Isolation and Identification of Dominant Bacteria from Raw Donkey Milk Produced in a Region of Morocco by QIIME 2 and Evaluation of Their Antibacterial Activity.

Recently, the interest in donkey milk has increased considerably because it proved high nutritive and functional values of their ingredients. Its chemical composition is widely studied, but its microbiota, especially lactic acid bacteria, remains less studied. This study focuses on analyzing, isolating, and identifying lactic acid bacteria and evaluating their capacity to produce biomolecules with antibacterial activity. Among 44 strains identified, 43 are Gram-positive, and most are catalase-negative and cocci-shaped. Five strains were selected to evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. Different induction methods allowed to amplify the antibacterial effects against these pathogenic strains.

Enzymatic synthesis of non-digestible oligosaccharide catalyzed by dextransucrase and dextranase from maltose acceptor reaction.

Non-digestible oligosaccharides have wide food industrial applications as dietary fibers and prebiotics. The aim of this study is to realize the effective biosynthesis of isomalto-oligosaccharides (IMOs) and reduce the production of by-product dextran. In the presence of acceptors improved the dextransucrase reaction shifting to oligosaccharides formation but a number of by-products dextran appeared. Maltose acceptor performed stronger inhibition behaviors in dextran synthesis than lactose and glucose acceptor due to its higher efficiencies. Acceptors had no influence on the structure of by-product dextran which mainly composed of α-(1,6)-glycosidic linkages and low α-(1,3)-glycosidic branch. In addition, the Mw and contents of IMOs and oligodextrans synthesized by dual-enzyme were hard to control. Addition of maltose acceptor in the dual-enzyme reaction, the adequate dextranase preferentially degraded dextran than the acceptor products to yield the IMOs. Results indicated that the combined use of the dual-enzyme and the maltose acceptor is a simple and effective method to promote the high-quality of functional IMOs.

Quantitative analysis of straight-chain/branched-chain Ratio During Enzymatic Synthesis of Dextran Based on Periodate Oxidation.

The applications of dextran depend not only on the molecular weight but also on the types and number of branches. In this study, dextran generated from Leuconostoc mesenteroides (L.M.CICC-20724) was characterized by fourier-transform infrared spectrum and nuclear magnetic resonance spectroscopy. Our analyses showed that dextran was a polysaccharide composed of d-glucose units with predominantly α(1 â†’ 6) linkages in the main chain and few α(1 â†’ 3) linkages in the branch. Periodate oxidation, a classic chemical method, was usually combined with Smith degradation and gas chromatography to analyze glycosidic linkages in polysaccharide quantitatively. In this study, we calculated the exact straight-chain/branched-chain ratio in the dextran using periodate oxidation only. The ratios obtained by periodate oxidation only were compared to the ratios obtained by nuclear magnetic resonance. The results showed that the ratios of the two groups were nearly equal, and the average relative error between the two groups was 0.83%. This method was evaluated and found to be accurate and stable. This technique provided a convenient and straightforward chemical method for the quantitative analysis of the straight-chains and branched-chains in polysaccharides which had a similar structure. The ratios during the enzymatic synthesis process of dextran were analyzed by this method and were found to be stable with a high level of approximately 95% on average.

Evaluation of the Efficiency of Ethanol Precipitation and Ultrafiltration on the Purification and Characteristics of Exopolysaccharides Produced by Three Lactic Acid Bacteria.

Exopolysaccharides (EPS) produced by three Lactic Acid Bacteria strains, Lactococcus lactis SLT10, Lactobacillus plantarum C7, and Leuconostoc mesenteroides B3, were isolated using two methods: ethanol precipitation (EPS-ETOH) and ultrafiltration (EPS-UF) through a 10 KDa cut-off membrane. EPS recovery by ultrafiltration was higher than ethanol precipitation for Lactococcus lactis SLT10 and Lactobacillus plantarum C7. However, it was similar with both methods for Leuconostoc mesenteroides B3. The monomer composition of the EPS fractions revealed differences in structures and molar ratios between the two studied methods. EPS isolated from Lactococcus lactis SLT10 are composed of glucose and mannose for EPS-ETOH against glucose, mannose, and rhamnose for EPS-UF. EPS extracted from Lactobacillus plantarum C7 and Leuconostoc mesenteroides B3 showed similar composition (glucose and mannose) but different molar ratios. The molecular weights of the different EPS fractions ranged from 11.6±1.83 to 62.4±2.94 kDa. Molecular weights of EPS-ETOH fractions were higher than those of EPS-UF fractions. Fourier transform infrared (FTIR) analysis revealed a similarity in the distribution of the functional groups (O-H, C-H, C=O, -COO, and C-O-C) between the EPS isolated from the three strains.

Comparison Between Fluorescent Probe and Ion-Selective Electrode Methods for Intracellular pH Determination in Leuconostoc mesenteroides.

The intracellular pH (pHin) of Leuconostoc mesenteroides subsp. mesenteroides 19D was evaluated by two different methods, fluorescent probe and ion-selective electrode. Two fluorescent probes 5 (and-6)-carboxyfluorescein diacetate succinimidyl ester (cFDASE) and 5 (and-6)-carboxy-2',7'-dichlorofluorescein diacetate succinimidyl ester (cDCFDASE) were tested to evaluate the intracellular pH (pHin) of living cells at a medium pH (pHex) ranged from 5.0 to 6.5. Salicylic acid was used as a probe for the ion-selective electrode method. Cells kept 60-80% of cFDASE probe at all pHex values against 5-10% of cDCFDASE probe at pHex ≤ 6.0. The pHin values measured by the ion-selective electrode were higher by 0.1-0.6 pH units at pHex ranged from 5.0 to 6.5 than those determinated by fluorescent probe method. The possibility to study the intracellular pH at a wide external pH range using a single probe, and the simplicity of the material and experimental protocol may make the ion-selective electrode method most useful and easy to measure the intracellular pH of lactic acid bacteria compared with the other techniques like fluorescent probes.

Identification and Characterization of l-Malate Dehydrogenases and the l-Lactate-Biosynthetic Pathway in Leuconostoc mesenteroides ATCC 8293.

One putative l-lactate dehydrogenase gene (l- ldh) and three putative d- ldh genes from Leuconostoc mesenteroides ATCC 8293 were overexpressed, and their enzymatic properties were investigated. Only one gene showed d-LDH activity, catalyzing pyruvate and d-lactate interconversion, whereas the other genes displayed l- and d-malate dehydrogenase (MDH) activity, catalyzing oxaloacetate and l- and d-malate interconversion, suggesting that strain ATCC 8293 may not harbor an l- ldh gene. Putative phosphoenolpyruvate carboxylase (PEPC)- and malolactic enzyme (MLE)-encoding genes were identified from strain ATCC 8293, and sequence analysis showed that they could exhibit PEPC and MLE activities, respectively. l-Lactate production and transcriptional expression of the mle gene in this strain were highly increased in the presence of l-malate. We propose that in strain ATCC 8293, which lacks an l- ldh gene, l-lactate is produced through sequential enzymatic conversions from phosphoenolpyruvate to oxaloacetate, then l-malate, and finally l-lactate by PEPC, l-MDH, and MLE, respectively.

Preparing the key metabolite of Z-ligustilide in vivo by a specific electrochemical reaction.

The key in vivo metabolites of a drug play an important role in its efficacy and toxicity. However, due to the low content and instability of these metabolites, they are hard to obtain through in vivo methods. Electrochemical reactions can be an efficient alternative to biotransformation in vivo for the preparation of metabolites. Accordingly, in this study, the metabolism of Z-ligustilide was investigated in vitro by electrochemistry coupled online to mass spectrometry. This work showed that five oxidation products of the electrochemical reaction were detected and that two of the oxidation products (senkyunolide I and senkyunolide H) were identified from liver microsomal incubation as well. Furthermore, after intragastric administration of Z-ligustilide in rats, senkyunolide I and senkyunolide H were detected in the rat plasma and liver, while 6,7-epoxyligustilide, a key intermediate metabolite of Z-ligustilide, was difficult to detect in vivo. By contrast, 6,7-epoxyligustilide was obtained from the electrochemical reaction. In addition, for the first time, 6 mg of 6,7-epoxyligustilide was prepared from 120 mg of Z-ligustilide. Therefore, electrochemical reactions represent an efficient laboratory method for preparing key drug metabolites.

Optimization, chain conformation and characterization of exopolysaccharide isolated from Leuconostoc mesenteroides DRP105.

Response surface methodology (RSM) was used to optimize the fermentation condition of exopolysaccharide (EPS) producing strain Leuconostoc mesenteroides DRP105. Result showed that the optimum condition was sucrose 86.83g/L, tryptone 15.47g/L, initial pH7.18 and maximum yield was 53.79±0.78g/L in 36h fermentation. Chain conformation was characterized by Congo red test, ß-elimination and circular dichroism (CD), which indicated that the EPS was O-linkage and exhibited random coil structure in aqueous solution. CD results concluded hydrogen-bond interaction and chirality might be connected with concentration. Purified EPS has a higher degradation temperature of 279.42°C, suggesting high thermal stability of the EPS. The absolute value of zeta potential and particle size were enhanced with increasing concentration. Crude EPS and its purified fraction were found to have moderate DPPH, hydroxyl, superoxide anion radicals scavenging activities and reducing power. This study provided a high yield EPS with unique characteristics for industrial applications.

Response surface methodology for optimisation of edible coatings based on dextran from Leuconostoc mesenteroides T3.

The aim of this study was to develop dextran-based edible films plasticized by sorbitol. In order to optimise the film-forming formulation, response surface methodology was used. The influence of dextran and sorbitol concentration on the mechanical and water vapour barrier properties of obtained films was investigated. The results showed that both parameters exhibited significant effect on the water vapour permeability of a film. Both dextran and sorbitol concentration had significant influence on tensile strength and elongation at break, whereas only sorbitol concentration had significant effect on Young's modulus. After optimisation by desirability approach, it was found that a film made of 3.40 wt% of dextran and 20.43 wt% of sorbitol showed the lowest water vapour permeability and the highest tensile strength and elasticity.

The effect of eubiotic feed additives on the performance of growing pigs and the activity of intestinal microflora.

The aim of this study was to compare the effect of probiotic bacteria, prebiotics, phytobiotics and their combinations on performance and microbial activity in the digestive tract of growing pigs. The experiment was conducted over 28 d on 48 male pigs of about 12 kg body weight (BW), which were allocated to following treatments.: (1) Control Group (Con) without additive, (2) Group I, addition of a prebiotic (inulin), (3) Group Ph, a phytobiotic (herbal water extracts), (4) Group P, a probiotic composed of four strains of lactic acid bacteria, (5) Group PhP, phytobiotic and probiotic bacteria and (6) Group PhPI, a phytobiotic, probiotic bacteria and a prebiotic. Animal performance was recorded and at d 28 six pigs from each group were euthanised to collect digesta samples. In all groups except for Group I, diarrhoea incidents were observed. Groups Ph and P had significantly higher daily gains and final BW, and Group Ph utilised feed better than other groups. The pH of ileal digesta was significantly lower in Group PhPI. In the caecal digesta of Groups I, P and PhP, the pH level was lower than in the other groups but dry matter contents was significantly higher in Groups Con and I. The short-chain fatty acids and particular acid content differed significantly only in the colonic digesta. The yeast and mould numbers in caecal digesta was highest in Group Con. No treatment effects were observed for the number of lactic acid bacteria, coli group bacteria or Clostridium. However, the observed significantly higher number of total bacteria suggests that a multi-component eubiotic treatment changes the bacterial composition and distribution more effectively. Our findings indicated that all used additives changed the intestinal microflora, but the multi-component eubiotics were not beneficial as feed additives offered separately. Moreover, supplementation of phytobiotics and probiotic bacteria also improved the animal performance significantly.

Dextran coated silver nanoparticles - Chemical sensor for selective cysteine detection.

A simple, fast and non-costly method for selective cysteine (Cys) detection, based on optical changes of silver colloids, is developed. For that purpose, stable colloids consisting of silver nanoparticles (Ag NPs) coated with polysaccharide dextran (Dex), isolated from bacterium species Leuconostoc mesenteroides T3, were prepared. The synthesized samples were thoroughly characterized including absorption and FTIR spectroscopy, as well as transmission electron microscopy and X-ray diffraction analysis. The silver colloids display high sensitivity and selectivity towards Cys detection in aqueous solutions. The Ag NPs coated with Dex provide possibility to detect Cys among a dozen amino acids and its detection limit was found to be 12.0µM. The sensing mechanism - red shift of optical absorption - is discussed in terms of the agglomeration of Ag NPs due to formation of hydrogen bonds between Cys molecules attached to different Ag NPs.

Peroxyl radical- and photo-oxidation of glucose 6-phosphate dehydrogenase generates cross-links and functional changes via oxidation of tyrosine and tryptophan residues.

Protein oxidation is a frequent event as a result of the high abundance of proteins in biological samples and the multiple processes that generate oxidants. The reactions that occur are complex and poorly understood, but can generate major structural and functional changes on proteins. Current data indicate that pathophysiological processes and multiple human diseases are associated with the accumulation of damaged proteins. In this study we investigated the mechanisms and consequences of exposure of the key metabolic enzyme glucose-6-phosphate dehydrogenase (G6PDH) to peroxyl radicals (ROO•) and singlet oxygen (1O2), with particular emphasis on the role of Trp and Tyr residues in protein cross-linking and fragmentation. Cross-links and high molecular mass aggregates were detected by SDS-PAGE and Western blotting using specific antibodies. Amino acid analysis has provided evidence for Trp and Tyr consumption and formation of oxygenated products (diols, peroxides, N-formylkynurenine, kynurenine) from Trp, and di-tyrosine (from Tyr). Mass spectrometric data obtained after trypsin-digestion in the presence of H216O and H218O, has allowed the mapping of specific cross-linked residues and their locations. These data indicate that specific Tyr-Trp and di-Tyr cross-links are formed from residues that are proximal and surface-accessible, and that the extent of Trp oxidation varies markedly between sites. Limited modification at other residues is also detected. These data indicate that Trp and Tyr residues are readily modified by ROO• and 1O2 with this giving products that impact significantly on protein structure and function. The formation of such cross-links may help rationalize the accumulation of damaged proteins in vivo.

Structural characterization of the immunostimulatory exopolysaccharide produced by Leuconostoc mesenteroides strain NTM048.

The exopolysaccharide (EPS) produced by probiotic Leuconostoc mesenteroides subsp. mesenteroides strain NTM048 has been reported to be an immunostimulant that enhances mucosal IgA production. In this study, we found that intranasal administration of mice with the EPS and an antigen (ovalbumin) resulted in secretion of antigen-specific IgA and IgG in the airway mucosa and the serum, suggesting that the EPS has the adjuvant activity for use with mucosal vaccination. Methylation analysis coupled to GC-MS, and 1D and 2D NMR spectroscopy revealed that 94% of the EPS consists of an α-(1 â†’ 6) glucan containing 4% of 1→3-linked α-glucose branches. To determine structures of minor components, we enzymatically digested the glucan with dextranase and used 2D NMR spectroscopy to identify the remaining polymer as a fructan (or fructans), containing both ß-(2 â†’ 6)- and ß-(2 â†’ 1)-linked fructofuranose residues. These residues may either enter into separate polymers of each linkage type or form a mixed fructan containing both linkage types.

Enzymatic Production of Melibiose from Raffinose by the Levansucrase from Leuconostoc mesenteroides B-512 FMC.

Melibiose, which is an important reducing disaccharide formed by α-1,6 linkage between galactose and glucose, has been proven to have beneficial applications in both medicine and agriculture. In this study, a characterized levansucrase from Leuconostoc mesenteroides B-512 FMC was further used to study the bioproduction of melibiose from raffinose. The reaction conditions were optimized for melibiose synthesis. The optimal pH, temperature, substrate concentration, ratio of substrates, and units of enzymes were determined as pH 6.0, 45 °C, 210 g/L, 1:1 (210 g/L:210 g/L), and 5 U/mL, respectively. The transfructosylation product of raffinose was determined to be melibiose by FTIR and NMR. A high raffinose concentration was found to strongly favor the production of melibiose. When 210 g/L raffinose and 210 g/L lactose were catalyzed using 5 U/mL purified levansucrase at pH 6.0 and 45 °C, the maximal yield of melibiose was 88 g/L.

Macromolecular and Elemental Composition Analyses of Leuconostoc mesenteroides ATCC 8293 Cultured in a Chemostat.

The cellular composition and metabolic compounds of Leuconostoc mesenteroides ATCC 8293 were analyzed after cultivation in an anaerobic chemostat. The macromolecular composition was 24.4% polysaccharide, 29.7% protein, 7.9% lipid, 2.9% DNA, and 7.4% RNA. Its amino acid composition included large amounts of lysine, glutamic acid, alanine, and leucine. Elements were in the order of C > O > N > H > S. The metabolites in chemostat culture were lactic acid (73.34 mM), acetic acid (7.69 mM), and mannitol (9.93 mM). These data provide a first view of the cellular composition of L. mesenteroides for use in metabolic flux analysis.

Isomelezitose formation by glucansucrases.

Several glucansucrases were surveyed for their ability to produce isomelezitose, a trisaccharide with the structure α-D-glucopyranosyl (1 â†’ 6) ß-D-fructofuranosyl (2 â†” 1) α-D-glucopyranoside. Nearly all strains tested, with one exception, produced at least trace levels of isomelezitose. Yields were low but significant, ranging from less than 1% to approximately 5% based on sucrose. This trisaccharide may arise in either of two ways: glucopyranosyl transfer to the 6Fru-OH position of sucrose, or to the anomeric OH position of isomaltulose. This study indicates that isomelezitose formation may be a general phenomenon of many glucansucrase reactions.

Dextran: Influence of Molecular Weight in Antioxidant Properties and Immunomodulatory Potential.

Dextrans (α-d-glucans) extracted from Leuconostoc mesenteroides, with molecular weights (MW) of 10 (D10), 40 (D40) and 147 (D147) kDa, were evaluated as antioxidant, anticoagulant and immunomodulatory drugs for the first time. None presented anticoagulant activity. As for the antioxidant and immunomodulatory tests, a specific test showed an increase in the dextran activity that was proportional to the increase in molecular weight. In a different assay, however, activity decreased or showed no correlation to the MW. As an example, the reducing power assay showed that D147 was twice as potent as other dextrans. On the other hand, all three samples showed similar activity (50%) when it came to scavenging the OH radical, whereas only the D10 sample showed sharp activity (50%) when it came to scavenging the superoxide ion. D40 was the single dextran that presented with immunomodulatory features since it stimulated the proliferation (~50%) of murine macrophages (RAW 264.7) and decreased the release of nitric oxide (~40%) by the cells, both in the absence and presence of lipopolysaccharides (LPS). In addition, D40 showed a greater scavenging activity (50%) for the hydrogen peroxide, which caused it to also be the more potent dextran when it came to inhibiting lipid peroxidation (70%). These points toward dextrans with a 40 kDa weight as being ideal for antioxidant and immunomodulatory use. However, future studies with the D40 and other similarly 40 kDa dextrans are underway to confirm this hypothesis.

Purification and characterization of a bacteriocin from an oenological strain of Leuconostoc mesenteroides subsp. cremoris.

Malolactic fermentation (MLF), which improves organoleptic properties and biologic stability of some wines, may cause wine spoilage if uncontrolled. Bacteriocins were reported as efficient preservatives to control MLF through their bactericidal effect on malolactic bacteria. Leuconostoc mesenteroides subsp. cremoris W3 isolated from wine produces an inhibitory substance that is bactericidal against malolactic bacteria in model wine medium. Treatment of the culture supernatant of strain W3 with proteases eliminated the inhibitory activity, which proved that it is a true bacteriocin and we tentatively termed it mesentericin W3. The bacteriocin inhibited the growth of food-borne pathogenic bacteria such as Enterococcus faecalis, Listeria monocytogenes, and malolactic bacteria. It was active over a wide pH range and stable to organic solvents and heat. Mesentericin W3 was purified to homogeneity by a pH-mediated cell adsorption-desorption method, cation exchange, hydrophobic interaction, and reverse-phase chromatography. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectroscopy (MS) and partial amino acid sequence analysis revealed that mesentericin W3 was identical to mesentericin Y105.