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The establishment of the health-promoting benefits of probiotics is challenged by the antimicrobial bio-barriers throughout the host's gastrointestinal (GI) tract after oral administration. Although microencapsulation has been frequently utilised to enhance the delivery of probiotics, microcapsules of sub-100 μm were found to be ineffective and therefore questioned as an effective delivery vehicle for viable probiotics despite the sensory advantage. In this study, four probiotics strains were encapsulated in chitosan-coated alginate microcapsules of sub-100 μm. Only a minor protective effect was observed from this original type of microcapsule. In order to enhance the survival of these probiotics, sucrose, a metabolisable sugar, and lecithin vesicles were added to the wall material. Both of the ingredients could be readily encapsulated with the probiotics, and protected them from stresses in the simulated GI fluids. The metabolisable sugar effectively increased the survival of the probiotics in gastric acid, mainly through energizing the membrane-bound F1F0-ATPases. The lecithin vesicles proved to alleviate the bile salt stress, and hence notably reduced the viability loss at the elevated bile salt concentrations. Overall, three out of the total four probiotics in the reinforced sub-100 μm microencapsules could significantly survive through an 8-h sequential treatment of the simulated GI fluids, giving less than 1-log drop in viable count. The most vulnerable strain of bifidobacteria also yielded a viability increase of 3-logs from this protection. In conclusion, the sub-100 μm microcapsules can be a useful vehicle for the delivery of probiotics, as long as suitable protectants are incorporated in the wall matrix.

Aims: To set-up an experimental and analytical methodology to evaluate the feasibility of developing simple, accurate and quantitative models based on Raman spectroscopy and multivariate analysis for the quantification of metal ions adsorbed to the bacterial surface of Lactobacillus kefir., Methods and Results: One millilitre cultures from two strains of Lact. kefir in the stationary phase were harvested and washed twice with ultra pure water. The bacterial pellets were resuspended into 1 ml solutions of Pb(+2), Cd(+2) or Ni(+2) ranging from 0 to 0·9 mmol l(-1). The suspensions were further incubated for 1 h at 30°C at pH 5·5. After centrifugation, the pellets were kept to register the Raman spectra and the supernatants were used for the analytical determination of Pb(+2) , Cd(+2) and Ni(+2). Micro-organisms nontreated with metal ions were used as controls. Principal component analysis (PCA) was performed over the preprocessed Raman spectra to evaluate whether the clusters obtained could be correlated with the concentration of metal ions attached to the bacterial biomass. After that, partial least squares (PLS) models were calibrated with the aim of quantifying the metal ions adsorbed to the bacterial surface. According to the analytical determinations, the maximum binding capacity of all the metals (q(max)) attained values that are comparable with those observed for other lactic acid bacteria (ca. 0·200 mmol g(-1)). The spectral analysis revealed that the main functional groups involved in the bacteria/metal interaction are carboxylates, phosphates and polysaccharides. In PCA, the first two principal components explain more than 72% variance of the spectral data set contained in the data structure, allowing a clear discrimination among samples of different concentrations. Based on this information and using as reference the results obtained by analytical methods, PLS prediction models were successfully defined for the quantification of Pb(+2), Cd(+2) and Ni(+2) attached to the bacterial surface., Conclusions: The calibration and validation of methods based on multivariate analysis allowed the definition of models for the quantification of Pb(+2), Cd(+2) and Ni(+2) attached to bacterial surfaces. The high percentages of explained variances in PCA gave a strong support to calibrate the prediction models, depicting very good correlations with the reference method (correlations ∼0·90 in all cases)., Significance and Impact of the Study: Lactobacillus kefir CIDCA 8348 and JCM 5818 bind Pb(+2), Cd(+2) and Ni(+2) in an efficient way. This fact gives support for their potential use as sequestrants of traces of these metals in products addressed to human and animal consume. The prediction models developed would be useful for the determination of the investigated metal ions in unknown samples giving at the same time, structural information about this interaction. This is certainly the most important contribution of this work., (© 2012 The Authors. Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.)

This study aimed at utilizing ultrasound treatment to further enhance the growth of lactobacilli and their isoflavone bioconversion activities in biotin-supplemented soymilk. Strains of lactobacilli (Lactobacillus acidophilus BT 1088, L. fermentum BT 8219, L. acidophilus FTDC 8633, L. gasseri FTDC 8131) were treated with ultrasound (30 kHz, 100 W) at different amplitudes (20%, 60% and 100%) for 60, 120 and 180 s prior to inoculation and fermentation in biotin-soymilk. The treatment affected the fatty acids chain of the cellular membrane lipid bilayer, as shown by an increased lipid peroxidation (P<0.05). This led to increased membrane fluidity and subsequently, membrane permeability (P<0.05). The permeabilized cellular membranes had facilitated nutrient internalization and subsequent growth enhancement (P<0.05). Higher amplitudes and longer durations of the treatment promoted growth of lactobacilli in soymilk, with viable counts exceeding 9 log CFU/mL. The intracellular and extracellular β-glucosidase specific activities of lactobacilli were also enhanced (P<0.05) upon ultrasound treatment, leading to increased bioconversion of isoflavones in soymilk, particularly genistin and malonyl genistin to genistein. Results from this study show that ultrasound treatment on lactobacilli cells promotes (P<0.05) the β-glucosidase activity of cells for the benefit of enhanced (P<0.05) isoflavone glucosides bioconversion to bioactive aglycones in soymilk., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A cell-surface 29-kDa protein (Lam29, cysteine-binding protein of the ABC transporter) from Lactobacillus mucosae ME-340 showed an adhesin-like property for human ABO blood group antigens expressed on the gastrointestinal mucosa. In addition, here we report that Lam29 also bound to an 18-kDa protein on human colonic mucus. By ligand blot assay and N-terminal amino acid sequence of the protein, it was identified as human histone H3. By ligand blot and microplate binding assays with recombinant histone H3, binding between Lam29 and histone H3 was confirmed. The adhesion of ME-340 cells to histone H3 was significantly inhibited by 26% after the addition of 2.5 mg/mL Lam29 as compared to the absence of Lam29 (p<0.01). By GHCl extraction and transcription attenuation of ME-340 cells, binding reduction of ME340 cells against histone H3 was detected at 12% and 13% respectively, as compared to control cells by the BIACORE assay (p<0.01). These data indicate that Lam29 shows multiple binding activities to blood group antigens and histone H3 in human colonic mucus. This is the first report to indicate that lactobacilli expressing Lam29 adhere to histone H3 on gastrointestinal mucosa.

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7-3.6logCFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry., (Copyright © 2011. Published by Elsevier GmbH.)

The aggregation and adhesion of bacterial cells is a serious disadvantage for electrophoretic separations of bacteria. In this study, lipid-based liquid crystalline nanoparticles were used as a pseudostationary phase to minimise the bacterial aggregation and adsorption to the inner walls of microchannels. Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Lactobacillus rhamnosus were selected as analytes and were separated by microchip electrophoresis (MCE) with laser-induced fluorescence (LIF) detection using 4.5 mM tris(hydroxymethyl) aminomethane (TRIS)-4.5 mM boric acid-0.1 mM ethylenediaminetetraacetate (EDTA) (TBE) containing poly(ethylene oxide) (PEO) and lipid-based nanoparticles as the running buffer. The mechanism of lipid-based nanoparticles affecting bacterial adhesion and aggregation was discussed and supported by zeta potential experiments. Under the optimal conditions, the three species of bacteria were identified with patterned peaks. This proposed MCE method using lipid-based nanoparticles as running buffer additives was also used to analyse a real yogurt sample, and valuable bacterial information was obtained by the electropherograms., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Previous studies have revealed that the C-terminal region of the S-layer protein from Lactobacillus is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of Lactobacillus crispatus K2-4-3 isolated from chicken intestine., Results: Multiple sequence alignment revealed that the C-terminal region (LcsB) of Lb. crispatus K2-4-3 SlpB had a high similarity with the cell wall binding domains SA and CbsA of Lactobacillus acidophilus and Lb. crispatus. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in Escherichia coli, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of gfp:lcsB was inserted into the Lactococcus lactis expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the nisA promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of L. lactis with the aid of the LcsB anchor., Conclusion: The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria in vitro and in vivo, and has also the potential for biotechnological application.

Many studies have investigated the immunostimulatory effects of bacteria, such as the anti-allergic effects of lactic acid bacteria (LAB) and LAB-fermented milk. Importantly, these anti-allergic effects have been observed for both viable and nonviable bacteria. However, there are no reported immunological effects of LAB isolated from kimoto, the traditional yeast starter culture used for brewing sake, which also involves spontaneous lactate fermentation. In this study, we determined whether the Leuconostoc mesenteroides and Lactobacillus sakei bacterial strains obtained from kimoto affected the production of interleukin-12 (IL-12), an inducer of the T-helper type-1 immune response. By incubating autoclaved bacteria with J774.1 macrophage-like cells, we found that L. sakei LK-117 induced a sustained increase in IL-12p40 production. The IL-12-inducing ability of LK-117 was unaffected by anti-TLR2 neutralization and was entirely inhibited when the LK-117 cells were treated with RNase. When LK-117 cells were treated with M-1, an N-acetylmuramidase, at varying concentrations and for different periods of time, the ability of the bacteria to induce IL-12 decreased quickly. Although an active fraction could be prepared by chromatography from the soluble products of enzymolysis, the fraction's induction ability was <2% of that of intact organisms, and induction ability disappeared completely upon anti-TLR2 neutralization after treating the active fraction with RNase. These results suggest that single-stranded RNA released from cells that were disrupted by autoclaving might act as a TLR ligand and provide a novel mechanism in which heat-killed LAB could be used to regulate immune activity., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Adherence to the gastrointestinal tract is a key element desirable for many of the proposed beneficial health effects of probiotic bacteria. The aims of this study were to determine the amounts of adhesion of 3 Lactobacillus salivarius strains (Lb6, Lb9, and Lb10) to porcine small intestinal mucins and to determine whether adhesion is a function of lectin-like activities. Dot and Western blot assays were performed to investigate bacterial adhesion. Several carbohydrates and glycoproteins were evaluated to determine whether they interfered with adhesion of the Lactobacillus strains to intestinal mucins and to determine whether they had lectin-like activities. The Lb9 and Lb10 strains had greater association with piglet mucins than did those from 22- to 24-wk-old finishing pigs (P = 0.021 and 0.037, respectively), whereas the Lb6 strain adhered to both (P = 0.138). Western blot assays showed that bacterial adhesion detected piglet mucosa from the duodenum, jejunum, and ileum. In finishing pigs, the adhesion was variable throughout the gastrointestinal tract. Galactose and mannose diminished the interaction of the Lb9 and Lb10 strains in intestinal mucosa (P = 0.028 and 0.026, respectively), whereas pig gastric mucin reduced the adhesion of the Lb6 strain (P = 0.013). Adhesion of the Lb9 and Lb10 strains to intestinal mucosa was less after protease treatment (P = 0.023 and 0.018, respectively), which indicates that proteins are needed for the Lb9 and Lb10 strains to recognize mucin. The Lb6 strain also demonstrated diminished adhesion after periodate treatment (P = 0.038). From these results, we suggest that the nature of the bacterial lectin-like substance is a surface protein that loosely binds to the bacterial cell surface. All the tested strains adhered to specific targets in the small intestinal mucosa of piglets, and the bacteria had lectin-like proteins involved in this adhesion.

This work showed the effect of pheromone plantaricin A (PlnA) on the proliferation and migration of the human keratinocytes NCTC 2544. PlnA was chemically synthesized and used as pure peptide or biologically synthesized during co-cultivation of Lactobacillus plantarum DC400 and Lactobacillus sanfranciscensis DPPMA174. The cell-free supernatant (CFS) was used as the crude preparation containing PlnA. The inductive effect of PlnA on the proliferation of NCTC 2544 cells was higher than that found for hyaluronic acid, a well known skin protective compound. As shown by scratch assay and image analyses, PlnA enhanced the migration of NCTC 2544 cells. Compared to the basal serum free medium (control), the highest inductive effect was found using 10μg/ml of chemically synthesized PlnA. Similar results (P>0.05) were found for CFS. In agreement, the percentage of the starting scratch area was decreased after treatment (24h) with PlnA. The expression of transforming growth factor-β1 (TGF-β1), keratinocyte growth factor 7 (FGF7), vascular endothelial growth factor (VEGF-A), and interleukin-8 (IL-8) genes was affected by PlnA. Compared to control, TGF-β1 gene was under expressed in the first 4h of treatments and up-regulated after 8-24h. On the contrary, FGF7 gene was strongly up-regulated in the first 4h of treatments. Compared to control, VEGF-A and IL-8 genes were always up-regulated during the 4-24h from scratching. Since capable of promoting the proliferation and migration of the human keratinocytes and of stimulating IL-8 cytokine, the use of PlnA for dermatological purposes should be considered., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Fibrinolytic system impairment contributes to the development of thrombotic disease such as cardiovascular disease and stroke. Therefore, an agent that increases fibrinolytic activity may be useful for the prevention of these diseases. In this study, to explore novel profibrinolytic agents, we examined the profibrinolytic effect of Enzamin, an extract of metabolic products from Bacillus subtilis AK and Lactobacillus in vitro and in vivo. Enzamin directly enhanced plasmin activity generated by tissue-type plasminogen activator (t-PA) by twofold but not by urokinase-type plasminogen activator (u-PA) in vitro, which was measured employing both the chromogenic substrate H-D: -Val-Leu-Lys-pNA (S-2251) and fibrin plate. Enzamin also increased plasmin activity generated by t-PA in the cell lysate and culture medium of endothelial cells, measured by fibrin zymography. Furthermore, the oral administration of a 1% concentration of Enzamin increased plasmin activity generated by t-PA by 1.7-fold but not by u-PA in the euglobulin fraction of mouse plasma. In conclusion, Enzamin has a unique ability to enhance the fibrinolytic activity through an increase in endogenous plasmin activity generated by t-PA released from endothelial cells, and may be a beneficial supplement for the prevention of thrombotic episodes.

A method of direct lipid analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in intact membranes, without prior extraction/separation steps, is described. Here, we demonstrate the efficacy of a strong base, 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-time-of-flight (TOF) MS analysis of whole cell bacteria. Initially, individual acidic low-molecular-weight analytes such as standard free fatty acids and phospholipids were analyzed using DMAN as matrix. Clear negative-mode MALDI-TOF MS spectra of all analytes show only deprotonated analyte signals at a low picomole limit of detection with the complete absence of matrix-related signals. These results indicate that DMAN represents a suitable matrix for MALDI-TOF MS analysis of mixtures of complex lipids as the intact membranes of microorganisms. DMAN was successfully applied to the analysis of Lactobacillus sanfranciscensis and L. plantarum microorganisms. Different components were sensitively detected in a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids, glycolipids, phosphatidylglycerols (PGs) and cardiolipins. This method might be of general application, offering the advantage of quickly gaining information about lipid components of other gram-positive bacterial membranes., (Copyright © 2011 John Wiley & Sons, Ltd.)

The methods of Gram-positive cells disintegration to obtain cell wall fragments have been described. It has been shown that the use of ultrasonic disintegrator UZD-500 at the frequency 22 kHz during 30 min is optimal for disintegration of lactic acid bacteria cells. Also the ability to use grinding of cells in liquid nitrogen (6 times to the complete evaporation) for obtaining the cell wall fragments has been demonstrated.

In this work, a method based on Raman spectroscopy in combination with Principal Component Analysis (PCA) and Partial Least Square-Discriminant Analysis (PLS-DA) has been developed for the rapid differentiation of heterofermentative related lactobacilli. In a first approach, Lactobacillus kefir strains were discriminated from other species of heterofermentative lactobacilli: Lb. parakefir and Lb. brevis. After this first approach, PCA allowed for a clear differentiation between Lb. parakefir and Lb.brevis. For the first level of discrimination, PCA was performed on the whole spectra and also on delimited regions, defined taking into consideration the loading values. The best regions allowing a clear differentiation between Lb. kefir and non-Lb. kefir strains were found to be: the 1700-1500 cm(-1), 1500-1185 cm(-1) and 1800-400 (whole spectrum) cm(-1) Raman ranges. In order to develop a classification rule, PLS-DA was carried out on the mentioned regions. This method permitted the discrimination and classification of the strains under study in two groups: Lb. kefir and non-Lb. kefir. The model was further validated using lactobacilli strains from different culture collections or strains isolated from kefir grains previously identified using molecular methods. The second approach based on PCA was also performed on the whole spectra and on delimited regions, being the regions 1700-1500 cm(-1), 1500-1185 cm(-1) and 1185-1020 cm(-1), i.e., those allowing the clearest discrimination between Lb. parakefir and Lb. brevis. The results obtained in this work, allowed a clear discrimination within heterofermentative lactobacilli strains, proteins being the biological structures most determinant for this discrimination.

For precise identification of a Lactobacillus K1 isolate, LC-MS/MS analysis of the putative surface layer protein was performed. The results obtained from LTQ-FT-ICR mass spectrometry confirmed that the analyzed protein spot is the surface layer protein originating from Lb. helveticus species. Moreover, the identified protein has the highest similarity with the surface layer protein from Lb. helveticus R0052. To evaluate the proteomic study, multilocus sequence analysis of selected housekeeping gene sequences was performed. Combination of 16S rRNA sequencing with partial sequences for the genes encoding the RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), translational elongation factor Tu (tuf), and Hsp60 chaperonins (groEL) also allowed to classify the analyzed isolate as Lb. helveticus. Further classification at the strain level was achieved by sequencing of the slp gene. This gene showed 99.8% identity with the corresponding slp gene of Lb. helveticus R0052, which is in good agreement with data obtained by nano-HPLC coupled to an LTQ-FT-ICR mass spectrometer. Finally, LC-MS/ MS analysis of surface layer proteins extracted from three other Lactobacillus strains proved that the proposed method is the appropriate molecular tool for the identification of S-layer-possessing lactobacilli at the species and even strain levels.

A sensitive bacteria biosensor was prepared for the detection of trace lactate. The sensitive bioelement, Lactobacillus bulgaricus and Streptococcus thermophilus mixed culture, and palygorskite, a perfect matrix for bacteria, was co-immobilized on the surface of oxygen electrode. The biosensor possesses fine selective specificity, good sensitivity and longer operational life time, which were due to the mutual help relationship of symbiotic bacteria and 240 days acclimation with lactate as the carbon source. Hydrodynamic amperometry, an advanced electrochemical method, is suitable for on-line monitoring the concentration change of dissolved oxygen that is closely accompanied with the metabolism of lactate. Electrochemical data show that the current is very sensitive to the changes of the concentration of lactate. The response current was linear with lactic acid concentration in the range from 0 to 300μmol L(-1), where the response time is no more than 240 s (R=0.9952), and the sensitivity was 1.87mA mol(-1)L. Experiments show the biosensor is also very useful for long time on-line monitoring of lactate, such as fermentation progress., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Lactobacilli have been barely studied in cows. We proposed isolate and characterize lactic acid bacteria from dairy cows as compared to those raised for meat production and elucidate the presence of strains with evident probiotic employment's potential. For this, isolation and quantification of LAB mainly lactobacilli were realized from vaginal cattle samples in MRS medium. Each selected microorganism was then briefly characterized. The MATH method was employed using hexadecane, xilene an toluene as solvent. According to the hydrophobic characteristics, strains were classified into three categories: high (71-100%), medium (36-70%) and low (0-35%). Hydrogen peroxide qualitative production was studies too, lactobacilli were streaked onto an MRS agar plate containing 5 mg of 3,3',5,5'-tetramethylbenzidine and 0.20 mg of horseradish peroxidase. Twenty-one sampled cows (78%) were positive for lactic acid microflora, 12 belonging to the dairy group and 17 of the meat group. Total LAB counting including dairy and meat cows were log 2,41 CFU/ml. Of overall identified strains, an 83% corresponded to lactobacilli. Most strains belonged to the heterofermentative facultative group (75%), with L. plantarum as the most frequent specie. The highest proportion of isolated vaginal strains (69%) had low hydrophobicity, the LAB with highest hydrophobic characteristics (3 strains) were found only in meat cows. In the qualitative evaluation of H(2)O(2) production, a positive reaction was observed in 13 of 29 strains (45%). The role of lactobacilli in vaginal microbiota is limited, and therefore the present work is interesting in incorporate knowledge of normal microflora of progesteronic healthy cows, in this case in production animals. The isolation and characterization data obtained are consistent in consider the study of particular strains with great potential in the development of a probiotic for production cows., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Eight strains belonging to Lactobacillus spp. and five to Enterococcus spp. were isolated from the gut of worker Apis mellifera L. bees. Studies based on 16S rRNA sequencing revealed that AJ5, IG9, A15 and CRL1647 strains had a 99% identity with Lactobacillus johnsonii, while SM21 showed a 99% similarity with Enterococcus faecium. L. johnsonii CRL1647, AJ5 and IG9 were high lactic acid producers (values were between 177 and 275 mM), and in vitro they inhibited different human food-borne pathogens and Paenibacillus larvae, the American foulbrood agent. This bacterium was the most sensitive to the lactic acid effect being inhibited by 44 mM of this metabolite. L. johnsonii CRL1647, AJ5 and IG9 also presented important surface properties. These cells showed between 77% and 93% of auto-aggregation. The preliminary study of the chemical nature of the aggregating factors revealed that the molecules involved in the surface of each L. johnsonii strain were quite complex; and something of a peptidic nature was mainly involved. E. faecium SM21 produced bacteriocin-like compounds with anti-Listeria effects. Furthermore, a band close to 6.0-7.5 kDA was detected by SDS-PAGE studies, and the entA, B and P structural genes were amplified by PCR reactions. For the first time, bee-gut associated L. johnsonii and E. faecium strains have been isolated, identified, cultivated and some of their functional properties reported., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Purpose of the Study: To verify the single dose bioavailability of two oral formulations of soy isoflavones, with and without lactobacilli, in menopausal women in antibiotic therapy., Methods: Twelve menopause women (mean age 54.3 years, BMI 25.0 kg/m2) participated in a controlled cross-over study. Reference and test treatments were: R = tablets containing soy isoflavones 60 mg (genistin 30 mg + daidzin 30 mg) + calcium and vitamin D3; E = R + 500 million vital spores of Lactobacillus sporogenes (E is Estromineral, a food supplement containing soy isoflavones 60 mg, calcium 141 mg and vitamin D3 5 microg). The design included 2 periods of 5 days of amoxicillin + clavulanate treatment with a 2-week wash-out. After each period alternatively a single dose of each formulation was given in randomised sequence. Genistein and daidzein were determined in plasma by HPLC, sampled 10 times within 24 h after dosing., Results: Genistein pharmacokinetics parameters were higher after E than after R administration: peak plasma concentration (Cmax) +24.3%, area under the concentration curve (AUC0-24) +24.4% and mean residence time +11.0%. Daidzein Cmax and AUC showed a larger variability on R, evidenced by higher scatter from the mean on the formulation without lactobacilli., Conclusion: A trend is shown for a greater absorption of genistein from a formulation containing lactobacilli.

It was previously established that Lactobacillus fermentum ACA-DC 179, Lactobacillus plantarum ACA-DC 287 and Lactobacillus plantarum ACA-DC 2350 exhibit antimicrobial activity against Salmonella enterica serovar Typhimurium. In order to further investigate the killing effect of these microorganisms against Salmonella cells, we employed Fourier transform infrared spectroscopy (FT-IR). Salmonella cells were incubated with different concentrated lactobacilli supernatants and their FT-IR spectra were recorded. The second derivative transformation of the original spectra revealed changes in spectral regions corresponding to absorptions of major cellular constituents (e.g. cell wall, cell membrane, and proteins of the cell) among the Salmonella cells treated with the supernatants and those treated with the control samples. Principal component analysis of the second derivative transformed spectra showed that the yet unidentified antimicrobial compound(s) produced by the lactobacilli tested clearly interfered with the fatty acids of the cell membrane, as well as the polysaccharides of the cell wall in Salmonella cells, pointing towards a dual killing mode. Our study shed light for the first time in the anti-Salmonella activity of the particular Lactobacillus strains., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Aims: To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity., Methods and Results: Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination., Conclusions: A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined., Significance and Impact of the Study: The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures., (© 2010 The Authors. © 2010 The Society for Applied Microbiology.)

Chitosan was used as a coating material to improve encapsulation of a probiotic and prebiotic in calcium alginate beads. Chitosan-coated alginate microspheres were produced to encapsulate Lactobacillus gasseri (L) and Bifidobacterium bifidum (B) as probiotics and the prebiotic quercetin (Q) with the objective of enhancing survival of the probiotic bacteria and keeping intact the prebiotic during exposure to the adverse conditions of the gastro-intestinal tract. The encapsulation yield for viable cells for chitosan-coated alginate microspheres with quercetin (L+Q and B+Q) was very low. These results, together with the study about the survival of microspheres with quercetin during storage at 4 degrees C, demonstrated that probiotic bacteria microencapsulated with quercetin did not survive. Owing to this, quercetin and L. gasseri or B. bifidum were microencapsulated separately. Microencapsulated L. gasseri and microencapsulated B. bifidum were resistant to simulated gastric conditions (pH 2.0, 2h) and bile solution (3%, 2h), resulting in significantly (p<0.05) improved survival when compared with free bacteria. This work showed that the microencapsulation of L. gasseri and B. bifidum with alginate and a chitosan coating offers an effective means of delivery of viable bacterial cells to the colon and maintaining their survival during simulated gastric and intestinal juice., (Copyright 2010 Elsevier B.V. All rights reserved.)

Lactobacillus sakei is a lactic acid bacterium mainly found in meat and meat products. In order to understand the factors favoring its adaptation to meat matrix, growth parameters and survival of the strain L. sakei 23K in the presence of sarcoplasmic or myofibrillar extracts were assessed. Cytosolic proteins putatively involved in the response of this strain to meat proteins were determined using 2D electrophoresis and the significantly regulated proteins were identified by Maldi Tof-MS analyses. From the 31 differentially expressed spots, 16 occurred in the presence of myofibrillar extract while 6 proteins were modulated by the sarcoplasmic extract. Two dipeptidases were overexpressed in the presence of sarcoplasmic proteins, in correlation to the protein degradation patterns obtained by SDS-PAGE. In the presence of the myofibrillar extract, L. sakei 23K overexpressed proteins related to energy and pyrimidine metabolism as well as ala- and tyr-tRNA synthetases, involved in translation, while others corresponding to general stress response, pyrimidine, vitamin and cofactor biosynthesis were down-regulated. The supplementary nutrients furnished by meat extracts modulated the overexpression of proteins related to translation, peptide/amino acid metabolism and energy production while the stress proteins were under regulated. The results obtained here suggest that meat proteins would not represent a stress environment per se for L. sakei 23K in contrast to the harsh conditions during meat processing. This study has extended the understanding of the molecular responses and growth mechanisms of L. sakei 23K in the presence of meat proteins. The transference of genomic information into useful biological insight is an important step for the selection of well-adapted strains for the achievement of high-quality fermented products., (Copyright 2010 Elsevier B.V. All rights reserved.)

A novel heterodimeric beta-galactosidase with a molecular mass of 105 kDa was purified from crude cell extracts of the soil isolate Lactobacillus pentosus KUB-ST10-1 using ammonium sulphate fractionation followed by hydrophobic interaction and affinity chromatography. The electrophoretically homogenous enzyme has a specific activity of 97 U(oNPG)/mg protein. The K(m), k(cat) and k(cat)/K(m) values for lactose and o-nitrophenyl-beta-D-galactopyranoside (oNPG) were 38 mM, 20 s(-1), 530 M(-1).s(-1) and 1.67 mM, 540 s(-1), 325 000 M(-1).s(-1), respectively. The temperature optimum of beta-galactosidase activity was 60-65 degrees C for a 10-min assay, which is considerably higher than the values reported for other lactobacillal beta-galactosidases. Mg(2+) ions enhanced both activity and stability significantly. L. pentosus beta-galactosidase was used for the production of prebiotic galacto-oligosaccharides (GOS) from lactose. A maximum yield of 31% GOS of total sugars was obtained at 78% lactose conversion. The enzyme showed a strong preference for the formation of beta-(1-->3) and beta-(1-->6) linkages, and the main transgalactosylation products identified were the disaccharides beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Gal, beta-D-Galp-(1-->3)-D-Gal, and the trisaccharides beta-D-Galp-(1-->3)-D-Lac, beta-D-Galp-(1-->6)-D-Lac.

The focus of this study was to monitor the survival of populations and the volatile compound profiles of selected Lactobacillus strains during long-term incubation in milk. The enumeration of cells was determined by both the Direct Epifluorescent Filter Technique using carboxyfluorescein diacetate (CFDA) staining and the plate method. Volatile compounds were analysed by the gas-chromatography technique. All strains exhibited good survival in cultured milks, but Lactobacillus crispatus L800 was the only strain with comparable growth and viability in milk, assessed by plate and epifluorescence methods. The significant differences in cell numbers between plate and microscopic counts were obtained for L. acidophilus strains. The investigated strains exhibited different metabolic profiles. Depending on the strain used, 3 to 8 compounds were produced. The strains produced significantly higher concentrations of acetic acid, compared to other volatiles. Lactobacillus strains differed from one another in number and contents of the volatile compounds.

Lactic acid bacteria (LAB) are a heterogeneous group of micro-organisms with high relevance for fermented food and beverages as well as human and animal health. Identification of LAB is complicated by species richness, a high degree of heterogeneity between species and the presence of very closely related species at the same time. In the present study we developed identification systems based on FTIR spectra and artificial neural networks (ANNs) for species identification of 92 LAB species out of nine genera and strain identification of probiotic and environmental Enterococcus faecium strains used as additives for animal feed. The reference databases established in this work permit reliable identification of LAB with accuracies of 93.2% at species and 97.1% at strain level for Ec. faecium, demonstrating the excellent suitability of FTIR spectroscopy as a technique applicable for simultaneous species and strain identification., ((c) 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

S-layer proteins from Lactobacillus kefir and Lactobacillus brevis are able to adsorb on the surface of positively charged liposomes composed by Soybean lecithin, cholesterol and stearylamine. The different K values for S-layer proteins isolated from L. kefir and L. brevis (4.22 x 10(-3) and 2.45 x 10(2) microM(-1) respectively) indicates that the affinity of the glycosylated protein isolated from L. kefir is higher than the non-glycosylated one. The attachment of S-layer proteins counteracts the electrostatic charge repulsion between stearylamine molecules in the membrane surface, producing an increase in the rigidity in the acyl chains as measured by DPH anisotropy. Laurdan generalized polarization (GP) shows that glycosylated causes a GP increase, attributed to a lowering in water penetration into the head groups of membrane phospholipids, with charge density reduction, while the non-glycosylated does not affect it. The octadecyl-rhodamine results indicate that S-layer coated liposomes do not show spontaneous dequenching in comparison with control liposomes without S-layer proteins, suggesting that S-layer protein avoid spontaneous liposomal fusion. It is concluded that the increase in stability of liposomes coated with S-layers proteins is due to the higher rigidity induced by the S-layer attachment by electrostatic forces., (Copyright 2010 Elsevier B.V. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease; moreover, it seems to be implicated in the development of Crohn's disease in humans. In the present study, fluorescence ratio imaging microscopy (FRIM) was used to assess changes in intracellular pH (pH(i)) of one strain of MAP after exposure to nisin and neutralized cell-free supernatants (NCSs) from five bacteriocin-producing lactic acid bacteria (LAB) with known probiotic properties. The evaluation of pH(i) by FRIM provides information about the physiological state of bacterial cells, bypassing the long and problematic incubations needed for methods relying upon growth of MAP such as determination of colony forming units. The FRIM results showed that both nisin and the cell-free supernatant from Lactobacillus plantarum PCA 236 affected the pH(i) of MAP within a few hours. However, monitoring the population for 24h revealed the presence of a subpopulation of cells probably resistant to the antimicrobial compounds tested. Use of nisin and bacteriocin-producing LAB strains could lead to new intervention strategies for the control of MAP based on in vivo application of probiotic cultures as feed additives at farm level., (Copyright 2010 Elsevier B.V. All rights reserved.)

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478bp with a G+C content of 37.2%. Ab initio annotation indicated four putative open reading frames (orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter 'wild' isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria., (2010 Elsevier B.V. All rights reserved.)

Lactobacillus salivarius, a probiotic bacterium, encounters acidic conditions in its passage through the gastrointestinal tract of human and animal hosts. We studied the effect of a rapid downshift in extracellular pH from 6.5 to 4 on cell growth. The maximum growth rate was higher in low pH medium with glutathione supplementation than without. Cells developed a GSH-mediated acid-tolerance response and, when grown with 0.5 mM GSH, reached a higher final density than with other conditions. These findings suggest that the increased growth rate is caused by uptake of GSH which acts as a nutrient source as well as having protective functions, allowing for continued growth.

Twelve lactobacilli previously isolated from newborn infants' gastrointestinal tract and Feta cheese were further characterized by pulse field gel eletrophoresis (PFGE). All strains exhibited distinct PFGE genotypic patterns with the exception of DC421 and DC423 strains possessing identical patterns. The strains DC421, 2035 and 2012 were found to posses certain cell surface traits such as hydrophobicity, autoaggregation and/or high adhesive capacity suggesting potential immunomodulatory activity. However, application of the dorsal mouse air pouch system revealed that only the DC421, DC429 and 2035 strains exhibited strong immunostimulatory activity such as increased chemotaxis of polymorphonuclear (PMN) cells in association with increased phagocytosis and cytokine production. The same strains also induced immunomodulatory activity in the gut associated lymphoid tissue in mice in the absence of any inflammatory response. All strains induced IgA production while reduced TNFalpha production by small intestine cells. The strains DC421 and DC429 exerted their effect on the intestine through Toll-like receptor TLR2/TLR4/TLR9 mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-gamma), interleukin (IL)-5, IL-6 and IL-10 are included. The strain 2035 induced similar cytokine profile through the synergy of TLR2/TLR4. This study further supports the eligibility of the air pouch model to discriminate presumptive probiotic Lactobacillus strains exhibiting immunostimulatory activity in the gut. Furthermore, evidence is provided that the cell surface traits examined may not be the only criteria but an alternative and important component of a complex mechanism that enables a microorganism to interact with the host gut to exert its immunoregulatory activity., (Copyright 2010 Elsevier B.V. All rights reserved.)

Fatty acids have been successfully used to trace the transfer of organic matter in coastal and estuarine food webs. To delineate these web connections, fatty acid profiles were analyzed in species of microbes (Azotobacter vinelandii, and Lactobacillus xylosus), prawns (Metapenaeus monoceros and Macrobrachium rosenbergii) and finfish (Mugil cephalus), that are associated with decomposing leaves of two mangrove species, Rhizophora apiculata and Avicennia marina. The fatty acids, except long chain fatty acids, exhibit changes during decomposition of mangrove leaves with a reduction of saturated fatty acids and an increase of monounsaturated fatty acids. The branched fatty acids are absent in undecomposed mangrove leaves, but present significantly in the decomposed leaves and in prawns and finfish, representing an important source for them. This revealed that the microbes are dominant producers that contribute significantly to the fishes and prawns in the mangrove ecosystem. This work has proved the fatty acid biomarkers as an effective tool for identifying the trophic interactions among dominant producers and consumers in this mangrove.

The importance of 3D structuring in the N- and C-terminal ends of the two peptides (39-mer LcnG-alpha and 35-mer LcnG-beta) that constitute the two-peptide bacteriocin lactococcin G was analysed by replacing residues in the end regions with the corresponding D-isomeric residues. When assayed for antibacterial activity in combination with the complementary wild-type peptide, LcnG-alpha with four D-residues in its C-terminal region and LcnG-beta with four d-residues in either its N- or its C-terminal region were relatively active (two- to 20-fold reduction in activity). 3D structuring of the C-terminal region in LcnG-alpha and the C- and N-terminal regions in LcnG-beta is thus not particularly critical for retaining antibacterial activity, indicating that the 3D structure of these regions is not vital for interpeptide interactions or for interactions between the peptides and cellular components. The 3D structure of the N-terminal region in LcnG-alpha may be more important, as LcnG-alpha with four N-terminal D-residues was the least active of these four peptides (10- to 100-fold reduction in activity). The results are consistent with a proposed structural model of lactococcin G in which LcnG-alpha and -beta form a transmembrane parallel helix-helix structure involving approximately 20 residues in each peptide, starting near the N terminus of LcnG-alpha and at about residue 13 in LcnG-beta. Upon expressing the lactococcin G immunity protein, sensitive target cells became resistant to all of these D-residue-containing peptides. The end regions of the two lactococcin G peptides are consequently not involved in essential structure-dependent interactions with the immunity protein. The relatively high activity of most of the D-residue-containing peptides suggests that bacteriocins with increased resistance to exopeptidases may be generated by replacing their N- and C-terminal residues with d-residues.

Lactobacillus intermedius NRRL B-3693 produced mannitol, lactic acid, and acetic acid when grown on fructose at 37 degrees C. The optimal pH for mannitol production from fructose by the heterofermentative lactic acid bacterium (LAB) in pH-controlled fermentation was at pH 5.0. It produced 160.7 +/- 1.1 g mannitol in 40 h with a volumetric productivity of 4.0 g l(-1) h(-1) in a simplified medium containing 250 g fructose, 50 g corn steep liquor (CSL), and 33 mg MnSO(4) per liter. However, the mannitol production by the LAB was severely affected by the variability of CSL. The supplementation of CSL with soy peptone (5 g/l), tryptophan (50 mg/l), tryptophan (50 mg/l) plus tyrosine (50 mg/l), or commercial protease preparation (2 ml/100 g of CSL) enhanced the performance of the inferior CSL and thus helped to overcome the nutrient limitations.

Aim: The study aimed for the complete purification and recharacterization of the highly hydrophobic circular bacteriocins, gassericin A and reutericin 6., Methods and Results: Gassericin A and reutericin 6 were purified to homogeneity using previously described method and reverse-phase HPLC with an octyl column and eluents of aqueous acetonitrile and 2-propanol. Mass analysis, N-terminal sequencing and bacteriocin assay of the HPLC-purified bacteriocins showed the two bacteriocins had identical seamless circular structures with the same m/z value (5651) of [M + H](+) and both had the same specific activity. D/L-amino acid composition analysis using two distinct methods with the chiral fluorescent derivatization reagents (+)-1-(9-fluorenyl)ethyl chloroformate and O-phthalaldehyde/N-acetyl-L-cystein revealed neither gassericin A nor reutericin 6 contained D-alanine residues contrary to our previous results., Conclusion: Purified gassericin A and reutericin 6 are chemically identical circular molecules containing no D-alanine residues., Significance and Impact of the Study: The HPLC conditions developed in this study will facilitate advanced purification and correct characterization of other highly hydrophobic bacteriocins.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.

Aims: The aim of this study was to determine the antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20 against several micro-organisms, including Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi., Methods and Results: Antimicrobial and antiadhesive activities were determined using the microdilution method in 96-well culture plates. The biosurfactant showed antimicrobial activity against all the micro-organisms assayed, and for twelve of the eighteen micro-organisms (including the pathogenic Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus agalactiae), the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were achieved for biosurfactant concentrations between 25 and 50 mg ml(-1). Furthermore, the biosurfactant showed antiadhesive activity against most of the micro-organisms evaluated., Conclusions: As far as we know, this is the first compilation of data on antimicrobial and antiadhesive activities of biosurfactants obtained from lactobacilli against such a broad group of micro-organisms. Although the antiadhesive activity of biosurfactants isolated from lactic acid bacteria has been widely reported, their antimicrobial activity is quite unusual and has been described only in a few strains., Significance and Impact of the Study: The results obtained in this study regarding the antimicrobial and antiadhesive properties of this biosurfactant opens future prospects for its use against micro-organisms responsible for diseases and infections in the urinary, vaginal and gastrointestinal tracts, as well as in the skin, making it a suitable alternative to conventional antibiotics.

In this study, the crude biosurfactant produced by a Lactobacillus paracasei strain isolated in a Portuguese dairy industry was characterized. The minimum surface tension (41.8mN/m) and the critical micelle concentration (2.5mg/ml) obtained were found to be similar to the values previously reported for biosurfactants isolated from other lactobacilli. The biosurfactant was found to be stable to pH changes over a range from 6 to 10, being more effective at pH 7, and showed no loss of surface activity after incubation at 60 degrees C for 120h. Although the biosurfactant chemical composition has not been determined yet, a fraction was isolated through acidic precipitation, which exhibited higher surface activity as compared with the crude biosurfactant. Furthermore, this isolated biosurfactant showed antimicrobial and anti-adhesive activities against several pathogenic microorganisms. In addition, L. paracasei exhibited a strong autoaggregating phenotype, which was maintained after washing and resuspending the cells in PBS, meaning that this attribute must be related to cell surface components and not to excreted factors. The autoaggregation ability exhibited by this strain, together with the antimicrobial and anti-adhesive properties observed for this biosurfactant opens the possibility for its use as an effective probiotic strain.

This paper describes the structure of neutral exopolysaccharide (EPS) produced by Lactobacillus johnsonii 142, strain of the lactic acid bacteria isolated from the intestine of mice with experimentally induced inflammatory bowel disease (IBD). Sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and (1)H,(13)C HSQC experiments revealed that the repeating unit of the EPS is a pentasaccharide: -->3)-alpha-D-Galp-(1-->3)-beta-d-Glcp-(1-->5)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1--> The rabbit antiserum raised against whole cells of L. johnsonii 142 reacted with homologous EPS, and cross-reacted with exopolysaccharide from Lactobacillus animalis/murinus 148 isolated also from mice with IBD, but not reacted with EPS of L. johnsonii 151 from healthy mice., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Lactic acid (LA) fermentation by Lactobacillus salivarius ATCC 11741 immobilized on loofa sponge (LS) was evaluated. To increase the surface area of LS for cell immobilization, H2O2 and chitosan were introduced as surface modifying reagents. Four chitosan of different molecular weight were separately coated on LS. All experiments were conducted in shaking flask mode at 100 rpm rotating speed and 37 degrees with 5% CaCO3 as a pH regulating agent. The effects of initial glucose concentration were investigated in the range of 20-100 g L-1 on LA fermentation by free cells. The results indicate that the maximum concentration of LA was produced with 50 g L-1 glucose concentration. The immobilized cell system produced 1.5 times higher concentration than free cells for 24 h of fermentation. Moreover, immobilized cells can shorten the fermentation time by 2 fold compared with free cells at the same level of LA concentration. At 1% w/v chitosan in 2% v/v acetic acid, Yp/s and productivities of various molecular weight of chitosan were insignificantly different. Repeated batch fermentations showed 5 effective recycles with Yp/s and productivity in the range of 0.55-0.85 and 0.90-1.20 g L-1 h-1, respectively. It is evident that immobilization of L. salivarius onto LS permits reuse of the system under these fermentation conditions. Scanning electron micrographs indicated that there were more intact cells on the chitosan-treated LS than on the untreated LS, thus confirming the effectiveness of the LS-chitosan combination when being utilized as a promising immobilization carrier for LA fermentation.

The surface properties of lactobacilli are of significant technological importance as they determine the interaction of the bacterial cells with the gastrointestinal mucosa, and therefore influence their location in the gut and their functionality. Studying the surface of the bacteria is critical for understanding the adhesion process better. This review compiles the knowledge from studies on the characterization Lactobacillus surfaces and evaluates the potential relationship between the cells' physicochemical characteristics and their adhesive abilities. It also discusses the effect that the production processes, such as fermentation and drying, can exert on the surface properties and adhesion abilities of lactobacilli., (Copyright 2010 Elsevier Inc. All rights reserved.)

Lactobacillus fermentum strain L23 and L. rhamnosus strain L60 were selected as an alternative treatment to prevent or treat urogenital infections based on their probiotic properties and production of bacteriocins. The objectives of the present work were to study the inhibitory activities of these two bacteriocin-producing strains, and to analyze the interactions between pairs of bacteriocins that inhibit urogenital pathogens. Antimicrobial activity tests of L23 and L60 were performed by a diffusion method with 207 bacterial strains, isolated from female patients presenting a urogenital infection. Inhibitory substances interaction tests were carried out by using a streak-diffusion method on agar plates. One hundred percent of the clinical isolates showed sensitivity to the antimicrobial substances produced by L23 and L60. The selected lactobacilli produced larger inhibition halos when compared to several antibiotics commonly used for treating these infections. Synergistic interactions and indifferent interactions were recorded in 68.6% and 31.4% of the cases, respectively. No antagonistic interactions were observed. In conclusion, the bacteriocin-producing strains L23 and L60 are potential candidates for probiotic prophylaxis and treatment of urogenital disorders in women.

Development of effective topical microbicides for the prevention of HIV-1 sexual transmission represents a primary goal for the control of the AIDS pandemic. The viral coreceptor CCR5, used by the vast majority of primary HIV-1 isolates, is considered a primary target molecule. RANTES and its derivatives are the most suitable protein-based compounds to fight HIV-1 via CCR5 targeting. Yet, receptor activation should be avoided to prevent pro-inflammatory effects and possibly provide anti-inflammatory properties. C1C5 RANTES is a chemokine mutant that exhibits high anti-HIV-1 potency coupled with CCR5 antagonism. However, the need for the formation of an N-terminal intramolecular disulfide bridge between non-natural cysteine residues at positions 1 and 5 represents a challenge for the correct folding of this protein in recombinant expression systems, a crucial step towards its development as a microbicide against HIV-1. We report here a rare case of superior folding in a prokaryote as compared to an eukaryotic expression system. Production of C1C5 RANTES was highly impaired in CHO cells, with a dramatic yield reduction compared to that of wild type RANTES and secretion of the molecule as disulfide-linked dimer. Conversely, a human vaginal isolate of Lactobacillus jensenii engineered to secrete C1C5 RANTES provided efficient delivery of the monomeric protein. This and other reports on successful secretion of complex proteins indicate that lactic acid bacteria are an excellent system for the expression of therapeutic proteins, which can be used as a platform for the engineering of conceptually novel RANTES mutants with potent anti-HIV-1 activity.

Aim: To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products., Methods and Results: An in vitro system, operated with batch bioreactor, was used for this assessment. Selected bacterial species were monitored at two pH values, over 24 h of batch culture incubation. The concentration of short chain fatty acids (SCFA) and lactate in the fermented material was also determined. The addition of L. agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 into vessel 2 (Cc + P) increased the total anaerobes, lactobacilli and bifidobacteria after 24 h incubation. Moreover, lactobacilli supplementation decreased the total aerobes and streptococci, but it did not have any effects on coliforms. The supplementation of lactobacilli in vessel 2 (Cc + P) was found to significantly increase the production of lactate, propionate and butyrate. Furthermore, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5.8., Conclusions: L. agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230, as probiotic bacteria, have the ability to re-establish proper microbial balance by the formation of lactate as well as propionate, and stimulate butyrate-producing bacteria to produce butyrate in the chicken cecum., Significance and Impact of the Study: This study was the first to report this under in vitro conditions, highlighting the probiotic roles of the two Lactobacillus strains in broiler cecal fermentation at different initial pH. These useful data can be helpful in improving the fermentation process in chicken cecum.

Lactobacillus crispatus (L. crispatus) ZJ001 is highly adhesive to epithelial cells and expresses S-layer proteins. In this study, S-layer genes were sequenced and expressed in E. coli to characterize the function of S-layer proteins with this particular strain. L. crispatus ZJ001 harbored two Slayer genes slpA and slpB, and only slpA gene was expressed in the bacterium, as revealed by RT-PCR and immunoassays. The mature SlpA showed 47% amino acid sequence identity to SlpB. The SlpA and SlpB of L. crispatus ZJ001 were highly homologous at the C-terminal region to other Lactobacillus S-layer proteins, but were substantially variable at N-terminal and middle regions. Electron microscopic analysis indicated that His-slpA expressed in E. coli was able to form a sheet-like structure similar to the natural S-layer, but His-slpB formed as disclike structures. In the cell binding experiments, HeLa cells were able to bind to both recombinant His-slpA and HisslpB proteins to the extent similar to the natural S-layer. The cell binding domains remain mostly in the N-terminal regions in SlpA and SlpB, as shown by high binding of truncated peptides SlpA2-228 and SlpB2-249. Our results indicated that SlpA was active and high binding to HeLa cells, and that the slpA gene could be targeted to display foreign proteins on the bacterial surface of ZJ001 as a potential mucosal vaccine vector.

Aims: To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells., Methods and Results: Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells., Conclusion: The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells., Significance and Impact of the Study: The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.

Aim: To assess sorption properties of Spherocelle beads consisting of particles of macroporous celiulose with various charges in relation to bacterial cells of manufacturing probiotic strains from different taxonomic groups., Materials and Methods: The following manufacturing strains: Bifidobacterium bifidum 1, Lactobacillus plantarum 8PA-3 and Escherichia coli M-17, as well as 3 variants of Spherocelles' matrix: neutral, with positive and negative charges, were used., Results: Spherocelle globules DEAE with a positive charge of the matrix were successively used for designing of immobilized probiotic preparations. Efficacy of sorbent is determined by sorption of > or =1000 viable cells as well as bacterial metabolites interacting in conditions of sorbent-regulated pH on each globule with diameter 100-180 microm. It provides, on the one hand, prolonged viability of probiotic bacteria in culture fluid within 6 months and, on the other hand, optimal pharmacokinetics of preparation due to gradual desorption of metabolites from sorbent globules., Conclusion: Sorbent Spherocell DEAE is biocompatible with cells of manufacturing strains of lactobacilli, bifidobacteria and E. coli and recommended for designing of immobilized probiotics.

Background: The possibility of using microbes to maintain health, and to prevent or treat disease is a topic as old as microbiology. The research of novel probiotic strains is important in order to satisfy the increasing request of the market and to obtain functional products in which the probiotic cultures are more active and with better probiotic characteristics than those already present on the market., Aim of the Study: In this study, the probiotic potential of Lactobacillus strains isolated from Italian elderly human faeces was investigated., Methods: The Lactobacillus strains were identified and examined for resistance to gastric acidity and bile toxicity, adhesion to HT-29 cells, antimicrobial activities, antibiotic susceptibility and plasmid profile. Survival of the strains through human intestine was examined in a 3 months human feeding trial., Results: Two strains, Lactobacillus rhamnosus IMC 501 and Lactobacillus paracasei IMC 502, tolerated well low pH and bile acids. In antimicrobial activity assays, both strains showed inhibitory properties towards selected potential harmful microorganisms, particularly against Candida albicans. The two selected strains expressed high in vitro adherence to HT-29 cells increasing this characteristic when they are used in combination and they were resistant to vamcomycin, colistin sulphate, gentamicin, oxolinic acid and kanamycin. Moreover, the two strains could be recovered from stools of volunteers after the feeding trials., Conclusions: Lactobacillus rhamnosus IMC 501 and L. paracasei IMC 502 present favourable strain-specific properties for their utilisation as probiotics in functional foods and the high adhesion ability of the L. rhamnosus IMC 501 and L. paracasei IMC 502 used in combination, confirmed by both in vitro and in vivo study, indicate that the two bacterial strains could be used as health-promoting bacteria.

The effect of cryoprotectants (maltodextrin+glycerol) and cryoprotectants+antioxidant [ascorbic acid and/or butylated hydroxytoluene (BHT)] mixtures on the survival, electrolyte leakage, and lipid degradation of freeze-dried Weissella paramesenteroides LC11 during storage was investigated and compared with that of the control (cells without additives) over a 90-day storage period at 4 or 20 degrees in glass tubes with water activity (a(w)) of 0.23. The survival, electrolyte leakage, and lipid degradation were evaluated through colony counts, electrical conductivity, and thiobarbituric acid reactive substances (TBARS) content, respectively. The fatty acids composition was determined by gas chromatography, in both the total lipid extract and the polar lipid fraction, and compared with that of the control after the 90-day storage period. As the storage proceeded, increases in leakage value and TBARS content, as well as a decrease in viability, were observed. After 90 days of storage, the major fatty acids found in both the total lipid extract and the polar lipid fraction were palmitic (16:0), palmitoleic (16:1), stearic (18:0), oleic (18:1), linoleic (18:2), and linolenic (18:3) acids. The survival, leakage value, TBARS content and 18:2/16:0 or 18:3/16:0 ratio were the greatest for the protected strain held at 4 degrees . Cells with the cryoprotectants+BHT mixture showed the highest percentage of survival and 18:2/16:0 or 18:3/16:0 ratio in both lipid extracts, as well as the lowest leakage value and TBARS content after the 90-day storage period. Drying cells with the cryoprotectants+BHT mixture considerably slowed down polar lipid degradation and loss of membrane integrity, resulting in improved viability during storage.