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In this work, we tried to investigate the impact of treatment with instant controlled pressure drop technology (DIC) on the allergenicity of milk proteins. In DIC treatment, milk proteins are exposed to a high-pressure saturated steam (0.4 MPa, 144 °C) during 25 s and ended with an abrupt pressure drop towards a vacuum (5 kPa, 32 °C). Immunoglobulin E (IgE)-binding ability is determined with indirect enzyme linked immuno spectrophotometric analysis and western blot test. Results indicate significant changes in the IgE binding of treated proteins with DIC. Treated caseins showed increase in the IgE-binding ability; this indicates that DIC enhance the antigenicity of treated caseins. In the opposite, treated whey proteins (ß-lactoglobulin and α-lactalbumin) showed decrease in the IgE binding of these proteins. DIC treatment at 0.4 MPa during 25 s causes a greater reduction of the allergenicity of whey proteins than treatment with 0.6 MPa for 25 s. [ABSTRACT FROM AUTHOR]

Abstract: Vitamin A deficiency is one of the major causes of mortality and morbidity in the developing World. This deficiency can be prevented by alimentary or pharmaceutical supplementation. However, both vitamin A oxidation and isomerization should be prevented, as these phenomenons result in loss of nutritional efficacy. The aim of this study was to investigate the effect of a food protein matrix, β-lactoglobulin (β-Lg) aggregates produced by high pressure (HP), on the stabilization of β-carotene during storage and gastro-duodenal digestion and therefore on its bioavailability. In vitro gastro-duodenal digestion of β-Lg aggregates entrapping β-carotene showed that up to 12% and 33% of total β-carotene was released after peptic and pancreatic digestion, respectively. Overall, our study showed that β-Lg aggregates are efficient for caging and stabilization of β-carotene during storage and digestion. Hence, it may be an interesting approach for the protection and the delivery of vitamin A. [Copyright &y& Elsevier]

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5–6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65–80 °C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the β-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in β-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein. [Copyright &y& Elsevier]

Examines binding and conformational properties of porcine odorant binding proteins (OBP). Existence of weak interactions of capsaicin with OBP; Hydration state of the protein binding sites and of ligand; Effect of pressure on the quaternary structure of oligomeric proteins; Occurrence of dissociation during protein denaturation.

The milk protein β-lactoglobulin has been widely studied since its discovery, both as a purified protein and in mixtures with other milk proteins, where its effect on the processing properties is of importance to the dairy industry. The protein can bind a variety of small hydrophobic molecules, which may allow its use as an oral delivery vehicle. In the present study we have examined the binding of odd-numbered fatty acids by isothermal calorimetry (ITC), X-ray crystallography and computer modelling to provide a clearer picture of the extent and variability of the central binding pocket. The Kd values for the fatty acids C13, C15, C16, C17 and C19 as determined by ITC are 1.93, 2.91, 3.05, 4.11 and 8.67 × 10 −7  M, respectively. The molecular structures revealed the ligands bound in the central cavity with generally well ordered lipophilic tails but significant positional variation at the carboxyl group end. In silico docking analyses identified the lipophilic interactions within the central cavity as the main driving force for binding with electrostatic interactions and H-bonds playing a minor role. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) have been used by mankind from immemorial times due to their technological properties and ability to improve sensorial properties of foods. Some LAB strains are also able to hydrolyze milk proteins, which increase their digestibility and contribute to the production of desirable flavors. Moreover, proteolytic activity of LAB on milk proteins may release bioactive peptides with different activities such as antihypertensive, antioxidant, antimicrobial, immunomodulatory and mineral-binding activities. This study aimed to evaluate the proteolytic activity of Enterococcus faecalis FT132 and Lactobacillus paracasei FT700, isolated from Brazilian dairy products. The proteolytic activity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high-performance liquid chromatography using milk proteins as substrates in different conditions. Both E. faecalis FT132 and L. paracasei FT700 were proteolytic at pH 6.5 in the range of 37-42 °C, and these activities were due to metalloproteases. L. paracasei FT700 could then be used as adjunct culture in fermented dairy products in order to increase their digestibility. On the contrary, E. faecalis FT132, which harbors three virulence genes asa1, ace and gelE and which is resistant to erythromycin and tetracycline, cannot be added to food products. However, the peptides produced after hydrolytic activity on milk proteins by both strains might be used as ingredients in purified formulas. [ABSTRACT FROM AUTHOR]

ABSTRACT Proteolytic activity of Lactobacillus helveticus A75 and effects of caseins hydrolysis by this strain on their immuno-reactivity were studied. Proteases of L. helveticus A75 hydrolyzed both αS1- and β-caseins and were inhibited by serine- and metallo-proteases inhibitors. Polymerase chain reaction amplification revealed the presence of prtH gene, which was 99% identical to prtH gene of L. helveticus CNRZ 32. Effect of proteolysis of αS1- and β-caseins by L. helveticus A75 on their immunoglobulin E (IgE) binding ability was studied by an enzyme-linked immunosorbent assay with the pool of eight sera from cow milk allergy patients. The IgE binding ability of hydrolyzed proteins was lower than that of intact proteins, which indicates that hydrolysis of αS1- and β-caseins by proteases of L. helveticus A75 decreases their recognition by specific IgE. Half maximal inhibitory concentration values were 2.01 and 2.28 µg/mL for αS1- and β-casein, respectively, and increased until 9.08 and 13.65 µg/mL, respectively, after their hydrolysis by L. helveticus A75. PRACTICAL APPLICATIONS The obtained results yield new information about proteolytic activity and potential application of lactobacilli isolated from traditional Azerbaijani cheeses. Isolated strain could represent new adjunct cultures for the dairy industry. [ABSTRACT FROM AUTHOR]

Abstract: The aims of this study were to characterize bacteriocin activity, some functional and probiotic properties, and to evaluate the safety of Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. The studied strain inhibited the growth of selected tested LAB, Listeria monocytogenes and Bacillus cereus strains. PCR amplification with specific primers indicated that E. faecium AQ71 carry genes encoding enterocins P, L50A, L50B and A. Bacteriocin(s) produced by the studied strain was/were heat stable and active in a broad pH range. Triton X-20, Triton X-80, Triton X-100, β-mercaptoethanol, Na-EDTA, SDS and NaCl did not affect the antimicrobial activity of the strain. The cell free supernatants of the strain caused the lysis of cells of Lactobacillus brevis F145 and inhibited the growth of L. monocytogenes. E. faecium AQ71 was negative for the tested virulence factors and did not present multi-resistance to antibiotics. The strain was resistant to physiological concentrations of bile salts and showed good auto-aggregation ability as well as co-aggregation ability with L. monocytogenes. E. faecium AQ71 exhibited good esterase, esterase lipase, acid phosphatase and aminopeptidase activities. [Copyright &y& Elsevier]

Abstract: A collection of cocci isolates (293) obtained from traditional Egyptian dairy products collected from four Egyptian regions yielded 151 lactic acid bacteria (LAB) cocci isolates. Among them, 24 isolates were characterised as highly proteolytic. SDS–PAGE showed that 6 isolates were the most proteolytically active, which were classified into Enterococcus faecalis HH22 (4 isolates) and Enterococcus faecium DO623 (2 isolates). The proteolytic activity of E. faecalis was higher than that of E. faecium (particularly on β-casein). The maximal degradation of milk proteins was achieved at pH 6.5–7.2 (E. faecalis) or pH 6.5 (E. faecium) and at 42°C for both strains. The proteolytic activities of the two strains were inhibited mostly by the presence of EDTA, showing that their proteases belong mainly to metalloproteases. A slight inhibition of proteolysis by PMSF in the case of E. faecalis HH22 suggests a limited inclusion of serine proteases in its protease system. [Copyright &y& Elsevier]

More than 200 isolates were obtained from 15 Egyptian traditional dairy products (Domiatti cheese, Ras cheese and Rayeb milk) collected from local markets of Alexandria, Tanta and Kafr El-Sheikh. Examination with optical microscope of these dairy samples allowed to classify 92 bacilli, 64 of which were identified as lactobacilli. The proteolytic activity of lactobacilli isolates was tested on skim milk agar. Eight isolates showing a high proteolytic activity were further tested on UHT skim milk. The strain showing the highest proteolytic activity was purified and identified as Lactobacillus fermentum IFO 3656. The specific proteolytic activity of this strain and the factors affecting it (pH, temperature and presence of inhibitors) were studied. The proteolysis targeted mainly caseins (73% of whole casein), especially β-casein (85%). Smaller portions of whey proteins were proteolyzed (20%) essentially β-lactoglobulin. The proteolysis process gave rise to medium-sized peptide populations. The optimum conditions for the proteolysis activity of the studied strains were pH 6.5 and 37 °C. Proteolytic activities were very slightly affected by the increase of the temperature to 42 °C or the pH to 8.2. The protease system of Lactobacillus fermentum IFO 3956 is most probably composed from a high amount of metalloproteases and small amount of cysteine and serine proteases. [ABSTRACT FROM AUTHOR]

Abstract: Ovine β-lactoglobulin (BLG, a mixture of variants A and B at a ratio of 46/54) and α-lactalbumin (ALA) were subjected to pepsin activity. The degree of peptic hydrolysis of native whole BLG reached 63%, 74%, 82% and 87% after 2, 4, 8 and 20h hydrolysis, respectively. BLG variant B was degraded completely after 2h of pepsin digestion while variant A was degraded gradually showing 19%, 44%, 61% and 73% hydrolysis after 2, 4, 8 and 20h, respectively. The main factors responsible for the exceptional pepsin susceptibility of ovine BLG are the slightly different tertiary structure of ovine BLG (compared with bovine BLG) as perceived from near circular dichroism spectra at pH 2, and its higher surface hydrophobicity, as demonstrated by a higher binding activity to 1-anilinonaphthalene-8-sulphonate. Reversed phase-high performance liquid chromatograms (RP-HPLC) profiles of the peptic hydrolysates of BLG showed the production of hydrophobic peptides at the early stages of hydrolysis, while more hydrophilic peptides appeared only at a later stage of hydrolysis. Mass spectroscopy analysis allowed the characterisation of 17 and 13 peptides after 2 and 20h hydrolysis, respectively. Most of the enzyme activity was oriented first towards the N-terminal part of the molecule and later towards the C-terminal part of the protein; little or no activity was observed in the central region of the molecule even after 20h hydrolysis. Native ovine ALA was almost completely degraded by pepsin, yielding 93%, 94%, 95% and 98% hydrolysis after 2, 4, 8 and 24h, respectively. The RP-HPLC profile of the ALA hydrolysate showed 5 major hydrophobic peptides and 7 minor more hydrophilic peptides, which did not change with the time of hydrolysis. [Copyright &y& Elsevier]

Sheep susceptibility to scrapie is governed by polymorphisms at two major sites, codons 136 and 171, of the prp gene. To get more insight into the prion protein (PrP) sequence-linked basis of differential scrapie susceptibility, a high yield one-step method for the purification (over 99% final purity) of the full-length recombinant sheep PrP was developed, based on the affinity of the conserved octapeptide repeats for transition-metal cations. Thermal and chemical denaturation experiments and limited proteolysis studies were performed on the natural variants (A136R171, V136Q171 and A136Q171) and a recombinant PrP mutated at position 136 (V136R171). Results revealed the influence of mutations in positions 136 and 171 on the folding thermodynamic parameters and on the conformation of the C-terminal domain. Together, our results show that the VQ cellular protein linked to higher scrapie susceptibility is intrinsically more compact and/or stable than the resistance-linked AR counterpart. This might lead to a lower in vivo clearance rate of VQ and a consequently higher probability of occurrence of pathological events. [ABSTRACT FROM AUTHOR]

Lactiplantibacillus plantarum species is known for the high diversity of the loci encoding bacteriocin (plantaricin) production. The aim of this work was to study the distribution of pln genes in French and Ukrainian isolates of L. plantarum originated from fermented plant material. PCR with the primers to plnA, plnB, plnC, plnD, plnEF, plnI, plnK, plnJ, plnG, plnN, plnW genes was performed to find out the peculiarities of L. plantarum strains. French and Ukrainian isolates shared none of the identical combinations of genes from plantaricin regulon indicating the diversity of pln loci in L. plantarum of fermented plant origin from the distant geographical regions. Certain combinations of genes from plantaricin regulon could be proposed as indicators marking the geographical origin of L. plantarum strains. Among the studied isolates, some resembled the described in the literature L. plantarum strains, and others contained unique combinations of pln genes. [ABSTRACT FROM AUTHOR]

Bouhezza is an Algerian traditional cheese made using different types of milk (goat, sheep and cow), ripened in a goat-skin or sheep-skin bag called Chekoua or Djeld of Bouhezza. The objective of the study was the characterization of Bouhezza cheese through ten cheese preparation trials using goat raw milk. The chemical and microbiological composition and proteolysis were monitored on ripened samples. The physicochemical characteristics showed that Bouhezza is acidic with a pH value of 3.95 and 2.36% for titratable acidity as lactic acid, 13.20% for protein and 12.74% for fat. At the end of ripening, Bouhezza was classified to be a ripened, soft and mid-fat cheese. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of Bouhezza samples showed low proteolysis after 30 days of ripening. Microbiological results showed a high content of lactic acid bacteria (LAB), 8 to 9 log cfu/ g, an absence of pathogenic bacteria, and a low level of contamination flora. This makes the cheese a healthy product and of an acceptable hygienic quality. Four proteolytic lactic acid bacteria were identified by 16S ribosomal deoxyribonucleic acid (rDNA) sequencing: two strains of Enterococcus faecalis, one E. faecium, and one Lactobacillus paracasei ssp. paracasei. Reversed phase-high performance liquid chromatography (RP–HPLC) of the soluble fraction at pH 4.4 showed changes occurred during ripening. This work highlights, characterizes, and improves the knowledge of cheese Bouhezza made with raw goat's milk. This product is rich in nutrients, such as proteins and minerals, that are useful to the consumer. [ABSTRACT FROM AUTHOR]

This study investigated the digestibility of proteins in a pea protein‐fortified sponge cake, as well as the impact of the degree of structure of the bolus produced by elderly subjects on the digestibility of proteins by combining ex vivo and in vitro approaches via the standardized protocol INFOGEST. The sponge cakes were consumed by a group of 20 elderly subjects with contrasting physiology, their boli were recovered just before swallowing, and their apparent viscosity was measured to delineate the bolus degree of structure. According to this criterion, two pools were formed with boli from subjects selected at the extremes: low viscosity and high viscosity, with apparent viscosity values (at 120 s−1) of 124 ± 18 and 208 ± 19 Pa s, respectively. The sponge cakes and the two pools underwent in vitro digestion. Protein hydrolysis kinetics was followed by measuring the released primary amino groups (NH2) and by sodium‐dodecyl‐sulfate polyacrylamide gel electrophoresis at different time points. For all samples, the representative bands of pea proteins disappear gradually during digestion, accompanied by the appearance of bands indicating the presence of proteins with MW < 15 kDa. In addition, the NH2 concentrations increase over time and do not differ between sponge cake and pea protein isolate. Moreover, the degree of structure of the food bolus has no significant effect on the concentration of NH2 released. These results showed that pea proteins in a fortified sponge cake are bioaccessible under standardized conditions and that the degree of structure of the bolus did not influence protein digestibility for these foods. [ABSTRACT FROM AUTHOR]

Evaluation of Lactobacillus plantarum strains perspective for plant protection or fermentation of food of plant origin should include tests on abilities to attach to plant surfaces and to form biofilms. Comparing RAPD-profiles with these properties and also with geographical origin of the strains is necessary to find out the possibility of rapid selection of the strains and tracking their dissemination. Biofilm formation and RAPD-PCR profiles of 30 strains of L. plantarum isolated from grape must and pickles originated from France and Ukraine were compared. All isolates were able to attach to Lepidium sativum seedlings and form biofilms of different types. Amount of attached cells varied from (7.8 ± 0.2) x 104 to (2.1 ± 0.3) x 10³ CFU/cm² . The most developed biofilms were formed on shoots, leaves, seed coat shells. On roots this ability was strain dependent: the majority of French isolates (77.0%) formed separated microcolonies without developed matrix layer. Among Ukrainian isolates, 47.1% of the strains showed the same tendency, and in 41.2% of the strains individual attached cells without formation of microcolonies were observed. The level of initial attachment of bacterial cells to roots did not coincide with the level of subsequent formation of biofilms. Gene plnA was detected in 28.1% of isolates. All plnA possessing strains except the one formed microcolonies or biofilms with developed matrix. Lactobacilli could eliminate the biofilms of Agrobacterium tumefaciens pJZ from tomato roots in 75.0 – 100% of tested samples depending on a strain and method of inoculation. No clear association between the geographical origin of the L. plantarum strains and clustering by RAPD-PCR analysis was found. Two strains with high level of biofilm formation were grouped in one subcluster. [ABSTRACT FROM AUTHOR]

A total of 230 isolates of lactic acid bacteria were isolated from gastrointestinal tracts (GIT) of indigenous chickens purchased from local market in Bang Rakam district, Phitsanulok, Thailand. Among 230 isolates, only 7 isolates, named CM6CR07, CM6CR11, CF1GI15, CF1GI14, CF1GI17-1, CF1GI17-2 and CF1GI 19 showed an inhibition zone against indicator strains. Bacteriocin activity was completely inactivated when Neutralized Cell Free Supernatants (NCFS) of the isolates were treated with proteases, confirming the proteinaceous nature of the bacteriocin component. NCFS of all selected bacteriocin producing lactic acid bacteria (SBP-LAB) retained their bacteriocin activity after heating at 100 °C for 30 min. All of SBP-LAB were identified as Enterococcus faecalis . These Ent . faecalis isolates harbored the same virulence genes. All strains were positive for gel E (gelatinase), efa A fs (cell wall adhesins), hyl (hyaluronidase), ace (adhesin of collagen protein), cyl L L (cytolysin structural subunit) and cyl L S (cytolysin structural subunit) and were negative for asa1 (aggregation substance), agg (aggregation protein involved in adherence to eukaryotic cells), and esp (enterococcal surface protein). These Ent . faecalis showed a wide range of inhibitory spectrum against food-spoilage and food-borne pathogens when studied by agar spot test. The CFS and NCFS also inhibited some of food-spoilage and food-borne pathogenic indicator strains when checked by agar well diffusion assay. All of these Ent . faecalis survived simulated stomach conditions, whereas all of them could survive in conditions of simulated intestinal juice. [ABSTRACT FROM AUTHOR]

Lactococcus lactis KTH0-1S isolated from Thai traditional fermented shrimp ( Kung- som) is able to produce heat-stable bacteriocin and inhibits food spoilage bacteria and food-borne pathogens. The inhibitory effect of bacteriocin remained intact after treatment with different pHs and after heating, but was sensitive to some proteolytic enzymes. Addition of bacteriocin KTH0-1S to Staphylococcus aureus cultures decreased viable cell counts by 2.8 log CFU/ml, demonstrating a bactericidal mode of action. Furthermore, the growth of S. aureus decreased significantly after 12-h co-cultivation with bacteriocinogenic strain. The molecular mass of bacteriocin KTH0-1S was found to be 3.346 kDa after ammonium sulfate precipitation, reversed phase (C Sep-Pak), cation-exchange chromatography, RP-HPLC on C column and mass spectrometry (MS/MS) analysis. Bacteriocin KTH0-1S was identified as nisin Z using PCR amplification and sequencing. The majority of tested virulence factors were absent, confirming the safety. Evidenced inhibitory effect of this strain, the absence of virulence factors creates the possibility for its application as protective culture to inhibit pathogenic bacteria in the several fermented seafood products. [ABSTRACT FROM AUTHOR]

Fungal growth in bakery products represents the most frequent cause of spoilage and leads to economic losses for industrials and consumers. Bacteria, such as lactic acid bacteria and propionibacteria, are commonly known to play an active role in preservation of fermented food, producing a large range of antifungal metabolites. In a previous study (Le Lay et al., 2016), an extensive screening performed both in vitro and in situ allowed for the selection of bacteria exhibiting an antifungal activity. In the present study, active supernatants against Penicillium corylophilum and Aspergillus niger were analyzed to identify and quantify the antifungal compounds associated with the observed activity. Supernatant treatments (pH neutralization, heating and addition of proteinase K) suggested that organic acids played the most important role in the antifungal activity of each tested supernatant. Different methods (HPLC, mass spectrometry, colorimetric and enzymatic assays) were then applied to analyze the supernatants and it was shown that the main antifungal compounds corresponded to lactic, acetic and propionic acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels such as phenyllactic, hydroxyphenyllactic, azelaic and caproic acids. Based on these results, various combinations of the identified compounds were used to evaluate their effect on conidial germination and fungal growth of P. corylophilum and Eurotium repens . Some combinations presented the same activity than the bacterial culture supernatant thus confirming the involvement of the identified molecules in the antifungal activity. The obtained results suggested that acetic acid was mainly responsible for the antifungal activity against P. corylophilum and played an important role in E. repens inhibition. [ABSTRACT FROM AUTHOR]

Lactococcus lactis subsp. lactis KT2W2L was used for biopreservation in cooked, peeled and ionized tropical shrimps (CPITS) during storage at 8 °C under modified atmosphere packaging (MAP). The neutralized and filtered cell-free supernatant produced by this strain exhibited a broad spectrum of inhibition against 23 strains from 24 indicator strains of food-spoilage bacteria and food-borne pathogens by agar well diffusion assay. The growth of Brochothrix thermosphacta CD274, Carnobacterium maltaromaticum CD263 and Listeria innocua CIP 80.11T was inhibited by L. lactis subsp. lactis KT2W2L when co-cultivated at 25 °C. However, only L. innocua CIP 80.11T was inhibited in a co-cultivation at 8 °C. L. lactis subsp. lactis KT2W2L was then used for biopreservation in CPITS. Growth of bacteria group inoculated in CPITS was monitored at regular intervals during storage period under MAP at 8 °C by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique. L. lactis subsp. lactis KT2W2L inhibited the growth of B. thermosphacta and L. innocua CIP 80.11T batches after 7 days of storage. However, it was inactive against C. maltaromaticum batch. The growth of L. lactis subsp. lactis KT2W2L alone or in the presence of indicators increased to reach about 8-9 log CFU/g within 7 days of storage and remained constant until the end of the experiment. In the batches without L. lactis subsp. lactis KT2W2L, all bacterial groups grew well on the cooked shrimp matrix, reaching their maximal levels after 2 weeks of storage. The pH of all homogenized suspensions of MAP-CPITS was quite stable at 6.0-6.7. The control batches were under the enumeration threshold (<1.70 ± 0.00 log CFU/g) until 14 days of storage, and then, an increase in growth was detected on agar plates. In addition, TTGE revealed to be an excellent tool to monitor the change of the microbial ecosystem in this product. [ABSTRACT FROM AUTHOR]

Nine lactic acid bacteria strains showing bacteriocin-like activity were isolated from various fresh fish viscera. The following species were identified based on 16S rDNA sequences: Enterococcus durans (7 isolates), Lactococcus lactis (1) and Enterococcus faecium (1). These strains were active against Listeria innocua and other LAB. Random amplified polymorphic DNA analyses showed four major patterns for the E. durans species. PCR analyses revealed a nisin gene in the genome of the Lc. lactis strain. Genes coding enterocins A, B and P were found in the genome of the E. faecium isolate. Enterocins A and B genes were also present in the genome of E. durans GM19. Hence, this is the first report describing E. durans strains producing enterocins A and B. Electrospray ionization mass spectrometry revealed that the purified bacteriocin produced by the E. durans GMT18 strain had an exact molecular mass of 6,316.89 Da. This bacteriocin was designated as durancin GMT18. Edman sequencing failed to proceed; suggesting that durancin GTM18 may contain terminal lanthionine residues. Overall, the results obtained revealed the presence of a variety of enterococci in Mediterranean fish viscera, as evidenced by their genetic profiles and abilities to produce different bacteriocins. These strains could be useful for food biopreservation or as probiotics. [ABSTRACT FROM AUTHOR]

Abstract: The aims of this study were to characterize inhibitory activity spectra, some probiotic properties and safety of Lactobacillus curvatus A61 for its future application in production of fermented foods. The studied strain was isolated from traditional homemade cheese manufactured in Azerbaijan. The cell-free supernatant of culture of Lb. curvatus A61 inhibited the growth of tested LAB, as well as of Listeria monocytogenes and Bacillus cereus strains. The strain presented antifungal activity and inhibited the growth of Cladosporium and Fusarium ssp. during co-cultivation on agar media. PCR amplification with specific primers revealed the presence of curvacin A encoding gene in Lb. curvatus A61. Bacteriocin produced by the studied strain was heat stable and active in a broad pH range, and in the presence of Triton X-20, Triton X-80, Triton X-100, β-mercaptoethanol, Na-EDTA, SDS and NaCl. The mode of action of bacteriocin against selected indicator strains was found to be bacteriostatic. Lb. curvatus A61 was resistant to physiological concentrations of bile salts and showed high auto-aggregation ability, as well as co-aggregation ability with pathogenic L. monocytogenes strains. It was sensitive to chloramphenicol, penicillin, tetracycline, ciprofloxacin and vancomycin, but resistant to ampicillin and gentamicin. [Copyright &y& Elsevier]

Abstract: Strain SMXD51, isolated from chicken ceca and identified as Lactobacillus salivarius, produced a component that inhibits the growth of Gram-positive and Gram-negative bacteria and especially Campylobacter jejuni. The active peptide from the cell-free supernatant of Lb. salivarius SMXD51 was purified in three steps: (i) precipitation with 80% saturated ammonium sulfate, (ii) elution on a reversed phase SPE UPTI-CLEAN cartridge using different concentrations of acetonitrile, (iii) final purification by reversed phase HPLC on a C18 column. The mode of action of this peptide of 5383.2 Da was identified as bactericidal, and its amino acid composition was established. This new bacteriocin SMXD51 appears potentially very useful to reduce Campylobacter in poultry prior to processing. [Copyright &y& Elsevier]

Effect of combined microwave (MW) and enzymatic hydrolysis on the human immunoglobulin E (IgE)-binding properties of β-lactoglobulin (β-lg) and other whey proteins (WP) was investigated. Separated β-lg and full whey protein isolate (WPI) were hydrolyzed with trypsin, chymotrypsin, mixture of trypsin/chymotrypsin, and pepsin at three microwave power levels: 50 W during 1 and 5 min, 100 and 200 W during 1 and 3 min. The immunoreactivity of the obtained hydrolysates resulting from combined microwave protease treatment was assessed using sera of young patients allergic to bovine whey proteins. The application of microwave treatment at 200 W enhances the hydrolysis of β-lg by pepsin in 3 min and decreases significantly its immunoreactivity. The extensive hydrolysis of the microwave-treated β-lg and WPI with trypsin, chymotrypsin, and the mixture of trypsin with chymotrypsin did not have an impact on the IgE binding of the products obtained in all the studied conditions. [ABSTRACT FROM AUTHOR]

The interest of consumers for diverse fermented foods has increased in recent years thanks to the positive perception of their impact on consumer health considered as beneficial. Hence, there is an evident need for search of novel ways and for new food preservation agents of natural origins. In this aspect, lactic acid bacteria are very good candidates. It should be highlighted also that the onset of food allergies is rising significantly in recent years. The reduction of the immunoreactivity of food proteins could be achieved thanks to pre-prandial proteolysis occurring in fermented dairy (and other food) systems changing the allergen presentation or cleaving the allergenic protein epitopes, and produce hypoallergenic products. [ABSTRACT FROM AUTHOR]

Abstract: The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41–K60; Y102–R124; L149–I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS–PAGE and RP-HPLC. Peptides released were identified by LC–MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3–5year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products. [Copyright &y& Elsevier]

collection of 147 isolates obtained from 23 samples of traditional Azerbaijani dairy products was screened for the presence of proteolytic enzymes. Six Enterococcus faecalis strains obtained from three cheese samples have been identified as proteinase-producing strains, according to their ability to hydrolyze caseins. RAPD-PCR profiles of their total DNA showed different patterns for strains isolated from different cheese samples. The proteolytic activities of these strains were studied during their growth in milk and in non-proliferative cells system. Isolated strains were able to hydrolyze α-, α-, β-caseins and BLG albeit to different extents, at optimal pH in the range 6.0-7.2 and optimal temperature in the range 37-45 °C, depending on the strain. Proteolysis was strongly inhibited in the presence of EDTA-specific inhibitor of metalloproteases-but the presence of other types of proteases cannot be excluded. The potential pathogenicity of the strains was evaluated investigating the presence of the genes coding different virulence factors and their resistance to antibiotics. The obtained results yield new information about technological characteristics and safety of studied Enterococci strains from Azerbaijani artisanal dairy products. Many from the isolated strains contribute certainly to the differences in flavor, texture, and taste of Azerbaijani traditional cheeses and could represent new adjunct cultures for the dairy industry. [ABSTRACT FROM AUTHOR]

Abstract: β-Casein (β-CN) is a milk protein widely used in food industries because of its mild emulsifying properties due to its amphiphilicity. However, the elements determining its micellization behavior in solution and interfacial behavior at the air–water interface are not well known. In order to study how the forced dimerisation influences functional properties of β-CN, recombinant wild-type β-CN was produced and distal cysteinylated forms of recombinant β-CN were engineered. We show that 1) cysteinylated β-CN formed mainly dimers bridged by disulfide bonds; 2) the process of dimerization adds to the micellization process with temperature and is poorly reversible; 3) covalent disulfide linkage forms at the air–water interface at a lower temperature than in bulk. In conclusion, the location of the cysteinylation in the C-terminus or N-terminus or both is of importance for the properties of β-CN. [Copyright &y& Elsevier]

Abstract: Due to the combination of flexibility and amphiphilicity, in solution β-casein (β-CN) acts as a surfactant molecule. It undergoes self-association and forms “micellar-like” aggregates in aqueous media. The aim of our study was to understand the role played by the charges borne by the N-terminal part of β-CN in its association behavior. β-CN polarity was modified in order to induce new aggregation properties and to understand better the main factors defining them. An unphosphorylated wild type recombinant form (WT A2) of β-CN has been obtained by heterologous expression in the prokaryotic host Escherichia coli. A mutant (MU A2) form of β-CN was engineered by duplicating the first 6 N-terminal amino acids residues (RELEEL) of WT A2, three additional negatively charged glutamic acids replacing some of the negative charges lost by the absence of phosphorylation in prokaryotic expression system. Dynamic light scattering and fluorimetry experiments showed that recombinant and native β-CN have different behaviors in temperature-dependent association. Micellisation was impaired by the absence of phosphate groups. Mutation conferred to the protein the ability to form aggregates whose size is inversely proportional to the concentration. These different behaviors during the association processes displayed by recombinant β-CN are correlated with modifications of the temperature-induced compaction of the proteins, what was revealed by circular dichroism measurements. WT A2 was less condensed at the air-water interface than native β-CN; this was in part reversed by the additive mutation. However, MU A2 compensating the lack of phosphate charges in recombinant caseins forms a more cohesive monolayer at the air–water interface than do the other β-CN, which means that the mutation reinforces monomers interaction at the surface. [Copyright &y& Elsevier]

Conformational changes of ewe's α-lactalbumin (ALA) upon removal of Ca 2+ were determined by surface hydrophobicity, calorimetry and circular dichroism. Native ewe's ALA resisted trypsinolysis, showing 4% maximum degradation after 20 h of hydrolysis. Removal of bound calcium by addition of either ethylenediaminetetraacetic acid or ethylene glycol bis β-aminoethyl ether-N,N,N,N-tetraacetic acid induced major protein conformational changes, enhancing its susceptibility to trypsinolysis, and leading to complete degradation of the protein. Reversed-phase high-performance liquid chromatography profiles of tryptic hydrolysate of Ca 2+ -free ALA were nearly the same through the whole enzymatic incubation period (24 h) showing the absence of sequential hydrolytic mechanism. They were characterized by the presence of five main peaks representing hydrophobic large-sized peptides. Cleaving the S-S bonds in the resulting hydrolysates with 2-mercaptoethanol gave rise to new peaks representing more hydrophilic and hydrophobic peptides. [ABSTRACT FROM AUTHOR]

In pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. In prion diseases the lethal evolution is associated with formation of PrPSc, a misfolded and amyloid form of the normal cellular prion protein PrP. Although several molecular mechanisms were proposed to account for the propagation of the infectious agent, the events responsible for cell death are still unclear. The correlation between PrPC expression level and the rate of disease evolution on one side, and the fact that PrPSc deposition in brain did not strictly correlate with the apparition of clinical symptoms on the other side, suggested a potential role for diffusible oligomers in neuronal death. To get better insight into the molecular mechanisms of PrPC oligomerization, we studied the heat-induced oligomerization pathway of the full-length recombinant ovine PrP at acidic pH. This led to the irreversible formation of two well-identified soluble oligomers that could be recovered by size-exclusion chromatography. Both oligomers displayed higher β-sheet content when compared to the monomer. A sequential two-step multimolecular process accounted for the rate of their formation and their ratio partition, both depending on the initial protein concentration. Small-angle X-ray scattering allowed the determination of the molecular masses for each oligomer, 12mer and 36mer, as well as their distinct oblate shapes. The two species differed in accessibility of polypeptide chain epitopes and of pepsin-sensitive bonds, in a way suggesting distinct conformations for their monomeric unit. The conversion pathway leading to these novel oligomers, displaying contrasted biochemical reactivities, might be a clue to unravel their biological roles. [Copyright &y& Elsevier]