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The present study aimed to evaluate the in vitro antibacterial and antioxidant activities and the in vivo wound healing performance of a noval exopolysaccharides (EPS-Ca 6 ) produced by Lactobacillus sp.Ca 6 strain. The results showed that EPS-Ca 6 had a potential antioxidant activity determined through four different assays: DPPH scavenging activity, reducing power, β-carotene bleaching by linoleic acid assay, and Metal chelating activities. It also exhibited significant antibacterial activity against pathogenic bacteria Salmonella enterica and Micrococcus luteus. The wound healing activity of the EPS-Ca 6 , using excision wound model in rats, showed that this novel EPS accelerated significantly wound healing activity as compared to the control group, and a total closure was achieved after 14days of wound induction. Furthermore, histological examination of biopsies showed fully re-epithelialized wound with a complete epidermal regeneration. Overall the finding indicates that the EPS-Ca 6 might be useful as a wound healing agent in modern medicine., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The ability to adsorb zearalenone by five strain of lactic acid bacteria was evaluated: four strains of Lactobacillus spp. isolated from pig rectal swabs and one commercial strain (Lactobacillus rhamnosus). Several factors affecting the adsorption capacity were evaluated in order to improve the adsorption of the mycotoxin by bacteria. The stability of the zearalenone-bacteria complex was analyzed. In every case, bacterial adsorption capacity was higher than 40.0%. The strain showing the highest adsorption (68.2%) was selected for the following steps of this research. The adsorption percentages obtained after processing 6.5 and 7.5mL MRS broth were 57.40%+3.53 and 64.46%+0.76, respectively. The stability of zearalenone-bacteria complex was evaluated by successively rinsing. In the first rinsing step 42.26%+0.414 was still bound. In the second rinsing step 25.12%+0.664 was still bound, whereas 15.82%+0.675 remained in the pellet after the third rinse. Results obtained demonstrated that Lactic Acid Bacteria has capacity to adsorb zearalenone. Finally adsorption was increased using a higher volume of initial broth. These results could be used to design a new lyophilized powder for detoxification, using lactic acid bacteria as potential zearalenone adsorbents., (Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.)

The objective of this study was to explore the antioxidant effect of the surface layer proteins (SLPs) and their mechanism. We investigated four SLPs which were extracted from L. casei zhang, L. rhamnosus, L. gasseri and L. acidophilus NCFM respectively using LiCl. The protective effect of SLPs on H 2 O 2 -induced HT-29 cells oxidative injury was investigated. As results, SLPs (100μg/mL) could significantly mitigate HT-29 cells cytotoxicity, improve the activities of total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase (SOD), decrease the contents of malondialdehyde (MDA) and lactate dehydrogenase (LDH), compared with H 2 O 2 -induced group (P<0.05). Furthermore, SLPs were also shown to attenuate the apoptosis rate (10.94-24.03%, P<0.01), suppress the elevation of intracellular reactive oxygen species (ROS) and calcium levels, restore mitochondrial membrane potential (MMP) and block the activation of apoptosis-related proteins of caspase-3 and caspase-9 (P<0.05). Considering all the parameters analyzed, we concluded that Lactobacillus SLPs play an essential role in the antioxidant capacity of HT-29 cells induced by H 2 O 2 , and the mechanism could be attributed to SLPs' ability to enhance the activity of the intracellular antioxidant enzyme system, reduce ROS accumulation and to inhibit apoptosis by regulating mitochondrial pathway., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bacteriocin TSU4 is a novel antimicrobial peptide isolated from Catla catla gut isolate Lactobacillus animalis TSU4. It has been reported for its potential antimicrobial activity against fish pathogens and food spoilage bacteria. In vivo safety evaluation is necessary to determine its immunogenicity, toxicity, and importance in real-life applications. The present study was designed to evaluate the immunogenicity, acute and sub-chronic toxicity of bacteriocin TSU4 in BALB/c mice to ensure its safety in industrial application. Male BALB/c mice were administered intraperitoneally for immunogenicity assessment, by oral gavage with 50, 100, and 200 mg/kg/body weight for acute test and 0.5 mg/kg/day dose of bacteriocin TSU4 for sub-chronic toxicity test. Neither mortality nor any infections were observed during experimental period. There was no major increase in antibody titer during the immunogenicity test, and no mortality was observed during acute or sub-chronic toxicity tests. The LD 50 value of bacteriocin TSU4 was found to be higher than 200 ± 0.45 mg/kg. No significant change in the serum biochemical markers, histopathological analysis and visual observation in spleen sizes was observed. These findings revealed that bacteriocin TSU4 is a non-immunogenic, safe, non-toxic, and could be a potential candidate for industrial applications in food preservation and aquaculture industries.

This study was aimed to evaluate the potential of high pressure homogenization for the microencapsulation of two probiotic lactic acid bacteria, Lactobacillus paracasei A13 and Lactobacillus salivarius subsp. salivarius CET 4063 to produce functional fermented milks. Microcapsules of the considered functional microorganisms were obtained by HPH treatments at 50MPa in the presence of sodium alginate and vegetable oil. The microencapsulated microorganisms were then inoculated as adjuncts to produce fermented milks. As controls were used fermented milks in which the two probiotic lactobacilli were inoculated without encapsulation. The viability of the strains was monitored during almost 2months of refrigerated storage. The survival of lactic acid bacteria after the gastric-duodenal simulated test was determined. Fermented milk texture parameters, the presence of exo-polysaccharides and the production of volatile molecules were also evaluated over storage. The microcapsules, for both the considered probiotic strains, were homogeneous and with a size<100μM and therefore did not adversely affect the sensory properties of the fermented milks. The encapsulation decreased the hyperacidity phenomena generally related to the inclusion of probiotic microorganisms in fermented milks. The lower acidity of the products due to the microencapsulation was fundamental for the improvement of the viability of the starter culture and the sensory characteristics of the products. The microencapsulation conditions increased the resistance to the simulated digestion processes, although the strain Lb. paracasei A13 generally showed a higher resistance to the gastric barrier respect to Lb. salivarius CECT 4063. By contrast, the data obtained showed a reduction of EPS production by the microencapsulation. The volatile profiles showed specific profiles in relation to the probiotic strain used and microencapsulation process. In conclusion, the results of this study underlined the applicative potential of HPH microencapsulation of probiotic microorganisms to produce fermented milk with improved functionality and with enhanced sensory properties., (Copyright © 2017. Published by Elsevier Ltd.)

In the present study 45 lactic acid bacteria (LAB) strains were isolated from Feta-type cheese and were screened for probiotic potential in a series of established in vitro tests, including resistance to low pH, resistance to pepsin and pancreatin and tolerance to bile salts. The strain K5, which displayed properties similar to or even better than the reference strain Lactobacillus plantarum ATCC 14917, was chosen for further analysis. Firstly, multiplex PCR analysis indicated that the novel strain belongs to the paracasei species. Secondly, the susceptibility against common antibiotics was determined to ensure a safe exploitation of the potentially probiotic strain. Additionally, the performance of L. paracasei K5 as starter in the fermentation of pomegranate juice was studied to evaluate its technological properties. Finally, a novel multiplex PCR assay, based on random amplified polymorphic DNA (RAPD) analysis was developed for its efficient and accurate detection in food products., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this study was to evaluate the effects of two species of poultry (cockerel and Japanese quail), two levels of enzyme (0 and 0.5 g/kg), and 6 processing methods (control (C), gamma irradiation 25 (GI25), and 50 kGy (GI50), autoclaving (AU), fermentation without lactobacillus (F), and fermentation with lactobacillus (FL)) on the nitrogen-corrected apparent metabolizable energy (AMEn) of barley. In this experiment, each processing method was tested at two levels of enzyme (0 and 0.5 g/kg) in six replications with two cockerels or four quails per replication. The results showed that the AMEn values by barley processing methods were: C-11.45 and 12.27; GI25-11.50 and 11.30; GI50-11.49 and 11.29; AU-11.70 and 11.46; F-12.90 and 12.30; FL-1295 and 12.51 MJ/kg in cockerels and quails, respectively. The AMEn (11.49 MJ/kg) of enzyme 0 was significantly lower than that of enzyme 0.5 (12.35 MJ/kg). There was a significant difference in the AMEn of processed barley between cockerels and quails. In conclusion, the most effective processing method of barley for AMEn is fermentation with lactobacillus and enzyme., (© 2017 Poultry Science Association Inc.)

In Gram-positive bacteria, such as lactic acid bacteria, general glycosylation systems have not been documented so far. The aim of this work was to characterize in detail the glycosylation of the S-layer protein of Lactobacillus kefiri CIDCA 83111. A reductive β-elimination treatment followed by anion exchange high performance liquid chromatography analysis was useful to characterize the O-glycosidic structures. MALDI-TOF mass spectrometry analysis confirmed the presence of oligosaccharides bearing from 5 to 8 glucose units carrying galacturonic acid. Further nanoHPLC-ESI analysis of the glycopeptides showed two O-glycosylated peptides: the peptide sequence SSASSASSA already identified as a signature glycosylation motif in L. buchneri, substituted on average with eight glucose residues and decorated with galacturonic acid and another O-glycosylated site on peptide 471-476, with a Glc 5-8 GalA 2 structure. As ten characteristic sequons (Asn-X-Ser/Thr) are present in the S-layer amino acid sequence, we performed a PNGase F digestion to release N-linked oligosaccharides. Anion exchange chromatography analysis showed mainly short N-linked chains. NanoHPLC-ESI in the positive and negative ion modes were useful to determine two different peptides substituted with short N-glycan structures. To our knowledge, this is the first description of the structure of N-glycans in S-layer glycoproteins from Lactobacillus species., Significance: A detailed characterization of protein glycosylation is essential to establish the basis for understanding and investigating its biological role. It is known that S-layer proteins from kefir-isolated L. kefiri strains are involved in the interaction of bacterial cells with yeasts present in kefir grains and are also capable to antagonize the adverse effects of different enteric pathogens. Therefore, characterization of type and site of glycosidic chains in this protein may help to understand these important properties. Furthermore, this is the first description of N-glycosidic chains in S-layer glycoprotein from Lactobacillus spp., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We studied as hydrogen peroxide, lactic acid, or surfactants from clinical isolates of vaginal lactobacilli and cell-free supernatants from probiotic strain LCR35 can influence on the sensitivity of opportunistic bacteria to antibiotics. We found that the most effective in increasing sensitivity to antibiotics were hydrogen peroxide and surfactants or their combination but no lactic acid. In some cases, the effect of the composition of hydrogen peroxide and surfactants was clearly higher than the sum of effects of these substances alone. With using of the supernatant of LCR35 was shown that the combination of surfactant and lactate has greater effect compared with surfactants alone. In concluding, metabolites of vaginal lactobacilli are suitable for the role of "antibiotic assistants" and it can help solve the problems the antibiotic resistance.

Background & Objectives: Shigella dysenteriae is one of the most virulent pathogens causing bacillary dysentery and is responsible for high mortality in infants. To reduce the load of antibiotic therapy for treating shigellosis, this study was carried out to assess the ex vivo effect of novel probiotic lactobacilli, isolated from infant's stool samples, on killing S. dysenteriae type 1 residing in the rat macrophages., Methods: Stool samples from infants were collected, processed for the isolation of lactobacilli and screened for the probiotic attributes (acid tolerance, bile tolerance, ability to adhere intestinal cells and anti-S. dysenteriae activity). The effect of cell-free supernatant of lactobacilli on Shigella- infected macrophages in terms of phagocytic ability, extent of lipid peroxidation, nitrite, superoxide dismutase and glutathione levels was evaluated., Results: Based on the probiotic attributes, three lactobacilli were isolated from the stool samples of infants. Using classical and molecular tools, these isolates were identified as Lactobacillus pentosus, L. Paraplantarum and L. rhamnosus. All the three lactobacilli had the ability to kill intramacrophage S. dysentriae type 1. The anti-Shigella activity of the probiotic lactobacilli was attributed to increased antioxidative ability and decreased free radical production by the infected macrophages., Interpretation & Conclusions: Probiotic cocktail of L. pentosus, L. paraplantarum and L. rhamnosus showed ex vivo killing of S. dysenteriae residing inside the rat macrophages significantly. This cocktail has the potential to be used as a natural alternative for treating S. dysenteriae infection, especially in infants, however, further studies need to be done to confirm these finding in vivo.

To alleviate adverse effects of heavy metal toxicity, diverse range of removing methods have been suggested, that is usage of algae, agricultural by-products and microorganisms. Here, we investigated lead (Pb) biosorption efficacy by two lactic acid bacteria species (LABs) in broiler chickens. In an in vitro study, Pb was added to culture medium of LABs (Lactobacillus pentosus ITA23 and Lactobacillus acidipiscis ITA44) in the form of lead acetate. Results showed that these LABs were able to absorb more than 90% of Pb from the culture medium. In follow-up in vivo study, LABs mixture was added to diet of broiler chickens contained lead acetate (200 mg/kg). Pb exposure significantly increased lipid peroxidation and decreased antioxidant activity in liver. The changes were recovered back to normal level upon LABs supplementation. Moreover, addition of LABs eliminated the liver tissue lesion and the suppressed performance in Pb-exposed chicks. Analysis of liver and serum samples indicated 48% and 28% reduction in Pb accumulation, respectively. In conclusion, results of this study showed that L. pentosus ITA23 and L. acidipiscis ITA44 effectively biosorb and expel dietary Pb from gastrointestinal tract of chickens.

Crystal structures of Lactobacillus buchneri isoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5'-phosphate (PLP), in complex with N-(5'-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of the L. buchneri isoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-ℇ-caprolactam racemase from Achromobactor obae and γ-aminobutyrate aminotransferase from Escherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved in L. buchneri isoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine.

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.

In this study, the potential of Fourier transform infrared (FTIR) spectroscopy for assessing putative biochemical and structural differences between the two variants, rough (R) and smooth (S), of Lactobacillus farciminis CNCM-I-3699, a pleomorphic strain, was investigated. The main differences observed were localized in the polysaccharide (1200-900 cm-1) and protein (1700-1500 cm-1) regions. Based on spectral information in these two spectral ranges, clustering resulted in a dendrogram that showed a clear discrimination between both morphotypes. Significant increases in favor of morphotype S compared to R at specific wavenumbers for polysaccharides (22.18% vs. 5.24% at 1068 cm-1) and capsular polysaccharides (16% vs. 13.17% at 1048 cm-1) were recorded. Compared to S, the morphotype R exhibits a 1.27-fold higher signal at the wavenumber of 1637 cm-1 assigned to the amide I β-sheet and a 2.71-fold higher signal at the wavenumber of 1513 cm-1 assigned to the tyrosine involved in the β-sheet arrangement of proteins. The FTIR analysis is efficient to separate and give data on mainly surface component differences observed previously between S colony morphotype (ropy and exopolysaccharide positive) and the R colony morphotype (non-ropy but highly autoaggregative)., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Several glucansucrases were surveyed for their ability to produce isomelezitose, a trisaccharide with the structure α-D-glucopyranosyl (1 → 6) β-D-fructofuranosyl (2 ↔ 1) α-D-glucopyranoside. Nearly all strains tested, with one exception, produced at least trace levels of isomelezitose. Yields were low but significant, ranging from less than 1% to approximately 5% based on sucrose. This trisaccharide may arise in either of two ways: glucopyranosyl transfer to the 6 Fru -OH position of sucrose, or to the anomeric OH position of isomaltulose. This study indicates that isomelezitose formation may be a general phenomenon of many glucansucrase reactions., (Published by Elsevier Ltd.)

This study was conducted to investigate the inhibitory effect of Lactobacillus cells and supernatants on the growth of the human colon cancer cell line HT-29. Our study results indicated that the PM153 strain exhibits the best adhesion ability and the highest survival in the gastrointestinal tract simulation experiment. Furthermore, after an 8-h co-culture of PM153 and HT-29 cells, the PM153 strain can induce the secretion of nitric oxide from the HT-29 cells. In addition, after the co-culture of the BCRC17010 strain (10⁸ cfu/mL) and HT-29 cells, the Bax/Bcl-2 ratio in the HT-29 cells was 1.19, which showed a significant difference from the other control and LAB groups ( p < 0.05), which therefore led to the inference that the BCRC17010 strain exerts a pro-apoptotic effect on the HT-29 cells. Upon co-culture with HT-29 cells for 4, 8 and 12 h, the BCRC14625 strain (10⁸ cfu/mL) demonstrated a significant increase in lactate dehydrogenase (LDH) activity ( p < 0.05), causing harm to the HT-29 cell membrane; further, after an 8-h co-culture with the HT-29 cells, it induced the secretion of nitric oxide (NO) from the HT-29 cells. Some lactic acid bacteria (LAB) strains have ability to inhibit the growth of the colorectal cancer cell line HT-29 Bax/Bcl-2 pathway or NO production. In summary, we demonstrated that the BCRC17010 strain, good abilities of adhesion and increased LDH release, was the best probiotic potential for inhibition of HT-29 growth amongst the seven LAB strains tested in vitro.

In the present study, we have obtained two exopolysaccharide (EPS)-producing thermophilic lactic acid bacteria (LAB) that were isolated from tropical fruits of Thailand. The two strains, designated LY45 and PY45, were identified as Pediococcus pentosaceus and Lactobacillus amylovorus, respectively. Both plant-derived LAB strains, which produce neutral EPSs together with the acidic one, can grow vigorously at 45°C and even at 50°C. Hyaluronidase (EC 3.2.1.35), which catalyzes the degradation of hyaluronic acid, activates an inflammatory reaction. Interestingly, EPSs produced by the LY45 and PY45 strains were found to inhibit hyaluronidase activity at the same order of IC 50 values as did sodium cromoglicate and dipotassium glycyrrhizinate, which are well-known as anti-inflammatory agents. The LY45-derived neutral EPS consists of glucose and mannose as monosaccharide components, whereas the acidic one contains mainly mannose, together with glucose and galactose. On the other hand, although Lactobacillus amylovorus PY45 also produces neutral and acidic EPSs, the main monosaccharide in both EPSs is mannose, and glucose is a minor component. Furthermore, the PY45 strain may be probiotically and industrially useful because the microorganism can utilize starch and glycogen as carbon sources.

The aim of this study was to investigate the antibacterial effect of the crude starter " kpètè-kpètè " and lactic acid bacteria used during the production of "tchoukoutou." To achieve this, a total of 11 lactic acid bacteria and 40 starter samples were collected from four communes. The samples were tested on 29 gram + and - strains by disk diffusion method. The minimum inhibitory and bactericidal concentrations of starter and lactic acid bacteria were determined by conventional methods. Organic acids, sugar, and volatile compounds were determined using the HPLC method. The "kpètè-kpètè" displays a high antibacterial activity against the tested strains. The most sensitive strain was S. epidermidis (12.5 mm) whereas the resistance strain was Proteus mirabilis (8 mm). All the tested ferment has not any inhibitory effect on Enterococcus faecalis . The lactic acid bacteria isolates of Parakou showed the highest (17.48 mm) antibacterial activity whereas the smallest diameter was obtained with the ferment collected from Boukoumbé (9.80 mm). The starters' chemical screening revealed the presence of tannins, anthocyanin flavonoids, triterpenes, steroids, reducing compounds, and mucilage O-glycosides. These compounds are probably the source of recorded inhibition effect. The lactic acid bacteria of the "kpètè-kpètè" could be used to develop a food ingredient with probiotic property.

The use of lactic acid bacteria (LAB) as probiotics in aquaculture may improve the quality of seed production and limit the use of antibiotics in fish hatcheries. This study attempted to further characterize the candidate probiotic Lactobacillus casei X2, and the immune and physiological responses of the sea bass larvae. L. casei X2 was confirmed as a good candidate, due to its wide antibacterial spectrum against both Gram-positive and Gram-negative bacteria, and its free radical scavenging activity. In addition, if the strain did not seem able to form biofilm on abiotic surfaces, it adhered strongly to Hep-2 cells. However, these characteristics did not seem efficient in vivo. At 20 days post-hatch (dph), the expression level of CAT gene was significantly different between group fed without probiotic and the two groups treated with either Pediococcus acidilactici or L. casei. This gene was upregulated in the group treated with strain X2 and downregulated in the group with a commercial probiotic strain P. acidilactici, suggesting a better antioxidant activity with the later strain. At the same sampling date, the IL-1β gene was upregulated in the group treated with P. acidilactici, and the HSP70 gene was overexpressed at 41 dph. As the stimulation of these two last genes, such transcriptomic indicators must be cautiously interpreted., (© 2016 John Wiley & Sons Ltd.)

The lactic acid bacteria (LAB) microbiota of Saudi chicken ceca was determined. From 60 samples, 204 isolates of lactic acid bacteria were obtained. Three isolates produced antimicrobial activities against Campylobacter jejuni, Listeria monocytogenes, and Bacillus subtilis. The isolate DN317, which had the highest activity against Campylobacter jejuni ATCC 33560, was identified as Lactobacillus curvatus (GenBank accession numbers: KX353849 and KX353850). Full inhibitory activity was observed after a 2-h incubation with the supernatant at pH values between 4 and 8. Only 16% of the activity was conserved after a treatment at 121 °C for 15 min. The use of proteinase K, pepsin, chymotrypsin, trypsin, papain, and lysozyme drastically reduced the antimicrobial activity. However, lipase, catalase, and lysozyme had no effect on this activity. The active peptide produced by Lactobacillus curvatus DN317 was purified by precipitation with an 80% saturated ammonium sulfate solution, and two steps of reversed phase HPLC on a C18 column. The molecular weight of this peptide was 4448 Da as determined by MALDI-ToF. N-terminal sequence analysis using Edman degradation revealed 47 amino acid residues (UniProt Knowledgebase accession number C0HK82) revealing homology with the amino acid sequences of sakacin P and curvaticin L442. The antimicrobial activity of the bacteriocin, namely curvaticin DN317, was found to be bacteriostatic against Campylobacter jejuni ATCC 33560. The use of microbial antagonism by LAB is one of the best ways to control microorganisms safely in foods. This result constitutes a reasonable advance in the antimicrobial field because of its potential applications in food technology.

Kefiran is a neutral polysaccharide constituted by glucose and galactose produced by Lactobacillus kefiranofaciens. It is included into kefir grains and has several health promoting properties. In the present work, shear and extensional properties of different kefiran aqueous dispersions (0.5, 1 and 2% wt.) were assessed and compared to other neutral gums commonly used in food, cosmetic and pharmaceutics industries (methylcellulose, locust bean gum and guar gum). Kefiran showed shear flow characteristics similar to that displayed by other representative neutral gums, although it always yielded lower viscosities at a given concentration. For each gum system it was possible to find a correlation between dynamic and steady shear properties by a master curve including both the apparent and complex viscosities. When studying extensional properties of selected gums at 2% wt. by means of a capillary break-up rheometer, kefiran solutions did not show important extensional properties, displaying a behaviour close the Newtonian., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Many species of Lactobacillus sp. possess Surface(s) layer proteins in their envelope. Among other important characteristics S-layer from Lactobacillus acidophilus binds to the cellular receptor DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin; CD209), which is involved in adhesion and infection of several families of bacteria. In this report we investigate the activity of new S-layer proteins from the Lactobacillus family (Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus helveticus and Lactobacillus kefiri) over the infection of representative microorganisms important to human health. After the treatment of DC-SIGN expressing cells with these proteins, we were able to diminish bacterial infection by up to 79% in both gram negative and mycobacterial models. We discovered that pre-treatment of the bacteria with S-layers from Lactobacillus acidophilus and Lactobacillus brevis reduced bacteria viability but also prevent infection by the pathogenic bacteria. We also proved the importance of the glycosylation of the S-layer from Lactobacillus kefiri in the binding to the receptor and thus inhibition of infection. This novel characteristic of the S-layers proteins may contribute to the already reported pathogen exclusion activity for these Lactobacillus probiotic strains; and might be also considered as a novel enzymatic antimicrobial agents to inhibit bacterial infection and entry to host cells., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Lactic acid bacteria are generally believed to have positive roles in maintaining good health and immune system in humans. A number of Lactobacilli spp. are known to produce important metabolites, among which biosurfactants in particular have shown antimicrobial activity against several pathogens in the intestinal tract and female urogenital tract partly through interfering with biofilm formation and adhesion to the epithelial cells surfaces. Around 46 reports are documented on biosurfactant production from Lactobacillus spp. of which six can be broadly classified as cell free biosurfactant and 40 as cell associated biosurfactants and only approximately 50% of those have reported on the structural composition which, in order of occurrence were mainly proteinaceous, glycolipidic, glycoproteins, or glycolipopeptides in nature. Due to the proteinaceous nature, most biosurfactant produced by strains of Lactobacillus are generally believed to be surlactin type with high potential toward impeding pathogens adherence. Researchers have recently focused on the anti-adhesive and antibiofilm properties of Lactobacilli-derived biosurfactants. This review briefly discusses the significance of Lactobacilli-derived biosurfactants and their potential applications in various fields. In addition, we highlight the exceptional prospects and challenges in fermentation economics of Lactobacillus spp.-derived biosurfactants' production processes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The purpose of this study was to investigate whether vaginal administration of probiotic Lactobacillus results in their colonization and persistence in the vagina and whether it promotes normalization and maintenance of pH and Nugent score. A single-arm, open-label controlled towards the baseline (pre-post) study including 35 apparently healthy women was conducted. Each woman was examined three times during the study. Women were instructed to receive daily for 7 days, the probiotic suppositories SYNBIO(®) gin (Lactobacillus rhamnosus IMC 501(®) and Lactobacillus paracasei IMC 502(®)). Vaginal swabs were collected during visit 1, 2, and 3 to determine the total lactobacilli count, the presence of the two administered bacteria, the measure of the pH, and the estimation of Nugent score. Evaluation of treatment tolerability was based on analysis of the type and occurrence of adverse events. The probiotic vaginal suppository was well tolerated and no side effects were reported. Intermediate Nugent score was registered in 40 % of women at visit 1 and these intermediate scores reverted to normal at day 7 (end of treatment) in 20 % of subjects. Administration of SYNBIO(®) gin contributed to a significant increase in the lactobacilli level at visit 2. Molecular typing revealed the presence of the two strains originating from SYNBIO(®) gin in 100 % of women at visit 2 and 34 % at visit 3. No significant changes were registered for pH between visits. The SYNBIO(®) gin product is safe for daily use in healthy women and it could be useful to restore and maintain a normal vaginal microbiota.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is well-known to be a powerful technique for the analysis of biological samples. By using glycerol-based liquid support matrices (LSMs) instead of conventional MALDI matrices the power of this technique can be extended further. In this study, we exploited LSMs for the identification of complex samples, that is, the Lactobacillus proteome and a bovine serum albumin (BSA) digest. Liquid and solid MALDI samples were manually and robotically prepared by coupling a nanoflow high-performance liquid chromatography (nanoHPLC) system to an automated MALDI sample spotting device. MS and MS/MS data were successfully acquired at the femtomole level using TOF/TOF as well as Q-TOF instrumentation and used for protein identification searching sequence databases. For the BSA digest analysis, liquid MALDI samples resulted in peptide mass fingerprints, which led to a higher confidence in protein identification compared with solid (crystalline) MALDI samples; however, postsource decay (PSD) MS/MS analysis of both the proteome of Lactobacillus plantarum WCFS1 cells and BSA digest showed that further optimization of the formation and detection of peptide fragment ions is still needed for liquid MALDI samples, as the MS/MS ion search score was lower than that for the solid MALDI samples, reflecting the poorer quality of the liquid MALDI-PSD spectra, which can be attributed to the differences in PSD parameters and their optimization that is currently achievable.

There is a growing interest in supporting human health by using prebiotics, such as oligosaccharides, and beneficial bacteria, also called probiotics. Combining these two components we can develop synbiotics. In order to create successful combination of synbiotic it is very important to evaluate the influence of prebiotic oligosaccharides to probiotic bacteria and their behavior, such as growth and secretion of health related biomolecules, including bacteriocins. In this study seven type strains of probiotic bacteria (five Lactobacillus sp. and two Lactococcus sp.) and two Lactobacillus sp. strains, isolated from probiotic yoghurt, were cultivated with various commercially available and extracted oligosaccharides (OS). The aim of this study was to evaluate the influence of these OS on type and isolated bacterial strains growth and antibacterial activity. Obtained results suggest that combination of certain OS with probiotic strains may considerably improve their growth and/or antibacterial activity. We also determined the antibacterial activity spectrum of investigated strains with combination of OS against common food borne pathogens. Results of this work show that prebiotic OS can be useful for modulating probiotic bacteria growth, antibacterial activity and even specificity of this activity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Edible and medicinal mushrooms contain bioactive compounds with promising effects on several cardiovascular risk biomarkers. However, strains of Ganoderma lucidum of Mexican origin have not yet been studied. Standardized extracts of G. lucidum (Gl) were given to C57BL/6 mice fed a high-cholesterol diet compared with the drug simvastatin. The effects of the extracts on serum biochemical parameters, liver lipid content, cholesterol metabolism, and the composition of gut microbiota were assessed. Acetylsalicylic acid (10 mM) added to the cultivation substrate modulated properties of Gl extracts obtained from mature basidiomata. Compared to the high-cholesterol diet group, the consumption of Gl extracts significantly reduced total serum cholesterol (by 19.2% to 27.1%), LDL-C (by 4.5% to 35.1%), triglyceride concentration (by 16.3% to 46.6%), hepatic cholesterol (by 28.7% to 52%) and hepatic triglycerides (by 43.8% to 56.6%). These effects were associated with a significant reduction in the expression of lipogenic genes (Hmgcr, Srebp1c, Fasn, and Acaca) and genes involved in reverse cholesterol transport (Abcg5 and Abcg8), as well as an increase in Ldlr gene expression in the liver. No significant changes were observed in the gene expression of Srebp2, Abca1 or Cyp7a1. In several cases, Gl-1 or Gl-2 extracts showed better effects on lipid metabolism than the drug simvastatin. A proposed mechanism of action for the reduction in cholesterol levels is mediated by α-glucans and β-glucans from Gl, which promoted decreased absorption of cholesterol in the gut, as well as greater excretion of fecal bile acids and cholesterol. The prebiotic effects of Gl-1 and Gl-2 extracts modulated the composition of gut microbiota and produced an increase in the Lactobacillaceae family and Lactobacillus genus level compared to the control group, high-cholesterol diet group and group supplemented with simvastatin. Mexican genetic resources of Gl represent a new source of bioactive compounds showing hypocholesterolemic properties and prebiotic effects.

Whole cell extracts of two Lactobacillus strains were tested with primary antibodies from two pooled sera from allergic patients. Fluorescently labelled anti-human IgG and anti-human IgE secondary antibodies applied in Western blotting, together with an appropriate image acquisition protocol facilitated imagining bacterial proteins that reacted with human IgG and IgE., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study was conducted to examine if adding microbial inoculants or propionic acid to alfalfa silages contaminated with Escherichia coli O157:H7 would inhibit the growth of the pathogen during or after ensiling. Alfalfa forage was harvested at the early bloom stage, wilted to a dry matter concentration of 54%, chopped to 19-mm lengths, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1×10(5) cfu/g of E. coli O157:H7 (EC); (3) EC and 1×10(6) cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1×10(6) cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 2.2g/kg of propionic acid (EC+PA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, 16, and 100d and analyzed for EC counts, pH, and organic acids. In addition, samples from d 100 were analyzed for chemical composition, ammonia-N, counts of yeasts and molds, and aerobic stability. Escherichia coli O157:H7 was detected in all silages until d 7, but by d 16 it was not detected in those treated with EC+LB and EC+LP, though it was still detected in EC and EC+PA silages. However, by d 100, the pathogen was not detected in any silage. The rate of pH decrease to 5.0 was fastest for the EC+LP silage (7d), followed by the EC+LB silage (16d). Nevertheless, all silages had attained a pH of or less than 5.0 by d 100. The rapid decrease in pH in EC+LP and EC+LB silages was observed due to higher lactate and acetate concentrations, respectively, relative to the other silages during the early fermentation phase (d 3-16). Propionic acid was only detected in the EC+PA silage. Yeast counts were lowest in EC+LB and EC+PA silages. Subsamples of all d-100 silages were reinoculated with 1×10(5) cfu/g of EC immediately after silo opening. When the pathogen was subsequently enumerated after 168h of aerobic exposure, it was not detected in silages treated with EC+PA, EC+LB, or EC+LP, which all had pH values less than 5.0. Whereas the EC silage had a pH value of 5.4 and 2.3 log cfu/g of the pathogen. Certain bacterial inoculants can hasten the inhibition of E. coli O157:H7 during ensiling, such as propionic acid, and they can also prevent its growth on silage contaminated with the pathogen after ensiling., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Three water-soluble, non-ionic extracellular polysaccharides (EPS) obtained from lactic acid bacteria (S. thermophilus THS, L. helveticus K16 and S. thermophilus ST1) were subjected to a comparative study by means of multidetector size-exclusion chromatography, providing distributions and averages of molar masses, radii of gyration and intrinsic viscosities. All polysaccharides displayed random coil character. Further analysis of the data reveals differences in chain stiffness and extension that could be well correlated to structural features. The calculated persistence lengths ranged from 5 to 10nm and fall within the range typical for many single-stranded bacterial or plant polysaccharides. The ST1 polysaccharide had the highest molar mass but the lowest persistence length, which is attributed to the presence of the flexible (1→6)-linkage in the main chain., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: To develop potent drug delivery agents to treat multidrug resistant (MDR) bacterial infections using gold nanoparticles (AuNPs) prepared by bacterial exopolysaccharide (EPS) being a reducing and stabilizing agent., Methods: Gold nanoparticles were prepared by EPS, extracted from Lactobacillus plantarum. AuNPs were characterized by UV-visible spectrophotometer, transmission electron microscope (TEM), zeta potential and Fourier transform infrared spectroscopy. Antibiotics used for functionalization of AuNPs were levofloxacin, cefotaxime, ceftriaxone and ciprofloxacin. The resulted functionalized AuNPs were tested against various MDR bacteria by employing different assays such as well diffusion assay, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time killing assay., Key Findings: The nanostructures exhibited excellent bactericidal activity and reduced MIC and MBC against MDR Gram positive and Gram negative bacteria compared to free drugs. Escherichia coli was the most susceptible MDR bacteria followed by Klebsiella pneumoniae and Staphylococcus aureus. TEM results revealed that the bactericidal activity of nanostructures could be mediated by penetration, loss of cytoplasmic contents and cell lysis., Significance: Antibiotic functionalized nanostructures were more potent than free drugs and could be used as potent drug delivery vehicles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The aim of this study was to formulate probiotics-loaded pellets in a tablet form to improve storage stability, acid tolerability, and in vivo intestinal protective effect. Bacteria-loaded pellets primarily prepared with hydroxypropyl methylcellulose acetate succinate were compressed into tablets with highly compressible excipients and optimized for flow properties, hardness, and disintegration time. The optimized probiotic tablet consisted of enteric-coated pellets (335 mg), microcrystalline cellulose (Avicel PH102, 37.5 mg), and porous calcium silicate (25 mg) and allowed whole survival of living bacteria during the compaction process with sufficient tablet hardness (13 kp) and disintegration time (14 minutes). The multiple-unit tablet showed remarkably higher storage stability under ambient conditions (25°C/60% relative humidity) over 6 months and resistance to acidic medium compared to uncoated strains or pellets. Repeated intake of this multiple-unit tablet significantly lowered plasma level of endotoxin, a pathogenic material, compared to repeated intake of bare probiotics or marketed products in rats. These results, therefore, suggest that the multiple-unit tablet is advantageous to better bacterial viability and gain the beneficial effects on the gut flora, including the improvement of intestinal barrier function.

Background: Lactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed., Results: We found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI)., Conclusions: The origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.

New strains are desirable to diversify flavour of fermented dairy products. The objective of this study was to evaluate the potential of Leuconostoc spp. and Lactobacillus spp. in the production of aroma compounds by metabolic fingerprints of volatiles. Eighteen strains, including five Lactobacillus species (Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus sakei) and three Leuconostoc species (Leuconostoc citreum, Leuconostoc lactis, and Leuconostoc mesenteroides) were incubated for 5 weeks in a curd-based slurry medium under conditions mimicking cheese ripening. Populations were enumerated and volatile compounds were analysed by headspace trap gas chromatography-mass spectrometry (GC-MS). A metabolomics approach followed by multivariate statistical analysis was applied for data processing and analysis. In total, 12 alcohols, 10 aldehydes, 7 esters, 11 ketones, 5 acids and 2 sulphur compounds were identified. Very large differences in concentration of volatile compounds between the highest producing strains and the control medium were observed in particular for diacetyl, 2-butanol, ethyl acetate, 3-methylbutanol, 3-methylbutanoic acid and 2-methylbutanoic acid. Some of the characterized strains demonstrated an interesting aromatizing potential to be used as adjunct culture.

Background: The genus Lactobacillus belongs to a large heterogeneous group of low G + C Gram-positive anaerobic bacteria, which are frequently used as probiotics. The health-beneficial effects, in particular the immunomodulation effect, of probiotics depend on the strain and dose used. Strain variations may be related to diversity of the cell surface architecture of bacteria and the ability to express specific antigens or secrete compounds. The use of Lactobacillus as probiotic requires a comprehensive understanding of its effect on host immune system. To evaluate the potential immunoreactive properties of proteins isolated from four Lactobacillus strains: L. johnsonii 142 and L. johnsonii 151, L. rhamnosus LOCK 0900 and L. casei LOCK 0919, the polyclonal sera obtained from mouse and human have been tested as well as with sera from rabbits immunized with whole lactobacilli cells., Results: The reactivity of isolated proteins detected by SDS-PAGE and Western blotting was heterogeneous and varied between different serum samples. The proteins with the highest immunoreactivity were isolated, purified and sequenced, in particular the fractions were identified as phosphoglycerate kinase (L. johnsonii 142), glyceraldehyde 3-phosphate dehydrogenase (L. johnosnii 142, L. rhamnosus LOCK 0900), hypothetic protein JDM1&#95;1307 (L. johnsonii 151) and fructose/tagatose-bisphosphate-aldolase (L. casei LOCK 0919)., Conclusion: The different prevalence of reactions against tested antigens in rabbit, mouse and human sera may indicate significant differences in immune system and commensal cross-talk in these groups. The identification of immunoreactive lactobacilli proteins opens the possibility to use them as an antigens for development of vaccines.

Exopolysaccharides (EPS)-producing lactic acid bacteria (LAB) are industrially important microorganisms in the development of functional food products and are used as starter cultures or coadjutants to develop fermented foods. There is large variability in EPS production by LAB in terms of chemical composition, quantity, molecular size, charge, presence of side chains, and rigidity of the molecules. The main body of the review will cover practical aspects concerning the structural diversity structure of EPS, and their concrete application in food industries is reported in details. To strengthen the food application and process feasibility of LAB EPS at industrial level, a future academic research should be combined with industrial input to understand the technical shortfalls that EPS can address.

The aim of this work was to develop a synbiotic vaginal insert containing the probiotic strain Lactobacillus crispatus BC5, the prebiotic substrate fructo-oligosaccharide and the antioxidant agent ascorbic acid, for the prophylaxis and therapy of vaginal infections. Mucoadhesive in situ gelling vaginal inserts based on hydroxypropyl methylcellulose were prepared by freeze-drying, stored at +2-8 °C for 90 days and characterized in terms of technological and functional properties. Complete survival of L. crispatus BC5 was found immediately after insert preparation (96.08%) as well as after 90 days of storage (95.82%) in the vaginal inserts containing fructo-oligosaccharide, ascorbic acid and skimmed milk. Synbiotic inserts showed improved mucoadhesion ability (from three- to five-fold) with respect to a standard formulation based on hydroxypropyl methylcellulose alone. Moreover, inserts allowed to modulate lactobacilli release in virtue of the different amounts of fructo-oligosaccharide. Finally, antimicrobial activity was exerted by L. crispatus BC5 released from the vaginal formulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Lactobacillus kefiranofaciens ZW3 was obtained from kefir grains, which have high lactose hydrolytic activity. In this study, a heterodimeric LacLM-type β-galactosidase gene (lacLM) from ZW3 was isolated, which was composed of two overlapping genes, lacL (1,884 bp) and lacM (960 bp) encoding large and small subunits with calculated molecular masses of 73,620 and 35,682 Da, respectively. LacLM, LacL, and LacM were expressed in Escherichia coli BL21(DE3) and these recombinant proteins were purified and characterized. The results showed that, compared with the recombinant holoenzyme, the recombinant large subunit exhibits obviously lower thermostability and hydrolytic activity. Moreover, the optimal temperature and pH of the holoenzyme and large subunit are 60°C and 7.0, and 50°C and 8.0, respectively. However, the recombinant small subunit alone has no activity. Interestingly, the activity and thermostability of the large subunit were greatly improved after mixing it with the recombinant small subunit. Therefore, the results suggest that the small subunit might play an important role in maintaining the stability of the structure of the catalytic center located in the large subunit.

Lactic acid bacteria with antifungal properties are applied for biopreservation of food. In order to further our understanding of their antifungal mechanism, there is an ongoing search for bioactive molecules. With a focus on the metabolites formed, bioassay-guided fractionation and comprehensive screening have identified compounds as antifungal. Although these are active, the compounds have been found in concentrations that are too low to account for the observed antifungal effect. It has been hypothesized that the formation of metabolites and consumption of nutrients during bacterial fermentations form the basis for the antifungal effect, i.e., the composition of the exometabolome. To build a more comprehensive view of the chemical changes induced by bacterial fermentation and the effects on mold growth, a strategy for correlating the exometabolomic profiles with mold growth was applied. The antifungal properties were assessed by measuring mold growth of two Penicillium strains on cell-free ferments of three strains of Lactobacillus paracasei pre-fermented in a chemically defined medium. Exometabolomic profiling was performed by reversed-phase liquid chromatography in combination with mass spectrometry in electrospray positive and negative modes. By multivariate data analysis, the three strains of Lb. paracasei were readily distinguished by the relative difference of their exometabolomes. The relative differences correlated with the relative growth of the two Penicillium strains. Metabolic footprinting proved to be a supplement to bioassay-guided fractionation for investigation of antifungal properties of bacterial ferments. Additionally, three previously identified and three novel antifungal metabolites from Lb. paracasei and their potential precursors were detected and assigned using the strategy.

Lactobacillus spp. from Mexican Cocido cheese were shown to produce bacteriocin-like substances (BLS) active against Staphylococcus aureus,Listeria innocua,Escherichia coli, andSalmonella typhimurium by using the disk diffusion method. Crude extracts of Lactobacillus fermentum showed strong inhibitory activity against Staph. aureus, L. innocua, E. coli, and Salmonella cholerae. Complete inactivation of antimicrobial activity was observed after treatment of crude extracts with proteinase K, pronase, papain, trypsin, and lysozyme, confirming their proteinaceous nature. However, antimicrobial activity was partly lost for some of the crude extracts when treated with α-amylase, indicating that carbohydrate moieties were involved. The antimicrobial activity of the crude extracts was stable at 65°C for 30min over a wide pH range (2-8), and addition of potassium chloride, sodium citrate, ethanol, and butanol did not affect antibacterial activity. However, antimicrobial activity was lost after heating at 121°C for 15min, addition of methanol or Tween 80. Fourteen out of 18 Lactobacillus spp. showed antimicrobial activity against different test microorganisms, and 12 presented bacteriocin-like substances. Generation time and growth rate parameters indicated that the antimicrobial activity of crude extracts from 3 different strains was effective against the 4 indicator microorganisms. One of the crude extracts showed inhibition not only against gram-positive but also against gram-negative bacteria. Bacteriocin-like substances produced by this specific Lactobacillus strain showed potential for application as a food biopreservative., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Campylobacter jejuni, one of the most common causes of gastroenteritis worldwide, is transmitted to humans through poultry. We previously reported that Lactobacillus gasseri SBT2055 (LG2055) reduced C. jejuni infection in human epithelial cells in vitro and inhibited pathogen colonization of chickens in vivo. This suggested that the LG2055 adhesion and/or co-aggregation phenotype mediated by cell-surface aggregation-promoting factors (APFs) may be important for the competitive exclusion of C. jejuni. Here, we show that cell surface-associated APF1 promoted LG2055 self-aggregation and adhesion to human epithelial cells and exhibited high affinity for the extracellular matrix component fibronectin. These effects were absent in the apf1 knockout mutant, indicating the role of APF1 in LG2055-mediated inhibition of C. jejuni in epithelial cells and chicken colonization. Similar to APF1, APF2 promoted the co-aggregation of LG2055 and C. jejuni but did not inhibit C. jejuni infection. Our data suggest a pivotal role for APF1 in mediating the interaction of LG2055 with human intestinal cells and in inhibiting C. jejuni colonization of the gastrointestinal tract. We thus provide new insight into the health-promoting effects of probiotics and mechanisms of competitive exclusion in poultry. Further research is needed to determine whether the probiotic strains reach the epithelial surface., (© 2015 John Wiley & Sons Ltd.)

The Lactobacillus acidophilus homology group comprises Gram-positive species that include L. acidophilus, L. helveticus, L. crispatus, L. amylovorus, L. gallinarum, L. delbrueckii subsp. bulgaricus, L. gasseri, and L. johnsonii. While these bacteria are closely related, they have varied ecological lifestyles as dairy and food fermenters, allochthonous probiotics, or autochthonous commensals of the host gastrointestinal tract. Bacterial cell surface components play a critical role in the molecular dialogue between bacteria and interaction signaling with the intestinal mucosa. Notably, the L. acidophilus complex is distinguished in two clades by the presence or absence of S-layers, which are semiporous crystalline arrays of self-assembling proteinaceous subunits found as the outermost layer of the bacterial cell wall. In this study, S-layer-associated proteins (SLAPs) in the exoproteomes of various S-layer-forming Lactobacillus species were proteomically identified, genomically compared, and transcriptionally analyzed. Four gene regions encoding six putative SLAPs were conserved in the S-layer-forming Lactobacillus species but not identified in the extracts of the closely related progenitor, L. delbrueckii subsp. bulgaricus, which does not produce an S-layer. Therefore, the presence or absence of an S-layer has a clear impact on the exoproteomic composition of Lactobacillus species. This proteomic complexity and differences in the cell surface properties between S-layer- and non-S-layer-forming lactobacilli reveal the potential for SLAPs to mediate intimate probiotic interactions and signaling with the host intestinal mucosa., (Copyright © 2015 Johnson et al.)

The present study was undertaken to evaluate the effect of Lactobacillus kefiri (PFT), a novel kefir product, on apoptosis of gastric cancer cells (AGS), breast cancer cells (4T1), and human peripheral blood mononuclear cells (PBMCs). Cells were cultured with PFT and apoptosis was determined by flow cytometry using 7-AAD dye and cytospin preparation. Mitochondrial dysfunction and expression of Bcl2 were monitored by flow cytometry. Results showed that PFT induced apoptosis in AGS gastric cancer cells in a dose-dependent manner. Apoptosis was detected at a concentration of 0.3 mg/ml (20.8%), increased to 25.8% at 0.6 mg/ml, 37% at 1.2 mg/ml, 53.1% at 2.5 mg/ml, and peaked at 66.3% at 5.0 mg/ml. Apoptosis is associated with the decreased polarization of mitochondrial membrane potential (MMP) and decreased Bcl2 expression. PFT-treated AGS cells manifested membrane blebbing, nuclear condensation, and fragmentation as identified in cytospin cytocentrifuge Giemsa stained preparations. On the other hand, flow cytometry analysis showed that PFT did not induce apoptosis in 4T1 breast cancer cells nor in PBMCs. These results suggest that PFT is safe for white blood cells and selectively induces apoptotic effects in gastric cancer cells. Hence, it may have potential as a therapeutic agent for the treatment of gastric cancers.

Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated or inoculated with 1 of 3 microbial inoculants and with or without exogenous protease addition. The inoculant treatments contained (1) Lactobacillus buchneri 40788 and Pediococcus pentosaceus, (2) L. buchneri 40788, and (3) a mixture of P. pentosaceus and Propionibacterium freudenreichii. Protease, but not inoculation, increased ivSD by 7.5 percentage units (44.4 vs. 51.9%). Protease addition increased ivSD in rehydrated corn and HMC. Microbial inoculation improved fermentation profiles but did not affect ivSD., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Fifteen sourdoughs produced in western Sicily (southern Italy) were analysed by classical methods for their chemico-physical characteristics and the levels of lactic acid bacteria (LAB). pH and total titratable acidity (TTA) were mostly in the range commonly reported for similar products produced in Italy, but the fermentation quotient (FQ) of the majority of samples was above 4.0, due to the low concentration of acetic acid estimated by high performance liquid chromatography (HPLC). Specific counts of LAB showed levels higher than 10(8) CFU g(-1) for many samples. The colonies representing various morphologies were isolated and, after the differentiation based on phenotypic characteristics, divided into 10 groups. The most numerous group was composed of facultative heterofermentative isolates, indicating a relevance of this bacterial group during fermentation. The genetic analysis by randomly amplified polymorphic DNA (RAPD)-PCR, 16S rRNA gene sequencing and species-specific PCRs identified 33 strains as Lactobacillus plantarum, Lactobacillus curvatus and Lactobacillus graminis. Due to the consistent presence of L. plantarum, it was concluded that this species codominates with obligate heterofermentative LAB in sourdough production in this geographical area. In order to evaluate the performances at the basis of their fitness, the 29 L. plantarum strains were investigated for several technological traits. Twelve cultures showed good acidifying abilities in vitro and L. plantarum PON100148 produced the highest concentrations of organic acids. Eleven strains were positive for extracellular protease activity. Bacteriocin-like inhibitory substances (BLIS) production and antifungal activity was scored positive for several strains, included L. plantarum PON100148 which was selected as starter for experimental sourdough production. The characteristics of the sourdoughs and the resulting breads indicated that the best productions were obtained in presence of L. plantarum PON100148., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Very limited information exists on the exclusion of pathogens by probiotics in the gut of the host challenged with pathogens. In this study, we tested probiotic characteristics in vitro and anticolonization ability of Lactobacillus paracasei FJ861111.1 in mice infected with selected pathogenic microorganisms. The in vitro results indicated that L. paracasei FJ861111.1 had a high survival in acidic conditions at pH 2.5 and bile salt concentration at 0.3%, and strong inhibition ability against common pathogens including Shigella dysenteriae, Staphylococcus aureus, Cronobacter sakazakii, Escherichia coli, and Candida albicans. The cell adhesion assays showed that L. paracasei FJ861111.1 exhibited strong adherence to HT-29 cells and excluded the adhesion of selected food-borne pathogens to HT-29 cells. The in vivo results showed that fermented milk with L. paracasei and viili (a Nordic yogurt product) significantly improved the population of total bacteria and of Lactobacillus in the feces of mice, and significantly inhibited the colonization of C. albicans to the intestines of mice post-C. albicans infection. Thus, it appears that this strain could be used as a probiotic organism for manufacturing functional fermented milk., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Freezing is widely used for preserving different types of cells. Frozen concentrates of lactic acid bacteria (LAB) are extensively used for manufacturing food, probiotic products and for green chemistry and medical applications. However, the freezing and thawing processes cause cell injuries that result in significant cell death. Producing homogeneous bacterial populations with high cryotolerance remains a real challenge. Our objective was to investigate the biochemical and physiological changes in a LAB model at the cell scale following fermentation and freezing in order to identify cellular biomarkers of cryotolerance. Infrared spectra of individual bacteria produced by applying different fermentation and freezing conditions were acquired using synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy to achieve sub-cellular spatial resolution. Fluorescent microscopy was concomitantly assessed, thus making possible to simultaneously analyse the biochemistry and physiological state of a single cell for the first time. Principal component analysis was used to evaluate changes in cell composition, with particular focus on lipids, proteins and polysaccharides. SR-FTIR results indicated that before freezing, freeze-resistant cells grown in a rich medium presented a high content of CH3 groups from lipid chains, of cell proteins in an α-helix secondary structure and of charged polymers such as teichoic and lipoteichoic acids that constitute the Gram-positive bacterial wall. Moreover, SR-FTIR microspectroscopy made it possible to reveal cell heterogeneity within the cluster of resistant cells, which was ascribed to the diversity of potential substrates in the growth medium. Freezing and thawing processes induced losses of membrane integrity and cell viability in more than 90% of the freeze-sensitive bacterial population. These damages leading to cell death were ascribed to biochemical modification of cell membrane phospholipids, in particular a rigidification of the cytoplasmic membrane following freezing. Furthermore the freeze-resistant cells remained viable after freezing and thawing but a modification of protein secondary structure was detected by SR-FTIR analysis. These results highlighted the potential application of bimodal analysis by SR-FTIR and fluorescence microscopy to increase our knowledge about mechanisms related to cell damage.

Bacterial infections causing fish diseases and spoilage during fish food processing and storage are major concerns in aquaculture. Use of bacteriocins has recently been considered as an effective strategy for prevention of bacterial infections. A novel bacteriocin produced by Catla catla gut isolates, Lactobacillus animalis TSU4, designated as bacteriocin TSU4 was purified to homogeneity by a three-step protocol. The molecular mass of bacteriocin TSU4 was 4117 Da determined by Q-TOF LC/MS analysis. Its isoelectric point was ~9. Secondary conformation obtained by circular dichroism spectroscopy showed molecular conformation with significant proportions of the structure in α-helix (23.7 %) and β-sheets (17.1 %). N-terminal sequencing was carried out by the Edman degradation method; partial sequence identified was NH2-SMSGFSKPHD. Bacteriocin TSU4 exhibited a wide range of antimicrobial activity, pH and thermal stability. It showed a bacteriocidal mode of action against the indicator strain Aeromonas hydrophila MTCC 646. Bacteriocin TSU4 is the first reported bacteriocin produced by fish isolate Lactobacillus animalis. The characterization of bacteriocin TSU4 suggested that it is a novel bacteriocin with potential value against infections of bacteria such as A. hydrophila MTCC 646 and Pseudomonas aeruginosa MTCC 1688 and application to prevent spoilage during food preservation.

Aim: Study the effect of lactobacilli exopolysaccharides (EPS)on cytokine and phagocyte activity in vitro and in mice organism during modelling of an infectious process., Materials and Methods: Lactobacillus delbrueckii subsp. delbrueckii B-1596 (laksaran 1596), L. delbrueckii B-1936 (laksaran 1936) and L. delbrueckii ssp. bulgaricus (laksaran Z) were used in the study. EPS were administered into white mice 1 hour after the Staphylococcus aureus 209-P infection. Index of phagocyte completion and index of killing activation (IKA) were calculated during phagocyte activity study. IL-1α, TNF-α, IFN-γ and IL-4 cytokine content was determined in blood sera and macrophage supernatants., Results: Laksaran 1596, 1936 and Z had ambiguous effect on cytokine production. Laksaran: Z and 1936, 6 hours after mice infection increased IL-1 content in blood sera. Laksaran Z had the most pronounced effect on macrophages, resulting in an increase of active macrophages, facilitating increased digestion of S. aureus 209-P and IKA increase, stimulated cytokine production., Conclusion: The results obtained allow to speak about a possibility of using laksaran Z as a prophylaxis immune modulating preparation for correction of animal cytokine status.

This study investigates the effects of different doses of serotonin, its precursor 5-hydroxytry-ptophan (5HTP), and m-hydroxybenzylhydrazine inhibitor (NSD1015), administered via intraperitoneal for 5 consecutive days, on behavior and average body weight of broilers. We also measured the humoral immune response and quantification of Salmonella Enteritidis in broilers chickens that received the drugs evaluated and a Lactobacillus pool. The study was divided into 3 experiments: Experiment 1--administration of pharmaceuticals with choice of dosage; Experiment 2--administration of pharmaceuticals and a Lactobacillus pool in birds that were not challenged with S. Enteritidis, and Experiment 3--administration of pharmaceuticals and a Lactobacillus pool in birds challenged with S. Enteritidis. The ELISA was used to scan dosages of intestinal IgA and serum IgY. We used colony-forming units to quantify S. Enteritidis. The concentrations of IgA and IgY did not show significant differences (P>0.05) in Experiment 2. In Experiment 3, NSD1015 associated with Lactobacillus determined higher IgA concentrations, promoting greater stimulus to the immune system than 5HTP. Regarding quantification of S. Enteritidis in the cecal content of birds, 5HTP associated to Lactobacillus determined the smallest number of bacteria, showing possible interaction of 5-hydroxytryptophan and Lactobacillus spp. with the immune system of broiler chickens., (© 2015 Poultry Science Association Inc.)