNA gene sequencing, and then characterized in vitro for their tolerance to gastrointestinal conditions, hemolytic activity, and antibiotics sensibility. The isolates were identified as Lactobacillus fermentum (06), Lactobacillus rhamnosus (04), Lactobacillus plantarum (02), and Lactobacillus salivarius (01). The adhesion to epithelial cells HT29 was shown to be a strain-dependent character. L. fermentum 88 and L. plantarum 9, being the ones showing higher adhesion values. They were further characterized by determining their antimicrobial activity, hydrophobicity, co-aggregation, antioxidant activity, as well as the ability to inhibit the adhesion of pathogens to the human epithelial cell line HT29. Moreover, these two strains were able to reduce the adhesion of Escherichia coli to HT29 cells, although they failed for inhibiting the adhesion of other pathogens such as Cronobacter sakazaki or Salmonella enterica. These results point out the importance of considering the ecological fitness of the strains in selecting probiotic bacteria and the potential of some of the analyzed strains for the development of food products.This work was funded by the Tunisian Ministry of Higher Education and Scientific research (laboratory project LR03ES03) and the Department of Microbiology and Biochemistry of Dairy products, IPLA-CSIC, Spain.Peer reviewe

Diversity and Antimicrobial Properties of Lactic Acid Bacteria Isolated from Rhizosphere of Olive Trees and Desert Truffles of Tunisia

A total of 119 lactic acid bacteria (LAB) were isolated, by culture-dependant method, from rhizosphere samples of olive trees and desert truffles and evaluated for different biotechnological properties. Using the variability of the intergenic spacer 16S-23S and 16S rRNA gene sequences, the isolates were identified as the genera Lactococcus, Pediococcus, Lactobacillus, Weissella, and Enterococcus. All the strains showed proteolytic activity with variable rates 42% were EPS producers, while only 10% showed the ability to grow in 9% NaCl. In addition, a low rate of antibiotic resistance was detected among rhizospheric enterococci. Furthermore, a strong antibacterial activity against plant and/or pathogenic bacteria of Stenotrophomonas maltophilia, Pantoea agglomerans, Pseudomonas savastanoi, the food-borne Staphylococcus aureus, and Listeria monocytogenes was recorded. Antifungal activity evaluation showed that Botrytis cinerea was the most inhibited fungus followed by Penicillium expansum, Verticillium dahliae, and Aspergillus niger. Most of the active strains belonged to the genera Enterococcus and Weissella. This study led to suggest that environmental-derived LAB strains could be selected for technological application to control pathogenic bacteria and to protect food safety from postharvest deleterious microbiota

Análisis comparativo de cepas tunecinas productoras de dextrano pertenecientes a los géneros Leuconostoc y Weisella

Resumen del trabajo presentado en el XI Workshop de la Sociedad Española de Microbiota, Probióticos y Prebióticos, celebrado en Granada (España), del 12 al 14 de febrero de 2020Introducción/Objetivos. Los exopolisacáridos (EPS) producidos por las bacterias ácido lácticas (BAL) poseen una amplia aplicación industrial. Así, los dextranos se emplean en la industria alimentaria para mejorar las propiedades reológicas de alimentos fermentados y poseen capacidad inmunomoduladora y antiviral. El objetivo de este trabajo fue identificar weisellas productoras de dextrano y caracterizar su naturaleza y el efecto de sus polímeros comparándolo con los de Leuconostoc lactis AV1n. Metodología. Los EPS se caracterizaron por análisis de la composición de azúcares y de metilación. Su tamaño se determinó por SEC-MALS. La producción de dextrano y el flujo metabólico se cuantificaron por el método del fenol sulfúrico y por cromatografía de gases. Se analizó la interacción bacteria-células Caco-2, y la formación de biopelícula por tinción con cristal violeta. Resultados. Se demostró que los EPS eran dextranos con elevadas masas moleculares (5,84x107-2,61x108 Da). Su producción se detectó durante la fase exponencial de crecimiento, con un consumo de sacarosa de > 95% por AV1n y de 57% por las weisellas. El EPS producido por W. confusa 11.3b disminuyó en la fase estacionaria. Este resultado indicativo de una actividad dextranasa, fue confirmado por la detección de halos de degradación de azul dextrano. W. confusa V30 mostró menor adherencia a los enterocitos en presencia de glucosa (38,1%) versus sacarosa (14,3%). El tipo de azúcar no afectó la adhesión de W. cibaria AV2ou (10,5±1,4%) y 11.3b (11,2±3,0%). AV1n mostró mayor adherencia en presencia de sacarosa (48,8%) versus glucosa (27,8%) y se categorizó como fuerte formador de biopelícula sobre poliestireno en presencia de sacarosa (3,38±0,38) versus glucosa (0,78±0,21). Dichos azúcares no influyeron diferencialmente en la formación de biopelícula por las weisellas. Conclusiones. Por primera vez, L. lactis AV1n ha evidenciado al dextrano como efector positivo de la adhesión y agregación de BAL, y se ha detectado una actividad dextranasa en una weisella

Inhibitory Effect of Lactobacillus plantarum FL75 and Leuconostoc mesenteroides FL14 against Foodborne Pathogens in Artificially Contaminated Fermented Tomato Juices

Tomatoes and tomato based-foods contain beneficial microorganisms and various organic acids that have important nutritional values for human. The objective of this study was to access the physiochemical properties of fermented tomatoes juices and to evaluate the competitiveness of lactic acid bacteria (LAB) against Listeria monocytogenes, Listeria innocua, and Salmonella spp., in artificially contaminated tomato juice. Microbial counting (LAB, fungi Salmonella spp., and Listeria spp.) was performed after fermentation and weekly during storage. Different organic acids (Lactic, succinic, and acetic) and ethanol were also monitored using HPLC method. Color parameters were also determined. The results showed an increase of lactic and acetic acid content, during fermentation and storage of juices inoculated with Lactobacillus plantarum and Leuconostoc mesenteroides at 25°C. Besides, citric acid and ethanol revealed higher content at the end of storage compared to that registered at 4°C. The pH from tomatoes juices decreased from an initial value of 4.5 to below 3.2. Alongside, foodborne pathogen population was significantly suppressed in tomatoes juices when the samples were coinoculated with LAB strains. Moreover, the inhibition of Salmonella species was faster compared to that of Listeria. After four weeks of storage at 4°C, Lb. plantarum and Lc. mesenteroides showed high survival rate, while pathogenic bacteria, yeasts, and molds cell numbers decreased drastically in all the contaminated vials. This work highlights the efficiency of Lb. plantarum and Lc. mesenteroides as potential starters for developing nutritious and safe fermented tomato juice products

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 PdsrLL 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 PdsrLL-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 PdsrLL-mrfp fusion or the PdsrLS 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 PdsrLL 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 PdsrLL. A different behavior was detected, when expression from PdsrLS 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.This work was supported by the Spanish Ministry of Economy and Competitiveness (Grants AGL2015-65010-C3-1-R and RTI2018-097114-B-100) and the Tunisian Ministry of Higher Education and Scientific Research.Peer Reviewe