Leuconostoc Isolates Research Articles

Screening for ability of locally isolates Leuconostoc spp. to produce biosurfactant and determination of optimum condition for its production

Fifteen samples of raw milk and yogurt were used to isolate Leuconostoc spp. ,Twenty eight Leuconostoc spp.were isolated. All isolates were tested for biosurfactant production , Producer isolates were then subjected to the biochemical examination and Vitek 2 system for identification to the species . Eleven of biosurfactantproducer isolates were identified from 28 Leuconostoc isolates. They are distributed as Leuconostoc mesenteroide sssp cremoris . Surface activity of Leuconostoc mesenteroides ssp cremor is supernatants were studied .The optimal conditions of biosurfactant production included growing at different temperatures; different pH, different incubation periods, aeration and inoculum ratio were also studied. The results showed that surface activity of supernatant was differed according to isolate, with oil displacement diameter 4-7 mm. Anaerobic and aerobic conditions for 24 hours at30o C and pH 6 and 2% inoculum were fitting to biosurfactant production.

Selection of Leuconostoc strains isolated from artisanal Serrano Catarinense cheese for use as adjuncts in cheese manufacture.

Serrano Catarinense cheese is a raw bovine milk cheese produced in the region of Santa Catarina, Brazil. Twelve representative strains of Leuconostoc isolated from 20 samples of this artisanal cheese were selected and submitted for evaluation of the acidifying, proteolytic, autolytic, aminopeptidase and lipolytic activities, NaCl and acid resistance, production of dextran and biogenic amines and antimicrobial activity. The aim was to genetically and technologically characterize the Leuconostoc strains in order to use them in mixed starter cultures for cheese manufacture. Leuconostoc mesenteroides subsp. mesenteroides was the species that accounted for the largest proportion of isolates of Leuconostoc genus. Two leuconostoc isolates stood out in the acidifying activity, with reduction in pH of 1.12 and 1.04 units. The isolates showed low proteolytic and autolytic activity. Most of the isolates were dextran producers, presented good resistance to the salt and pH conditions of the cheese and showed antimicrobial activity against cheese pathogen bacteria, and none of them produced biogenic amines. These results allowed the selection of five strains (UEL 04, UEL 12, UEL 18, UEL 21 and UEL 28) as good candidates for use as adjunct cultures for cheese manufacture. © 2018 Society of Chemical Industry.

Leuconostoc spp. Associated with Root Rot in Sugar Beet and Their Interaction with Rhizoctonia solani.

Rhizoctonia root and crown rot is an important disease problem in sugar beet caused by Rhizoctonia solani and also shown to be associated with Leuconostoc spp. Initial Leuconostoc studies were conducted with only a few isolates and the relationship of Leuconostoc with R. solani is poorly understood; therefore, a more thorough investigation was conducted. In total, 203 Leuconostoc isolates were collected from recently harvested sugar beet roots in southern Idaho and southeastern Oregon during 2010 and 2012: 88 and 85% Leuconostoc mesenteroides, 6 and 15% L. pseudomesenteroides, 2 and 0% L. kimchi, and 4 and 0% unrecognized Leuconostoc spp., respectively. Based on 16S ribosomal RNA sequencing, haplotype 11 (L. mesenteroides isolates) comprised 68 to 70% of the isolates in both years. In pathogenicity field studies with commercial sugar beet 'B-7', all Leuconostoc isolates caused more rot (P < 0.0001; α = 0.05) when combined with R. solani than when inoculated alone in both years. Also, 46 of the 52 combination treatments over the 2 years had significantly more rot (P < 0.0001; α = 0.05) than the fungal check. The data support the conclusion that a synergistic interaction leads to more rot when both Leuconostoc spp. and R. solani are present in sugar beet roots.

Antimicrobial potential of Leuconostoc species against E. coli O157:H7 in ground meat

Ground beef is risky by foodborne pathogens such as E. coli O157:H7 due to the cross-contamination during grinding. The objective of this study was to evaluate the antagonistic activities of Leuconostoc species isolated from ground beef product in order to limit the growth of E. coli O157:H7. While Leuconostoc has been known as spoilage bacteria, the Leuconostoc isolates showed antimicrobial activity on foodborne pathogens such as E. coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and meat-spoilage bacteria Brochothrix thermosphacta. Antimicrobial activity of cell-free supernatant (CFS) was evaluated by heat, enzyme, and pH adjustment and antagonistic activity by cell competitive growth. In addition, CFS concentration was screened for the presence of organic acids for the antimicrobial properties. Regulation of virulence genes was investigated by inhibition of locus of enterocyte effacement expression by CFS. The result showed that Leuconostoc isolates inhibited the growth of E. coli O157:H7 by active antimicrobial agent including organic acids and the growth of background microorganisms, and E. coli O157:H7 in ground beef was inhibited with the supplement of the CFS.

English

In the present study, a total of 56 isolates were isolated from different root vegetables. Out of these, 17 isolates were identified as Leuconostoc spp. All the 17 isolates were checked for antibiotic sensitivity against different antibiotics. Results revealed that majority of the isolates were resistant to Penicillin G, Vancomycin, Oxacillin and Ceftazidime. Four isolates (S-9, S-13, S-37 and S-42) were resistant to methicillin. However, all the isolates were highly sensitive to Imipenum. Carbenicillin and Amoxicillin sublactam showed antibacterial sensitivity against all the isolates except S-13 and S-B2C2, respectively. Electrophorogram revealed that among the different 17 Leuconostoc isolates, S-B2C2 showed the presence of multiple plasmids (six) corresponding to the molecular weights of 1.5, 1.9, 2.0, 2.6, 3.2 and 10 kb, respectively. Endonuclease restriction analysis study was carried out with purified plasmid using four endonucleases (Alu I, Bam HI, Hae III and Hind III). Treatment with Alu I resulted in the disappearance of all the 6 plasmid bands, indicating complete digestion of the plasmids. Restriction analysis of plasmid DNA of isolate S-B2C2 revealed complete digestion of two plasmids (2.6 and 1.5 kb) when treated with Hind III. However, a new band of molecular weight equivalent to 1.7 kb did appear. Data presented in the paper indicates the multiple plasmid availability in bacteria and their diversity in response to restriction sites available on them. Key words: Antibiotic resistance, plasmid, restriction digestion, root vegetables.&nbsp

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial-resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P=0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm-made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm-made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to >512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial-resistant LAB in imported and Japanese farm-made cheeses on the Japanese market, but not in Japanese commercial cheeses.

Identification, typing, and functional characterization of Leuconostoc spp. strains from traditional, starter-free cheeses

Selected Leuconostoc strains are required as aroma producers in dairy starters. In this work, 42 Leuconostoc isolates from different raw milk traditional cheeses made without the addition of commercial starters were identified by molecular methods. These isolates were assigned to the species Leuconostoc citreum (24), Leuconostoc mesenteroides (13), and Leuconostoc lactis (5). Typing the isolates by repetitive extragenic profiling (Rep-PCR) identified 22 strains, out of which 14 were selected for further characterization based on their behavior in milk. The biochemical, technological, and food safety variables recorded were examined to select appropriate candidates for use as adjunct cultures. In agreement with the wide genetic diversity recorded by Rep-PCR, wide phenotypic biodiversity was seen among the species and strains in terms of enzyme and carbohydrate fermentation profile, acidification rate, the production of volatile compounds in milk, and growth under different pH, salt, and temperature conditions. No production of biogenic amines was recorded even when suitable amino acid precursors were present in the growth medium. All 14 strains proved to be susceptible or intrinsically resistant to a set of 16 antibiotics except for one Leuc. citreum strain that showed an atypical resistance to ciprofloxacin. The remaining 13 strains could be confidently used as components of adjunct cultures in the dairy industry.

Microbial diversity and dynamics of Pecorino di Filiano PDO, a traditional cheese of Basilicata region (Southern Italy)

The evolution of microbial populations of ‘Pecorino di Filiano’ (PF) cheese was investigated during ripening in natural cave and storeroom. 62.5% of isolates grow at 45 and 15 °C and 77.7% showed high salt concentrations tolerance. Brevibacterium linens was dominant in surface samples. Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus paracasei subsp. paracasei were more frequently isolated both surface and core samples, while Leuconostoc lactis and Leuconostoc mesenteroides subsp. mesenteroides prevailed among Leuconostoc isolates. Our results suggest the importance of the ripening environment of cheeses and how a biological ecosystem affects and produces the typical features of artisanal products.

Surface properties of Lactobacillus and Leuconostoc isolates from homemade cheeses showing auto-aggregation ability

Ten lactobacilli and one leuconostoc showing auto-aggregation ability were isolated from artisanal cheeses. Furthermore, non-aggregation strains were isolated from the same cheese sample, if existed. The analysis of factor(s) possibly involved in auto-aggregation was performed. The pretreatment of cells with proteinase K resulted in the disappearance of auto-aggregation ability. Moreover, cells also lost aggregation ability after three-times, successive washing in distilled water. Testing the ability of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8 and its aggregation-deficient derivative BGSJ2-81 to co-aggregate with Listeria innocua ATCC33090, Escherichia coli ATCC25922 or Salmonella typhimurium TR251 showed that strain BGSJ2-8 co-aggregated with these strains, but derivative BGSJ2-81 was not. However, the treatment of L. paracasei subsp. paracasei BGSJ2-8 with proteinase K prior to co-aggregation tests resulted in losing co-aggregation ability. Surface properties of selected strains were analyzed by MATS (microbial adhesion to solvents) method. It was noticed that the strains with auto-aggregation ability were highly hydrophobic in comparison with aggregation-deficient ones. Comparative analyses of the surface features of strain L. paracasei subsp. paracasei BGSJ2-8 and its derivative BGSJ2-81 revealed notable difference.

Genotypic and technological characterization of Leuconostoc isolates to be used as adjunct starters in Manchego cheese manufacture

Twenty-seven Leuconostoc (Ln.) isolates from Manchego cheese were characterized by phenotypic and genotypic methods, and their technological abilities studied in order to test their potential use as dairy starter components. While phenotypic diversity was evaluated by studying the biochemical characteristics of technological interest (i.e. acidifying and aminopeptidase activities), genotypic diversity was evidenced by using Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). Additional technological abilities such as lipolytic, proteolytic and autolytic activities, salt and pH tolerance and production of dextran, flavour compounds and biogenic amines, were investigated. The marked differences among strains reflected the existing biodiversity in naturally fermented products. After statistically evaluating their performance, strains C0W2, belonging to Ln. lactis, and C16W5 and N2W5, belonging to Ln. mesenteroides subsp. dextranicum, revealed the best properties to be used in mixed dairy starter cultures. This study evidences the fact that natural environments can be considered as a proper source of useful strains, for the dairy industry.

Microfluidic technology applied to cell-wall protein analysis of olive related lactic acid bacteria

A fully automated electrophoretic method for separating cell-wall proteins was applied to 102 isolates from olive phylloplane and brine previously phenotypically identified as Lactobacillus plantarum (10), Leuconostoc mesenteroides subsp. mesenteroides (60), Leuconostoc spp. (12) and Enterococcus faecium (19). The high sensitivity of this technique allowed to discriminate isolates and to distinguish closely related strains within the same species. L. plantarum was characterized by proteins at 70.4 and 163.9 kDa, while 3 proteins (20.4, 52.9, 68. kDa) were typical for E. faecium. No characteristic proteins were found for Leuconostoc isolates except for that at 31.8 kDa mainly related to the ecological origin since only isolates from brine showed this protein. However, Leuc. mesenteroides subsp. mesenteroides and Leuconostoc spp. were differentiated by additional proteins. Sequence analysis of the 16S rRNA gene of selected strains was in agreement with the phenotypic identification and allowed to identify all Leuconostoc spp. isolates as Leuc. mesenteroides. Cell-wall protein fingerprinting performed by microfluidic technology completed the phenotypic characterization of olive-related lactic acid bacteria allowing their strain typing.

Succession of dominant and antagonistic lactic acid bacteria in fermented cucumber: Insights from a PCR-based approach

The goal of the investigation was to study the succession of major groups of lactic acid bacteria (LAB) and their antagonism in salt-fermented cucumber using PCR. In a direct detection method as well as a short enrichment process, PCR enabled detection of Leuconostoc and Lactobacillus during early hours of fermentation. Subsequently, Lactobacillus and Pediococcus emerged as the dominant genera. Nucleic acid sequence of culture-independent clones confirmed the detection of Pediococcus as a dominant genera emerging during late stages of fermentation. PCR also revealed time-dependent emergence of mesentericin, pediocin and plantaricin A producers and accounted for the LAB succession in the fermenting samples. A total of 328 LAB isolates were obtained collectively from 30 cucumber samples, of which PCR could identify an overwhelming 186 Lactobacillus isolates followed by 113 Pediococcus and 29 Leuconostoc isolates, respectively. Based on antimicrobial assay against target strain Leuconostoc mesenteroides NRRL B640, 28% of the LAB were bacteriocin producers, of which pediocin producers were substantial, followed by plantaricin A and mesentericin producers. The bacteriocins elaborated by the isolates were active against a large number of Gram-positive target LAB strains and pathogenic bacteria including Bacillus cereus, Enterobacter aerogenes, Enterococcus faecalis, Escherichia coli, Listeria monocytogenes and Staphylococcus aureus.

Antimicrobial susceptibility of lactic acid bacteria isolated from a cheese environment

In the production of the Spanish traditional blue-veined Cabrales cheese, lactic acid bacteria strains free of antibiotic resistance that have a transferrable capacity are necessary as components of a specific starter. To select for these bacteria, the minimum inhibitory concentration (MIC) of 12 antibiotics and 2 mixtures (containing beta-lactamase inhibitor and penicillin) were determined by microbroth and agar dilution techniques in 146 strains belonging to the genera Lactococcus, Enterococcus, Lactobacillus, and Leuconostoc. The antibiotic-resistance profiles of Lactococcus and Enterococcus species were different from those of Lactobacillus and Leuconostoc, but clear genus- or species-associated patterns were not observed. Cefoxitin and metronidazole were not effective against bacteria of these genera. The MICs of beta-lactam antibiotics for lactobacilli and leuconostoc isolates were higher than those for lactococci and enterococci, but no strain was clinically resistant. All lactobacilli and leuconostoc isolates were resistant to high levels of vancomycin, a type of resistance not seen among the tested members of the genera Lactococcus and Enterococcus. The majority of the observed resistance appeared to be either intrinsic or nonspecific, although some strains of Lactococcus lactis, Enterococcus spp., and Lactobacillus spp. were resistant to antibiotics, such as chloramphenicol, erythromycin, clindamycin, or tetracycline.

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

A polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method was developed to detect and identify typical Leuconostoc species. This method utilises a set of specific primers for amplification of the 16S rDNA region of typical Leuconostoc species. All Leuconostoc-type strains, all Leuconostoc isolates from kimchi, Korea's traditional, fermented vegetable product, and strains from closely related genera were examined to verify the identification by this method. The primers resulted in amplification only for nine typical Leuconostoc spp., but not for any other genera tested. The size of the amplified products was 976 bp and the amplicons of the different species could be differentiated from each other with MseI, HaeIII and Tsp509I endonucleases, except for the species Leuconostoc argentinum and Leuconostoc lactis, which were indistinguishable. A PCR-RFLP method for the typical Leuconostoc species was optimized to identify a large number of isolates from fermented vegetable product. This PCR-RFLP method enables the rapid and reliable identification of Leuconostoc species and to distinguish them from the other phylogenetically related lactic acid bacteria in food samples.

Technological characterization of lactic acid bacteria from Tenerife cheese

Summary One hundred and thirty lactic acid bacteria (Lactococcus, Lactobacillus and Leuconostoc), isolated from Tenerife cheese, were technologically characterized. Interesting differences within genera, species and strains were found. None of the strains showed lipolytic activity on tributyrin agar while the majority exhibited proteolytic activity on calcium caseinate agar. Citrate was metabolized exclusively by 98.3% of the lactobacilli isolates. Lactococcus lactis ssp. lactis showed the highest acidifying and proteolytic activities in milk, although considerable inter‐strain variations were found. Lactobacilli had the highest aminopeptidolytic activity. Dipeptidase activity was displayed by most lactic acid bacteria isolates with the higher specific activity exhibited by Lc. lactis ssp. lactis strains. Leuconostoc isolates had the highest esterolytic and β‐galactosidase activity.

The microbiology of South African traditional fermented milks

A total of 15 samples of traditional fermented milk were collected from individual households in South Africa and Namibia. Lactic acid bacteria dominated the microflora of these samples, especially the genera Leuconostoc, Lactococcus and Lactobacillus. Other groups identified included pyogenic streptococci and enterococci. The dominant lactococci species was Lactococcus lactis subsp. lactis. Eighty-three percent of the leuconostoc isolates were identified as Leuconostoc mesenteroides subsp. dextranicum. Other species identified included Leuconostoc citreum, Leuconostoc lactis, Lactobacillus delbrueckii subsp. lactis and Lactobacillus plantarum.

Biochemical and microbiological characterization of artisanal `Peñamellera' cheese: analysis of its indigenous lactic acid bacteria

Abstract Three batches of artisanal `Penamellera' cheese manufactured at different times of the year have been analysed. Biochemical parameters fluctuated widely between batches due to unpredictable changes in temperature and humidity during manufacture. The profile of volatile compounds in `Penamellera' cheese was found to be simple, with ethanol, 2-heptanone and 3-methyl butanol as the predominant components. The microbiota of the three batches was independently monitored on the surface and in the interior of the cheeses through a 30 d ripening period. Lactococci became dominant early after manufacture and remained so during ripening (they almost reached values of 10 9 cfu g −1 ). Most of the isolates were classified as Lactococcus lactis subsp. lactis . Lactobacilli remained at a subdominant level (around 5.0×10 7 cfu g −1 ) and a high proportion of heterofermentative strains was found. Dextran-producing Leuconostoc isolates were found at a level of approximately 10 6 cfu g −1 . Strains of lactococci were examined for several technologically important traits as a first approach to select suitable strains to be used in starter cultures.

Nutritional value of the non-protein N that accumulates during growth of proteinase-positive strains of Lactococcus lactis in milk for dairy lactococcal and leuconostoc isolates

To estimate the suitability of cultured milk for the subsequent growth of dairy lactic acid bacteria in cheese manufacturing, control milk was first cultured until the end of the exponential phase with one of four proteolytic (Prt+) strains of Lactococcus lactis differing in their proteolytic enzymes, and pasteurized after readjusting the pH to its initial value. Nineteen non-proteolytic (Prt−) strains of Lc. lactis and nine Leuconostoc mesenteroides strains of dairy origin were then grown in the precultured milk until the stationary phase and their growth was compared with that in control milk. Despite the accumulation of non-protein N (NPN) during preculture, the growth of most Prt− strains of Lc. lactis in precultured milk was either reduced or unchanged whereas that of Ln. mesenteroides strains was unchanged or slightly stimulated. This reduction in growth was reversed by adding an NPN source to precultured milk, indicating that it was due to the exhaustion of assimilable NPN in precultured milk. Thus, preculturing milk with Prt+ strains of Lc. lactis could not be recommended for promoting the subsequent growth of starter cultures in cheese manufacturing.

Effect of Sodium Nitrite and Sodium Chloride on Growth of Lactic Acid Bacteria

The effect of NaNO2 and NaCl on the growth of 24 lactic acid bacteria strains isolated from vacuum-packed cooked ring sausages were examined by analyzing different growth parameters with Bioscreen. NaNO2 had a very limited effect on the growth of lactic acid bacteria at 50 and 100 mg/l but at 400 mg/l a more pronounced inhibitory effect was found. Bacterial growth was enhanced by 1-2% (w/v) of added NaCl, while NaCl concentrations above 3% (w/v) had a clear inhibitory effect. Leuconostoc isolates seemed to be more sensitive to sodium nitrite and sodium chloride than homofermentative lactobacilli strains. Among homofermentative lactobacilli, the strains resembling Lactobacillus curvatus were more sensitive to NaCl than those resembling Lactobacillus sake.

Characteristics of Salt-Tolerant Lactic Acid Bacteria, in Particular Lactobacilli, Leuconostocs and Pediococci, Isolated from Salted Raw Milk

One hundred and fourty five lactobacilli, leuconostocs and pediococci were isolated from salted raw milks incubated at 30°C for 4 to 21 d. Of 126 lactobacilli isolated, mostly from 9 to 12% salted milk, 115 were identified as homofermentative, nonthermophilic lactobacilli-73 were classified as Lactobacillus plantarum, 31 Lactobacillus casei, 8 strains were motile and 3 Lactobacillus xylosus. The remaining 11 isolates were heterofermentative lactobacilli-8 were Lactobacillus cellobiosus and 3 Lactobacillus brevis-buchneri. Strains of L. plantarum fermented many oligosaccharides, produced DL lactate and gas from L(+) but not from D(−) tartrate and their cell wall peptidoglycan was of the mesodiaminopimelic acid type. Eight strains of L. casei proved to be subsp. pseudoplantarum on the basis of inactive lactic acid production; 8 were subsp. rhamnosus and 2 subsp. alactosus, according to their pattern of sugar fermentation. L. xylosus simulated L. casei morphologically but differed from it in fermentation of xylose. The motile strains fermented arabinose and mostly sucrose but not lactose and produced 73.2 to 94 μmoles ml−1 inactive lactate from 1 % glucose. None of 10 Leuconostoc isolates produced dextran from sucrose but they fermented trehalose and were identified with Leuconostoc paramesenteroides. Three strains belonged to Pediococcus and produced 62 μmoles ml−1 of inactive lactate, whereas other six strains were atypical pediococci. Nineteen strains representing L. plantarum, L. casei, motile strains and Pediococcus gave, on examination for isomers of lactic acid, 32.8 to 171 μmoles ml−1 inactive lactate; the L(+) enanthiomorph generally predominated.