The novel bacteriocin BacYB1 produced by Leuconostoc mesenteroides YB1: From recent analytical characterization to biocontrol Verticillium dahliae and Agrobacterium tumefaciens.

Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases.

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides.

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway. KEY POINTS: Co-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer. L. mesenteroides existing enhances T cells viability and activity. GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

Leuconostoc mesenteroides utilizes glucose fermentation to produce electricity and ameliorates high-fat diet-induced abdominal fat mass.

Bacteria capable of producing electricity in intestinal microbiota have been discovered. However, no studies have explored butyric acid which generated by electrogenic bacteria on the host organism have significant physiological impacts on certain organs. We found that the capacity for electrical current generation by the commensal gut Leuconostoc mesenteroides EH-1 (L. mesenteroides EH-1) during glucose fermentation. The electricity production was essential for the gut colonization of L. mesenteroides EH-1 since the inhibition of electricity production by cyclophilin A inhibitor (TMN355) significantly diminished the number of bacteria attached to the human gut epithelial cell surface. The adipocyte differentiation contributes to the increased 4-hydroxy-2-nonenal (4-HNE), considered as a biomarker of reactive oxygen species (ROS). The effect of intestinal electrogenic microbiota in the high-fat diet (HFD)-induced 4-HNE and abdominal fat accumulation in mice was investigated in this study. The oral administration of glucose with a butyric acid-producing L. mesenteroides EH-1 bacterium attenuated the expression of 4-HNE and abdominal fat. The level of 4-HNE and abdominal fat depot were markedly increased in mice administered with cyclophilin A inhibitor-pretreated bacteria or GLPG-0974, an antagonist of free fatty acid receptor 2 (Ffar2). Our studies suggest a novel means by which the probiotic bacteria can modulate fat mass deposition and oxidative stress via the cyclophilin A-mediated electron production and the butyric acid-activated Ffar2 pathway.

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

Three-level hybrid modeling for systematic optimization of biocatalytic synthesis: α-glucosyl glycerol production by enzymatic trans-glycosylation from sucrose.

Mechanism-based kinetic models are rigorous tools to analyze enzymatic reactions, but their extension to actual conditions of the biocatalytic synthesis can be difficult. Here, we demonstrate (mechanistic-empirical) hybrid modeling for systematic optimization of the sucrose phosphorylase-catalyzed glycosylation of glycerol from sucrose, to synthesize the cosmetic ingredient α-glucosyl glycerol (GG). The empirical model part was developed to capture nonspecific effects of high sucrose concentrations (up to 1.5 M) on microscopic steps of the enzymatic trans-glycosylation mechanism. Based on verified predictions of the enzyme performance under initial rate conditions (Level 1), the hybrid model was expanded by microscopic terms of the reverse reaction to account for the full-time course of GG synthesis (Level 2). Lastly (Level 3), the application of the hybrid model for comprehensive window-of-operation analysis and constrained optimization of the GG production (~250 g/L) was demonstrated. Using two candidate sucrose phosphorylases (from Leuconostoc mesenteroides and Bifidobacterium adolescentis), we reveal the hybrid model as a powerful tool of "process decision making" to guide rational selection of the best-suited enzyme catalyst. Our study exemplifies a closing of the gap between enzyme kinetic models considered for mechanistic research and applicable in technologically relevant reaction conditions; and it highlights the important benefit thus realizable for biocatalytic process development.

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.

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.

Production of electricity and reduction of high-fat diet-induced IL-6 by glucose fermentation of Leuconostoc mesenteroides.

Electrogenic bacteria can mediate electron transfer to conserve energy and promote growth. To examine bacterial electrogenicity, an L. mesenteroides EH-1 strain was cultured in rich media in the presence and absence of 2% glucose. After 12 h incubation, glucose triggered fermentation of L. mesenteroides EH-1 to produce >10 mmol/l acetate and elicit electricity measured by voltage changes. The electricity production was mediated by glucose fermentation since pre-treatment of L. mesenteroides EH-1 with furfural, a fermentation inhibitor, completely diminished the voltage increases. The deficiency of furfural pre-treated L. mesenteroides EH-1 in electricity production can be restored by the external addition of acetate into the bacterial culture, suggesting the function of acetate as an electron donor. Oral administration of HFD-fed mice with L. mesenteroides EH-1 in the presence or absence of glucose significantly attenuated the high level of pro-inflammatory IL-6 cytokine in blood. Bacterial electricity can be elicited by fermentation. Supplementation of fermenting and electrogenic L. mesenteroides EH-1 may provide a novel approach for the reduction of pro-inflammatory IL-6 cytokine that increased in chronic inflammation, autoimmune diseases, cancers, and infections.

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.

Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates.

The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d. Subtype 2a RNAs are riboswitches that sense the bacterial alarmone ppGpp and typically regulate amino acid biosynthesis genes. Subtype 2b riboswitches sense the purine biosynthetic intermediate PRPP and frequently partner with guanine riboswitches to regulate purine biosynthesis genes. In this study, we examined ykkC subtype 2c RNAs, which are found upstream of genes encoding hydrolase enzymes that cleave the phosphoanhydride linkages of nucleotide substrates. Subtype 2c representatives mostly recognize adenosine and cytidine 5'-diphosphate molecules in either their ribose or deoxyribose forms (ADP, dADP, CDP, and dCDP). Other nucleotide-containing compounds, especially nucleoside 5'-triphosphates, are strongly rejected by some members of this putative riboswitch class. High ligand concentrations in vivo are predicted to turn on expression of hydrolase enzymes, which presumably function to balance cellular nucleotide pools. These results further showcase the striking functional diversity derived from the structural scaffold shared among all ykkC motif RNAs, which has been adapted to sense at least five different types of natural ligands. Moreover, riboswitches for nucleoside diphosphates provide additional examples of the numerous partnerships observed between natural RNA aptamers and nucleotide-derived ligands, including metabolites, coenzymes, and signaling molecules.

Effect of potential probiotic Leuconostoc mesenteroides FB111 in prevention of cholesterol absorption by modulating NPC1L1/PPARα/SREBP-2 pathways in epithelial Caco-2 cells.

Mustard kimchi consumption reduces cholesterol levels in rats. To identify lactic acid bacteria (LAB) in kimchi which exert this effect, 20 LAB isolates were evaluated for cholesterol reduction in an in vitro screen. The FB111 strain showed the highest cholesterol-lowering activity and was identified as Leuconostoc mesenteroides. This strain was characterized as a potential probiotic through sequential analyses for resistance to gastrointestinal digestion and bile salts, and adhesion to Caco-2 cells. The Caco-2 cells treated with L. mesenteroides FB111 (6-8 log CFU/mL) showed toxicological effect. The reduction of cholesterol uptake in these cells was inhibited by 48.6% compared to the control and significantly higher than that of the Lactobacillus rhamnosus GG (LGG) strain-treated group after 2-h incubation. The levels of NPC1L1, ABCG5, ABCG8, SREBP-1, SREBP-2, and PPARα gene expression were determined by reverse transcription-quantitative polymerase chain reaction analysis. The L. mesenteroides FB111 and LGG inhibited the mRNA expression of NPC1L1 (P < 0.05), whereas the expression of PPARα was increased. Moreover, the FB111 strain also inhibited the expression of SREBP-2 mRNA. Overall, we found that L. mesenteroides FB111 has efficient cholesterol-lowering effects and might be useful as a probiotic in the food industry.

A genome-scale metabolic network of the aroma bacterium Leuconostoc mesenteroides subsp. cremoris.

Leuconostoc mesenteroides subsp. cremoris is an obligate heterolactic fermentative lactic acid bacterium that is mostly used in industrial dairy fermentations. The phosphoketolase pathway (PKP) is a unique feature of the obligate heterolactic fermentation, which leads to the production of lactate, ethanol, and/or acetate, and the final product profile of PKP highly depends on the energetics and redox state of the organism. Another characteristic of the L. mesenteroides subsp. cremoris is the production of aroma compounds in dairy fermentation, such as in cheese production, through the utilization of citrate. Considering its importance in dairy fermentation, a detailed metabolic characterization of the organism is necessary for its more efficient use in the industry. To this aim, a genome-scale metabolic model of dairy-origin L. mesenteroides subsp. cremoris ATCC 19254 (iLM.c559) was reconstructed to explain the energetics and redox state mechanisms of the organism in full detail. The model includes 559 genes governing 1088 reactions between 1129 metabolites, and the reactions cover citrate utilization and citrate-related flavor metabolism. The model was validated by simulating co-metabolism of glucose and citrate and comparing the in silico results to our experimental results. Model simulations further showed that, in co-metabolism of citrate and glucose, no flavor compounds were produced when citrate could stimulate the formation of biomass. Significant amounts of flavor metabolites (e.g., diacetyl and acetoin) were only produced when citrate could not enhance growth, which suggests that flavor formation only occurs under carbon and ATP excess. The effects of aerobic conditions and different carbon sources on product profiles and growth were also investigated using the reconstructed model. The analyses provided further insights for the growth stimulation and flavor formation mechanisms of the organism.

Aroma formation in retentostat co-cultures of Lactococcus lactis and Leuconostoc mesenteroides.

Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc mesenteroides are considered to be the main aroma producers in Dutch-type cheeses. Both species of lactic acid bacteria were grown in retentostat mono- and co-cultures to investigate their interaction at near-zero growth rates and to determine if co-cultivation enhances the aroma complexity compared to single species performance. During retentostat mono-cultures, the growth rates of both species decreased to less than 0.001 h-1 and a large fraction of the cells became viable but not culturable. Compared to Lc. mesenteroides, L. lactis reached a 3.4-fold higher biomass concentration caused by i) a higher ATP yield on substrate, ii) a higher biomass yield on ATP and iii) a lower maintenance requirement (mATP). Dynamic models estimated that the mATP of both species decreased approximately 7-fold at near-zero growth rates compared to high growth rates. Extension of these models by assuming equal substrate distribution resulted in excellent prediction of the biomass accumulation in retentostat co-cultures with L. lactis dominating (100:1) as observed in ripened cheese. Despite its low abundance (∼1%), Lc. mesenteroides contributed to aroma production in co-cultures as indicated by the presence of all 5 specific Lc. mesenteroides compounds. This study provides insights in the production of cheese aroma compounds outside the cheese matrix by co-cultures of L. lactis and Lc. mesenteroides, which could be used as food supplements in dairy or non-dairy products.

In situ riboflavin fortification of different kefir-like cereal-based beverages using selected Andean LAB strains.

Cereal-based functional beverages represent social, economic, and environmental sustainable opportunities to cope with emerging trends in food consumption and global nutrition. Here we report, for the first time, the polyphasic characterization of three cereal-based kefir-like riboflavin-enriched beverages, obtained from oat, maize and barley flours, and their comparison with classical milk-based kefir. The four matrices were successfully fermented with commercial starters: i) milk-kefir and ii) water-kefir, proving the potential of cereal ingredients in the formulation of dairy-like fermented beverages with milk-kefir starter behavior better in these matrices. In the light of their potentiality, seven riboflavin-producing Andean Lactic Acid Bacteria (LAB) were tested for tolerance to food stresses commonly encountered during food fermentation. Moreover, the LAB strains investigated were screened for spontaneous riboflavin overproducing derivatives. Lactobacillus plantarum M5MA1-B2 with outstanding response to stress, was selected to improve riboflavin content in an in situ fortification approach. The combination of L. plantarum M5MA1-B2 riboflavin overproducing strain with milk kefir starter in oat, lead to cover, for one serving of 100 g, 11.4% of Recommended Dietary Allowance (RDA). Besides, addition of L. plantarum M5MA1-B2 improved performance of water kefir in oat and maize matrices. Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) analysis provided the on-line Volatile Organic Compounds profiles supporting the best combination of starter, LAB and cereal matrix for novel functional foods development.

Molecular and Functional Study of a Branching Sucrase-Like Glucansucrase Reveals an Evolutionary Intermediate between Two Subfamilies of the GH70 Enzymes.

Glucansucrases (GSs) in glycoside hydrolase family 70 (GH70) catalyze the synthesis of α-glucans from sucrose, a reaction that is widely seen in lactic acid bacteria (LAB). These enzymes have been implicated in many aspects of microbial life. Products of GSs have great commercial value as food supplements and medical materials; therefore, these enzymes have attracted much attention from both science and industry. Certain issues concerning the origin and evolution of GSs are still to be addressed, although an increasing number of GH70 enzymes have been characterized. This study describes a GS enzyme with the appearance of a branching sucrase (BrS). Structural analysis indicated that this GS enzyme produced a type of glucan composed of an α-(1→6) glucosidic backbone and α-(1→4) branches, as well as a considerable amount of α-(1→3) branches, distinguishing it from the GSs identified so far. Moreover, sequence-based analysis of the catalytic core of this enzyme suggested that it might be an evolutionary intermediate between the BrS and GS subgroups. These results provide an evolutionary link between these subgroups of GH70 enzymes and shed new light on the origination of GSs.IMPORTANCE GH70 GSs catalyze the synthesis of α-glucans from sucrose, a reaction that is widely seen in LAB. Products of these enzymes have great commercial value as food supplements and medical materials. Moreover, these enzymes have attracted much attention from scientists because they have potential in tailored synthesis of α-glucans with desired structures and properties. Although more and more GSs have been characterized, the origin and evolution of these enzymes have not been well addressed. This study describes a GS with the appearance of a BrS (i.e., high levels of similarity to BrSs in sequence analysis). Further analysis indicated that this enzyme synthesized a type of insoluble glucan composed of an α-(1→6) glucosidic backbone and many α-(1→4)- and α-(1→3)-linked branches, the linkage composition of which has rarely been reported in the literature. This BrS-like GS enzyme might be an evolutionary intermediate between BrS and GS enzymes.

Immunostimulatory effect on dendritic cells of the adjuvant-active exopolysaccharide from Leuconostoc mesenteroides strain NTM048.

This study evaluated the immunostimulative effect on bone marrow-derived dendritic cells (DCs) of adjuvant-active exopolysaccharide (EPS) produced by Leuconostoc mesenteroides strain NTM048. EPS stimulation increased IL-6, IL-10, IL-12, and retinal dehydrogenase (RALDH) gene expression levels and induced retinoic acid-synthesizing RALDH-active DCs, which play a crucially important role in controlling adaptive immune responses in mucosa.

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut.

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett-Burman (PB) experiment, sucrose, Ca2+, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca2+ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.

Enzyme-resistant isomalto-oligosaccharides produced from Leuconostoc mesenteroides NRRL B-1426 dextran hydrolysis for functional food application.

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1426 was produced and purified using polyethylene glycol fractionation. In our earlier study, it was reported that L. mesenteroides dextransucrase synthesizes a high-molecular mass dextran (>2 × 10(6)  Da) with ∼85.5% α-(1→6) linear and ∼14.5% α-(1→3) branched linkages. Isomalto-oligosaccharides (IMOs) were synthesized through depolymerization of dextran by the action of dextranase. The degree of polymerization of IMOs was 2-10 as confirmed by mass spectrometry. The nuclear magnetic resonance spectroscopic analysis revealed the presence of α-(1→3) linkages in the synthesized IMOs. The IMOs were resistant to dextranase, α-glucosidase, and α-amylase, and therefore can have potential application as food additives in the functional foods.

Mode of action of leucocin K7 produced by Leuconostoc mesenteroides K7 against Listeria monocytogenes and its potential in milk preservation.

OBJECTIVES: To investigate the mode of action of leucocin K7 against Listeria monocytogenes and to assess its inhibitory effect on Lis. monocytogenes in refrigerated milk. RESULTS: A bacteriocin-producing strain, Leuconostoc mesenteroides K7, was isolated from a fermented pickle. The bacteriocin, leucocin K7, exhibited antagonistic activity against Lis. monocytogenes with an MIC of 28 µg/ml. It was sensitive to proteaseS and displayed good thermal stability and broad active pH range. Leucocin K7 had no effect on the efflux of ATP from Lis. monocytogenes but triggered the efflux of K(+) and the intracellular hydrolysis of ATP. It also dissipated the transmembrane electrical potential completely and transmembrane pH gradient partially. It 80 AU/ml inhibited the growth of Lis. monocytogenes by 2.3-3.9 log units in milk; when combined with glycine (5 mg/ml), it completely eliminated viable Lis. monocytogenes over 7 days CONCLUSION: Leucocin K7 shows different mode of action from nisin and may have potential application in milk preservation.

Exploitation of Leuconostoc mesenteroides strains to improve shelf life, rheological, sensory and functional features of prickly pear (Opuntia ficus-indica L.) fruit puree.

Strains of Leuconostoc mesenteroides were identified from raw prickly pear (Opuntia ficus-indica L.). Five autochthonous strains were selected based on the kinetics of growth and acidification on prickly pear fruit juice, and the capacity to synthesize exo-polysaccharides. All selected Leuc. mesenteroides strains showed an in vitro mucilage-degrading capability. A protocol for processing and storage of fermented prickly pear fruit puree (FP) was set up. Unstarted FP and chemically acidified FP were used as the controls. Starters grew and remained viable at elevated cell numbers during 21 days of storage at 4 °C. Contaminating Enterobacteriaceae and yeasts were found only in the controls. Viscosity and serum separation distinguished started FP compared to the controls. Colour parameters, browning index, sensory attributes, antimicrobial activity, vitamin C and betalains levels were positively affected by lactic acid fermentation. Increase of free radical scavenging activity in ethyl acetate soluble extract suggested an effect of selected strains on phenolic profiles. Started FP markedly inhibited the inflammatory status of Caco-2/TC7 cells, and also contributed to maintaining the integrity of tight junctions. Started FP scavenged the reactive oxygen species generated by H2O2 on Caco-2 cells. All selected strain variously affected the immunomodulatory activity towards anti- and pro-inflammatory cytokines.