Thomas Haertlé

Improvement of functional properties of β-lactoglobulin glycated through the Maillard reaction is related to the nature of the sugar

The improvement of functional properties of proteins available in large quantity using non-toxic chemical modifications is of a great interest for the food industry. In this study, the Maillard reaction was used to improve the functional properties (solubility, heat stability, emulsifying and foaming properties) of β-lactoglobulin (BLG) glycated with several sugars (arabinose, galactose, glucose, lactose, rhamnose or ribose). Protein samples were heated in the presence or absence (heated control) of different sugars for three days at 60°C. Subsequent glycation induced a modification of the solubility profile shifting minimum solubility towards more acidic pH. Glycated proteins exhibited a better thermal stability when heated at pH 5 as compared to native or heated control. Glycation of BLG with arabinose or ribose (the most reactive sugars) improved its emulsifying properties. Foaming properties were better when BLG was glycated with glucose or galactose (moderately reactive sugars). These results suggest that the nature of the sugar is an essential factor for improving the functional properties of glycated proteins by processes of Maillard reaction.

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants.

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 degrees 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 beta-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 beta-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.

Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis

Aims:  The aim of this study was to develop a method for microbial degradation of indigenous keratin wastes and to compare it with a method of alkaline hydrolysis.

Induction of New Physicochemical and Functional Properties by the Glycosylation of Whey Proteins

Nucleophilic primary amino groups of whey proteins (β-lactoglobulin and α-lactalbumin) were modified with reducing sugars in mild heat conditions. After 49 hr of heating (60°C) at pH 6.5, 20–30% of β-lactoglobulin amino groups were substituted with aldohexoses (galactose, mannose, glucose) and lactose, whereas up to 70% and 90% of β-lactoglobulin amino groups were modified with ribose and glyceraldehyde, respectively. Gel electrophoresis and reversed-phase HPLC coupled with electrospray ionization mass spectrometry of glycosylated proteins indicated that the substitution was random. Consequently, highly heterogeneous families of glycosylated proteins were generated. Proteins substituted with hexoses and lactose exhibited higher solubility and improved emulsifying properties as compared with nonglycosylated proteins, in the whole pH range studied. In contrast, proteins glycosylated with ribose and glyceraldehyde showed lower solubility close to their isoelectric points. β-Lactoglobulin modified with ribose and glyceraldehyde displayed substantial differences in denaturation behavior as compared with native protein. When compared with β-lactoglobulin, glycosylation of α-lactalbumin was quicker. There was no difference in glycosylation yields nor rates of α-lactalbumin in presence and absence of calcium.

Improvement of the antimicrobial and antioxidant activities of camel and bovine whey proteins by limited proteolysis.

The compositions and structures of bovine and camel milk proteins are different, which define their functional and biological properties. The aim of this study was to investigate the effects of enzymatic hydrolysis of camel and bovine whey proteins (WPs) on their antioxidant and antimicrobial properties. After enzymatic treatment, both the antioxidant and the antimicrobial activities of bovine and camel WPs were improved. The significantly higher antioxidant activity of camel WPs and their hydrolysates as compared with that of bovine WPs and their hydrolysates may result from the differences in amounts and/or in accessibilities of antioxidant amino acid residues present in their primary structures and from the prevalence of alpha-lactalbumin and beta-lactoglobulin as proteolytic substrates in camel and bovine whey, respectively. The results of this study reveal differences in antimicrobial and antioxidant activities between WP hydrolysates of bovine and camel milk and the effects of limited proteolysis on these activities.

Biological activity of camel milk casein following enzymatic digestion

The aim of this study was to investigate the effects of enzymatic hydrolysis with digestive enzymes of camel whole casein and beta-casein (β-CN) on their antioxidant and Angiotensin Converting Enzyme (ACE)-inhibitory properties. Peptides in each hydrolysate were fractionated with ultra-filtration membranes. The antioxidant activity was determined using a Trolox equivalent antioxidant capacity (TEAC) scale. After enzymatic hydrolysis, both antioxidant and ACE-inhibitory activities of camel whole casein and camel β-CN were enhanced. Camel whole casein and β-CN showed significant ACE-inhibitory activities after hydrolysis with pepsin alone and after pepsinolysis followed by trypsinolysis and chymotrypsinolysis. Camel β-CN showed high antioxidant activity after hydrolysis with chymotrypsin. The results of this study suggest that when camel milk is consumed and digested, the produced peptides start to act as natural antioxidants and ACE-inhibitors.

Glycodelin and β‐lactoglobulin, lipocalins with a high structural similarity, differ in ligand binding properties

Human glycodelin, a lipocalin with a high amino acid similarity to β‐lactoglobulins, appears as various glycoforms with different biological activities in endometrium (glycodelin‐A) and seminal plasma (glycodelin‐S). We found that the structures of these glycodelins and β‐lactoglobulin are similar. Despite this structural similarity, unlike β‐lactoglobulin, glycodelin‐A binds neither retinoic acid nor retinol. It was impossible to detect any endogenous retinoids or steroids in any of the two purified glycodelins. Both their glycoforms share similar thermodynamic parameters of reversible denaturation suggesting that native folding of glycodelin‐A and glycodelin‐S is not influenced by the differences in glycosylation or by ligand binding.

Effect of Pulsed-Light Treatment on Milk Proteins and Lipids

Pulsed-light treatment offers the food industry a new technology for food preservation. It allows the inactivation of numerous micro-organisms including most infectious foodborne pathogens. In addition to microbial destruction, one can also question whether pulsed-light treatment induced conformational changes in food components. To investigate this question, the influence of pulsed-light treatment on protein components of milk was evaluated by using UV spectroscopy, spectrofluorometry, electrophoresis, and determination of amino acid composition. Pulsed-light treatment resulted in an increase of UV absorbance at 280 nm. The intrinsic tryptophan fluorescence of beta-lactoglobulin (BLG) showed a 7 nm red shift after 10 pulses. SDS-PAGE showed the formation of dimers after treatment of BLG by 5 pulses and more. No significant changes in the amino acid composition of proteins and lipid oxidation were observed after pulsed-light treatment. The obtained results indicated changes in the polarity of the tryptophanyl residue microenvironment of BLG solutions or changes in the tryptophan indole structure and some aggregation of studied proteins. Hence, pulsed-light treatment did not lead to very significant changes in protein components; consequently, it could be applied to process protein foods for their better preservation.

Improving safety of salami by application of bacteriocins produced by an autochthonous Lactobacillus curvatus isolate.

The aims of this study were to isolate LAB with anti-Listeria activity from salami samples, characterize the bacteriocin/s produced by selected isolates, semi-purify them and evaluate their effectiveness for the control of Listeria monocytogenes during manufacturing of salami in a pilot scale. Two isolates (differentiated by RAPD-PCR) presented activity against 22 out of 23 L. monocytogenes strains for bacteriocin MBSa2, while the bacteriocin MBSa3 inhibited all 23 strains in addition to several other Gram-positive bacteria for both antimicrobials and were identified as Lactobacillus curvatus based on 16S rRNA sequencing. A three-step purification procedure indicated that both strains produced the same two active peptides (4457.9 Da and 4360.1 Da), homlogous to sakacins P and X, respectively. Addition of the semi-purified bacteriocins produced by Lb. curvatus MBSa2 to the batter for production of salami, experimentally contaminated with L. monocytogenes (10(4)-10(5) CFU/g), caused 2 log and 1.5 log reductions in the counts of the pathogen in the product after 10 and 20 days respectively, highlighting the interest for application of these bacteriocins to improve safety of salami during its manufacture.

Molecular Characterization of a Novel Bacteriocin and an Unusually Large Aggregation Factor of Lactobacillus paracasei subsp. paracasei BGSJ2-8, a Natural Isolate from Homemade Cheese

Screening the collection of natural isolates from semi-hard homemade cheese resulted in isolation and characterization of strain Lactobacillus paracasei subsp. paracasei BGSJ2-8. The strain BGSJ2-8 harbors several important phenotypes, such as bacteriocin production, aggregation phenomenon, and production of proteinase. Bacteriocin SJ was purified by three-step chromatography. Mass spectrometry established molecular mass of the active peptide at 5372 Da. The auto-aggregation phenotype of wild-type (WT) strain was mediated by secreted aggregation-promoting factor (protein of molecular mass > 200 kDa), probably acting in cooperation with other cell surface protein(s). Comparative study of WT and its spontaneous nonaggregating derivative revealed that aggregation factor was responsible for the observed differences in the bacteriocin and proteinase activities. Bacteriocin SJ activity and resistance to different stresses were higher in the presence of aggregating factor. In contrast, proteinase activity was stronger in the nonaggregating derivative.