Comparison of SYBR® Green qPCR assay and plate count method to describe growth of Weissella viridescens and Leuconostoc mesenteroides in pure and mixed cultivation.

The current study was conducted to statistically compare the SYBR® Green quantitative polymerase chain reaction (qPCR) assay and the conventional plate counting (PC) method to construct growth curves of a cocktail of Weissella viridescens in pure culture under different isothermal storage conditions (4, 8, 14, and 30 °C) and in mixed culture with Leuconostoc mesenteroides at 8 °C. The efficiency and specificity of the qPCR standard curves were confirmed, and both methods were adequate to quantify the growth kinetics of W. viridescens at all isothermal temperatures, demonstrating a good correlation and agreement. The efficiencies of the standard curves varied between 98% and 102%. The SYBR® Green qPCR assay was also able to differentiate the growth curves of W. viridescens and L. mesenteroides in the mixed culture at 8 °C. Additionally, the SYBR® Green qPCR method was considered a faster and more sensitive alternative to construct growth curves under different isothermal conditions and differentiate morphologically similar lactic acid bacteria. Overall, the results suggest that the SYBR® Green qPCR method is a reliable and efficient tool to study microbial growth kinetics in pure and mixed cultures.

Leuconostoc mesenteroides and Liquorilactobacillus mali strains, isolated from Algerian food products, are producers of the postbiotic compounds dextran, oligosaccharides and mannitol.

Six lactic acid bacteria (LAB) isolated from Algerian sheep's milk, traditional butter, date palm sap and barley, which produce dextran, mannitol, oligosaccharides and vitamin B2 have been characterized. They were identified as Leuconostoc mesenteroides (A4X, Z36P, B12 and O9) and Liquorilactobacillus mali (BR201 and FR123). Their exopolysaccharides synthesized from sucrose by dextransucrase (Dsr) were characterized as dextrans with (1,6)-D-glucopyranose units in the main backbone and branched at positions O-4, O-2 and/or O-3, with D-glucopyranose units in the side chain. A4X was the best dextran producer (4.5 g/L), while the other strains synthesized 2.1-2.7 g/L. Zymograms revealed that L. mali strains have a single Dsr with a molecular weight (Mw) of ~ 145 kDa, while the Lc. mesenteroides possess one or two enzymes with 170-211 kDa Mw. As far as we know, this is the first detection of L. mali Dsr. Analysis of metabolic fluxes from sucrose revealed that the six LAB produced mannitol (~ 12 g/L). The co-addition of maltose-sucrose resulted in the production of panose (up to 37.53 mM), an oligosaccharide known for its prebiotic effect. A4X, Z36P and B12 showed dextranase hydrolytic enzymatic activity and were able to produce another trisaccharide, maltotriose, which is the first instance of a dextranase activity encoded by Lc. mesenteroides strains. Furthermore, B12 and O9 grew in the absence of riboflavin (vitamin B2) and synthesized this vitamin, in a defined medium at the level of ~ 220 µg/L. Therefore, these LAB, especially Lc. mesenteroides B12, are good candidates for the development of new fermented food biofortified with functional compounds.

Leuconostoc performance in soy-based fermentations - Survival, acidification, sugar metabolism, and flavor comparisons.

Leuconostoc spp. is often regarded as the flavor producer, responsible for the production of acetoin and diacetyl in dairy cheese. In this study, we investigate seven plant-derived Leuconostoc strains, covering four species, in their potential as a lyophilized starter culture for flavor production in fermented soy-based cheese alternatives. We show that the process of lyophilization of Leuconostoc can be feasible using a soy-based lyoprotectant, with survivability up to 63% during long term storage. Furthermore, the storage in this media improves the subsequent growth in a soy-based substrate in a strain specific manner. The utilization of individual raffinose family oligosaccharides was strain dependent, with Leuconostoc pseudomesenteroides NFICC99 being the best consumer. Furthermore, we show that all investigated strains were able to produce a range of volatile flavor compounds found in dairy cheese products, as well as remove certain dairy off-flavors from the soy-based substrate like hexanal and 2-pentylfuran. Also here, NFICC99 was strain producing most cheese-related volatile flavor compounds, followed by Leuconostoc mesenteroides NFICC319. These findings provide initial insights into the development of Leuconostoc as a potential starter culture for plant-based dairy alternatives, as well as a promising approach for generation of stable, lyophilized cultures.

Leuconostoc Citreum Inhibits Adipogenesis and Lipogenesis by Inhibiting p38 MAPK/Erk 44/42 and Stimulating AMPKα Signaling Pathways.

Probiotics provide a range of health benefits. Several studies have shown that using probiotics in obesity treatment can reduce bodyweight. However, such treatments are still restricted. Leuconostoc citreum, an epiphytic bacterium, is widely used in a variety of biological applications. However, few studies have investigated the role of Leuconostoc spp. in adipocyte differentiation and its molecular mechanisms. Therefore, the objective of this study was to determine the effects of cell-free metabolites of L. citreum (LSC) on adipogenesis, lipogenesis, and lipolysis in 3T3-L1 adipocytes. The results showed that LSC treatment reduced the accumulation of lipid droplets and expression levels of CCAAT/ enhancer-binding protein-α & ß (C/EBP-α & ß), peroxisome proliferator-activated receptor-γ (PPAR-γ), serum regulatory binding protein-1c (SREBP-1c), adipocyte fatty acid binding protein (aP2), fatty acid synthase (FAS), acetyl CoA carboxylase (ACC), resistin, pp38MAPK, and pErk 44/42. However, compared to control cells, adiponectin, an insulin sensitizer, was elevated in adipocytes treated with LSC. In addition, LSC treatment increased lipolysis by increasing pAMPK-α and suppressing FAS, ACC, and PPAR-γ expression, similarly to the effects of AICAR, an AMPK agonist. In conclusion, L. citreum is a novel probiotic strain that can be used to treat obesity and its associated metabolic disorders.

Microbial communities and metabolite profiles during the fermentation of Chinese Dongbei suancai with Chinese baijiu as supplementary material.

BACKGROUND: In industrial production of suancai, baijiu is commonly used to inhibit the spoilage bacteria and enhance the flavor. However, the effects of baijiu on the microbial diversity and metabolic pathways of suancai are rarely reported in the literature. This study aimed to explore the microbial community, its predicted functional roles, and the metabolites formed during fermentation of Chinese Dongbei suancai fermented using a mixed starter with Chinese baijiu as supplementary material. RESULTS: Results showed that Lactobacillus, Enterobacter, and Leuconostoc were the major bacterial genera in the Dongbei suancai fermented by adding baijiu. Linear discriminant analysis effect size indicated that Leuconostoc was the major biomarker in the early stage of fermentation, whereas Lactococcus, Weissella, and Lactobacillus plantarum were biomarkers in the middle and later stages of fermentation. A total of 638 metabolites were detected in suancai fermented by adding baijiu. However, the principal component analysis showed that baijiu significantly affected the metabolites of suancai in the early and later stages of fermentation. Furthermore, 58, 22, and 26 significantly differential metabolites (P < 0.01) were found on day 0, day 2, and day 30 of fermentation respectively. Moreover, Lactobacillus, Lactococcus, and Enterobacter had positive correlations with amino acids, nucleotides, organic acids, alcohols, and esters. Functional analysis implied that carbohydrate, amino acid, energy, and nucleotide metabolism were the major determinants of the characteristics of suancai fermented with baijiu as supplementary material. CONCLUSION: Baijiu changed the metabolites of inoculated fermented Dongbei suancai. © 2023 Society of Chemical Industry.

Pangenome and genomic taxonomy analyses of Leuconostoc gelidum and Leuconostoc gasicomitatum.

BACKGROUND: Leuconostoc gelidum and Leuconostoc gasicomitatum have dual roles in foods. They may spoil cold-stored packaged foods but can also be beneficial in kimchi fermentation. The impact in food science as well as the limited number of publicly available genomes prompted us to create pangenomes and perform genomic taxonomy analyses starting from de novo sequencing of the genomes of 37 L. gelidum/L. gasicomitatum strains from our culture collection. Our aim was also to evaluate the recently proposed change in taxonomy as well as to study the genomes of strains with different lifestyles in foods. METHODS: We selected as diverse a set of strains as possible in terms of sources, previous genotyping results and geographical distribution, and included also 10 publicly available genomes in our analyses. We studied genomic taxonomy using pairwise average nucleotide identity (ANI) and calculation of digital DNA-DNA hybridisation (dDDH) scores. Phylogeny analyses were done using the core gene set of 1141 single-copy genes and a set of housekeeping genes commonly used for lactic acid bacteria. In addition, the pangenome and core genome sizes as well as some properties, such as acquired antimicrobial resistance (AMR), important due to the growth in foods, were analysed. RESULTS: Genome relatedness indices and phylogenetic analyses supported the recently suggested classification that restores the taxonomic position of L. gelidum subsp. gasicomitatum back to the species level as L. gasicomitatum. Genome properties, such as size and coding potential, revealed limited intraspecies variation and showed no attribution to the source of isolation. The distribution of the unique genes between species and subspecies was not associated with the previously documented lifestyle in foods. None of the strains carried any acquired AMR genes or genes associated with any known form of virulence. CONCLUSION: Genome-wide examination of strains confirms that the proposition to restore the taxonomic position of L. gasicomitatum is justified. It further confirms that the distribution and lifestyle of L. gelidum and L. gasicomitatum in foods have not been driven by the evolution of functional and phylogenetic diversification detectable at the genome level.

Postbiotic metabolites, antioxidant and anticancer activities of probiotic Leuconostoc pseudomesenteroides strains in natural pickles.

In this study, five strains of Leuconostoc pseudomesenteroides were thought to have probiotic properties and anticancer activity isolated from natural pickles and identified by performing the 16S rRNA sequence analysis. The probiotic properties, postbiotic amounts, the capacity to adhere to the L-929, HT-29 and Caco-2 cell lines, the effects of postbiotic and bacterial extracts on cell viability and biochemical activities were investigated in the strains. In the results, Leu. pseudomesenteroides Y6 strain was detected to have the best resistance to the stomach and intestinal environments, and the quantities of postbiotic metabolites are similar to each other. The bacterial adhesion capacities were found to be in the range of 1.66-8.5%. Furthermore, postbiotic metabolites of all isolates had good anticancer activity (27.67-86.05%) and the activity of bacterial extractions increased depending on concentration. Leu. pseudomesenteroides Y4 and Y6 strains generally showed better activity than controls and all strains were strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavengers in the antioxidant studies. In conclusion, the Y6 strain, which had the best probiotic feature, was found to show significantly good biological activity. It is thought that this isolate will be supported by new in vivo studies and eventually be brought to the food and health industry.

Comparative genomics analysis of genus Leuconostoc resolves its taxonomy and elucidates its biotechnological importance.

Genus Leuconostoc consists of a diverse range of lactic acid bacteria (LAB) from dairy, food and environmental ecology. Even though the species of Leuconostoc are commercially significant, their taxonomy is largely based on old, low-resolution classical methods. Several taxonomic reclassifications in the past were inadequate for microbiologist and food industry professionals to demarcate any new strain of genus Leuconostoc. The current taxonomy of the genus is largely based on classical approaches, which are in utmost need of reinvestigation by whole genome-based approaches. In the present study, the taxono-phylogenomic analysis depicted sixteen species, including three novel genomospecies and several reshufflings across the species, namely, L. mesenteroides, L. pseudomesenteroides, L. gelidum and L. lactis. Genus-wide T3PKS, CAZymes, and vector plasmids supports its biotechnological potential. However, detection of the antibiotic resistance genes in such an important LAB genus raises concern over their utility in industry. Present, large-scale in-depth genome-based study can shed light on the genome dynamics of the member species, help to obtain a more robust taxonomy and elucidate its biotechnology importance.

Pilot-scale production of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 and its application in set yogurt.

Exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a linear dextran that is built by glucose units via α-1,6 glycosidic bond. The primary objective of this study was to investigate the yield of XG5 EPS and its application in set yogurt. In laboratory scale, the culture conditions of XG5 EPS production were optimized using the L9 (33) orthogonal test. Here, the optimized yield of XG5 EPS was 26.02 g/L under the conditions of 100 g/L sucrose, initial pH 7.0, 25°C incubation, and 100 rpm for 36 h in a shaking flask. Based on the optimized parameters of laboratory scale, a pilot fed-batch fermentation was performed in a 50-L bioreactor with an adjusted agitation speed of 20 rpm. The XG5 EPS yield reached 40.07 g/L in fed-batch fermentation, which was 54% higher than that achieved in laboratory scale. In addition, XG5 EPS was added into set yogurt to investigate its effect on the stability of set yogurt. Our data demonstrated that the XG5 EPS improved the water-holding capacity, texture profile, and viscosity of set yogurt during cold storage compared with the controls. In particular, addition of 0.5% XG5 EPS increased the structure of 3-dimensional network of set yogurt, which eventually improved the physical stability of the set yogurt. Overall, this study provided new insights for exploring the pilot scale production and application of dextran.

Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides Strain NTM048.

Exopolysaccharides (EPSs) occur widely in natural products made by bacteria, fungi and algae. Some EPSs have intriguing biological properties such as anticancer and immunomodulatory activities. Our group has recently found that EPSs generated from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 (NTM048 EPS) enhanced a production of mucosal immunoglobulin A (IgA) of mouse. Herein, we described the synthesis and evaluation of the tetrasaccharide fragments of NTM048 EPS to obtain information about the molecular mechanism responsible for the IgA-inducing activity.

Engineering of Leuconostoc citreum for Efficient Bioconversion of Soy Isoflavone Glycosides to Their Aglycone Forms.

Soy isoflavones are phytochemicals that possess various beneficial physiological properties such as anti-aging, anti-tumor, and antioxidant properties. Since soy isoflavones exist in glycoside forms, their bioavailability requires initial hydrolysis of the sugar moieties bound to them to be efficiently absorbed through the gut epithelium. Instead of conventional chemical hydrolysis using acids or organic solvents, alternative strategies for enhancing the bioavailability of soy isoflavones using biological methods are gaining attention. Here, we engineered Leuconostoc citreum isolated from Korean kimchi for efficient bioconversion of soy isoflavone glycosides into their aglycone forms to enhance their bioavailability. We first constructed an expression module based on the isoflavone hydrolase (IH)-encoding gene of Bifidobacterium lactis, which mediates conversion of isoflavone glycosides to aglycone forms. Using a high copy number plasmid and bicistronic expression design, the IH was successfully synthesized in L. citreum. Additionally, we determined enzymatic activity of the IH using an in vivo ß-glucosidase assay and confirmed its highly efficient bioconversion efficiency for various types of isoflavone glycosides. Finally, we successfully demonstrated that the engineered L. citreum could convert isoflavone glycosides present in fermented soymilk into aglycones.

Statistical optimization of exopolysaccharide production by Leuconostoc pseudomesenteroides JF17 from native Atlantic Forest juçara fruit.

Leuconostoc pseudomesenteroides belongs to a group of lactic acid bacteria normally isolated from fruits, which has the capacity to produce exopolysaccharides (EPS). The present study aimed to optimize the EPS production of L. pseudomesenteroides JF17, isolated from juçara fruits (palm trees threatened with extinction in the Atlantic Forest), using the response surface methodology (RSM), besides evaluating the fermentation kinetics. The maximum production of EPS 53.77 mg/mL was obtained under ideal conditions of MRS broth supplemented with sucrose at 18%, w/v, fermentation temperature of 20 °C and initial pH of 7.30. The Luedeking-Piret model suggested that the production of EPS by the JF17 strain appeared to be associated with the cell growth of the microorganism, in addition to having high efficiency in the production of the polysaccharide from the substrate (Yp/s = 17.85 ± 0.74 mg EPS/log CFU ). Thus, the ideal optimization conditions and kinetic parameters can be useful for increasing the scale up of the fermentation process in the industrial production of EPS by L. pseudomesenteroides JF17.

Long-term drench of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 protects against type 1 diabetes of NOD mice via stimulating GLP-1 secretion.

BACKGROUND: Type 1 diabetes is an autoimmune disease that results in the specific destruction of insulin-producing beta cells in the pancreas. The aim of this study was to investigate the mechanism of exopolysaccharide from Leuconostoc pseudomesenteroides XG5 (XG5 EPS) against type 1 diabetes. RESULTS: Long-term drench of XG5 EPS delayed the onset of autoimmune diabetes and had fewer islets with high-grade infiltration (an insulitis score of 3 or 4) than untreated NOD mice. Oral administration of 50 mg kg-1  d-1 XG5 EPS increased the insulin and glucagon-like peptide-1 (GLP-1) levels of serum, stimulated GLP-1 secretion and upregulated gcg mRNA expression of colon in NOD mice. Moreover, oral administration of 50 mg kg-1  d-1 XG5 EPS significantly increased total short-chain fatty acids levels in the colon contents, especially those of acetic acid and butyric acid. In NCI-H716 cells, 500 and 1000 µmol L-1 sodium butyrate promoted the secretion of GLP-1 and upregulated the mRNA expression of gcg and PC3, while XG5 EPS and sodium acetate did not stimulate the GLP-1 secretion. Therefore, long-term drench of XG5 EPS delayed the onset of autoimmune diabetes, which may be directly correlated with the increase of butyrate in the colon of NOD mice. CONCLUSION: Long-term drench of 50 mg kg-1  d-1 XG5 EPS promoted the expression and secretion of GLP-1 by increasing the production of butyric acid, thereby delaying T1D onset in NOD mice. © 2021 Society of Chemical Industry.

In vitro and genetic screening of probiotic properties of lactic acid bacteria isolated from naturally fermented cow-milk and yak-milk products of Sikkim, India.

A total of 272 isolates of lactic acid bacteria (LAB) were isolated from 22 samples of naturally fermented milk products of Sikkim in India viz. dahi, soft-variety chhurpi, hard-variety chhurpi, mohi and philu, out of which, 68 LAB isolates were randomly grouped on the basis of phenotypic characteristics, and were identified by 16S rRNA gene sequence analysis. Leuconostoc mesenteroides was the most dominant genus, followed by Leuc. mesenteroides subsp. jonggajibkimchii, Lactococcus lactis subsp. cremoris, Lc. lactis, Lc. lactis subsp. hordniae, Lc. lactis subsp. tructae, Enterococcus faecalis, E. italicus and E. pseudoavium. LAB strains were tested for probiotics attributes by in vitro and genetic screening, based on marker genes. LAB strains showed tolerance to pH 3.0, bile salt, resistance to lysozyme and ß-galactosidase activity. Enterococcus faecalis YS4-11 and YS4-14 and Lactococcus lactis subsp. cremoris SC3 showed more than 85% of hydrophobicity. Genes clp L and tdc encoding for low pH tolerance, agu A and Ir1516 encoding for bile tolerance, LBA1446 gene encoding for BSH activity, map A, apf, mub 1 and msa encoding for mucosal binding property were detected. Gene mesY for bacteriocin production was detected only in Leuconostoc spp. Based on the in vitro and genetic screening of probiotic attributes, Leuc. mesenteroides; Leuc. mesenteroides subsp. jonggajibkimchii and Lc. lactis subsp. cremoris were tentatively selected for possible probiotic candidates.

A specific oligosaccharide-binding site in the alternansucrase catalytic domain mediates alternan elongation.

Microbial α-glucans produced by GH70 (glycoside hydrolase family 70) glucansucrases are gaining importance because of the mild conditions for their synthesis from sucrose, their biodegradability, and their current and anticipated applications that largely depend on their molar mass. Focusing on the alternansucrase (ASR) from Leuconostoc citreum NRRL B-1355, a well-known glucansucrase catalyzing the synthesis of both high- and low-molar-mass alternans, we searched for structural traits in ASR that could be involved in the control of alternan elongation. The resolution of five crystal structures of a truncated ASR version (ASRΔ2) in complex with different gluco-oligosaccharides pinpointed key residues in binding sites located in the A and V domains of ASR. Biochemical characterization of three single mutants and three double mutants targeting the sugar-binding pockets identified in domain V revealed an involvement of this domain in alternan binding and elongation. More strikingly, we found an oligosaccharide-binding site at the surface of domain A, distant from the catalytic site and not previously identified in other glucansucrases. We named this site surface-binding site (SBS) A1. Among the residues lining the SBS-A1 site, two (Gln700 and Tyr717) promoted alternan elongation. Their substitution to alanine decreased high-molar-mass alternan yield by a third, without significantly impacting enzyme stability or specificity. We propose that the SBS-A1 site is unique to alternansucrase and appears to be designed to bind alternating structures, acting as a mediator between the catalytic site and the sugar-binding pockets of domain V and contributing to a processive elongation of alternan chains.

Levan from Leuconostoc citreum BD1707: production optimization and changes in molecular weight distribution during cultivation.

BACKGROUND: Levan is a well-known homopolymer of fructose composed predominantly of ß-(2, 6) fructofuranosyl linkages in the backbone with occasional ß-(2, 1) linkages in the branch chains with varied applications. However, high production cost due to low yield of microbial levan has become a bottleneck for its practical applications. Furthermore, factors affecting the molecular mass of the synthesized levan by Leuconostoc spp. during prolonged cultivation is not fully elucidated. METHODS: The cultivation condition for Leuconostoc citreum BD1707 to synthesize levan was optimized by single-factor experiments and subsequently with response surface methodology (RSM). The average molecular weight (Mw) of levan synthesized by the strain L.citreum BD1707 under the optimized cultivation conditions was monitored by high-performance size exclusion chromatography (HPSEC). Finally, the enzyme with levan-degrading activity was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The levan yield of BD1707 reached 34.86 g/L with a corresponding productivity of 7.47 g/L/d under the optimal cultivation conditions deduced by RSM, i.e., cultivation at 26 °C and 200 rpm for 112 h in tomato juice supplemented with 172 g/L sucrose with an initial pH value of 6.12. The Mw of levan reached a peak value of 2.320 × 107 Da at 6 h of cultivation under the optimized cultivation conditions and then gradually decreased to 8.809 × 106 Da after 120 h of cultivation. CONCLUSION: The levan yield of the strain L.citreum BD1707 could be sufficiently enhanced via cultivation condition optimization. The decrease in molecular mass of the synthesized levan was attributed predominantly to the hydrolytic activity of levansucrase secreted by L.citreum BD1707 during cultivation, with an estimated Mw of 130 KD by SDS-PAGE, while the effect of acid hydrolysis could be nearly neglected.

Systematic engineering of Saccharomyces cerevisiae for D-lactic acid production with near theoretical yield.

D-lactic acid is a chiral three-carbon organic acid that can improve the thermostability of polylactic acid. Here, we systematically engineered Saccharomyces cerevisiae to produce D-lactic acid from glucose, a renewable carbon source, at near theoretical yield. Specifically, we screened D-lactate dehydrogenase (DLDH) variants from lactic acid bacteria in three different genera and identified the Leuconostoc pseudomesenteroides variant (LpDLDH) as having the highest activity in yeast. We then screened single-gene deletions to minimize the production of the side products ethanol and glycerol as well as prevent the conversion of D-lactic acid back to pyruvate. Based on the results of the DLDH screening and the single-gene deletions, we created a strain called ASc-d789M which overexpresses LpDLDH and contains deletions in glycerol pathway genes GPD1 and GPD2 and lactate dehydrogenase gene DLD1, as well as downregulation of ethanol pathway gene ADH1 using the L-methionine repressible promoter to minimize impact on growth. ASc-d789M produces D-lactic acid at a titer of 17.09 g/L in shake-flasks (yield of 0.89 g/g glucose consumed or 89% of the theoretical yield). Fed-batch fermentation resulted in D-lactic acid titer of 40.03 g/L (yield of 0.81 g/g glucose consumed). Altogether, our work represents progress towards efficient microbial production of D-lactic acid.

Rejection of the reclassification of Leuconostoc gasicomitatum as Leuconostoc gelidum subsp. gasicomitatum based on whole genome analysis.

In 2014, Rahkila et al. transferred Leuconostoc gasicomitatum to Leuconostoc gelidum as L. gelidum subsp. gasicomitatum comb. nov. based on a 75 % DNA-DNA hybridization value. In the present study, the taxonomic status of L. gelidum subsp. gasicomitatum is re-evaluated by a polyphasic approach, including 16S rRNA, pheS, rpoA, recA, and atpA gene sequence analyses, phylogenomic treeing, analyses of ANI (average nucleotide identity) and dDDH (digital DNA-DNA hybridization), fatty acid methyl ester analysis and a phenotypic characterization. On the basis of the ANI and dDDH values, we propose to reject the proposal of Rahkila et al. to reclassify L. gasicomitatum as L. gelidum subsp. gasicomitatum.

Leuconostoc falkenbergense sp. nov., isolated from a lactic culture, fermentating string beans and traditional yogurt.

In the present study, the taxonomic positions of five strains (C, 17-2, LMG 10779T, LMG 18969 and LMG 11483) of Leuconostoc pseudomesenteroides were re-evaluated by a polyphasic approach, including the analyses of 16S rRNA, pheS and rpoA gene sequences, cellular fatty acids, average nucleotide and amino acid identities (ANI and AAI), digital DNA-DNA hybridization (dDDH), and phenotypic features. Based on rpoA sequence analysis, the five strains and L. pseudomesenteroides LMG 11482T were divided into two groups: strains C, LMG 10779T and LMG 18969; strains 17-2, LMG 11483 and LMG 11482T. Each of the two groups had almost identical rpoA sequences. The rpoA sequence similarity between strain LMG 10779T and L. pseudomesenteroides LMG 11482T was 95.6 %. Strains LMG 11483 and 17-2 had 98.1 and 97.2 % ANI values, 83.5 and 73.2 % dDDH values, and a 97.0 % AAI value with L. pseudomesenteroides LMG 11482T, greater than the threshold for species demarcation, indicating that strains LMG 11483 and 17-2 belong to L. pseudomesenteroides. Strains LMG 18969 and C shared 97.1 and 98.2 % ANI values, 73.4 and 83.2 % dDDH values, and 96.9 and 96.6 % AAI values with strain LMG 10779T, greater than the threshold for species demarcation, indicating that strains LMG 10779T, LMG 18969 and C represent the same species. The ANI, dDDH and AAI values between strain LMG 10779T and the type strains of phylogenetically related species were 75.2-92.5, 20.0-48.2 and 75.3-93.9 %, respectively, below the thresholds for species demarcation, indicating that strain LMG 10779T represents a novel species within the genus Leuconostoc. On the basis of the results presented here, (i) strains 17-2 and LMG 11483 belong to L. pseudomesenteroides, and (ii) strains LMG 10779T, LMG 18969 and C are considered to represent a novel species within the genus Leuconostoc, for which the name Leuconostoc falkenbergense sp. nov. is proposed with the type strain LMG 10779T (=CCUG 27119T).

Brewers' spent grain as substrate for dextran biosynthesis by Leuconostoc pseudomesenteroides DSM20193 and Weissella confusa A16.

BACKGROUND: Lactic acid bacteria can synthesize dextran and oligosaccharides with different functionality, depending on the strain and fermentation conditions. As natural structure-forming agent, dextran has proven useful as food additive, improving the properties of several raw materials with poor technological quality, such as cereal by-products, fiber-and protein-rich matrices, enabling their use in food applications. In this study, we assessed dextran biosynthesis in situ during fermentation of brewers´ spent grain (BSG), the main by-product of beer brewing industry, with Leuconostoc pseudomesenteroides DSM20193 and Weissella confusa A16. The starters performance and the primary metabolites formed during 24 h of fermentation with and without 4% sucrose (w/w) were followed. RESULTS: The starters showed similar growth and acidification kinetics, but different sugar utilization, especially in presence of sucrose. Viscosity increase in fermented BSG containing sucrose occurred first after 10 h, and it kept increasing until 24 h concomitantly with dextran formation. Dextran content after 24 h was approximately 1% on the total weight of the BSG. Oligosaccharides with different degree of polymerization were formed together with dextran from 10 to 24 h. Three dextransucrase genes were identified in L. pseudomesenteroides DSM20193, one of which was significantly upregulated and remained active throughout the fermentation time. One dextransucrase gene was identified in W. confusa A16 also showing a typical induction profile, with highest upregulation at 10 h. CONCLUSIONS: Selected lactic acid bacteria starters produced significant amount of dextran in brewers' spent grain while forming oligosaccharides with different degree of polymerization. Putative dextransucrase genes identified in the starters showed a typical induction profile. Formation of dextran and oligosaccharides in BSG during lactic acid bacteria fermentation can be tailored to achieve specific technological properties of this raw material, contributing to its reintegration into the food chain.