T. Haertlé

Probing the fatty acid binding site of β-lactoglobulins

The interactions of fatty acids with porcine and bovine β-lactoglobulins were measured using tryptophan fluorescence enhancement. In the case of bovine β-lactoglobulin, the apparent binding constants for most of the saturated and unsaturated fatty acids were in the range of 10−7 M at neutralpH. Bovine β-lactoglobulin displays only one high affinity binding site for palmitate with an apparent dissociation constant of 1·10−7 M. The strength of the binding was decreasing in the following way: palmitate > stearate > myristate > arachidate > laurate. Caprylic and capric acids are not bound at all. The affinity of β-lactoglobulin for palmitate decreased as thepH of the incubation medium was lowered and BLG/palmitate complex was not observed atpHs lower than 4.5. Surprisingly, chemically modified bovine β-lactoglobulin and porcine β-lactoglobulin did not bind fatty acids in the applied conditions.

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag.

Aims:  The aim of this study was to isolate and identify bacteriocin‐producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mares milk), and to purify and characterize bacteriocins produced by these LAB.

Isolation and partial biochemical characterization of a proteinaceous anti-bacteria and anti-yeast compound produced by Lactobacillus paracasei subsp. paracasei strain M3.

New proteinaceous active substance produced by Lactobacillus paracasei subsp. paracasei strain M3 used as a starter for Bulgarian yellow cheese was identified and studied. It displayed bactericidal and fungistatic activities. Its activity was checked against over 60 bacterial and yeast strains. It was efficient against Bacillus subtilis ATCC 6633, several L. delbrueckii species, Helicobacter pylori NCIPD 230 and some yeast species, for example Candida albicans, C. pseudointermedia NBIMCC 1532, C. blankii NBIMCC 85 and Saccharomyces cerevisiae NBIMCC 1812. The synthesis of the substance by producing strain was detected in the late logarithmic growth phase during batch fermentation. Anion exchange chromatography, reversed phase chromatography (RPC) on C4 column and HPLC on C18 column were used for partial purification of this antimicrobial compound. The gene responsible for the synthesis of the active substance is located on the bacterial chromosome.

β-Lactoglobulin binds retinol and protoporphyrin IX at two different binding sites

Measurement of tryptophan fluorescence quenching and the excitation energy transfer from tryptophanyl residues to the bound ligand indicates that β‐lactoglobulin binds tightly to hemin and protoporphyrin IX in a ligand‐to‐protein stoichiometric ratio. The apparent dissociation constants of hemin‐β‐lactoglobulin and protoporphyrin IX‐β‐lactoglobulin complexes are 2.5 × 10−7 M and 4 × 10−7 M, respectively. The addition of β‐lactoglobulin (final concentration = 10 μM, phosphate buffer 50 mM, pH 7.1) to the solution containing retinol and protoporphyrin IX triggers an energy transfer between β‐lactoglobulin tryptophan and protoporphyrin IX as well as between retinol and protoporphyrin IX. The efficiency of energy transfer depends on the distance between the donor (retinol) and the acceptor (protoporphyrin IX). Using the Förster theory, a retinol‐protoporphyrin IX distance of 25 Å was calculated. These results indicate that retinol and protoporphyrin IX are bound to the β‐lactoglobulin monomer at two different sites.

High-pressure effects on β-lactoglobulin interactions with ligands studied by fluorescence

The effects of pressure (0.1 MPa to 400 MPa) on intrinsic fluorescence of beta-lactoglobulin and on its binding of retinol and cis-parinaric acid have been studied at neutral and acid pHs. In neutral pH, fluorescence emission spectra of beta-lactoglobulin tryptophanes are characterized by an irreversible 14 nm red-shift indicating pressure-induced folding changes. The intensity of the fluorescence of retinol in beta-lactoglobulin-retinol complex is enhanced by a pressure increase up to 150 MPa. It decreases at higher pressures and disappears altogether at 300 MPa. beta-Lactoglobulin-retinol complex does not reassociate after decompression at neutral pH. At acid pH condition, the fluorescence quenching by pressure of beta-lactoglobulin tryptophans is coupled with a 2 nm spectral shift and is fully reversible demonstrating almost complete restoration of globulin folding. The evolution of retinol fluorescence in beta-lactoglobulin-retinol complex is also entirely reversible between 0.1 MPa and 400 MPa and the complex never dissociates in the studied pressure range. beta-lactoglobulin-cis-parinaric acid complexes at neutral and acid pH values dissociate irreversibly at 200 MPa and 350 MPa, respectively.

Binding of retinoids and β-carotene to β-lactoglobulin. Influence of protein modifications

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.

Proteolysis of β-lactoglobulin and β-casein by pepsin in ethanolic media

Abstract Limited proteolysis of β-lactoglobulin and β-casein by pepsin was performed in the presence of varying concentrations of ethanol. β-Lactoglobulin started to be cleaved by pepsin only in ethanol concentrations greater than 20%, when its secondary structure began to change. In 25% ethanol, the rate of hydrolysis of β-lactoglobulin was slow (40% remained intact after 40 h of hydrolysis) and many short and hydrophilic peptides were observed. The rate of hydrolysis of β-lactoglobulin reached its maximum in 30 and 35% ethanol (80% of β-lactoglobulin was hydrolysed after 10 h), and a mixed population of hydrophilic and hydrophobic peptides of different lengths was observed. Large hydrophobic peptides appeared first, then some shorter products. The rate of hydrolysis of β-lactoglobulin decreased at ethanol concentrations equal to or higher than 40%, when only a few long, hydrophobic peptides were produced. As seen by circular dichroism, the addition of ethanol to β-casein induced α-helix formation and reduced the rate of casein hydrolysis without changing the peptide profile. The only exception was the yield of a single peptide (Pro81 − Met93).

Thermal modifications of structure and codenaturation of α-lactalbumin and β-lactoglobulin induce changes of solubility and susceptibility to proteases

Study of heat denaturation of major whey proteins (beta-lactoglobulin or alpha-lactalbumin) either in separated purified forms, or in forms present in fresh industrial whey or in recomposed mixture respecting whey proportions, indicated significant differences in their denaturation depending on pH, temperature of heating, presence or absence of other codenaturation partner, and of existence of a previous thermal pretreatment (industrial whey). alpha-Lactalbumin, usually resistant to tryptic hydrolysis, aggregated after heating at > or = 85 degrees C. After its denaturation, alpha-lactalbumin was susceptible to tryptic hydrolysis probably because of exposure of its previously hidden tryptic cleavage sites (Lys-X and Arg-X bonds). Heating over 85 degrees C of beta-lactoglobulin increased its aggregation and exposure of its peptic cleavage sites. The co-denaturation of alpha-lactalbumin with beta-lactoglobulin increased their aggregation and resulted in complete exposure of beta-lactoglobulin peptic cleavage sites and partial unveiling of alpha-lactalbumin tryptic cleavage sites. The exposure of alpha-lactalbumin tryptic cleavage sites was slightly enhanced when the alpha-lactalbumin/beta-lactoglobulin mixture was heated at pH 7.5. Co-denaturation of fresh whey by heating at 95 degrees C and pH 4.5 and above produced aggregates stabilized mostly by covalent disulfide bonds easily reduced by beta-mercaptoethanol. The aggregates stabilized by covalent bonds other than disulfide arose from a same thermal treatment but performed at pH 3.5. Thermal treatment of whey at pH 7.5 considerably enhanced tryptic and peptic hydrolysis of both major proteins.

Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens

Aims:  The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food‐borne pathogens, identify inhibitory substances and study the kinetics of their production.

Binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid to β-lactoglobulin: Influence of protein modifications

The binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated β-lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified β-lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10−8 M for ligand/β-lactoglobulin complexes. The studied, chemically modified β-lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of ε-NH2 lysyl residues of β-lactoglobulin increases the apparent molar ratios of benzo(α)pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native β-lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand-β-lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of β-lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo(α)pyrene, ellipticine, and cis-parinaric acid, respectively.