Lactobacillus fermentum: a bacterial species with potential for food preservation and biomedical applications.

Lactic acid-producing bacteria are the most commonly used probiotics that play an important role in protecting the host against harmful microorganisms, strengthening the host immune system, improving feed digestibility, and reducing metabolic disorders. Lactobacillus fermentum (Lb. fermentum) is a Gram-positive bacterium belonging to Lactobacillus genus, and many reportedly to enhance the immunologic response as well as prevent community-acquired gastrointestinal and upper respiratory infections. Additionally, Lb. fermentum strains produce diverse and potent antimicrobial peptides, which can be applied as food preservative agents or as alternatives to antibiotics. Further functions attributed to probiotic Lb. fermentum strains are their abilities to decrease the level of blood stream cholesterol (as cholesterol-lowering agents) and to potentially help prevent alcoholic liver disease and colorectal cancer among humans. Finally, Lb. fermentum is a key microorganism in sourdough technology, contributing to flavor, texture, or health-promoting dough ingredients, and has recently been used to develop new foods stuffs such as fortified and functional foods with beneficial attributes for human health. Development of such new foodstuffs are currently taking important proportions of the food industry market. Furthermore, an increasing awareness of the consumers prompts the food-makers to implement alternative environmental friendly solutions in the production processes and/or suitable biological alternative to limit the use of antibiotics in feed and food. Here, we give an account on the application of Lb. fermentum strains in the biomedical and food preservation fields, with a focus on probiotic features such as bacteriocin production. We also summarize the use of Lb. fermentum as cell factories with the aim to improve the efficacy and health value of functional food.

Synergistic effect between colistin and bacteriocins in controlling Gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells.

Pathogens resistant to most conventional antibiotics are a harbinger of the need to discover novel antimicrobials and anti-infective agents and develop innovative strategies to combat them. The aim of this study was to assess the in vitro activity of colistin alone or in combination with two bacteriocins, nisin A and pediocin PA-1/AcH, against Salmonella choleraesuis ATCC 14028, Pseudomonas aeruginosa ATCC 27853, Yersinia enterocolitica ATCC 9610, and Escherichia coli ATCC 35150 (O157:H7). The strain most sensitive to colistin was enterohemorrhagic E. coli O157:H7, which was inhibited at a concentration of about 0.12 µg/ml. When nisin A (1.70 µg/ml) or pediocin PA-1/AcH (1.56 µg/ml) was combined with colistin, the concentrations required to inhibit E. coli O157:H7 were 0.01 and 0.03 µg/ml, respectively. The in vitro antigenotoxic effect of colistin was determined by using the comet assay method to measure the level of DNA damage in freshly isolated human peripheral blood leukocytes (PBLs) incubated with colistin for 1 h at 37°C. Changes in the tail extents of PBLs of about 69.29 ± 0.08 µm were observed at a final colistin concentration of about 550 ng/ml. Besides the synergistic effect, the combination of colistin (1 mg/ml) and nisin (2 mg/ml) permitted us to re-evaluate the toxic effect of colistin on Vero (monkey kidney epithelial) cells.

Required characteristics of Paenibacillus polymyxa JB-0501 as potential probiotic.

The ability of Paenibacillus polymyxa to inhibit the growth of Escherichia coli generic ATCC 25922 (Escherichia coli ATCC 25922) and to adhere to monolayers of the enterocyte-like human cell line Caco-2 was evaluated. P. polymyxa JB-0501 (P. polymyxa JB-0501), found in a livestock feed probiotic supplement, was compared to P. polymyxa reference strain ATCC 43685 and ATCC 7070 (P. polymyxa ATCC) in terms of carbohydrate utilization and resistance to lysozyme, acid, bile salts, and hydrogen peroxide. JB-0501 grew at pH 4.5 and at H2O2 concentrations less than 7.3 µg/ml and presented a higher affinity to hexadecane and decane. Bile salts at 0.2 % inhibited the growth of all three strains. P. polymyxa JB-0501 and P. polymyxa ATCC 43865 adhered to Caco-2 cell monolayers. The percentage of cells that adhered ranged from about 0.35 to 6.5 % and was partially proportional to the number applied. Contact time (from 15 min to 1 h) had little impact on adhesion. P. polymyxa JB-0501 inhibited the growth of E. coli ATCC 25922, as proven by the diffusion tests in agar. Taken together, these results suggested that P. polymyxa JB-0501 has the potential probiotic properties to justify its consideration as a livestock feed supplement.

Incorporation of omega-3 polyunsaturated fatty acids into soybean lecithin: effect of amines and divalent cations on transesterification by lipases.

The transesterification of soybean lecithin with methyl esters of EPA and DHA in an organic solvent (hexane) using various commercially available lipases was studied. Lipases produced by Candida antarctica, Pseudomonas fluorescens, Burkholderia cepacia, Mucor miehei, Thermomyces lanuginosus and Rhizomucor miehei were compared, in the absence or presence of histidine, arginine, urea, Ca²âº, Mg²âº, or a combination of urea and divalent cations (additives at 5 % of the total lipid mass). Transesterification using the R. miehei enzyme reached 11.32 and 12.30 % in the presence of Ca²âº or Mg²âº respectively, and 8.58 and 9.31 % when urea was also added. These were the greatest degrees of transesterification obtained. The results suggest the potential use of this immobilized lipase as a catalyst for interesterification reactions in organic solvent systems with low water content.

Potential Probiotic Lactic Acid Bacteria with Anti-Penicillium expansum Activity from Different Species of Tunisian Edible Snails.

This study aimed to isolate lactic acid bacteria (LAB) from the digestive tract, meat and slime of edible snails (Helix lucorum, Helix aspersa and Eobania vermiculata) and investigate their antagonistic activity against Penicillium expansum. They were then characterized for their probiotic potential. Among 900 bacterial isolates, 47 LAB exhibiting anti-P. expansum activity were identified through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as Levilactobacillus brevis (25), Lactococcus lactis (3), Enterococcus faecium (12), Enterococcus faecalis (4), Enterococcus casseliflavus (1), and Enterococcus mundtii (2). Sixty-two percent of the strains were tolerant to 100 mg/L of lysozyme. Seventy two percent of the isolates were able to survive at pH 3 and most of them tolerate 2.5% bile salt concentration. Moreover, 23% of the strains displayed bile salt hydrolase activity. Interestingly, all strains were biofilm strong producers. However, their auto- and co-aggregation properties were time and pH dependent with high aggregative potentiality at pH 4.5 after 24 h. Remarkably, 48.94% of the strains showed high affinity to chloroform. The safety assessment revealed that the 47 LAB had no hemolytic activity and 64% of them lacked mucin degradation activity. All isolated strains were susceptible to gentamycin, streptomycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Overall, 43 LAB strains showed inhibitory activity against a broad spectrum of pathogenic Gram-positive and gram-negative bacteria, fungi, and yeast. Our findings suggest that L. brevis (EVM12 and EVM14) and Ent. faecium HAS34 strains could be potential candidates for probiotics with interesting antibacterial and anti-P. expansum activities.

Colistin A and colistin B among inhibitory substances of Paenibacillus polymyxa JB05-01-1.

Recently, we isolated and reported the antagonism of Paenibacillus polymyxa JB05-01-1 (P. polymyxa JB05-01-1) against Gram-negative bacteria. Here, we provide more insights and attribute the abovementioned antagonism to the production of colistins A and B, which were purified by Amberlite column exchange, C18 column hydrophobicity, superdex 75 16/60 gel filtration chromatography connected to fast protein liquid chromatography and identified by MALDI TOF/TOF, and manual nanospray analysis. The amount of colistin A and colistin B recovered from 500 ml of culture supernatant was about 0.05 mg. The specific activity and the average recovery of the eluted substances were 5,120 AU/mg and 1.1%, respectively. The minimal inhibitory concentrations of the purified colistins against Escherichia coli O157:H7 and Pseudomonas fluorescens LRC R73 were 0.13 and 0.62 µg/ml, respectively.

Paenibacillus polymyxa JB05-01-1 and its perspectives for food conservation and medical applications.

The aim of this study was to isolate a novel bacterial strain with strong and broad spectrum antibacterial activity from a livestock feed prebiotic supplement. A novel strain, termed Paenibacillus polymyxa JB05-01-1, was isolated using traditional microbiological methods and identified on the basis of its phenotypic and biochemical properties as well as its 16S rRNA gene sequence. This strain was able to inhibit growth of gram-negative bacteria including Escherichia coli RR1, Pseudomonas fluorescens R73, Pantoea agglomerans BC1, Butyrivibrio fibrisolvens OR85, and Fibrobacter succinogenes. The above antagonism against the aforementioned bacteria was attributed to production of an antimicrobial substance(s) termed "JB05-01-1." Its production was optimal during the stationary phase. JB05-01-1 has a molecular weight of 2.5 KDa, its mode of action is bactericidal, and the divalent cations, Ca(2+) and Mn(2+), reduced its lethality. The antibacterial activity was heat-stable and was effective at a pH range of 2-9. Enzymes like trypsin, α-chymotrypsin, and proteinase K have abolished the antibacterial activity of JB05-01-1 indicating a proteinaceous motif. This type of naturally occurring bacteria and inhibitory substance(s) could represent an additional value in livestock feed supplements. The natural presence of antibacterial activity indicates an opportunity to decrease the addition of antibiotics.

SciDBMaker: new software for computer-aided design of specialized biological databases.

BACKGROUND: The exponential growth of research in molecular biology has brought concomitant proliferation of databases for stocking its findings. A variety of protein sequence databases exist. While all of these strive for completeness, the range of user interests is often beyond their scope. Large databases covering a broad range of domains tend to offer less detailed information than smaller, more specialized resources, often creating a need to combine data from many sources in order to obtain a complete picture. Scientific researchers are continually developing new specific databases to enhance their understanding of biological processes. DESCRIPTION: In this article, we present the implementation of a new tool for protein data analysis. With its easy-to-use user interface, this software provides the opportunity to build more specialized protein databases from a universal protein sequence database such as Swiss-Prot. A family of proteins known as bacteriocins is analyzed as 'proof of concept'. CONCLUSION: SciDBMaker is stand-alone software that allows the extraction of protein data from the Swiss-Prot database, sequence analysis comprising physicochemical profile calculations, homologous sequences search, multiple sequence alignments and the building of new and more specialized databases. It compiles information with relative ease, updates and compares various data relevant to a given protein family and could solve the problem of dispersed biological search results.

Purification of pediocin PA-1 by immunoaffinity chromatography.

A one-step purification procedure for purifying pediocin PA-1, a class IIa bacteriocin produced by Pediococcus acidilactici UL5, was developed based on column immunoaffinity chromatography with specific antipediocin PA-1 polyclonal antibodies coupled to cyanogen bromide-activated SepharoseTM. About 13.3 microg/mL purified pediocin PA-1 was obtained from 15 mL P. acidilactici UL5 culture supernatant, as measured by enzyme-linked immunosorbent assay. The specific activity and average recovery of the eluted pediocin PA-1 were about 6602 AU/mg and 53.3%, respectively. This is the first report of successful purification of pediocin PA-1 by immunoaffinity using pediocin PA-1-specific polyclonal antibodies.

Bacteriocins: Not Only Antibacterial Agents.

This commentary was aimed at shedding light on the multifunction of bacteriocins mainly those produced by lactic acid bacteria. These antibacterial agents were first used to improve food safety and quality. With the increasing antibiotic resistance concern worldwide, they have been considered as viable agents to replace or potentiate the fading abilities of conventional antibiotics to control human pathogens. Bacteriocins were also shown to have potential as antiviral agents, plant protection agents, and anticancer agents. Bacteriocins were reported to be involved in shaping bacterial communities through inter- and intra-specific interactions, conferring therefore to producing strains a probiotic added value. Furthermore, bacteriocins recently were shown as molecules with a fundamental impact on the resilience and virulence of some pathogens.

Incorportation of Ethyl Esters of EPA and DHA in Soybean Lecithin Using Rhizomucor miehei Lipase: Effect of Additives and Solvent-Free Conditions.

The transesterification of soybean lecithin with ethyl esters of polyunsaturated fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) using immobilized lipase from Rhizomucor miehei was tested in the presence or absence of organic solvent (hexane) and additives (urea with Ca(2+) or Mg(2+)). The reaction was carried out at a water concentration of 4 % and ethyl ester to phospholipid mass ratio of 3:1. After 24 h of reaction without solvent or additive, fatty acid incorporation reached 29.1 % and thereafter increased only slightly. After 48 h, incorporation was highest in the presence of Mg(2+), urea, and solvent. After 72 h, it was highest with Mg(2+) and urea in the presence or absence of solvent (56.8 and 45.7 %, respectively). Incorporation of EPA and DHA was thus initially fast without solvent and additive, but was increased after prolonged reaction in the presence of Mg(2+) and urea with or without solvent. These results are innovative and promising since they show that immobilized Rhizomucor miehei lipase has potential as a biocatalyst for interesterification reactions without solvent.

Antibiotic and antimicrobial peptide combinations: synergistic inhibition of Pseudomonas fluorescens and antibiotic-resistant variants.

Variants resistant to penicillin G (RvP), streptomycin (RvS), lincomycin (RvL) and rifampicin (RvR) were developed from a colistin-sensitive isolate of Pseudomonas fluorescens LRC-R73 (P. fluorescens). Cell fatty acid composition, K(+) efflux and sensitivity to antimicrobial peptides (nisin Z, pediocin PA-1/AcH and colistin) alone or combined with antibiotics were determined. P. fluorescens was highly sensitive to kanamycin, tetracycline and chloramphenicol at minimal inhibitory concentrations of 0.366, 0.305 and 0.732 µg/ml respectively. P. fluorescens, RvP, RvS, RvL and RvR were resistant to nisin Z and pediocin PA-1/AcH at concentrations ≥100 µg/ml but sensitive to colistin at 0.076, 0.043, 0.344, 0.344 and 0.258 µg/ml respectively. A synergistic inhibitory effect (FICI ≤0.5) was observed when resistant variants were treated with peptide/antibiotic combinations. No significant effect on K(+) efflux from the resistant variants in the presence of antibiotics or peptides alone or combined was observed. The proportion of C16:0 was significantly higher in antibiotic-resistant variants than in the parent strain, accounting for 32.3%, 46.49%, 43.3%, 40.1% and 44.1% of the total fatty acids in P. fluorescens, RvP, RvS, RvL and RvR respectively. Combination of antibiotics with antimicrobial peptides could allow reduced use of antibiotics in medical applications and could help slow the emergence of bacteria resistant to antibiotics.

Antibacterial activity of class I and IIa bacteriocins combined with polymyxin E against resistant variants of Listeria monocytogenes and Escherichia coli.

Development of resistance could render antimicrobial peptides ineffective as bio-preservatives in food. Variants resistant to nisin A or pediocin PA-1 were developed from Listeria monocytogenes and a variant resistant to polymyxin E was developed from Escherichia coli RR1. Inhibition of these organisms by these agents alone or in combination was assessed using critical dilution microassay and optical density measurement. The combination of pediocin or nisin with polymyxin E was synergistic against all five strains. The polymyxin/nisin combination at 9.3/32, 4.7/62.5 and 0.6/15.6 µg/ml inhibited growth of nisin-resistant L. monocytogenes, pediocin-resistant L. monocytogenes and polymyxin-resistant E. coli, respectively, by factors of 94%, 97% and 74% compared to controls. The pediocin/nisin combination was effective against L. monocytogenes and its variants, but not E. coli or its variant. Polymyxin (0.21 µg/ml) and polymyxin/nisin (0.3/7.8 µg/ml) reduced E. coli growth measured in the log phase by, respectively, 31.25% and 93.54%. L. monocytogenes growth in the logarithmic and stationary phases was reduced, respectively, by 90.46% and 77.52% by polymyxin/pediocin at 4.7/25 µg/ml. Our results suggest that the effective concentration of bacteriocin for control of resistant L. monocytogenes and E. coli variants could be lowered considerably by combination with polymyxin E. This suggests searching for polymyxin-like compounds to increase the efficiency of bacteriocins and slow the emergence of resistant mutants. This could be an important step towards the expanded use of bacteriocins in the medical arena.

Nisin A and Polymyxin B as Synergistic Inhibitors of Gram-positive and Gram-negative Bacteria.

Nisin A and polymyxin B were tested alone and in combination in order to test their antagonism against Listeria innocua HPB13 and Escherichia coli RR1, respectively. While the combination of both antibacterial substances was synergistically active against both target bacteria, nisin A alone did not show any inhibition of E. coli RR1. The nisin A/polymyxin B combination at 1.56/2.5 µg ml(-1) caused lysis of about 35.86 ± 0.35 and 73.36 ± 0.14% of L. innocua HPB13 cells after 3 and 18 h, respectively. Polymyxin B at 0.12 µg ml(-1) and nisin A/polymyxin B at 4.64/0.12 µg ml(-1) decreased the numbers of viable E. coli RR1 cells by about 0.23 and 0.65 log10 CFU ml(-1), respectively, compared to the control. Our data suggest that the concentration of nisin A required for the effective control of pathogenic strains Listeria spp. could be lowered considerably by combination with polymyxin B. The use of lower concentrations of nisin A or polymyxin B should slow the emergence of bacterial populations resistant to these agents.

Pediocin PA-1 production during repeated-cycle batch culture of immobilized Pediococcus acidilactici UL5 cells.

Pediocin PA-1 production by Pediococcus acidilactici UL5 cells immobilized in kappa-carrageenan/locust bean gum gel beads was studied during repeated-cycle batch (RCB) culture with pH control in Man Rogosa and Sharpe (MRS) broth supplemented with 1% glucose and whey permeate (SWP) medium. The pediocin PA-1 production by free P. acidilactici cells pH-controlled batch culture has reached 2048 and 4096 AU ml(-1) after 11 and 12 h of incubation, with volumetric productivities of 187 and 342 AU ml(-1) h(-1) in SWP and MRS media, respectively. In RCB culture, immobilized cells reached a maximum concentration of 7.3+/-0.2 x 10(10) and 4.3+/-0.9 x 10(10) cfu g(-1) of beads in MRS and SWP media, respectively. The maximum pediocin PA-1 activity obtained during RCB fermentation was 4096 AU ml(-1); it was attained after only 0.75 and 2 h of incubation in MRS and SWP media, respectively. The corresponding volumetric productivities were 5461 and 2048 AU ml(-1) h(-1). Pediocin PA-1 production in the RCB culture was highly stable over 12 fermentation cycles carried out over 3 d in SWP media.

Effect of antimicrobial peptides divergicin M35 and nisin A on Listeria monocytogenes LSD530 potassium channels.

The aim of this work was to study the effect of antimicrobial peptides: divergicin M35 and nisin A on Listeria monocytogenes LSD 530 potassium (K+) channels: ATP-sensitive (K ATP), calcium-activated (BK Ca), and depolarization-activated (Kv) types. Increase on K+ efflux and inhibition of cellular growth were observed after adding K+ channel activators pinacidil, NS1619, and cromakalim to divergicin M35. Increase in K+ efflux from log-phase cells was about 18 +/- 1.1, 11 +/- 0.63, and nmol mg(-1) of cell dry weight (CDW) for pinacidil and NS1619, respectively, over the efflux obtained with divergicin M35 alone. Increases in K+ efflux obtained by adding the same K+ channel activators to nisin A fit a completely different profile. Divergicin M35 activates K+ channels, particularly of the Kv and BK Ca types and to a lesser extent the K ATP type, causing K+ efflux and consequently cell death.

Class I/Class IIa bacteriocin cross-resistance phenomenon in Listeria monocytogenes.

Variants resistant to nisin A (vA), nisin Z (vZ), pediocin PA-1 (vP), divergicin M35 (vD) and to bacteriocin-like compounds produced by Bifidobacterium thermophilum subsp. infantis RBL67 (vB) were developed from Listeria monocytogenes LSD530. Lactic acid production, specific growth rate, potassium ion efflux, susceptibility to 13 antibiotics, cell-envelope fatty acid composition and bacteriocin cross-resistance were evaluated. Lactic acid production decreased to 75% or less of that by strain LSD530 for vP, vD and vB and to 20% or less for vA and vZ. Specific growth rates also decreased for all five variants. Acquired resistance to nisin A or Z increased resistance to pediocin and divergicin while vD showed increased resistance to nisin Z but decreased resistance to nisin A and vP exhibited increased resistance to nisin Z, pediocin and divergicin but decreased resistance to nisin A. Acquired bacteriocin resistance generally decreased antibiotic sensitivity, particularly to ampicillin, chloramphenicol, erythromycin and tetracycline. Palmitic acid (C(16:0)) in the cell wall fraction of all variants was significantly higher than in strain LSD530, accounting for 18%, 43%, 32%, 26%, 53% and 44% of the total fatty acids for LSD530, vP, vD, vB, vA, and vZ, respectively. The relationship between the acquisition of bacteriocin resistance, cross-resistance and pathogenicity of Listeria monocytogenes should be studied.

Evidence on correlation between number of disulfide bridge and toxicity of class IIa bacteriocins.

The aim of this work was to study in the same microbiological conditions and for the first time the inhibitory action of eight class IIa bacteriocins against 23 Gram-positive bacteria in which some of them are industrially used as starters and against the eight class IIa bacteriocins producing strains themselves. The sensitivity of each strain was determined after MPA using the mathematical model described by Cabo et al. (1999, J. Appl. Microbiol. 87, 907-914). Therefore, the principal component analysis realized led to a strong correlation between spectrum and extra-disulfide bridge and a minor correlation between spectrum and isoelectric point of each bacteriocin.