Physicochemical and structural characterisation of a branched dextran type exopolysaccharide (EPS) from Weissella confusa S6 isolated from fermented sausage (Sucuk).

Exopolysaccharide (EPS) producing Lactic Acid Bacteria (LAB) species can be presented in distinct environments. In this study, Turkish fermented sausage (sucuk) was tested for the presence of EPS producer LAB strains and slimy-mucoid colonies were selected for further tests. Among the isolates, Weissella confusa strain S6 was identified and tested for the physicochemical characterisation of its EPS. This strain was found to produce 0.74 g L-1 of EPS in modified BHI medium conditions. Structural characterisation of EPS S6 by 1H and 13C NMR demonstrated that EPS S6 was a highly branched dextran type glucan formed by mainly (1 â†’ 2)-linked α-d-glucose units together with low levels of (1 â†’ 3)-linked α-d-glucose units as branching points. This structure was further confirmed by methylation analysis detected by GC-MS. An average molecular weight of 8 × 106 Da was detected for dextran S6. The FTIR analysis supported the dextran structure and revealed the presence of distinct functional groups within dextran S6 structure. A strong thermal profile was observed for dextran S6 detected by DSC and TGA analysis and dextran S6 revealed a degradation temperature of 289 °C. In terms of physical status, dextran S6 showed amorphous nature detected by XRD analysis. SEM analysis of dextran S6 demonstrated its rough, compact and porous morphology whereas AFM analysis of dextran S6 detected in its water solution showed the irregularity with no clear cross-link within the dextran chains. These technological features of dextran S6 suggests its potential to be used for in situ or ex situ application during meat fermentations.

Multilocus sequence typing of L. bulgaricus and S. thermophilus strains from Turkish traditional yoghurts and characterisation of their techno-functional roles.

In this study, Streptococcus thermophilus and Lactobacillus bulgaricus strains from traditional Turkish yoghurts were isolated, identified by 16S rRNA sequencing and genotypically 14 S. thermophilus and 6 L. bulgaricus strains were obtained as distinct strains by MLST analysis. Lactic acid production levels of the L. bulgaricus strains were higher than S. thermophilus strains. HPLC analysis showed that EPS monosaccharide composition of the strains mainly consisted of glucose and galactose. In general, all strains were found to be susceptible for antibiotics, except some strains were resistance to gentamicin and kanamycin. Apart from two strains of S. thermophilus, all strains displayed strong auto-aggregation level greater than 95% at 24 h incubation. S. thermophilus strains showed higher cell surface hydrophobicity than L. bulgaricus strains. This study demonstrated the isolation, identification, genotypic discrimination and techno-functional features of wild type yoghurt starter cultures which can potentially find place in industrial applications. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01366-2.

Physicochemical Characterization of Dextran HE29 Produced by the Leuconostoc citreum HE29 Isolated from Traditional Fermented Pickle.

In this study, lactic acid bacteria (LAB) strains were isolated from traditional fermented pickles, and among the identified strains, Leuconostoc citreum HE29 with a strong slimy colony profile was further selected to determine the physicochemical and techno-functional properties of its exopolysaccharide (EPS). Glucose was the only sugar monomer in the core unit of EPS HE29 detected by HPLC analysis, and glucan HE29 revealed 7.3 kDa of molecular weight. Structural characterization of glucan HE29 by 1H and 13C NMR spectroscopy analysis demonstrated that EPS HE29 was a dextran-type EPS containing 5.3% levels of (1 → 3)-linked α-D-glucose units. This structural configuration was also supported by FT-IR analysis, which also demonstrated the functional groups within the dextran HE29 structure. In terms of thermal properties detected by TGA and DSC analysis, dextran HE29 demonstrated a degradation temperature of around 280 °C, showing its strong thermal features. A semi-crystalline nature was observed for dextran HE29 detected by XRD analysis. Finally, AFM and SEM analysis revealed tangled network-like properties and web-like branched structures for dextran HE29, respectively. These findings suggest the importance of plant-based fermented products as LAB sources in obtaining novel EPS structures with potential techno-functional roles.

Structural characterization of potato starch modified by a 4,6-α-glucanotransferase B from Lactobacillus reuteri E81.

The recent reports have revealed that increase in amount of α-1,6 linkages by modification of potato starch with enzyme (glycosyltransferases) treatment gains slowly digestible properties to the starch; however, the formation of new α-1,6-glycosidic linkages diminish the thermal resistance of the starch granules. In this study, a putative GtfB-E81, (a 4,6-α-glucanotransferase-4,6-αGT) from L. reuteri E81 was firstly used to produce a short length of α-1,6 linkages. NMR results revealed that external short chains mostly comprised of 1-6 glucosyl units were newly produced in potato starch, and the α-1,6 linkage ratio was significantly increased from 2.9 % to 36.8 %, suggesting that this novel GtfB-E81 might have potentially an efficient transferase activity. In our study, native and GtfB-E81 modified starches showed fundamental similarities with respect to their molecular properties and treatment of native potato starch with GtfB-E81 did not remarkably change thermal stability of the potato starch, which seems to be very prominent for the food industry given the significantly decreased thermal stability results obtained for the enzyme modified starches reported in the literature. Therefore, the results of this study should open up emerging perspectives for regulating slowly digestible characteristics of potato starch in future studies without a significant change in the molecular, thermal, and crystallographic properties.

Utilisation of an active branching sucrase from Lactobacillus kunkeei AP-37 to produce techno-functional poly-oligosaccharides.

Glucansucrase AP-37 was extracted from the culture supernatant of Lactobacillus kunkeei AP-37 and characteristics of the glucan produced by the active glucansucrase in terms of structural and functional roles were determined in this study. A molecular weight around 300 kDa was observed for glucansucrase AP-37 and its acceptor reactions with maltose, melibiose and mannose were also conducted to unveil the prebiotic potential of the poly-oligosaccharides formed via these reactions. The core structure of glucan AP-37 was determined by 1H and 13C NMR and GC/MS analysis which revealed that glucan AP-37 was a highly branched dextran composing of high levels of (1 â†’ 3)-linked α-d-glucose units with low levels of (1 â†’ 2)-linked α-d-glucose units. The structural features of the glucan formed, demonstrated that glucansucrase AP-37 was an α-(1 â†’ 3) branching sucrase. Dextran AP-37 was further characterised by FTIR analysis and XRD analysis demonstrated its amorphous nature. A fibrous compact morphology was observed for dextran AP-37 with SEM analysis whereas TGA and DSC analysis revealed its high stability as no degradation was observed up to 312 °C. Finally, the prebiotic potential of the dextran AP-37 and the gluco-oligosaccharides produced with the acceptor reaction of α-(1 â†’ 3) branching sucrase AP-37 were determined and promising results were found for the gluco-oligosaccharides to act as prebiotics.

Bioactive and technological properties of an α-D-glucan synthesized by Weissella cibaria PDER21.

A slimy-mucinous-type colony of EPS-producing Weissella cibaria PDER21 was isolated and identified. The monomer composition was glucose, showing that the EPS is a glucan type homopolysaccharide, The core structure of (1 â†’ 6)-linked α-d-glucose units including (1 â†’ 3)-linked α-d-glucose branches at a ratio of 93.4/6.6 was revealed by 1H and 13C NMR spectra and confirmed by FTIR analysis. The glucan showed a superior thermal stability with almost no degradation in structure up to 300 °C. XRD analysis revealed the amorphous structure while SEM analysis confirmed the layer-like morphology. The glucan had an antioxidant activity (89.5%), water-holding capacity (103.7%) and water solubility index (80.7%) values, suggesting that the glucan had a strong level of antioxidant properties; good water binding capacity and excellent solubility. The glucan PDER21 is a polysaccharide possessing a good combination of technical and functional attributes, suggesting a great deal of potential for use in the food industry.

Structural and bioactive characteristics of a dextran produced by Lactobacillus kunkeei AK1.

In this study, structural and techno-functional characteristics of an exopolysaccharide (EPS) produced by Lactobacillus kunkeei AK1 were determined. High-performance liquid chromatography (HPLC) analysis demonstrated that EPS AK1 was composed of only glucose units. 1H and 13C Nuclear magnetic resonance (NMR) analysis revealed that EPS AK1 was a dextran type EPS containing 4.78% (1 â†’ 4)-linked α-d-glucose branches. The molecular weight of EPS AK1 was determined to be 45 kDa by Gel Permeation Chromatography (GPC) analysis. A high level of thermal stability up to 280 °C was determined for dextran AK1 detected by Differential scanning calorimetry (DSC) and Thermogravimetric analysis (TGA). Dextran AK1 appeared as regular spheres with compact morphology and as irregular particles in the solution with no clear cross-linking between the chains of the polysaccharide observed by Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) analysis, respectively. X-ray diffraction analysis (XRD) analysis demonstrated that dextran AK1 had a crystalline structure. A relatively strong antioxidant activity was observed for dextran AK1 determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging and cupric reducing antioxidant capacity (CUPRAC) tests. Finally, only a digestion ratio of 3.1% was observed for dextran AK1 following the in vitro simulated gastric digestion test.

Synthesis of alternan-stabilized zinc nanoparticles: morphological, thermal, antioxidant and antimicrobial characterization.

This study aimed to synthesize zinc nanoparticles with antimicrobial and antioxidant activities using alternan (ZnNPs-Alt) produced by Lactobacillus reuteri E81 as the stabilizing agent. For the characterization of the ZnNPs-Alt, UV-vis spectroscopy, SEM, TEM and EDX analysis, XRD, FTIR and DSC analysis were applied. The functional role of ZnNPs-Alt was tested by determination of their antioxidant activities by DPPH and CUPRAC methodologies and their antibacterial and antifungal activities. Results of this study demonstrated that alternan was utilized as a successful stabilizer to produce crystalline, thermally stabile ZnNPs-Alt with a particle size of ∼ 100 nm. Importantly, strong antioxidant and antimicrobial activities were observed for ZnNPs-Alt in a concentration dependent manner. These findings confirmed the role of alternan as a stabilizing agent for the production of ZnNPs-Alt with functional roles.

Synthesis of silver nanoparticles prepared with a dextran-type exopolysaccharide from Weissella cibaria MED17 with antimicrobial functions.

Microbial polysaccharides can find distinct applications as stabilizer agents including synthesis of nanoparticles. In this study, a dextran-type exopolysaccharide (EPS) was used as the stabilizer agent for the green synthesis of silver nanoparticles (AgNPs-Dex) with antimicrobial characteristics. UV-vis spectroscopy analysis was used to test the formation of AgNPs-Dex. The uniform morphology at around 10 nm size was observed for AgNPs-Dex by TEM analysis and importantly EDX analysis demonstrated the embedment of Ag+ ions within dextran as the stabilizer agent. XRD analysis confirmed the crystalline nature of AgNPs-Dex and FTIR analysis demonstrated the interactions of dextran functional groups with silver. DSC and TGA analysis showed the alteration in the thermal stability of AgNPs-Dex compared to the stabilizer dextran. The antibacterial and antifungal activities of AgNPs-Dex were determined against food originated pathogenic bacteria and fungi and important inhibition levels were observed at 1 mg ml-1 concentration of AgNPs-Dex and this activity was observed to be concentration dependent.

Structural and physicochemical characterisation and antioxidant activity of an α-D-glucan produced by sourdough isolate Weissella cibaria MED17.

An exopolysaccharide (EPS) producer slimy-mucoid type colony was isolated from sourdough and identified as Weissella cibaria MED17. The 1H and 13C NMR spectra of EPS MED17 demonstrated that this EPS was a dextran type glucan ((1 â†’ 6)-linked α-D-glucose core structure) containing (1 â†’ 3)-linked α-D-glucose branches and proportion of (1 â†’ 6)-linked α-D-glucose units to (1 â†’ 3)-linked α-D-glucose units was 94.3:5.7%. The FTIR analysis also confirmed the (1 â†’ 6)-linked α-D-glucose linkage. A high level of thermal stability was observed for glucan MED17 as no degradation up to 300 °C was observed by TGA and DSC analysis. The XRD analysis of glucan MED17 showed its semi- crystalline nature and its compact sheet-like morphology was observed by SEM analysis. Finally, antioxidant characteristics of glucan MED17 were determined by ABTS and DPPH radical scavenging activity tests that revealed a moderate antioxidant activity of glucan MED17. These findings show potential techno-functional characteristics of glucan MED17.

Determining the optimum model parameters for oligosaccharide production efficiency using response surface integrated particle swarm optimization method: an experimental validation study.

Glucansucrases (GTFs) catalyzes the synthesis of α-glucans from sucrose and oligosaccharides in the presence of an acceptor sugar by transferring glucosyl units to the acceptor molecule with different linkages. The acceptor reactions can be affected by several parameters and this study aimed to determine the optimal reaction parameters for the production of glucansucrase-based oligosaccharides using sucrose and maltose as the donor and acceptor sugars, respectively via a hybrid technique of Response Surface Method (RSM) and Particle Swarm Optimization (PSO). The experimental design was performed using Central Composite Design and the tested parameters were enzyme concentration, acceptor:donor ratio and the reaction period. The optimization studies showed that enzyme concentration was the most effective parameter for the final oligosaccharides yields. The optimal values of the significant parameters determined for enzyme concentration and acceptor:donor ratio were 3.45 U and 0.62, respectively. Even the response surface plots for input parameters verified the PSO results, an experimental validation study was performed for the reverification. The experimental verification results obtained were also consistent with the PSO results. These findings will help our understanding in the role of different parameters for the production of oligosaccharides in the acceptor reactions of GTFs.

Preparation of gentiobiose-derived oligosaccharides by glucansucrase E81 and determination of prebiotic and immune-modulatory functions.

Gentiobiose-derived oligosaccharides were synthesized by the acceptor reaction of glucansucrase E81 obtained from Lactobacillus reuteri E81 with sucrose and gentiobiose as donor-acceptor sugars, respectively. The reaction products were monitored by TLC analysis and gentiobiose-derived oligosaccharides up to DP 8 were formed during the acceptor reaction as determined by ESI-MS/MS analysis. The glycosylation of the gentiobiose with α-(1 → 6) linkages and α-(1 → 3) linkages was shown by 1H and 13C NMR analysis confirming the structure of these gentiobiose-derived oligosaccharides. The in vitro prebiotic function of the oligosaccharides was determined in which probiotic strains were stimulated whereas no growth was observed in pathogen strains. Gentiobiose-derived oligosaccharides showed immune-modulatory functions in vitro and triggered the production of IL-4, IL12 and TNF-α cytokines in HT29 cells in a dose dependent manner. This study showed the production and functional characterisation of gentiobiose-derived oligosaccharides establishing a promising avenue for future applications.

Characterization of a 4,6­α­glucanotransferase from Lactobacillus reuteri E81 and production of malto-oligosaccharides with immune-modulatory roles.

A wide number of Lactic Acid Bacteria (LAB) species produce α-glucans with their ability to synthesize glucansucrases (GS) which use sucrose as substrate for the glucan production. Recently another group of enzymes in LAB gained special interest for their ability to produce α-glucans targeting the substrates containing α1-4-linkages and synthesizing new (α1-6) or (α1-3)-linkages as α­glucanotransferases. In this study, a putative 4,6­α­glucanotransferase (GTFB) from sourdough isolate Lactobacillus reuteri E81 was identified and expressed in Escherichia coli. The biochemical characterization of the GTFB-E81 confirmed its function as it cleaved the α1-4-linkages in different substrates and produced new gluco-oligomers/polymers containing α1-6 linkages together with the α1-4-linkages detected by NMR analysis. GTFB-E81 produced malto-oligosaccharides targeting maltose and maltoheptaose as substrates with up to DP 8 detected by TLC and ESI-MS/MS analysis. The functional roles of these malto-oligosaccharides were determined by testing their immune-modulatory functions in HT29 cells and they triggered the production of anti-inflammatory 1L-4 and pro-inflammatory IL-12 cytokines.

Glucan type exopolysaccharide (EPS) shows prebiotic effect and reduces syneresis in chocolate pudding.

Exopolysaccharides (EPS) of lactic acid bacteria are important biopolymers that can improve the physicochemical properties of food products and act as prebiotics. In this study the physicochemical role and the prebiotic effects of a glucan type EPS with (α1-3) and (α1-6) linkages were assessed in chocolate pudding containing Lactobacillus rhamnosus GG as a probiotic strain. The functions of EPS were determined by developing three different formulations: control, probiotic (Lactobacillus GG) and symbiotic pudding (Lactobacillus GG + EPS) samples. The pH and acidity of the symbiotic pudding sample were higher than the probiotic and the control samples during the 28-day of storage period. Similarly, an important level of increment in Lactobacillus GG levels in symbiotic sample was observed compared to the probiotic sample suggesting the prebiotic role of the α-glucan. Importantly, the syneresis in symbiotic pudding sample reduced significantly compared to other pudding samples which is related with the physicochemical role of glucan type EPS. This study reveals the prebiotic and physicochemical roles of α-glucan type EPS in a chocolate pudding model.

Characterization of functional properties of Enterococcus faecium strains isolated from human gut.

The aim of this work was to characterize the functional properties of Enterococcus faecium strains identified after isolation from human faeces. Of these isolates, strain R13 showed the best resistance to low pH, bile salts, and survival in the simulated in vitro digestion assay, and demonstrated an important level of adhesion to hexadecane as a potential probiotic candidate. Analysis of the antibiotic resistance of E. faecium strains indicated that in general these isolates were sensitive to the tested antibiotics and no strain appeared to be resistant to vancomycin. Examination of the virulence determinants for E. faecium strains demonstrated that all strains contained the virulence genes common in gut- and food-originated enterococci, and strain R13 harboured the lowest number of virulence genes. Additionally, no strain contained the genes related to cytolysin metabolism and showed hemolytic activity. The antimicrobial role of E. faecium strains was tested against several pathogens, in which different levels of inhibitory effects were observed, and strain R13 was inhibitory to all tested pathogens. PCR screening of genes encoding enterocin A and B indicated the presence of these genes in E. faecium strains. Preliminary characterization of bacteriocins revealed that their activity was lost after proteolytic enzyme treatments, but no alteration in antimicrobial activity was observed at different pHs (3.5 to 9.5) and after heat treatments. In conclusion, this study revealed the functional characteristics of E. faecium R13 as a gut isolate, and this strain could be developed as a new probiotic after further tests.

Impact of exopolysaccharide production on functional properties of some Lactobacillus salivarius strains.

The aim of this work was to characterize functional properties of Lactobacillus salivarius strains isolated from chicken feces. Detection of genes responsible for exopolysaccharide (EPS) production revealed that all strains harbored a dextransucrase gene, but p-gtf gene was only detected in strain E4. Analysis of EPS production levels showed significant alterations among strains tested. Biofilm formation was found to be medium composition dependant, and there was a negative correlation with biofilm formation and EPS production. Autoaggregation properties and coaggregation of L. salivarius strains with chicken pathogens were appeared to be specific at strain level. An increment in bacterial adhesion to chicken gut explants was observed in L. salivarius strains with the reduction in EPS production levels. This study showed that strain-specific properties can determine the functional properties of L. salivarius strains, and the interference of these properties might be crucial for final selection of these strains for technological purposes.