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.

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.

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.

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.

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.

Assessment of the immunoglobulin E-mediated immune response to milk-specific proteins in allergic patients using microarrays.

BACKGROUND: Cow's milk allergy (CMA) is one of the most widespread human allergies, especially in young children. Although CMA is intensively studied, little is known about the recognition patterns of milk allergens in allergic patients, and the determination these patterns is a prerequisite for the development of efficient diagnostic and prognostic tools. Several factors present difficulties for such a determination, because (i) milk contains a large number of potential allergens; (ii) the majority of these allergens consist of complex suspensions rather than solutions; (iii) the major allergens, such as caseins, cannot be highly purified in large amounts; and (iv) most of the time, very small amount of young patients' sera are readily available. METHODS: To overcome these difficulties, we developed a sensitive microarray assay that, in combination with near-infrared fluorescence detection, was used to study the immune response to milk and purified native milk proteins. RESULTS: This new assay allowed us to assess the binding ability of IgE to milk allergens from a large number of young patients using reduced amounts of clinical material. The data show that bovine lactoferrin can be classed as a strong milk allergen. We confirmed that bovine caseins are the main allergens in milk and that alpha(S1)-casein is more allergenic than alpha(S2)-, beta- and kappa-caseins, which were recognized with almost a similar frequency by the sera of patients. CONCLUSION: Microarray methods, in combination with near-infrared fluorescence detection, can be useful for the in vitro diagnosis of food allergies.

Toxicity to the pea aphid Acyrthosiphon pisum of anti-chymotrypsin isoforms and fragments of Bowman-Birk protease inhibitors from pea seeds.

Aphids feed on a protein-poor diet and are insensitive to several serine protease inhibitors. However, among the Bowman-Birk family of plant trypsin inhibitors (BBI), some members display significant toxicity to the pea aphid Acyrthosiphon pisum. A BBI isoform purified from pea seeds (PsTI-2) displays an IC50 of 41 microM and a LC50 of 48 microM at 7 days. Our data show that the chymotrypsin-directed active site from these bifunctional inhibitors is responsible for this activity, and that artificial cyclic peptides bearing the Bowman-Birk anti-chymotrypsin head induce much greater toxicity and growth inhibition than their anti-trypsin counterparts. The toxic syndrome included a rapid behavioural response of aphids on diets containing the toxic peptides, with induced restlessness after only 1 h of exposure to the chymotrypsin inhibitor. Nevertheless, chymotrypsin activity was not detected in aphid guts, using two chromogenic chymotrypsin substrates, and the physiological target of the chymotrypsin inhibitor remains unknown. These data show for the first time that plant chymotrypsin inhibitors, still widely unexplored, may act as paradoxical toxicants to aphids and serve as defensive metabolites for phloem-feeding insects.

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution.

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.

Disulfide bond assignment, lipid transfer activity and secondary structure of a 7-kDa plant lipid transfer protein, LTP2.

The 7-kDa lipid transfer proteins, LTP2s, share some amino-acid sequence similarities with the 9-kDa isoforms, LTP1s. Both proteins display an identical cysteine motif and, in this regard, LTP2s have been classified as lipid transfer proteins. However, in contrast with LTP1s, no data are available on their structure, cysteine pairings, lipid transfer and lipid binding properties. We reported on the isolation of two isoforms of 7-kDa lipid transfer protein, LTP2, from wheat seeds and showed for the first time that they indeed display lipid transfer activity. Trypsin and chymotrypsin digestions of the native LTP2 afforded the sequence of both isoforms and assignment of disulfide bonds. The cysteine pairings, Cys10--Cys24, Cys25--Cys60, Cys2--Cys34, Cys36--Cys67, revealed a mismatch at the Cys34-X-Cys36 motif of LTP2 compared to LTP1. Moreover, the secondary structure as determined by circular dichroism suggested an identical proportion of alpha helices, beta sheets and random coils. By analogy with the structure of the LTP1, we discussed what structural changes are required to accommodate the LTP2 disulfide pattern.

Why has porcine VEG protein unusually high stability and suppressed binding ability?

Von Ebner gland protein (VEGP) and odorant-binding protein (OBP) were purified from porcine lingual epithelium and nasal mucosa, respectively. Both VEGP and OBP preparations were homogeneous as indicated by SDS-PAGE, isoelectric focusing, gel-filtration and electrospray mass spectrometry. However, high-sensitivity differential scanning calorimetry (HS-DSC) yielded multiphasic denaturation thermograms for both proteins indicating their conformational heterogeneity. The unfolding transition of VEGP is observed at extremely high temperatures (about 110 degrees C), which is unexpected for a protein with significant structural homology to OBP and other lipocalins. Isothermal titration calorimetry (ITC) did not detect the binding of either aspartame or denatonium saccharide to VEGP nor did it detect binding of 2-isobutyl-3-methoxypyrazine (IBMP) to OBP. Extraction of OBP with mixed organic solvents eliminated the conformational heterogeneity and the protein showed a reversible two-state transition in HS-DSC thereafter. ITC also showed that the extracted OBP was able to bind IBMP. These results imply that tightly bound endogenous ligands increase the thermal stability of OBP and block the binding of other ligands. In contrast to OBP, the extraction of VEGP with organic solvents failed to promote binding or to establish thermal homogeneity, most likely because of the irreversible denaturation of VEGP. Thus, the elucidation of the functional behaviour of VEGP is closely related to the exhaustive purging of its endogenous ligands which otherwise very efficiently mask ligand binding sites of this protein.

Purification of a 41 kDa cod-allergenic protein.

Cod fish is one of the foods most frequently involved in allergy. Only the cod allergen Gad c I, a 12.3 kDa parvalbumin, has been purified and characterized. Recently, we have detected allergen bands which have not previously been described, in particular a 41 kDa protein, by Western-blot. In the present work, this protein has been purified from a crude cod extract by ammonium sulfate fractionation, hydroxyapatite chromatography and preparative electrophoresis; a single band with an Mr of 41 x 10(3) was found in silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition and the isoelectric point of the protein were determined. The purified protein (p41) was shown to bind specifically to reaginic IgE from sera of cod-allergic individuals and to a monoclonal anti-parvalbumin which recognizes specifically the first calcium binding site of parvalbumins. p41 may therefore contain a calcium binding site corresponding to an IgE-epitope similar to that of Gad c I.

Autoantibodies in pernicious anemia type I patients recognize sequence 251-256 in human intrinsic factor.

Pernicious anemia is an organ-specific autoimmune disease characterized by cobalamin deficiency, megaloblastic anemia, neuropathy, and autoimmune gastritis with anti-intrinsic factor autoantibodies. Type 1 anti-intrinsic factor autoantibodies block the cobalamin binding site of the intrinsic factor, a gastric protein required for the assimilation of cobalamin. The aim of our study was to identify the epitope domain of type 1 antibodies. Different series of peptides derived from the intrinsic factor sequence were synthesized and tested for antibody binding in enzyme-linked immunosorbent assay, radioisotope assay, gel filtration, and SDS-PAGE autoradiography. One of these peptides, named IF-R7 (the intrinsic factor aminoacid sequence 251-265), showed a type 1 antibody binding activity and inhibited, in vitro, their blocking activity with Ki at 2.3 microM. The cross-linking of IF-R7 to beta-lactoglobulin produced type 1 anti-intrinsic factor antibodies in immunized sheep. In vivo Schilling tests performed on guinea pigs also revealed IF-R7 peptide inhibition of type 1 antibody blocking activity. 256Ser, 258Lys, 262Tyr and 265Val of the IF-R7 were essential for the epitope recognition. Reactivity with type 1 antibodies was found in IF-R7 homologous peptides from herpesvirus Saimiri and from pathogenic Escherichia coli. In conclusion, the epitope of type 1 anti-intrinsic factor autoantibodies is located in the 251-265 amino acid sequence of the protein. The identification of this epitope will enable the definition of an experimental animal model of anti-IF autoimmunity in order to study the pathogenesis of pernicious anemia.

Mapping of HCG-receptor complexes.

Molecular forms of the porcine LH/CG receptor (pLHR) and complexes between hCG and either the full-length pLHR or its extracellular domain (ectodomain) have been produced in various recombinant systems. In COS cells and in the baculovirus insect cells system, the co-expression of the ecto- and endo-domains reconstituted a functional receptor where the association of the two domains seems to depend upon the presence of disulfide bridges. According to previous observations [39], synthetic peptides mimicking three regions of the ectodomain (21-38, 100-115, 250-272) were found to inhibit hormone binding and stimulation of cAMP production. Antisera raised against these peptides contained anti-peptide antibodies (Ab) able to interfere with hormone signalling. Moreover, the results of peptide mapping indicated that some peptides stretches may be more involved in signalling rather than in binding. Immunochemical mapping based on monoclonal antibodies (mAbs) was used to probe the hCG-ectodomain complex. It appeared that mAbs directed to epitopes present on the 'beta-tip' of hCG (assembled from the beta subunit loops 3 and 1, and previously designated site IIIb) and on the 'alpha-tip' (alpha subunit loops 1 and 3, site IIIa) bound to hCG-receptor complexes, whereas a conformational epitope (defined by the alpha-beta interface between beta seat belt C-terminus and alpha loop 2, site II) was masked. Interestingly, we and others previously reported that, in the hCG-full length receptor complex, site IIIa was shielded to mAb binding. A peptide mimicking the second extracellular loop (EL2) of the receptor endodomain was found to prevent the binding of a mAb directed to site IIIa, suggesting that this region of the endodomain may be interacting with the 'alpha-tip'. In the full-length, membrane anchored pLHR, the EL2 peptide inhibited hCG-induced cAMP production, but not binding. The possibility of inhibiting stimulation without inhibition of binding gives support to the 'negative specificity' hypothesis [6]. Thus, the ectodomain of the glycoprotein hormone receptors might be considered as a screening device preventing access of any glycoprotein hormone to the signalling peptide keys of the endodomain, which otherwise would be sensitive to any alpha subunit stimulation. Finally, antibody binding to site IIIa on the hCG-ectodomain complex was also hindered by an anti-peptide mAb directed against a peptide encoded by the eighth exon (pE x 8) of the LHR. This suggests that pEx8 is vicinal to the alpha-tip of hCG and to EL2 in the hCG-full length receptor complex. Altogether, these observations help to build up a topological model of the hCG-receptor complex.

Immunization against exon 1 decapeptides from the lutropin/choriogonadotropin receptor or the follitropin receptor as potential male contraceptive.

Pituitary gonadotropin hormones lutropin (LH) and follitropin (FSH) control steroidogenesis and gametogenesis in male and female gonads through interaction with G protein-coupled receptors, LHR and FSHR. In the male, LH acts on leydig cells and is mostly responsible for the acquisition of puberty and the production of androgens while FSH, together with androgens, regulates spermatogenesis within Sertoli cells. We have engineered filamentous phages displaying mouse LHR and human FSHR decapeptides chosen in hormone binding regions. Peptides from both receptors displayed on phages belong either to the receptor specific exon 1 (amino acids 18-27) or to the homologous exon 4 (amino acids 98-107). Vaccination of prepubertal BALB/c male mice with hybrid phages using sub-cutaneous or intraperitoneal injections induced immunity against receptors. Anti-receptor immunization produced agonist or antagonist effects depending only on the circulating levels of the antibodies. Both anti-LHR and anti-FSHR vaccines induced efficient as well as reversible male contraception, through different mechanisms: targeting LH receptors inhibited or hyperstimulated Leydig cell testosterone production while targeting FSH receptors did not affect testosterone levels.

A defined epitope on the human choriogonadotropin alpha-subunit interacts with the second extracellular loop of the transmembrane domain of the lutropin/choriogonadotropin receptor.

The monoclonal antibody, HT13 recognizes human choriogonadotropin (CG) bound to the extracellular domain of its receptor, but not to the full-length receptor. The HT13 epitope is located in the regions of residues 15-17 and 73-75 of the human CG alpha-subunit. Only one synthetic peptide, lutropin (LH)/CG-receptor-(481-497)-peptide (EL2 peptide), which spans the second putative extracellular loop of the LH/CG-receptor endodomain, prevents recognition of human CG by HT13 mAb. EL2 peptide decreases hormone-induced cAMP production, but not high-affinity binding. An anti-EL2 serum also displays the capacity to inhibit human CG-stimulated cAMP production. These results suggest that the second extracellular loop of the receptor is in contact with the HT13 epitope of human CG alpha-subunit and is involved in signal transduction. A relative orientation of the hormone versus the endodomain is proposed.