The effects of ultraviolet, gamma- and X-ray irradiation on the growth, antibacterial activity and radio-protective of Lactobacillus rhamnosus.

Ionizing radiation is widely applied in food production as preservation technology and for correction of the gut microbiome of cancer patients, rescuers, astronauts etc. Lactic acid bacteria (LAB) can be used for the same reason. The main goal of this study was to investigate the effect of irradiation on some activities of Lactobacillus rhamnosus MDC 9661 and its effect on the survival of irradiated rats. The results indicate that both ultraviolet (during 45 min) and X-ray irradiations (with 2 Sv) decreased the CFU and the antibacterial activity of the strain. Higher than 700 Sv dose of X-ray irradiation resulted in the total inhibition of antibacterial activity with the total reduction of colony forming units less than 10 cells ml-1 , while irradiated with 1000 Sv dose L. rhamnosus MDC 9661 did not lose its proteolytic activity. It was also shown that L. rhamnosus MDC 9661 was not immunogenic in the organism of the rats and cannot lead to the development of autoimmune responses. L. rhamnosus MDC 9661 demonstrated the necessary properties for probiotics and can be effectively used for the correction of the gut microbiome of all target groups. The co-aggregation of the cells is one of the mechanisms for resistance of LAB to irradiation.

Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

Adenosine deaminase inhibition.

Adenosine deaminase is a critical enzyme in purine metabolism that regulates intra and extracellular adenosine concentrations by converting it to inosine. Adenosine is an important purine that regulates numerous physiological functions by interacting with its receptors. Adenosine and consequently adenosine deaminase can have pro or anti-inflammatory effects on tissues depending on how much time has passed from the start of the injury. In addition, an increase in adenosine deaminase activity has been reported for various diseases and the significant effect of deaminase inhibition on the clinical course of different diseases has been reported. However, the use of inhibitors is limited to only a few medical indications. Data on the increase of adenosine deaminase activity in different diseases and the impact of its inhibition in various cases have been collected and are discussed in this review. Overall, the evidence shows that many studies have been done to introduce inhibitors, however, in vivo studies have been much less than in vitro, and often have not been expanded for clinical use.

Polymyxins interaction to the human serum albumin: A thermodynamic and computational study.

Polymyxin B and E (colistin), are a group of cationic charged cyclic antibiotic lipopeptides that are frequently used in the clinics to treat infections caused by the multidrug-resistant gram-negative bacteria. Since the interactions with the blood plasma drug-transport proteins may play a critical role in determining their pharmacological and pharmacokinetic profiles, we studied the binding properties of polymyxins to the human serum albumin (HSA) under simulated physiological conditions by the combination of biophysical approaches, such as isothermal titration calorimetry (ITC), fluorescence anisotropy, circular dichroism (CD) buttressed by computational studies. The HSA binding to the polymyxins was relatively strong (Ka ≈ 1.0 × 107 M-1). Molecular docking indicated that polymyxins bind to the cleft of HSA between domains I and III via the electrostatic interactions. This evidence was further confirmed by the entropy-driven interaction for the polymyxins bound HSA. Far UV-CD experiments showed that the secondary structure of HSA doesn't alter and its stable structure is preserved. Collectively, these investigations revealed that the polymyxins bind preferentially to the partially unfolded intermediate forms of the protein structure; however, HSA molecule does not undergo any significant conformational changes upon binding. This is promising as it may limit the unfavorable side effects of the medicine. On the whole, the results provide quantitative and qualitative insight of the binding interaction between HSA and polymyxins, which is important in understanding their effect as therapeutic agents.

Brazilian artisanal ripened cheeses as sources of proteolytic lactic acid bacteria capable of reducing cow milk allergy.

AIM: The objective was to obtain lactic acid bacteria (LAB) capable of hydrolysing immunoreactive proteins in milk, to optimize the hydrolysis, to determine the proteolysis kinetics and to test the safety of the best hydrolytic strain. METHODS AND RESULTS: Brazilian cheese was used as source of LAB capable of hydrolysing main milk allergens. Proteolytic isolates were submitted to RAPD-PCR for the characterization of clonal diversity. Optimized hydrolysis was strain and protein fraction dependent. 16S rDNA sequencing identified three proteolytic strains: Enterococcus faecalis VB43, that hydrolysed αS1 -, αS2 - and ß-caseins, α-lactalbumin and ß-lactoglobulin (partial hydrolysis), and Pediococcus acidilactici VB90 and Weissella viridescens VB111, that caused partial hydrolysis of αS1 - and αS2 -caseins. Enterococcus faecalis VB43 tested negative for virulence genes asa1, agg, efaA, hyl, esp, cylLL and cylLS but positive for genes ace and gelE. Ethylenediamine tetra-acetic acid inhibited the proteolysis, indicating that the main proteases of E. faecalis VB43 are metalloproteases. CONCLUSION: Brazilian artisanal cheese is a good source of LAB capable of hydrolysing allergenic proteins in milk. One isolate (E. faecalis VB43) presented outstanding activity against these proteins and lacked most of the tested virulence genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococcus faecalis VB43 presents good potential for the manufacture of hypoallergenic dairy products.

Soymilk fermentation by Enterococcus faecalis VB43 leads to reduction in the immunoreactivity of allergenic proteins ß-conglycinin (7S) and glycinin (11S).

Food allergies represent a serious problem affecting human health and soy proteins rank among the most allergenic proteins from food origin. The proteolytic enzymes produced by lactic acid bacteria (LAB) can hydrolyse the major allergens present in soybean, reducing their immunoreactivity. Many studies have reported the ability of LAB to ferment soy-based products; while the majority of them focus on the improvement of the sensory characteristics and functionality of soy proteins, a lack of information about the role of lactic fermentation in the reduction of immunoreactivity of these proteins exists. The aim of the present study was to evaluate the capability of the proteolytic strain Enterococcus faecalis VB43 to hydrolyse the main allergenic proteins present in soymilk and to determine the immunoreactivity of the obtained hydrolysates. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results of fermented soymilk demonstrated complete hydrolysis of the ß-subunit from ß-conglycinin and the acidic polypeptide from glycinin. Reversed phase high performance liquid chromatography (RP-HPLC) analysis of the peptides released after hydrolysis revealed the appearance of new peptides and the disappearance of non-hydrolysed proteins, indicating extensive hydrolysis of the substrate. Results from competitive enzyme-linked immunosorbent assay (ELISA) tests clearly indicated a reduction in the immunoreactivity (more than one logarithmic unit) in the fermented sample as compared to the non-fermented control. Our results suggest that the soymilk fermented by E. faecalis VB43 may induce lower allergic responses in sensitive individuals. The strain E. faecalis VB43 may be considered as an excellent candidate to efficiently reduce the immunoreactivity of soymilk proteins.

Inhibition of Chaperonin GroEL by a Monomer of Ovine Prion Protein and Its Oligomeric Forms.

The possibility of inhibition of chaperonin functional activity by amyloid proteins was studied. It was found that the ovine prion protein PrP as well as its oligomeric and fibrillar forms are capable of binding with the chaperonin GroEL. Besides, GroEL was shown to promote amyloid aggregation of the monomeric and oligomeric PrP as well as PrP fibrils. The monomeric PrP was shown to inhibit the GroEL-assisted reactivation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The oligomers of PrP decelerate the GroEL-assisted reactivation of GAPDH, and PrP fibrils did not affect this process. The chaperonin GroEL is capable of interacting with GAPDH and different PrP forms simultaneously. A possible role of the inhibition of chaperonins by amyloid proteins in the misfolding of the enzymes involved in cell metabolism and in progression of neurodegenerative diseases of amyloid nature is discussed.

Antiamyloidogenic Effects of Ellagic Acid on Human Serum Albumin Fibril Formation Induced by Potassium Sorbate and Glucose.

Oxidative stress has the main role in protein conformational changes and consequent direct involvement in different kind of diseases. Potassium sorbate as a widespread industrial preservative and glucose are two important oxidants that can be involved in oxidative stress. In this study the effect of ellagic acid as a phenolic antioxidant on amyloid fibril formation of human serum albumin upon incubation of potassium sorbate and glucose was studied using thioflavin T assay, surface tension, atomic force microscopy, Amadori product, and carbonyl content assays. The thioflavin T assay and atomic force microscopy micrographs demonstrated the antiamyloidogenic effect of ellagic acid on the human serum albumin fibril formation. This antioxidant also had the repair effect on surface tension of the modified human serum albumin (amyloid intermediates), which was destructed, caused by potassium sorbate and glucose. This mechanism takes place because of potent carbonyl stress suppression effect of ellagic acid, which was strengthening by potassium sorbate in the presence and absence of glucose.

Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and ß-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

Binding assessment of two arachidonic-based synthetic derivatives of adrenalin with ß-lactoglobulin: Molecular modeling and chemometrics approach.

A computational approach to predict the main binding modes of two adrenalin derivatives, arachidonoyl adrenalin (AA-AD) and arachidonoyl noradrenalin (AA-NOR) with the ß-lactoglubuline (BLG) as a nano-milk protein carrier is presented and assessed by comparison to the UV-Vis absorption spectroscopic data using chemometric analysis. Analysis of the spectral data matrices by using the multivariate curve resolution-alternating least squares (MCR-ALS) algorithm led to the pure concentration calculation and spectral profiles resolution of the chemical constituents and the apparent equilibrium constants computation. The negative values of entropy and enthalpy changes for both compound indicated the essential role of hydrogen bonding and van der Waals interactions as main driving forces in stabilizing protein-ligand complex. Computational studies predicted that both derivatives are situated in the calyx pose and remained in that pose during the whole time of simulation with no any significant protein structural changes which pointed that the BLG could be considered as a suitable carrier for these catecholamine compounds.

Protective effect of Enterococcus faecalis DAPTO 512 on the intestinal tract and gut mucosa: milk allergy application.

The allergenicity of ß-lactoglobulin (ß-Lg) was studied by using Ussing chamber in a murine model of ß-Lg allergy supplemented with hydrolysates obtained after fermentation of milk for 48 h at 37 (°)C with Enterococcus faecalis DAPTO 512, isolated from cow milk and identified by 16S rDNA sequence analysis. Balb/c mice were sensitised intraperitoneally with ß-Lg. Three groups of mice were formed: group 1, composed of naive mice used as control received only NaCl; group 2, positive control composed of mice sensitised intraperitoneally with ß-Lg; group 3, formed by mice which were given hydrolysates of 48 h then sensitised with ß-Lg. After 48 h of fermentation ß-casein and ß-Lg were degraded by E. faecalis DAPTO 512. ß-Lg immunisation was associated with strong IgG and IgE production in case of positive controls and a significant increase in short current circuit (Isc) and high conductance (G) responses were observed. The control and the hydrolysate groups showed a significant decrease in the production of IgG and IgE anti ß-Lg compared to the positive control. The allergenic potential of ß-Lg was markedly reduced in the group that received hydrolysates (Isc and G remained unchanged after intestine challenge with ß-Lg). The histological scrutiny showed villi atrophy, lymphocyte hyperplasia and a significant chorion detachment in the positive control group. In the group administered with hydrolysates of fermented milk, inflammatory signs were lower, the villi were long and thin and lymphocytes were less dense. The results showed that feeding of milk fermented with E. faecalis DAPTO 512 during 18 days prior to ß-Lg allergy induction exerts a protecting effect on the murine intestine and induces a significant decrease in the ß-Lg allergenicity.

Purification and characterization of the bacteriocin produced by Lactobacillus sakei MBSa1 isolated from Brazilian salami.

AIMS: The study aimed at determining the biochemical characteristics of the bacteriocin produced by Lactobacillus sakei MBSa1, isolated from salami, correlating the results with the genetic features of the producer strain. METHODS AND RESULTS: Identification of strain MBSa1 was performed by 16S rDNA sequencing. The bacteriocin was tested for spectrum of activity, heat and pH stability, mechanism of action, molecular mass and amino acid sequence when purified by cation-exchange and reversed-phase HPLC. Genomic DNA was tested for bacteriocin genes commonly present in Lact. sakei. Bacteriocin MBSa1 was heat-stable, unaffected by pH 2·0 to 6·0 and active against all tested Listeria monocytogenes strains. Maximal production of bacteriocin MBSa1 (1600 AU ml(-1)) in MRS broth occurred after 20 h at 25°C. The molecular mass of produced bacteriocin was 4303·3 Da, and the molecule contained the SIIGGMISGWAASGLAG sequence, also present in sakacin A. The strain contained the sakacin A and curvacin A genes but was negative for other tested sakacin genes (sakacins T-α, T-ß, X, P, G and Q). CONCLUSIONS: In the studied conditions, Lact. sakei MBSa1 produced sakacin A, a class II bacteriocin, with anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and characterization of the bacteriocin produced by a lactic acid bacteria isolated from salami (Lact. sakei MBSa1), linking genetic and expression information. Its heat-resistance, pH stability in acid conditions (pH 2·0-6·0) and activity against L. monocytogenes food isolates bring up a potential technological application to improve food safety.

Synergic study of α-glucosidase inhibitory action of aloin and its antioxidant activity with and without camel ß-casein and its peptides.

Regular consumption of natural antioxidants reduces the risk of developing diseases. Aloin is one of the main active phenolic components of Aloe vera. The main disadvantage of aloin is its concentration limit of use that causes cell damage. One of the aims of this study was to investigate the antioxidant activity of aloin in the presence and absence of camel ß-casein ( ß-CN) and its peptide fractions. The mixture of aloin, ß-CN and peptides showed a very high antioxidant activity in a synergistic manner as compared to each component alone. The alpha ( α)-glucosidase inhibitory activity of aloin was also investigated in the presence and absence of ß-CN and its peptides. Aloin alone is a potent inhibitor of α-glucosidase. The α-glucosidase inhibitory activity of aloin is reduced in the presence of ß-CN or its peptides. The combination of aloin and ß-CN or its peptides makes a high antioxidant functional ingredient.

Antifungal properties of durancins isolated from Enterococcus durans A5-11 and of its synthetic fragments.

The aim of this work was to study the antifungal properties of durancins isolated from Enterococcus durans A5-11 and of their chemically synthesized fragments. Enterococcus durans A5-11 is a lactic acid bacteria strain isolated from traditional Mongolian airag cheese. This strain inhibits the growth of several fungi including Fusarium culmorum, Penicillium roqueforti and Debaryomyces hansenii. It produces two bacteriocins: durancin A5-11a and durancin A5-11b, which have similar antimicrobial properties. The whole durancins A5-11a and A5-11b, as well as their N- and C-terminal fragments were synthesized, and their antifungal properties were studied. C-terminal fragments of both durancins showed stronger antifungal activities than other tested peptides. Treatment of D. hansenii LMSA2.11.003 strain with 2 mmol l(-1) of the synthetic peptides led to the loss of the membrane integrity and to several changes in the ultra-structure of the yeast cells. Chemically synthesized durancins and their synthetic fragments showed different antimicrobial properties from each other. N-terminal peptides show activities against both bacterial and fungal strains tested. C-terminal peptides have specific activities against tested fungal strain and do not show antibacterial activity. However, the C-terminal fragment enhances the activity of the N-terminal fragment in the whole bacteriocins against bacteria.

[Self-association and secondary structure of beta-casein].

The secondary structure alterations, accompanying isothermal and temperature guided beta-casein micellization have been studied by dynamic light scattering, circular dichroism and Fourier transform infrared spectroscopy techniques. Micelle formation induced by increase of protein concentration at constant temperature is accompanied by the formation of scanty number of additional peptide hydrogen bonds, preliminary assigned to intraprotein beta-structure. Heating results in more pronounced but qualitatively different changes consisted in dehydration of peptide groups and disruption of polyproline II helix segments with subsequent conversion to random and beta-turns. Nevertheless, in both cases the total number of residues involved in transition is quite few and cannot be regarded as a decisive factor for casein micellization.

[Chaperone-like activity of beta-casein and thermal stability of alcohol dehydrogenase].

To elucidate the correlation of structural peculiarities of beta-casein and their chaperon-like activity the modified forms of the protein (with cysteinyl residues introduced in polypeptide chain) were investigated. The aggregation of native and recombinant beta-caseins was studied as well as their chaperon-like activity towards alcohol dehydrogenase thermal aggregation. It was shown that physico-chemical and chaperone-like properties ofdimeric and oligomeric forms ofbeta-casein (which formation is due to intermolecular disulfide bonds) differ significantly from monomeric forms. It was found that thermal stability of alcohol dehydrogenase depends on beta-casein concentration.

Characterization of two safe Enterococcus strains producing enterocins isolated from Egyptian dairy products.

Five bacterial cocci isolates were selected from a wide pool of 503 isolates collected from traditional Egyptian dairy products on the basis of their inhibitory activities against Lactobacillus brevis F145, Lactobacillus bulgaricus 340, Enterococcus faecium HKLHS, Listeria ivanovii ATCC, Listeria innocua CIP 80.11 and Listeria monocytogenes EGDe 107776. These 5 isolates were identified as E. faecium TX1330 and E. faecium E980 by 16S rDNA amplification and sequencing. The antibacterial activity of the two strains was not affected by treatment of the cell free culture supernatant with catalase but their activities disappeared completely when digested with protease K, α-chymotrypsin and trypsin. The antimicrobial substance was stable over a wide range of pH (2-10) and was active after heating at 100 °C for 10 min. Bacteriocin yield in two strains reached a maximum (1,600 AU/ml) at the end of the exponential phase (6 h) and remained stable until the end of 24 h-incubation period when the medium reached pH 5.5. Maximal production of bacteriocin was obtained when growing the bacterial cells at temperatures ranging between 30 and 37 °C. Bacteriocin production was unaffected when the bacterial cells grew under severe conditions of pH (9.6) and in high salt (6.5% NaCl). Thanks to PCR gene amplification the bacteriocins produced by E. faecium TX1330 could be identified as enterocins A and B structural genes, while the bacteriocins produced by E. faecium E980 could be identified as enterocins P and L50A structural genes, which can be classified into two enterocin subclasses (IIa and IIc), respectively. PCR amplification demonstrated that the two studied strains did not contain virulence factors asal, cyl A and B, ace, efaAfs and espfm. These two strains were sensitive to most of the tested antibiotics but were resistant to tetracycline. E. faecium E980 was also resistant to chloramphenicol.

A newly discovered bacteriocin produced by Enterococcus faecalis 3915.

Different Enterococci, isolated from starters for the production of the traditional Bulgarian yellow cheese 'Kashkaval' were screened for bacteriocin production, and one of them, Enterococcus faecalis 3915 demonstrated bacteriocin activity. In this study, we investigated the growth parameters of the producer strain as well as the production kinetics and preliminary characterisation of the produced bacteriocin named enterocin 3915. For the growth modelling, the logistic model was used, while bacteriocin production was monitored. Experiments on inducibility were conducted, and strain was checked for the presence of plasmids. The peptide was crudely purified by ammonium sulphate precipitation followed by preparative PAGE. The approximate molecular mass was determined electrophoretically, and the activity was visualised by electrophoresis and agar overlay technique. It was found that E. faecalis 3915 produces a bacteriocin with constitutive synthesis and chromosomal localisation of its genetic determinants. The peptide revealed to be relatively heat-stable with a molecular mass of about 6.5 kDa. As E. faecalis 3915 originates from cheese starter it can be classified as generally recognised as safe (GRAS). The inhibitory activity of enterocin 3915 comprises commensals or pathogens, so properties generally accepted as probiotic could be attributed to the producer, and potential application in the dairy industry is not to be excluded.

[Keratinase of an anaerobic thermophilic bacterium Thermoanaerobacter sp. strain 1004-09 isolated from a hot spring in the Baikal Rift zone].

A thermophilic anaerobic bacterial strain 1004-09 belonging to the genus Thermoanaerobacter and capable of growth on protein substrates such as albumin, gelatin, casein, and alpha and beta-keratins was isolated from the Urinskii hot spring (Barguzin river valley, Republic of Buryatia, Russia). A 150-kDa serine proteinase was revealed in the strain supernatant; it exhibited optimal activity at 60 degrees C and pH 9.3 and was capable of keratin hydrolysis. A number of characteristics for the strain 1004-09 keratinase were established including activation by SDS and NaCl and residual activity (15% to the activity of the intact protein) in the presence of 10% ethanol and acetone.