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.

DNAzyme-based quantitative loop-mediated isothermal amplification for strain-specific detection of starter kimchi fermented with Leuconostoc mesenteroides WiKim32.

Leuconostoc spp. are generally utilized as kimchi starters because of their beneficial effects on kimchi fermentation and sensory characteristics. We developed a DNAzyme-based colorimetric method for measuring the abundance of the kimchi starter Leuconostoc mesenteroides WiKim32. A primer set for loop-mediated isothermal amplification and target-specific DNAzyme was designed based on the WiKim32 nucleotide sequence. In the presence of the target amplicon, DNAzyme bound to it, resulting in negligible amounts of green product. In contrast, with the addition of hemin and in the absence of the target amplicon, DNAzyme fragments not bound to the target amplicon formed G-quadruplex-hemin conjugates, generating a visible green product by oxidizing 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt. There was no cross-reaction with other strains. The method had high detection sensitivity and quantitative capacity in kimchi samples without a requirement for DNA isolation. This strategy provides a rapid, sensitive, and simple detection method with possible industrial applications.

Meat Native Lactic Acid Bacteria Capable to Inhibit Salmonella sp. and Escherichia coli.

In this study, lactic acid bacteria (LAB) strains were isolated from ground beef, and it was analyzed if they have any effect on the growth of two reference bacteria (Salmonella sp. and Escherichia coli). It was found that five isolates showed an inhibitory effect in both reference bacteria by spot at the lawn assay. These bacteria were selected to perform growth kinetics in co-culture to determine if they modify the growth parameters of the reference bacteria. Subsequently, LAB cultures and three treatments (crude extract, thermally treated and thermally treated with neutral pH) of cells free supernatants (CFS) were screened by the agar well diffusion assay. In co-culture, selected LAB altered the growth rate and reduce the maximum population of both reference bacteria. While, LAB cultures and CFS also showed antimicrobial activity, and there was no significant difference among CFS treatments. LAB isolated from ground beef showed an antimicrobial effect against the reference bacteria that could be used for meat biopreservation purposes.

Isolation and Characterization of Kimchi Starters Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104 for Manufacture of Commercial Kimchi.

This study was focused on developing and obtaining a kimchi starter for use in commercial kimchi production. Kimchi varieties made with selected starters are of high quality, have high levels of mannitol, and extended shelf life. The starters were screened for properties such as mannitol production, low gas/acid production, and acid resistance. Finally, kimchi fermentation testing was performed using selected LAB starters. Kimchi samples were prepared with lactic acid bacteria (LAB) starters, including Leuconostoc mesenteroides PBio03 and Leuconostoc mesenteroides PBio104. The LAB starters are isolated from kimchi and can grow under pH 3.0 and low temperature conditions of 5°C. Four kimchi samples were fermented and stored for 28 days at 5°C. The kimchi samples made with starters (PBio03 and PBio104) had better quality (production of mannitol and maintenance of heterofermentative LAB dominance) than the non-starter kimchi samples. In the starter kimchi, Leu. mesenteroides was the dominant LAB, comprising 80% and 70% of total LAB counts at 7 and 21 days, respectively. Mannitol content of the kimchi with Leu. mesenteroides PBio03 was 1,423 ± 19.1 mg/ 100 g at 28 days, which was higher than that of the non-starter kimchi sample (1,027 ± 12.2 mg/100 g). These results show the possibility of producing kimchi with improved qualities using Leu. mesenteroides PBio03 and PBio104 as starters.

Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level.

Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

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.

Bacteremia by Leuconostoc mesenteroides in an immunocompetent patient with chronic Chagas disease: a case report.

BACKGROUND: The Leuconostoc mesenteroides are members of the Streptococcae family and currently has been recognized as potential pathogens. This case describes a bacteremia caused by L. mesenteroides in an immunocompetent patient affected by Chagas disease. CASE PRESENTATION: A 67-year-old female patient with chagasic megaesophagus and megacolon was submitted to a Heller myotomy for achalasia in 2000 and endoscopic dilatation in 2015. Patient was admitted to the Nutrology Ward in May 2016 with protein-calorie malnutrition associated with achalasia and receiving enteral nutrition. In July 2016, the patient underwent a Serra-Doria surgery. In the third postoperative day she presented an important abdominal distension. She was submitted to a new surgical intervention, and then a terminal ileum perforation was detected, leading the surgeon to perform an enterectomy with side-to-side anastomosis. The next day after the surgery (4th postoperative day) the patient presented a decreased level of consciousness (Glasgow coma scale = 8), hypotension and hypoxemia. In two samples of blood cultures there was growth of Leuconostoc mesenteroides. Susceptibility pattern was evaluated by the diffusion disk method. The microorganism was susceptible to penicillin, ampicillin, chloramphenicol, erythromycin, and fluoroquinolones, but resistant to rifampin, tetracycline, vancomycin and teicoplanin. CONCLUSION: We concluded that infections caused by L. mesenteroides is serious and should be considered not only in settings of immunosuppression and prolonged antimicrobial use, but also in immunocompetent patients undergoing surgeries involving the gastrointestinal tract.

Pro-technological and functional characterization of lactic acid bacteria to be used as starters for hemp (Cannabis sativa L.) sourdough fermentation and wheat bread fortification.

Lactic acid bacteria were isolated from hemp (Cannabis sativa L.) flour, spontaneously fermented dough, and type I sourdough. Isolates were identified and further selected based on pro-technological, nutritional and functional properties. Lactobacillus plantarum/s5, Pediococcus acidilactici/s5, and Leuconostoc mesenteroides/s1 were used as mixed starter to produce hemp sourdough. Significant decreases of the concentration of phytic acid, condensed tannins, and total saponins were observed during fermentation. The in vitro protein digestibility increased up to 90%. Experimental wheat breads were made adding 5% to 15% (w/w) hemp sourdough to the formula, characterized, and compared to baker's yeast wheat bread manufactured without hemp sourdough. The use of hemp sourdough improved the textural features of wheat bread, without adversely affect the sensory profile. Proportionally to the fortification with hemp sourdough, protein digestibility of the breads increased, while the predicted glycemic index significantly decreased (87 vs 100%). This work demonstrated that the fermentation with selected starters improved nutritional functionality of hemp flour, allowing its large-scale use in different food applications, meeting the consumers and producers request for novel fermented baked goods with a well-balanced nutritional profile.

Lactobacillus curvatus KFP419 and Leuconostoc mesenteroides subsp. mesenteroides KDK411 Isolated from Kimchi Ameliorate Hypercholesterolemia in Rats.

Western-style diets increase the risk for cardiovascular diseases. It is suggested that the risk could be prevented by lowering cholesterol concentrations in blood. In the present study, hypocholesterolemic effects of the probiotics isolated from kimchi (Lactobacillus curvatus KFP419, Leuconostoc paramesenteroides KJP421, and Leuconostoc mesenteroide subsp. mesenteroides KDK411) were investigated in hypercholesterolemia-induced rats. There was no difference in growth performance between the rats fed high cholesterol diet (HCD) and normal diet (ND). However, blood total cholesterol, low-density lipoprotein cholesterol, and hepatic cholesterol were elevated by the HCD compared to ND, and those concentrations were decreased by dietary supplementation of KFP419 and KDK411. It was concomitant with an increase in fecal excretion of neutral sterols (cholesterol, coprostanol, and coprostanone) in the rats fed HCD compared to ND and was even greater with KDK411 supplementation. These findings indicate that probiotics L. curvatus KFP419 and L. mesenteroide subsp. mesenteroides KDK411 isolated from kimchi ameliorate hypercholesterolemia in rats by assimilating and excreting cholesterol in feces.

Composition of lactic acid bacteria during spontaneous curly kale (Brassica oleracea var. sabellica) fermentation.

The present work is the first report on spontaneous fermentation of curly kale and characteristics of autochthonous lactic acid bacteria (LAB). Our results indicate that curly kale fermentation is the new possibility of the technological use of this vegetable. Bacteria representing ten different species were isolated from three phases of curly kale fermentation and identified by MALDI-TOF mass spectrometry and 16S rRNA gene sequencing. Among them, four species were identified as Lactobacillus spp. (Lb. plantarum 332, Lb. paraplantarum G2114, Lb. brevis R413, Lb. curvatus 154), two as Weissella spp. (W. hellenica 152, W. cibaria G44), two as Pediococcus spp. (P. pentosaceus 45AN, P. acidilactici 2211), one as Leuconostoc mesenteroides 153, and one as Lactococcus lactis 37BN. The functional properties of isolates, i.e. acid, NaCl and bile salt tolerance, enzyme activities, adhesion to hydrocarbons, and antibiotic resistance, were examined. Among the tested strains, Lb. plantarum 332, Lb. paraplantarum G2114, P. pentosaceus 2211, and Lb. brevis R413 exhibited the best hydrophobicity value and high tolerance to bile salts, NaCl, and low pH.

Kocuria rosea, kocuria kristinae, leuconostoc mesenteroides as caries-causing representatives of oral microflora.

INTRODUCTION: Recently, opportunistic microflora are increasingly known to be involved in the development of pathological processes in various systems and organs. This situation promotes interest in their detailed study as causative agents of bacterial infections. THE AIM: To study the microbial species residing in carious cavities in acute profound caries. MATERIALS AND METHODS: The study involved 14 people with a diagnosis of acute profound caries. Microbiological methods included determining species of microorganisms' cultures from carious cavities in acute profound caries. Final identification was carried out by automatic bacteriological analyzer Vitec-2compact bioMérieux. CONCLUSION: Among the bacteria isolated, Kocuria rosae, Kocuria kristinae, and Leuconostoc mesenteroides are the focus of the authors' attention due to their identification rate in the patients. These microbial species are little studied due to the lack of data on their cariogenic associations.The meticulous study of the microorganisms, isolated from carious cavities in patients with acute profound caries by automatic bacteriological analyzer Vitec-2 Systems bioMérieux, and findings on their biochemical properties allow us to conclude that Kocuria rosae, Kocuria kristinae, and Leuconostoc mesenteroides are among the microorganisms making up the microflora of carious cavities under acute profound caries and are involved in the development of the caries process.

Emergence of Leuconostoc mesenteroides as a causative agent of oozing in carrots stored under non-ventilated conditions.

Long-term storage and transport of post-harvest carrots (Daucus carota L.) require a low-temperature, high-relative-humidity environment, usually with low ventilation. Following long-term storage, a slimy exudate (oozing) often appears on the carrots, leading to severe spoilage. We characterized the environmental conditions leading to these symptoms and identified the causative agent. Simulation of non-ventilated storage conditions revealed accumulation of CO2 (to 80%) and ethanol (to 1000 ppm); then, a transparent exudate appeared on the carrot surface which, upon ventilation, developed into tissue browning and soft rot. Peels from oozing carrots contained over 10-fold the total bacterial counts of healthy carrots. The total peel microbiome was determined by 16S rDNA sequencing. During oozing stage, the surface of carrots incubated in a CO2 -rich (98%) environment harboured a bacterial population dominated by Lactobacillales and Enterobacteriales, differing markedly from those incubated in air. Three prevalent bacterial isolates from the oozing carrots were identified as Pantoea agglomerans, Rahnella aquatilis and Leuconostoc mesenteroides. Inoculation of carrot discs with L. mesenteroides, but not the others, induced oozing under high CO2 , suggesting that this bacterium is responsible for oozing of stored carrots. These findings should enable development of approaches to preventing carrot spoilage during long-term storage.

Isolation and Characterization of Some Promoter Sequences from Leuconostoc mesenteroides SY2 Isolated from Kimchi.

Some promoters were isolated and characterized from the genome of Leuconostoc mesenteroides SY2, an isolate from kimchi, a Korean traditional fermented vegetable. Chromosomal DNA of L. mesenteroides SY2 was digested with Sau3AI and ligated with BamHI-cut pBV5030, a promoter screening vector containing a promoterless cat-86. Among E. coli transformants (TFs) resistant against Cm (chloramphenicol), 17 were able to grow in the presence of 1,000 µg/ml Cm and their inserts were sequenced. Transcription start sites were examined for three putative promoters (P04C, P25C, and P33C) by primer extension. Four putative promoters were inserted upstream of a promoterless α-amylase reporter gene in pJY15α. α-Amylase activities of E. coli TFs containing pJY15α (control, no promoter), pJY03α (pJY15α with P03C), pJY04α (with P04C), pJY25α (with P25C), and pJY33α (with P33C) were 66.9, 78.7, 122.1, 70.8, and 99.3 U, respectively. Cells harboring pJY04α showed 1.8 times higher activity than the control. Some promoters characterized in this study might be useful for construction of foodgrade expression vectors for Leuconostoc sp. and related lactic acid bacteria.

Lactic acid bacteria involved in cocoa beans fermentation from Ivory Coast: Species diversity and citrate lyase production.

Microbial fermentation is an indispensable process for high quality chocolate from cocoa bean raw material. lactic acid bacteria (LAB) are among the major microorganisms responsible for cocoa fermentation but their exact role remains to be elucidated. In this study, we analyzed the diversity of LAB in six cocoa producing regions of Ivory Coast. Ribosomal 16S gene sequence analysis showed that Lactobacillus plantarum and Leuconostoc mesenteroides are the dominant LAB species in these six regions. In addition, other species were identified as the minor microbial population, namely Lactobacillus curieae, Enterococcus faecium, Fructobacillus pseudoficulneus, Lactobacillus casei, Weissella paramesenteroides and Weissella cibaria. However, in each region, the LAB microbial population was composed of a restricted number of species (maximum 5 species), which varied between the different regions. LAB implication in the breakdown of citric acid was investigated as a fundamental property for a successful cocoa fermentation process. High citrate lyase producer strains were characterized by rapid citric acid consumption, as revealed by a 4-fold decrease in citric acid concentration in the growth medium within 12h, concomitant with an increase in acetic acid and lactic acid concentration. The production of citrate lyase was strongly dependent on environmental conditions, with optimum production at acidic pH (pH<5), and moderate temperature (30-40°C), which corresponds to conditions prevailing in the early stage of natural cocoa fermentation. This study reveals that one of the major roles of LAB in the cocoa fermentation process involves the breakdown of citric acid during the early stage of cocoa fermentation through the activity of citrate lyase.

Potential use of lactic acid bacteria Leuconostoc mesenteroides as a probiotic for the removal of Pb(II) toxicity.

It has been demonstrated that certain lactic acid bacteria (LAB) can sequester metal ions by binding them to their surfaces. In the present study, lead (Pb)-resistant LAB were isolated from kimchi, a Korean fermented food. A total of 96 different LAB strains were isolated, and 52 strains showed lead resistance. Among them, an LAB strain-96 (L-96) identified as Leuconostoc mesenteroides showed remarkable Pb resistance and removal capacity. The maximum adsorption capacity of this strain calculated using the Langmuir isotherm was 60.6 mg Pb/g. In an in vivo experiment, young male mice were provided with water (A), Pb-water (B), or Pb-water+ L-96 (C) during puberty. Lower glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in Pb-exposed male mice that received strain L-96 as a probiotic were suggestive of reduced hepatotoxicity. Moreover, feces from mice treated with L-96 contained more Pb than feces from untreated mice. Increased Pb elimination likely reduced internal accumulation, and this hypothesis was supported by significantly lower Pb concentrations in kidneys and testes of the mice treated with strain L-96. The motility and ATP content of epididymal spermatozoa were partially restored if strain L-96 was administered. In conclusion, isolated L-96 LAB had lead-biosorption activity and efficiently detoxified lead-poisoned male mice, resulting in recovering male reproductive function. These results suggest the potential use of LAB as a probiotic to protect humans from the adverse effects of Pb exposure.

Leuconostoc mesenteroides periprosthetic knee infection, an unusual fastidious Gram-positive bacteria: a case report.

BACKGROUND: Periprosthetic joint infections are mainly caused by Gram-positive cocci. Leuconostoc mesenteroides is a rare microorganism mainly causing bloodstream infections. At times, it might be confused with another type of cocci and give rise to misdiagnosed infections. Molecular diagnosis and biofilm production comprise important techniques to guide antibiotic treatment. CASE PRESENTATION: A 68-year-old Hispanic female with a previous history of bilateral knee arthroplasty presented with acute right-knee inflammation and gait impairment. Blood tests showed inflammatory response and knee x-ray revealed no prosthesis loosening. Irrigation and debridement was performed. Gram-positive cocci were obtained from cultures, and then biochemical and molecular identification revealed L. mesenteroides. Susceptibility and biofilm production were performed. The patient was treated with IntraVenous (IV) Ceftriaxone for ten days and was then switched to Amoxicillin-Clavulanate for 3 months with clinical and laboratory success. CONCLUSIONS: Microbiology diagnosis of fastidious microorganisms is mandatory to treat periprosthetic joint infections adequately. L. mesenteroides may infect non-immunocompromised persons; however, treatment guidelines are lacking.

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.

Production of a bacteriocin-like inhibitory substance by Leuconostoc mesenteroides subsp. dextranicum 213M0 isolated from Mongolian fermented mare milk, airag.

Strain 213M0 was selected with productivity of a bacteriocin-like inhibitory substance (BLIS) among 235 strains of lactic acid bacteria (LAB) isolated from Mongolian fermented milk 'airag'. Strain 213M0 was species-identified as Leuconostoc mesenteroides subsp. dextranicum by morphological observation, carbohydrate fermentation profiling and sequencing the 16S rRNA gene. Incubation temperature proper to produce the BLIS was 25°C rather than 30 and 37°C, and the production actively proceeded during the exponential growth phase of the producer cells. Antibacterial effect of BLIS 213M0 was limited to all nine strains of Listeria sp. bacteria and seven strains of LAB cocci among 53 tested strains, which corresponds to a typical feature of the class IIa pediocin-like bacteriocins. BLIS 213M0 was not inactivated in every broad pH range solution (pH 2.0-11.0), and was stable against storage at 25°C for 1 week and heating at 121°C for 15 min under pH 4.5. Peptide frame of BLIS 213M0 was confirmed by inactivation with some peptidases, and then its molecular weight was estimated to be 2.6-3.0 kDa using an in situ activity assay following sodium dodecyl sulfate polyacrylamide gel electrophoresis. The estimated size was different from the other Leuconostoc bacteriocins already reported. These results suggest that BLIS 213M0 would be a novel listericidal bacteriocin.