Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

Functional Properties of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains Isolated from Maasai Traditional Fermented Milk Products in Kenya

Lactobacillus plantarum was the major species among the lactic acid bacterial strains isolated from traditional fermented milk of the Maasai in Kenya. Selected strains were characterized for their functional properties using in vitro standard procedures. All strains expressed acid tolerance at pH 2.0 after 2-h exposure of values that ranged from 1% to 100%, while bile tolerance of acid-stressed cells at 0.3% oxgal varied from 30% to 80%. In vitro adhesion to the mucus-secreting cell line HT 29 MTX and binding capacity to extracellular protein matrices was demonstrated for several strains. The four strains tested in a simulated stomach duodenum passage survived with recovery rates ranging from 17% to 100%. Strains were intrinsically resistant to several antibiotics tested. From these in vitro studies, a number of Lb. plantarum strains isolated from the Maasai traditional fermented milk showed probiotic potential. The strains are good candidates for multifunctional starter culture development.     

Analysis of functional properties of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis>

Metabolites from Lactobacillus acidophilus were analysed. The results showed that Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid produced respectively 12.73 g and 13.33 g lactic acid l⁻¹ after incubating in skim milk at 37 °C for 36 h; and 2.229 unit and 1.808 unit β-galactosidase l⁻¹ in an MRS medium. The proteolytic activity of Lactobacillus acidophilus was high and the content of 17 free amino acids in the fermented milk of Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid was 394.4 mg l⁻¹ and 563.2 mg l⁻¹, respectively. Meantime, Lactobacillus acidophilus reduced cholesterol level in an MRS medium supplemented with cholesterol. Furthermore, Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid showed antimicrobial activity against Bacillus anthracis and Escherichia coli.     

Evaluation of the probiotic potential and effect of encapsulation on survival for <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ST16Pa isolated from papaya

Capability to produce antilisterial bacteriocins by lactic acid bacteria (LAB) can be explored by the food industry as a tool to increase the safety of foods. Furthermore, probiotic activity of bacteriogenic LAB brings extra advantages to these strains, as they can confer health benefits to the consumer. Beneficial effects depend on the ability of the probiotic strains to maintain viability in the food during shelf-life and to survive the natural defenses of the host and multiply in the gastrointestinal tract (GIT). This study evaluated the probiotic potential of a bacteriocinogenic Lactobacillus plantarum strain (Lb. plantarum ST16Pa) isolated from papaya fruit and studied the effect of encapsulation in alginate on survival in conditions simulating the human GIT. Good growth of Lb. plantarum ST16Pa was recorded in MRS broth with initial pH values between 5.0 and 9.0 and good capability to survive in pH 4.0, 11.0 and 13.0. Lb. plantarum ST16Pa grew well in the presence of oxbile at concentrations ranging from 0.2 to 3.0%. The level of auto-aggregation was 37%, and various degrees of co-aggregation were observed with different strains of Lb. plantarum, Enterococcus spp., Lb. sakei and Listeria, which are important features for probiotic activity. Growth was affected negatively by several medicaments used for human therapy, mainly anti-inflammatory drugs and antibiotics. Adhesion to Caco-2 cells was within the range reported for other probiotic strains, and PCR analysis indicated that the strain harbored the adhesion genes mapA, mub and EF-Tu. Encapsulation in 2, 3 and 4% alginate protected the cells from exposure to 1 or 2% oxbile added to MRS broth. Studies in a model simulating the transit through the GIT indicated that encapsulated cells were protected from the acidic conditions in the stomach but were less resistant when in conditions simulating the duodenum, jejunum, ileum and first section of the colon. To our knowledge, this is the first report on a bacteriocinogenic LAB isolated from papaya that presents application in food biopreservation and may be beneficial to the consumer health due to its potential probiotic characteristics.     

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.     

Effect of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Tennozu-SU2 on <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium Infection in Human Enterocyte-Like HT-29-Luc Cells and BALB/c Mice

The probiotic properties and inhibitory effect on Salmonella Typhimurium adhesion on human enterocyte-like HT-29-Luc cells of three Lactobacillus plantarum strains isolated from fermented fish, beach sand and a coastal plant were determined. Compared with the type strain L. plantarum NBRC 15891^T, which was isolated from pickled cabbage, L. plantarum Tennozu-SU2 isolated from the acorn of a coastal tree showed high autoaggregation in de Man, Rogosa and Sharpe (MRS) broth and an antagonistic effect against S. Typhimurium in brain heart infusion (BHI) broth. Furthermore, heat-killed L. plantarum Tennozu-SU2 cells inhibited S. Typhimurium adhesion on HT-29-Luc cells. Both live and heat-killed L. plantarum Tennozu-SU2 cells showed an inhibitory effect on gut colonisation in BALB/c mice, as assessed by viable Salmonella count in faecal samples and by invasion into liver and spleen tissues. The properties shown in this study suggest that L. plantarum Tennozu-SU2 is useful as a starter and probiotic bacteria in functional food material.     

Viability and Stress Response of Putative Probiotic <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in Honey Environment

Due to problem of preservation of dairy products which serve as a matrix for probiotics, it is challenging to use these probiotics as food supplements in many developing countries. To determine the suitability of the Lactobacillus strains for exploitation as probiotics in honey, we investigated the effect of their storage on the viability, functionality, and the mechanism associated with their protective effect. Three isolates obtained from our laboratory collection were identified through amplification of the 16S rRNA gene. The viability of the strains in honey at different storage conditions was studied. Three genes (hdc, gtf, and clpL) responsible for the resistance of bacteria in acidic environments were screened. SDS-PAGE analysis of total protein was performed to observe protein profile changes of the strains after exposure to honey. All the three isolates, namely, GGU, GLA51, and GLP56, were identified as Lactobacillus plantarum strains. After 28 days of storage in honey at 4 °C, viable cell concentrations of the three strains were higher than 2.04?×?10⁶ CFU/ml. During the same period at room temperature, only the Lactobacillus plantarum GLP56 strain remained viable with a cell concentration of 1.86?×?10⁴ CFU/ml. The clpL gene coding for ATPase was detected in all the three strains. The protein of molecular weight ~?50 kDa was absent in the protein profile of Lactobacillus plantarum GGU after 60 days of storage in honey at 4 °C. The Lactobacillus plantarum GLP56, Lactobacillus plantarum GLA51, and Lactobacillus plantarum GGU strains exposed to honey can withstand acidic environmental stress but their viability declines over time.     

<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.     

Influence of carbohydrates on cell properties of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis>

Lactobacilli represent normal commensals of the human body, particularly in the gut and vagina where they protect these environments from incoming pathogens via a variety of mechanisms. The influence of the carbohydrate source present in reconstituted MRS growth medium on the different cell properties of two Lactobacillus rhamnosus strains were examined. Two human vaginal isolates, BGHV719 and exopolysaccharide producer strain BGHV954 were analyzed. The results demonstrated that unlike in reconstituted MRS with glucose as a carbon source, the presence of fructose, mannose, or rhamnose, significantly reduced cell surface hydrophobicity of both strains. In addition, differences in cell wall protein composition of L. rhamnosus BGHV719 and alterations in colony mucoidity of L. rhamnosus BGHV954 were also demonstrated. Light and SEM microscopy revealed differences on the cellular level when BGHV719 was cultivated in the presence of different sugars. The results of this study point out the importance of complex relationships between growth medium composition and the different aspects of bacterial behavior, and call for more detailed analyses of versatile bacterial responses to the changes in the environment, including vaginal ecosystem. This is especially important since lactobacilli are amongst the most widely used of probiotics.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> isolated from cheese: production and partial characterization of bacteriocin B391

Lactobacillus plantarum B391, a strain isolated from an artisanal French cheese, is a producer of a bacteriocin, expressing activity against Enterococcus faecalis NCTC 775, Clostridium perfringens NCTC 13170 and several Listeria monocytogenes strains. High stability was recorded after heat treatment at 121 °C for 20 min and when stored at 4 °C for more than 40 days. A challenge test performed in milk for 11 days showed potential for the control of L. monocytogenes. In the presence of the lytic bacteriocin B391, L. monocytogenes cells present numerous morphology modifications of cell shape and surface structure as well as in the cell division pattern, resulting ultimately in lysis. The high level of Listeria growth inhibition obtained in the presence of Lb. plantarum B391, and the stability of B391 bacteriocin for a long period of time, make this strain potentially interesting to use in milk products to increase food safety.     

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.     

Selection of Potential Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> with Inhibitory Activity Against <Emphasis Type="Italic">Salmonella</Emphasis> and Fecal Coliform Bacteria

Three hundred and sixty presumptive lactic acid bacteria (LAB) isolated from pregnant sows, newborn, suckling, and weaned piglets were preliminarily screened for anti-Salmonella activity. Fifty-eight isolates consisting of Lactobacillus reuteri (n = 32), Lactobacillus salivarius (n = 10), Lactobacillus mucosae (n = 8), Lactobacillus johnsonii (n = 5), and Lactobacillus crispatus (n = 3) were selected and further characterized for probiotic properties including production of antimicrobial substances, acid and bile tolerance, and cell adherence to Caco-2 cells. Eight isolates including Lact. johnsonii LJ202 and Lact. reuteri LR108 were identified as potential probiotics. LJ202 was selected for further use in co-culture studies of two-bacterial and multiple-bacterial species to examine its inhibitory activity against Salmonella enterica serovar Enteritidis DMST7106 (SE7106). Co-culture of LJ202 and SE7106 showed that LJ202 could completely inhibit the growth of SE7106 in 10 h of co-culture. In co-culture of multiple-bacterial species, culturable fecal bacteria from pig feces were used as representative of multiple-bacterial species. The study was performed to examine whether interactions among multiple-bacterial species would influence antagonistic activity of LJ202 against SE7106 and fecal coliform bacteria. Co-culture of SE7106 with different combinations of fecal bacteria and probiotic (LJ202 and LR108) or non-probiotic (Lact. mucosae LM303) strains revealed that the growth of SE7106 was completely inhibited either in the presence or in the absence of probiotic strains. Intriguingly, LJ202 exhibited notable inhibitory activity against fecal coliform bacteria while LR108 did not. Taken together, the results of co-culture studies suggested that LJ202 is a good probiotic candidate for further study its inhibitory effects against pathogen infections in pigs.     

Enhancement of Nisin Production by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.     

Interference of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in the In Vitro Conjugative Transfer of R-Plasmids

Probiotic compounds, which are often constituted of lactobacilli, exert a number of health benefits through maintenance of the intestinal ecosystem balance. Among the important interactions that occur in the gut microbiota, plasmid transfer by mating is an increasing cause of concern, particularly when antibiotic-resistant genes are involved. Because lactobacilli seem to be able to influence this mechanism, the aim of the present work was to investigate the in vitro capability of two Lactobacillus plantarum strains (one bacteriocin producer and one nonproducer) to interfere with the conjugation processes. For this purpose different matings were performed adding to the donor and recipient cells L. plantarum 35d bac+ and L. plantarum 396/1 bac– as agents of interference. Conjugations added with a Staphylococcus aureus strain or without any agent of interference were used as controls. The results of our experiments demonstrated that both lactobacillus strains were able to decrease mating frequency. Statistically significant differences in the viable transconjugants were obtained in the presence and in the absence of the lactobacilli. The effect was almost the same with the two L. plantarum independent of bacteriocin production. In the trial performed with S. aureus, no decrease in mating frequency was observed, confirming that the capability to interfere with R-plasmid transfer ability could be a property of the tested L. plantarum strains.     

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.     

Yeasts in malting,with special emphasis on <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> (synonym <Emphasis Type="Italic">Pichia anomala</Emphasis>)

Malted barley is a major raw material of beer, as well as distilled spirits and several food products. The production of malt (malting) exploits the biochemical reactions of a natural process, grain germination. In addition to germinating grain, the malting process includes another metabolically active component: a diverse microbial community that includes various types of bacteria and fungi. Therefore, malting can be considered as a complex ecosystem involving two metabolically active groups. Yeasts and yeast-like fungi are an important part of this ecosystem, but previously the significance of yeasts in malting has been largely underestimated. Characterization and identification of yeasts in industrial processes revealed 25 ascomycetous yeasts belonging to 10 genera, and 18 basidiomycetous yeasts belonging to 7 genera. In addition, two ascomycetous yeast-like fungi belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Several ascomycetous yeast strains showed strong antagonistic activity against field and storage moulds, Wickerhamomyces anomalus (synonym Pichia anomala) being the most effective species. Malting studies revealed that W. anomalus VTT C-04565 effectively restricted Fusarium growth and hydrophobin production during malting and prevented beer gushing. In order to broaden the antimicrobial spectrum and to improve malt brewhouse performance, W. anomalus could be combined with other starter cultures such as Lactobacillus plantarum. Well-characterized microbial mixtures consisting of barley and malt-derived microbes open up several possibilities to improve malt properties and to ensure the safety of the malting process.     

The <Emphasis Type="Italic">dnaK</Emphasis> gene as a molecular marker for the classification and discrimination of the <Emphasis Type="Italic">Lactobacillus casei</Emphasis> group

It is hard to accurately identify specific species of the Lactobacillus casei group using phenotypic techniques alone. Some strains of this species group are considered to be probiotic and are widely applied in the food industry. In this study, we compared the use of two phylogenetic markers, the 16S rRNA and dnaK genes, for species discrimination of the members of the L. casei group using sequencing and RFLP. The results showed that L. casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus could be clearly distinguished based on the dnaK gene. The average sequence similarity for the dnaK gene (87.8%) among type strains was significantly less than that of the 16S rRNA sequence (99.1%). Therefore, the dnaK gene can be proposed as an additional molecular phylogenetic marker for L. casei that provides higher resolution than 16S rRNA. Species-specific RFLP profiles of the Lactobacillus strains were obtained with the enzyme ApoI. Our data indicate that the phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or RFLP assays.     

Mannitol production by heterofermentative <Emphasis Type="BoldItalic">Lactobacillus reuteri</Emphasis> CRL 1101 and <Emphasis Type="BoldItalic">Lactobacillus fermentum</Emphasis> CRL 573 in free and controlled pH batch fermentations

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.     

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.