Characterization and electrotransformation of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Lactobacillus paraplantarum</Emphasis> isolated from fermented vegetables

Aims of the study were to characterize two Lactobacillus plantarum-related strains, Lact. plantarum and Lactobacillus paraplantarum isolated from fermented vegetables and, for their potential use as starter strains, compare their growth in various food matrices. Species-level identification of the strains belonging to the Lact. plantarum group was performed by multiplex-PCR with species-specific primers and generation of distinct genotypic profiles was carried out by PFGE-based DNA-fingerprinting. Growth profiles were determined in various food and feed matrices. Compared to Lact. plantarum, Lact. paraplantarum reached higher cell densities in all plant-based matrices and MRS broth. On the contrary to the good growth in plant-based matrices and MRS, poor growth was observed in unprocessed milk. Supplemented lactose did not improve the growth of either tested strain, while predigestion of milk proteins with Lactobacillus helveticus or addition of casitone proved to be an effective means to enhance growth. To find out the applicability of molecular methods, the strains were transformed with replicative plasmids by electroporation. To our knowledge, this is a first report of the electrotransformation of Lact. paraplantarum with a recombinant plasmid.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and Its Probiotic and Food Potentialities

The number of studies claiming probiotic health effects of Lactobacillus plantarum is escalating. Lb. plantarum is a lactic acid bacterium found in diverse ecological niches, highlighting its particular capabilities of adaptation and genome plasticity. Another function that needs to be underlined is the capabilities of Lb. plantarum to produce diverse and potent bacteriocins, which are antimicrobial peptides with possible applications as food preservative or antibiotic complementary agents. Taken together, all these characteristics design Lb. plantarum as a genuine model for academic research and viable biological agent with promising applications. The present review aims at shedding light on the safety of Lb. plantarum and run through the main studies underpinning its beneficial claims. The mechanisms explaining probiotic-related features are discussed.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI activity was stimulated by Mn²⁺, Fe³⁺, Fe²⁺, Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, Pb²⁺. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K _m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production corresponds to a 39% equilibrium.     

In Vivo PCR-DGGE Analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.     

Expression of the Malolactic Enzyme Gene (<Emphasis Type="Italic">mle</Emphasis>) from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Under Winemaking Conditions

Malolactic fermentation (MLF) plays an important role in the production of wine, especially red wines, resulting in microbial stability, deacidification, as well as contributing to the aroma profile. MLF can be influenced by a number of factors. In this study, the influence of pH and ethanol on expression of the structural malolactic enzyme gene (mle) from Lactobacillus plantarum was investigated in a synthetic wine media, as well as in wine using quantitative PCR. Expression of mle was shown to be inducible by the presence of malic acid, with increased expression in the middle of MLF. Expression of mle was also shown to be increased at low pH values and decreased in the presence of ethanol. This indicates the role of MLF in acid tolerance and the negative impact of ethanol on the completion of MLF. The results therefore provide further evidence that L. plantarum should be applied as co-inoculation for MLF where alcohol will initially not have a negative impact on the malic acid degradation.     

Two-step production of gamma-aminobutyric acid from cassava powder using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

Production of gamma-aminobutyric acid (GABA) from crop biomass such as cassava in high concentration is desirable, but difficult to achieve. A safe biotechnological route was investigated to produce GABA from cassava powder by C. glutamicum G01 and L. plantarum GB01-21. Liquefied cassava powder was first transformed to glutamic acid by simultaneous saccharification and fermentation with C. glutamicum G01, followed by biotransformation of glutamic acid to GABA with resting cells of L. plantarum GB01-21 in the reaction medium. After optimizing the reaction conditions, the maximum concentration of GABA reached 80.5 g/L with a GABA productivity of 2.68 g/L/h. This is the highest yield ever reported of GABA production from cassava-derived glucose. The bioprocess provides the added advantage of employing nonpathogenic microorganisms, C. glutamicum and L. plantarum, in microbial production of GABA from cassava biomass, which can be used in the food and pharmaceutical industries.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Physiological responses to folate overproduction in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> WCFS1

Background  

Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells.     

Heterologous Expression of Oxalate Decarboxylase in <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> NC8

Lactic acid bacteria (LABs) are being used as a probiotic very often for various enteric problems. Many genetically modified LABs are created by different workers for various novel applications. In this study we examine the expression of heterologous oxalate decarboxylase (oxdc) in Lactobacillus plantarum NC8. Generally, this enzyme is not present in Lactobacillus spp. Oxdc gene from Bacillus subtilis was polymerase chain reaction-amplified and cloned in a shuttle vector pSIP400 series, downstream of the inducible promoter, P_orfx. In the presence of an inducing peptide, Sakacin-P, the expression of OxdC was observed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The cell-free extract and the purified protein from the recombinant LABs showed the presence of OxdC activity. The above recombinant LABs, with desired modifications, can be used as a possible probiotic for the degradation of intestinal dietary oxalate for preventing enteric hyperoxaluria.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Effects of heat-killed <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> against influenza viruses in mice

The potential use of dietary measures to treat influenza can be an important alternative for those who lack access to influenza vaccines or antiviral drugs. Lactobacillus plantarum (Lp) is one of many lactic acid bacteria that grow in ‘kimchi’, an essential part of Korean meal, and several strains of Lp reportedly show protective effects against influenza. Using heat-killed Lp (nF1) isolated from kimchi, which is known for its immunomodulatory effects, we investigated whether regular oral intake of nF1 could influence the outcome of influenza virus infection in a mouse model. In a lethal challenge with influenza A (H1N1 and H3N2 subtypes) and influenza B (Yamagata lineage) viruses, daily oral administration of nF1 delayed the mean number of days to death of the infected mice and resulted in increased survival rates compared with those of the non-treated mice. Consistent with these observations, nF1 treatment also significantly reduced viral replication in the lungs of the infected mice. Taken together, our results might suggest the remedial potential of heatkilled Lactobacillus probiotics against influenza.     

Regulation of stipule development by <Emphasis Type="Italic">COCHLEATA</Emphasis> and <Emphasis Type="Italic">STIPULE</Emphasis>-<Emphasis Type="Italic">REDUCED</Emphasis> genes in pea <Emphasis Type="Italic">Pisum sativum</Emphasis>

Pisum sativum L., the garden pea crop plant, is serving as the unique model for genetic analyses of morphogenetic development of stipule, the lateral organ formed on either side of the junction of leafblade petiole and stem at nodes. The stipule reduced (st) and cochleata (coch) stipule mutations and afila (af), tendril-less (tl), multifoliate-pinna (mfp) and unifoliata-tendrilled acacia (uni-tac) leafblade mutations were variously combined and the recombinant genotypes were quantitatively phenotyped for stipule morphology at both vegetative and reproductive nodes. The observations suggest a role of master regulator to COCH in stipule development. COCH is essential for initiation, growth and development of stipule, represses the UNI-TAC, AF, TL and MFP led leafblade-like morphogenetic pathway for compound stipule and together with ST mediates the developmental pathway for peltate-shaped simple wild-type stipule. It is also shown that stipule is an autonomous lateral organ, like a leafblade and secondary inflorescence.     

Selection of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> TN627 as a new probiotic candidate based on <Emphasis Type="Italic">in vitro</Emphasis> functional properties

Nine lactobacilli previously selected for high antagonism against food borne bacterial pathogens were identified via 16S rRNA gene sequencing and screened for probiotic potential for use in poultry production. The lactobacilli were subjected to a subtractive in vitro analysis system using a certified probiotic as reference. This allowed for selection of a milk-derived Lactobacillus plantarum strain, termed TN627. This organic acid-producing bacterium was free of harmful enzymatic activity and sensitive to several antibiotics. It also showed good growth at pH 4 and in the presence of bile. L. plantarum TN627 also exhibited high efficacy of adhesion to chicken enterocytes, which correlated with detecting genes encoding the mucusbinding, adhesion-promoting proteins (Mub and MapA) and the adhesion-like factor EF-Tu, commonly involved in adherence of lactobacilli to mucosal surfaces. Taken together, our findings suggest that TN627 is a promising probiotic candidate with high potential for application as a supplement in the animal feed industry.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

Safety of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ST8Sh and Its Bacteriocin

Total DNA extracted from Lb. plantarum ST8Sh was screened for the presence of more than 50 genes related to production of biogenic amines (histidine decarboxylase, tyrosine decarboxylase, and ornithine decarboxylase), virulence factors (sex pheromones, gelatinase, cytolysin, hyaluronidase, aggregation substance, enterococcal surface protein, endocarditis antigen, adhesion of collagen, integration factors), and antibiotic resistance (vancomycin, tetracycline, erythromycin, gentamicin, chloramphenicol, bacitracin). Lb. plantarum ST8Sh showed a low presence of virulence genes. Only 13 genes were detected (related to sex pheromones, aggregation substance, adhesion of collagen, tetracycline, gentamicin, chloramphenicol, erythromycin, but not to vancomycin, and bacitracin) and may be considered as indication of safety for application in fermented food products. In addition, interaction between Lb. plantarum ST8Sh and drugs from different groups were determined in order to establish possible application of the strain in combination with commercial drugs. Cytotoxicity of the semi-purified bacteriocins produced by Lb. plantarum ST8Sh was depended on applied concentration—highly cytotoxic when applied at 25 μg/mL and no cytotoxicity at 5 μg/mL.     

Effects of Dietary <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on Growth Performance,Digestive Enzymes and Gut Morphology of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

A 15-day feeding trial was conducted to investigate the effect of dietary Lactobacillus plantarum on growth performance, digestive enzyme activities and gut morphology of juvenile Pacific white shrimp, Litopenaeus vannamei (initial body weight = 7.96 ± 0.59 g). Four microbound diets were formulated to contain fermentation supernatant (FS), live bacteria (LB), dead bacteria (DB), and cell-free extract (CE) of L. plantarum. Results indicated that final weight was significantly higher in FS, DB, and CE group in comparison to the control group (P < 0.05). The maximum weight gain rate (WGR) and specific growth rate (SGR) of the CE diet group were significantly higher than that of other groups (P < 0.05). The FCR of CE diet group was lower than that of the control, LB, DB, and FS diets groups (P < 0.05). The highest digestive enzyme activities (amylase, lipase, and pepsin activity) in the hepatopancreas and gut of shrimp were observed in the CE diet group. Histological study revealed that dietary CE diet could significantly increase the enterocytes height of shrimp. The administration of cell-free extract of L. plantarum could effectively improve the growth performance of L. vannamei via the improvement of digestive enzyme activities and the enterocytes height of shrimp. The results of this study will be essential to promote application of probiotics in shrimp aquaculture.     

Effect of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> on the proliferation of <Emphasis Type="Italic">Propionibacterium acnes</Emphasis> and <Emphasis Type="Italic">Staphylococcus epidermidis</Emphasis>

While it is generally accepted that Propionibacterium acnes is involved in the development of acne, other bacteria including Staphylococcus epidermidis have also been isolated from the acne lesion. The interaction between Lactobacillus reuteri, a probiotic bacterium, and acnegenic bacteria is unclear. This study examined the effects of L. reuteri on the proliferation of P. acnes and S. epidermidis. Human-derived L. reuteri strains (KCTC 3594 and KCTC 3678) and rat-derived L. reuteri KCTC 3679 were used. All strains exhibited significant inhibitory effects on the growth of P. acnes and S. epidermidis. The proliferation of P. acnes was decreased by 2-log scales after incubation with L. reuteri for 24 h. In addition, the proliferation of S. epidermidis was decreased by 3-log scales after incubation with L. reuteri for 24 h, whereas the growth of L. reuteri was unaffected by P. acnes or S. epidermidis. Among the L. reuteri strains examined, L. reuteri KCTC 3679 had the strongest inhibitory effect on the growth of P. acnes and S. epidermidis, followed by L. reuteri KCTC 3594 and L. reuteri KCTC 3678. Interestingly, reuterin, an antimicrobial factor, was produced only by L. reuteri KCTC 3594. The most pronounced the antibacterial activities of L. reuteri were attributed to the production of organic acids. Overall, these results suggest that L. reuteri may be a useful probiotic agent to control the growth of bacteria involved in acne inflammation and prevent acne.