In vitro assessment of biofunctional properties of Lactiplantibacillus plantarum strain Jb21-11 and the characterization of its exopolysaccharide.

ABSTACT: The microbiota of traditional food provides a rich reservoir of biodiversity to find new strains with interesting features for novel functional food formulation. Therefore, this study aimed to investigate the biofunctional potential of the lactic acid bacteria (LAB) strain Jb21-11 isolated from Jben, a traditional Algerian fresh cheese. This isolate was selected out of a collection of 154 LAB based on its exopolysaccharide (EPS) phenotype and was preliminarily identified by polyphasic characterization as Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) and its biofunctional properties were then assessed in vitro. The tested strain demonstrated good resistance to gastric juice, acidity around pH 2, and 2% (v/v) bile salts, which are important characteristics for potential biofunctional LAB candidates. It also showed a good production of ropy EPS with 674 mg/L on MRS medium. However, this ability appears to compromise the adhesion of the strain to Caco-2 cells (less than 1%), which according to our results, seems not to be related to autoaggregation and hydrophobicity (44.88 ± 0.028% and 16.59 ± 0.012%). Furthermore, promising antimicrobial activity against three pathogenic bacteria (Escherichia coli, Staphylococcus aureus, and Salmonella) was detected probably due to antimicrobial metabolites excreted during fermentation process into the medium. Moreover, the strain L. plantarum Jb21-11 displayed a therapeutic functionality with both anti-inflammatory and immunomodulatory action using RAW 264.7 cells. The chemical features of the novel ropy Jb21-11-EPS were also investigated revealing the presence of three monosaccharides, namely, mannose, galactose, and glucose, with a molar ratio of 5.42:1.00:4.52 linked together by α- and ß-glycosidic bonds, presenting a relatively high molecular weight of 1.08 × 105 Da of interest for a texturing potential. Therefore, the new producing EPS strain Jb21-11 is a promising candidate for use as an adjunct culture for improving the texture of functional food.

Characterization of Lactic Acid Bacteria Strains Isolated from Algerian Honeybee and Honey and Exploration of Their Potential Probiotic and Functional Features for Human Use.

Using culture enrichment methods, 100 strains of bacilli of lactic acid bacteria (LAB) were isolated from honeybee Apis mellifera intermissa and fresh honey, collected from apiaries located in the north-east of Algeria. Amongst all of the isolated LAB, 19 selected strains were closely affiliated to four species-Fructobacillus fructosus (10), Apilactobacillus kunkeei (5), Lactobacillus kimbladii and/or Lactobacillus kullabergensis (4)-using phylogenetic and phenotypic approaches. The in vitro probiotic characteristics (simulated gastrointestinal fluids tolerance, autoaggregation and hydrophobicity abilities, antimicrobial activity and cholesterol reduction) and safety properties (hemolytic activity, antibiotic resistance and absence of biogenic amines) were evaluated. The results indicated that some strains showed promising potential probiotic properties. In addition, neither hemolytic activity nor biogenic amines were produced. The carbohydrate fermentation test (API 50 CHL) revealed that the strains could efficiently use a broad range of carbohydrates; additionally, four strains belonging to Apilactobacillus kunkeei and Fructobacillus fructosus were found to be exopolysaccharides (EPS) producers. This study demonstrates the honeybee Apis mellifera intermissa and one of her products as a reservoir for novel LAB with potential probiotic features, suggesting suitability for promoting host health.

Characterization and biological activities of carotenoids produced by three haloarchaeal strains isolated from Algerian salt lakes.

Halophilic archaea represent a promising natural source of carotenoids. However, little information is available about these archaeal metabolites and their biological effects. In the present work, carotenoids of strains Haloferax sp. ME16, Halogeometricum sp. ME3 and Haloarcula sp. BT9, isolated from Algerian salt lakes, were produced, extracted and identified by high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Analytical results revealed a variation in the composition depending on the strain with a predominance of bacterioruberin. The evaluation of antioxidant capacity using ABTS [(2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays showed that these extracts have a strong antioxidant potential, in particular those of Haloferax sp. ME16 which displayed antioxidant power significantly higher than that of ascorbic acid used as standard. Antibacterial activity of carotenoid extracts against four human-pathogenic strains and four fish-pathogenic strains was evaluated by agar disk diffusion method. The results showed a good antibacterial activity. These findings suggest that the C50 carotenoids from the studied strains offer promising prospects for biotechnological applications.

Selection of Exopolysaccharide-Producing Lactobacillus Plantarum (Lactiplantibacillus Plantarum) Isolated from Algerian Fermented Foods for the Manufacture of Skim-Milk Fermented Products.

The exopolysaccharide (EPS)-producing Lactobacillus plantarum (renamed as Lactiplantibacillus plantarum) LBIO1, LBIO14 and LBIO28 strains, isolated from fermented dairy products typical from Algeria, were characterized to evaluate the impact of the polymers in milk fermentations. Their genomes revealed the presence of two complete eps clusters of the four described for the reference strain WCFS1. Besides, the three strains presented identical sequences of eps3 and eps4 clusters, but LBIO1 and LBIO28 harbour three genes belonging to eps2 which are absent in the LBIO14 genome. The EPS purified from fermented skim-milks manufactured with the strains showed identical nuclear magnetic resonance (1H-NMR) and size exclusion chromatography coupled with a multiangle laser light scattering detector (SEC-MALLS) profiles for polymers LBIO1 and LBIO28, whereas LBIO14 EPS was different due to the lack of the high-molecular weight (HMW)-EPS and the absence of specific monosaccharide's peaks in the anomeric region of its proton NMR spectrum. The presence of the HMW-EPS correlated with optimal sensorial-physical characteristics of the fermented skim-milks (ropy phenotype). Their microstructures, studied by confocal scanning laser microscopy (CSLM), also showed differences in the organization of the casein-network and the distribution of the bacteria inside this matrix. Therefore, the strain LBIO1 can be proposed for the manufacture of dairy products that require high whey retention capability, whereas LBIO28 could be applied to increase the viscosity.

Bioprospecting and characterization of pigmented halophilic archaeal strains from Algerian hypersaline environments with analysis of carotenoids produced by Halorubrum sp. BS2.

A set of 110 extremely halophilic archaeal strains were isolated from seven distinct saline habitats located in different regions of Algeria. The physicochemical characterization of the samples showed that these habitats were thalassohaline. The carotenoid production from isolated strains varied from 0.1 to 3.68 µg/ml. Based on their physiological characteristics and pigment production, 43 strains were selected and identified by means of phenotypic tests and 16S ribosomal RNA gene sequencing. Phylogenetic analysis indicated that the isolates corresponded to the class Halobacteria and were closely related to genera Halorubrum, Haloarcula, Haloferax, Natrinema, Halogeometricum, Haloterrigena, and Halopiger. Carotenoids of the highest producer, strain Halorubrum sp. BS2 were identified using high-performance liquid chromatography-diode array detector and liquid chromatography-mass spectrometry. Bacterioruberin and bisanhydrobacterioruberin were the predominant carotenoids. The scavenging activity of these carotenoids reached 99% at a concentration of 18 µg/ml, which was much higher than that of ascorbic acid used as a reference compound. These carotenoids also exhibited significant antibacterial activities against four human-pathogenic strains and four fish-pathogenic strains. Variations in salinity, agitation rate, temperature, and light intensity were found to influence growth and carotenoid production of Halorubrum sp. BS2. Our results suggest that halophilic archaea represent a potential source for carotenoids, which are characterized by high antioxidant and antibacterial activities.

Streptococcus macedonicus strains isolated from traditional fermented milks: resistance to gastrointestinal environment and adhesion ability.

In this study, Streptococcus macedonicus (S. macedonicus) strains were identified from Algerian traditional fermented milks (Lben and Rayeb). Important prerequisites of probiotic interest such as acidity, bile salts tolerance, and adhesion ability to epithelial cells were investigated. A combination of phenotypic (ability to grow on Bile Esculin Azide medium, BEA; on high salt content medium NaCl 6.5%; on alkaline medium pH 9.6) and genotypic approaches (16S rRNA, ITS genes sequencing and MLST technique) allowed to identify four genetically distinct strains of S. macedonicus. These four strains and two references, Streptococcus thermophilus LMD-9 and Lactobacillus rhamnosus GG (LGG), were tested for their capacity to survive at low pH values, and at different concentrations of an equimolar bile salts mixture (BSM). Two different cell lines, Caco-2 TC7 and HT29-MTX, were used for the adhesion study. The results show that S. macedonicus strains selected constitute a distinct genetic entity from the Greek strain S. macedonicus ACA-DC-198. They were able to survive up to pH 3 and could tolerate high concentrations of bile salts (10 mM), unlike LMD-9 and LGG strains. Our strains also display in vitro adhesion similar to the LGG strain on Caco-2 TC7 and higher adhesion than the LMD-9 strain to Caco-2 TC7 and HT29-MTX cell models. This first characterization allows considering S. macedonicus as a potential candidate for possible probiotic effects that need to be investigated.

Production and Characterization of an Extracellular Acid Protease from Thermophilic Brevibacillus sp. OA30 Isolated from an Algerian Hot Spring.

Proteases have numerous biotechnological applications and the bioprospection for newly-thermostable proteases from the great biodiversity of thermophilic microorganisms inhabiting hot environments, such as geothermal sources, aims to discover more effective enzymes for processes at higher temperatures. We report in this paper the production and the characterization of a purified acid protease from strain OA30, a moderate thermophilic bacterium isolated from an Algerian hot spring. Phenotypic and genotypic study of strain OA30 was followed by the production of the extracellular protease in a physiologically-optimized medium. Strain OA30 showed multiple extracellular proteolytic enzymes and protease 32-F38 was purified by chromatographic methods and its biochemical characteristics were studied. Strain OA30 was affiliated with Brevibacillus thermoruber species. Protease 32-F38 had an estimated molecular weight of 64.6 kDa and was optimally active at 50 °C. It showed a great thermostability after 240 min and its optimum pH was 6.0. Protease 32-F38 was highly stable in the presence of different detergents and solvents and was inhibited by metalloprotease inhibitors. The results of this work suggest that protease 32-F38 might have interesting biotechnological applications.

Draft genome sequence of Thermoactinomyces sp. strain AS95 isolated from a Sebkha in Thamelaht, Algeria.

The members of the genus Thermoactinomyces are known for their protein degradative capacities. Thermoactinomyces sp. strain AS95 is a Gram-positive filamentous bacterium, isolated from moderately saline water in the Thamelaht region of Algeria. This isolate is a thermophilic aerobic bacterium with the capacity to produce extracellular proteolytic enzymes. This strain exhibits up to 99 % similarity with members of the genus Thermoactinomyces, based on 16S rRNA gene sequence similarity. Here we report on the phenotypic features of Thermoactinomyces sp. strain AS95 together with the draft genome sequence and its annotation. The genome of this strain is 2,558,690 bp in length (one chromosome, but no plasmid) with an average G + C content of 47.95 %, and contains 2550 protein-coding and 60 RNA genes together with 64 ORFs annotated as proteases.

The Geobacillus Pan-Genome: Implications for the Evolution of the Genus.

The genus Geobacillus is comprised of a diverse group of spore-forming Gram-positive thermophilic bacterial species and is well known for both its ecological diversity and as a source of novel thermostable enzymes. Although the mechanisms underlying the thermophilicity of the organism and the thermostability of its macromolecules are reasonably well understood, relatively little is known of the evolutionary mechanisms, which underlie the structural and functional properties of members of this genus. In this study, we have compared 29 Geobacillus genomes, with a specific focus on the elements, which comprise the conserved core and flexible genomes. Based on comparisons of conserved core and flexible genomes, we present evidence of habitat delineation with specific Geobacillus genomes linked to specific niches. Our analysis revealed that Geobacillus and Anoxybacillus share a high proportion of genes. Moreover, the results strongly suggest that horizontal gene transfer is a major factor deriving the evolution of Geobacillus from Bacillus, with genetic contributions from other phylogenetically distant taxa.

Draft Genome Sequence of Thermophilic Geobacillus sp. Strain Sah69, Isolated from Saharan Soil, Southeast Algeria.

Geobacillus spp. are potential sources of novel enzymes, such as those involved in the degradation of recalcitrant polymers. Here, we report a Geobacillus genome that may help reveal genomic differences between this strain and publicly available representatives of the same genus from diverse niches.

Marinobacter oulmenensis sp. nov., a moderately halophilic bacterium isolated from brine of a salt concentrator.

A Gram-negative, aerobic, moderately halophilic bacterium, designated Set74(T), was isolated from brine of a salt concentrator at Ain Oulmene, Algeria. The strain grew optimally at 37-40 °C, at pH 6.5-7.0 and with 5-7.5 % (w/v) NaCl and used various organic compounds as sole carbon, nitrogen and energy sources. Ubiquinone 9 (Q-9) was the major lipoquinone. The main cellular fatty acids were C16:0, C18:1ω9c, summed feature 7 (ECL 18.846; C19:0 cyclo ω10c and/or C19:1ω6c), C12:0 3-OH, C16:1ω9c, C18:0 and C12:0. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The G+C content of the genomic DNA was 57.4 mol%. The 16S rRNA gene sequence analysis indicated that strain Set74(T) was a member of the genus Marinobacter. The closest relatives of strain Set74(T) were Marinobacter santoriniensis NKSG1(T) (97.5 % 16S rRNA gene sequence similarity) and Marinobacter koreensis DD-M3(T) (97.4 %). DNA-DNA relatedness between strain Set74(T) and M. santoriniensis DSM 21262(T) and M. koreensis DSM 17924(T) was 45 and 37 %, respectively. On the basis of the phenotypic, chemotaxonomic and phylogenetic features, a novel species, Marinobacter oulmenensis sp. nov., is proposed. The type strain is Set74(T) ( = CECT 7499(T)  = DSM 22359(T)).

Marinobacter lacisalsi sp. nov., a moderately halophilic bacterium isolated from the saline-wetland wildfowl reserve Fuente de Piedra in southern Spain.

A Gram-negative, non-spore-forming, motile, moderately halophilic, aerobic, rod-shaped bacterium, designated strain FP2.5(T), was isolated from the inland hypersaline lake Fuente de Piedra, a saline-wetland wildfowl reserve located in the province of Málaga in southern Spain. Strain FP2.5(T) was subjected to a polyphasic taxonomic study. It produced colonies with a light-yellow pigment. Strain FP2.5(T) grew at salinities of 3-15 % (w/v) and at temperatures of 20-40 degrees C. The pH range for growth was 5-9. Strain FP2.5(T) was able to utilize various organic acids as sole carbon and energy source. Its major fatty acids were C(16 : 0), C(18 : 1)omega9c and C(16 : 1)omega9c. The DNA G+C content was 58.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain FP2.5(T) appeared to be a member of the genus Marinobacter and clustered closely with the type strains of Marinobacter segnicrescens, Marinobacter bryozoorum and Marinobacter gudaonensis (levels of 16S rRNA gene sequence similarity of 98.1, 97.4 and 97.2 %, respectively). However, DNA-DNA relatedness between the new isolate and the type strains of its closest related Marinobacter species was low; levels of DNA-DNA relatedness between strain FP2.5(T) and M. segnicrescens LMG 23928(T), M. bryozoorum DSM 15401(T) and M. gudaonensis DSM 18066(T) were 36.3, 32.1 and 24.9 %, respectively. On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, strain FP2.5(T) is considered to represent a novel species of the genus Marinobacter, for which the name Marinobacter lacisalsi sp. nov. is proposed. The type strain is FP2.5(T) (=CECT 7297(T)=LMG 24237(T)).

Halomicrobium katesii sp. nov., an extremely halophilic archaeon.

Two extremely halophilic archaea, strains Al-5(T) and K-1, were isolated from Lake Tebenquiche (Atacama Saltern, Chile) and Ezzemoul sabkha (Algeria), respectively. Cells of the two strains were short-rod-shaped and Gram-negative; colonies were orange-pigmented. They grew optimally at 37-40 degrees C and pH 7.0-7.5 in the presence of 25 % (w/v) NaCl. Magnesium was not required. Polar lipid analysis revealed the presence of phosphatidylglycerol and phosphatidylglycerophosphate methyl ester, the absence of phosphatidylglycerosulfate, and the presence of sulfated diglycosyl diether and diether diglycosyl as the sole glycolipids. DNA G+C contents of strains Al-5(T) and K-1 were 52.4 and 52.9 mol% (T(m) method), respectively. 16S rRNA gene sequence comparison with database sequences showed that strains Al-5(T) and K-1 were most closely related to Halomicrobium mukohataei DSM 12286(T) (similarities of 97.5 and 96.9 %, respectively). DNA-DNA hybridization indicated that strains Al-5(T) and K-1 were members of a single species. However, DNA-DNA relatedness to Halomicrobium mukohataei was 55.7+/-2.5 %. A comparative analysis of phenotypic characteristics and DNA-DNA hybridization between the isolates and Halomicrobium mukohataei DSM 12286(T) supported the conclusion that Al-5(T) and K-1 represent a novel species within the genus Halomicrobium, for which the name Halomicrobium katesii sp. nov. is proposed. The type strain is Al-5(T) (=CECT 7257(T)=DSM 19301(T)).

Salicola salis sp. nov., an extremely halophilic bacterium isolated from Ezzemoul sabkha in Algeria.

A novel, extremely halophilic bacterium was isolated from brine samples collected from Ezzemoul sabkha in north-east Algeria. Cells of this isolate, designated B2T, were Gram-negative, rod-shaped and motile. Growth occurred between 10 and 25% (w/v) NaCl and the isolate grew optimally at 15-20% (w/v) NaCl. The pH range for growth was 6.0-9.0 with an optimum at pH 7.0-7.5. The predominant fatty acids were C16:0 and C18:1omega9c. Other fatty acids present were C16:1omega9c, C18:0 10-methyl, C12:0 3-OH, C10:0 and C12:0. The G+C content of the genomic DNA was 56.0 mol%. 16S rRNA gene sequence analysis indicated that strain B2T was closely related to Salicola marasensis in the Gammaproteobacteria. The level of 16S rRNA gene sequence similarity between strain B2T and the type strain of Salicola marasensis was 99%. DNA-DNA hybridization experiments between strain B2T and Salicola marasensis indicated a level of relatedness of 52%. The phenotypic characteristics of strain B2T allowed its differentiation from recognized species of the genus Salicola. Strain B2T was able to hydrolyse starch but not aesculin. It was unable to use carbohydrates and could not use citrate, pyruvate or succinate as sole carbon and energy sources. On the basis of the polyphasic data presented, strain B2T is considered to represent a novel species of the genus Salicola, for which the name Salicola salis sp. nov. is proposed. The type strain is B2T (=CECT 7106T=LMG 23122T).

Halorubrum ezzemoulense sp. nov., a halophilic archaeon isolated from Ezzemoul sabkha, Algeria.

A novel extremely halophilic archaeon was isolated from Ezzemoul sabkha, Algeria. The strain, designated 5.1(T), was neutrophilic, motile and Gram-negative. At least 15 % (w/v) NaCl was required for growth. The isolate grew at pH 6.5-9.0, with optimum growth at pH 7.0-7.5. Mg(2+) was required for growth. Polar lipids were C(20)C(20) derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate and sulfated diglycosyl diether. The genomic DNA G+C content of strain 5.1(T) was 61.9 mol% (T(m)). Phylogenetic analysis based on comparison of 16S rRNA gene sequences revealed that strain 5.1(T) clustered with Halorubrum species. The results of DNA-DNA hybridization and biochemical tests allowed genotypic and phenotypic differentiation of strain 5.1(T) from other Halorubrum species. The name Halorubrum ezzemoulense sp. nov. (type strain 5.1(T)=CECT 7099(T)=DSM 17463(T)) is proposed.