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International audience; The use of plant proteins to design colloidal food systems is a hot topic in the current context of the protein transition. However, replacing animal-derived proteins (in particular, dairy proteins) that have been traditionally used for this purpose by plant proteins is a challenge from various perspectives, and in particular, because of drastically different solubility and functionality. A possible route to mitigate these issues is to combine plant and dairy proteins, providing that their interactions can be understood from the molecular to the macroscopic scale. This review addresses the major advances that have occurred in the field of such blend-based systems, all the way from their behaviour in aqueous dispersions to their potential applications in gels, foams and emulsions.

International audience; Gelation of whey protein fractal aggregates was induced by simultaneously adding salt (CaCl 2 or NaCl) and acid (HCl). Further, simultaneous addition of both specifically binding (Ca 2þ) and non-binding (Na þ) cations was studied at a fixed protein charge density. The effect of adding mixtures of ions on pH, gelation kinetics, elastic modulus and gel microstructure was investigated. For all studied systems the time of gelation (t g) had an Arrhenius dependence on temperature, characterised by an activation energy (E a). The value of E a depended on the type and concentration of added ions. The elastic modulus of gels was found to be independent of the ion composition, reaching 0.2e0.3 kPa at a protein concentration of 40 g L À1 and 1 kPa at 60 g L À1. At conditions of strong electrostatic repulsion between aggregates , addition of NaCl reduced t g. However, when the repulsions were weak, further addition of NaCl increased t g .

Inulin is a prebiotic polysaccharide that is being increasingly used in food emulsions as fat replacer, due to its thickening effect. In the present work, oil in water (O/W) emulsions (5/95) have been prepared by means of a pressure homogenizer, using β-lactoglobulin as an emulsifier (0.5 %), and varying the polysaccharide content up to 10 %. The zeta potential measurements suggest that there are no significant protein-polysaccharide interactions in the continuous phase. An increase in viscoelastic properties and viscosity of these pseudoplastic systems is observed as inulin content increases. Although inulin has no surface activity, its presence seems to favor protein adsorption kinetics at the interface. Likewise, the distribution of droplet sizes study for emulsions throughout the storage time points out how bimodal distributions obtained remain constant, indicating their stability, which is reinforced by backscattering results. These prebiotic emulsions may enjoy a great demand by the food industry. La inulina es un polisacárido prebiótico que se utiliza cada vez más en emulsiones alimentarias como sustituyente de grasa, al tener un efecto espesante sobre las mismas. En el presente trabajo, se han preparado emulsiones aceite/agua (5/95) mediante un homogeneizador de presión, utilizando b-lactoglobulina como emulsionante a una concentración fija de 0,5 % (p/p), y variando el contenido en polisacárido desde 0 al 10 % (p/p). Las medidas de popotencial zeta sugieren que no existen interacciones importantes entre la proteína y el polisacárido en la fase continua, al tiempo que se observa un aumento de las propiedades viscoelásticas y viscosidad de estos sistemas pseudopláticos. Aunque la inulina no presenta actividad superficial, su presencia parece favorecer la cinética de adsorción de la proteína en la interfase. Asimismo, el estudio de la distribución de tamaños de gota de las emulsiones a lo largo del tiempo de almacenamiento permite observar cómo las distribuciones bimodales obtenidas permanecen constantes, indicando la estabilidad de las mismas. Esto queda reforzado por los resultados obtenidos por medio de un analizador óptico de emulsiones con detectores de luz transmitida y retrodispersa. Emulsiones con ingredientes prebióticos como las estudiadas tiene un gran potencial en la industria alimentaria. La inulina és un polisacàrid prebiòtic que s’utilitza cada vegada més en emulsions alimentàries com substituent del greix, en tenir un efecte espessidor sobre les mateixes. En el present treball, s’han preparat emulsions oli / aigua (5/95) mitjançant un homogeneïtzador de pressió, utilitzant β-lactoglobulina com emulsionant a una concentració fixa de 0,5% (p/p), i variant el contingut en polisacàrid des de 0 al 10% (p/p). Les mesures de potencial zeta suggereixen que no hi ha interaccions importants entre la proteïna i el polisacàrid a la fase contínua, alhora que s’observa un augment de les propietats viscoelàstiques i de la viscositat d’aquests sistemes pseudoplàstics. Tot i que la inulina no presenta activitat superficial, la seva presència sembla afavorir la cinètica d’adsorció de la proteïna a la interfase. Així mateix, l’estudi de la distribució de mides de gota de les emulsions al llarg del temps d’emmagatzematge permet observar com les distribucions bimodals obtingudes romanen constants, indicant l’estabilitat de les mateixes. Això queda reforçat pels resultats obtinguts per mitjà d’un analitzador òptic de emulsions amb detectors de llum transmesa i retrodispersa. Emulsions amb ingredients prebiòtics com les estudiades tenen un gran potencial en la indústria alimentària. Universidad de Sevilla (España) III Plan Propio de Docencia

This work reports the influence of glyoxal (GO)-derived glycation on the gastrointestinal enzymatic hydrolysis of β-lactoglobulin and β-casein. Reduced digestibility of glycated proteins was found in both gastric and intestinal stage. Distribution of Maillard reaction products in digests with different molecular weight ranges was investigated subsequently. The colorless and brown MRPs largely presented in the digests smaller than 20 kDa. However, the resistance of fluorescent advanced glycation end products (AGEs) to enzymatic hydrolysis gradually increased during glycation, rendering fluorescent AGEs largely present in the digests larger than 20 kDa. No free N (e)-carboxymethyllysine (CML) was detected in digests. The relative amount of CML in digests larger than 1 kDa was higher than that of Lys, demonstrating the hindrance of CML to enzymatic hydrolysis. This study highlights the resistance of GO-derived AGEs to digestive proteases via blockage of tryptic cleavage sites or steric hindrance, which is a barrier to the absorption of dietary AGEs.

International audience; Several prochiral NCN pincer complexes of palladium(II) including hemilabile ligands and a long aliphatic chain were synthesized and characterized spectroscopically. In some of the complexes, the presence of two different substituents on the N donor atoms made them stereogenic so that they were isolated as a mixture of diastereoisomers, which could be differentiated by 1 H NMR. Binding of some of these complexes to bovine b-lactoglobin by insertion within its inner cavity was theoretically investigated by molecular docking simulations and experimentally confirmed by CD spectroscopy. Adjunction of H-bond donor substituents on the ligand framework gave more stable supramolecular protein – complex assemblies. These constructs were shown to catalyze aldol condensation reactions in aqueous medium affording in some cases the less favorable cis-product. Since the corresponding complexes gave exclusively the trans product in the absence of b-lactoglobulin, this unusual diastereoselectivity was ensued by the second sphere of coordination brought by the protein host.

beta-Lactoglobulin (BLG) is an important nutrient of dairy products and an important allergen in cow's milk allergy. The aim of this study was to investigate the potential of laccase to cross-link BLG in the presence of an apple phenolic extract (APE) and to characterize the obtained products for their digestibility by pepsin and pancreatin. The composition of the apple phenolics used for cross-linking was determined by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The apple phenolic extract contained significant amounts of quercetin glycosides, catechins and chlorogenic acid. The laccase cross-linked BLG in the presence of apple phenolics. The polymerization rendered the protein insoluble in the reaction mixture. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the cross-linking reaction mixture revealed a heterogeneous mixture of high molecular masses (cross-linked BLG), with a fraction of the BLG remaining monomeric. Enzymatic processing of BLG by laccase and apple polyphenols as mediators can decrease the biphasal pepsin pancreatin digestibility of the monomeric and cross-linked protein, thus decreasing its nutritional value. In addition, reduced BLG digestibility can decrease its allergenic potential. Apple polyphenols can find usage in the creation of new, more functional food products, designed to prevent obesity and hypersensitivity-related disorders. β-Laktoglobulin (BLG) je važan nutrijent mlečnih proizvoda i važan alergen kod alergija na kravlje mleko. Cilj ove studije je bilo ispitivanje potencijala lakaze da unakrsno poveže BLG u prisustvu fenolnog ekstrakta jabuke (APE), kao i karakterizacija dobijenih proizvoda sa aspekta njihove digestibilnosti pepsinom i pankreatinom. Kompozicija fenola jabuke korišćenih za unakrsno povezivanje određena je pomoću LC-ESI-MS. Fenolni ekstrakt jabuke sadrži znatne količine glikozida kvercetina, katehine i hlorogensku kiselinu. BLG je unakrsno povezan lakazom u prisustvu fenola jabuke, pri čemu je polimerizacija učinila BLG nerastvornim u reakcionoj smeši. SDS-PAGE analiza pokazala je da reakciona smeša sadrži heterogenu smešu velikih molekulskih masa (unakrsno povezan BLG), kao i deo zaostalog monomernog BLG. Enzimsko procesovanje BLG lakazom, u prisustvu polifenola jabuke kao medijatora, može smanjiti bifaznu pepsin-pankreatinsku digestibilnost kako monomernog, tako i unakrsno povezanog BLG, i na taj način smanjiti njegovu nutritivnu vrednost. Takođe, smanjena digestibilnost BLG može smanjiti njegov alergeni potencijal. Polifenoli jabuke mogu se koristiti za kreiranje novih, funkcionalnijih prehrambenih proizvoda, dizajniranih za prevenciju kako gojaznosti, tako i bolesti vezanih za preosetljivost.

The interactions effect between (+)-catechin, β-lactoglobulin and two types of polysaccharides (pectin and chitosan) were studied at pH 4. Dynamic light scattering (DLS) revealed that pectin formed smaller complexes in the presence of (+)-catechin and β-lg, while chitosan presented a reduced number of populations with higher particle size. Dynamic interfacial data (obtained by a drop tensiometer) revealed that the complexes formed between pectin (+)-catechin and β-lg slowed down the β-lg migration to the oil/water interface; contrarily to the chitosan mixtures where protein migrated faster. The surface dilatational modulus of β-lg was ~45 mN/m and increased to ~60 mN/m when mixing with pectin and (+)-catechin, while the values of the surface dilatational modulus for the chitosan mixed interfacial films decreased to ~35 mN/m. The free (+)-catechin content decreased 12% and 10% when interacting with pectin and chitosan, respectively, as compared to pure (+)-catechin. However, the (+)-catechin antioxidant activity were not affected by the interactions. Complexes formed between polyphenols, proteins and polysaccharides could be used as a good alternative to understand and consequently improve the phytochemicals stability in food matrices. Fil: Oliveira, Ana Lúcia Silva. Universidade Católica Portuguesa; Portugal Fil: Von Staszewski, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pizones Ruiz Henestrosa, Víctor Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pintado, Manuela. Universidade Católica Portuguesa; Portugal Fil: Pilosof, Ana Maria Renata. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

In this paper we describe the rheological behaviour of Ca2+-induced cold-set gels of whey protein mixtures. Cold- set gels are important applications for products with a low thermal stability. In previous work [1], we determined the state diagram for whey protein mixtures that were heated for 10 h at pH 2 at 80°C. Under these conditions, the major whey protein, β-lactoglobulin (β-lg), forms fibrils. When whey protein mixtures are heated at protein concentrations in the liquid solution regime of the state diagram, cold-set gels can be formed by adding Ca2+ ions at pH 7. We studied the rheological behaviour of cold-set gels for various sample compositions for whey protein mixtures. When keeping the total whey protein concentration constant, the elastic modulus, G’, for the cold-set gels decreased for increasing a-lactalbumin and bovine serum albumin ratios, because less material (β- lg fibrils) was available to form a gel network. In the cold-set gels the interactions between the β-lg fibrils induced by the calcium ions are dominant. The β-lg fibrils are forming the cold-set gel network and therefore determine the gel strength. a-Lactalbumin and bovine serum albumin are not incorporated in the stress-bearing structure of the gels.

The avarone/avarol quinone/hydroquinone couple shows considerable antitumor activity. In this work, covalent modification of b-lactoglobulin by avarone and its derivatives as well as by the synthetic steroidal quinone 2,5(10)-estradiene-1,4,17-trione and its derivatives were studied. The techniques for studying chemical modification of b-lactoglobulin by quinones were: UV/Vis spectrophotometry, SDS PAGE and isoelectrofocusing. SDS PAGE results suggest that polymerization of the protein occurs. It could be seen that the protein of 18 kD gives the bands of 20 kD, 36 kD, 40 kD, 45 kD, 64 kD and 128 kD depending on modification agent. The shift of the pI of the protein (5.4) upon modification toward lower values (from pI 5.0 to 5.3) indicated that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.

International audience; The decrease in medium permittivity due to temperature rise could play an important role in the denaturation/polymerization ofheat-treated b-lactoglobulin. The permittivity of a b-lactoglobulin suspension was decreased, either by heat treatment or by addingorganic solvent, and the rate of loss of the native b-lactoglobulin was estimated by SDS-PAGE analysis. Setting the permittivity to63.8 by addition of organic solvent, induced b-lactoglobulin polymerization, but it differed from that induced by change in permittivity upon thermal treatment itself (heating to 70 C). To explain the protein polymerization process induced by heating, other physical parameters should be taken into account, such as change in water organization around the protein.

Protein and polysaccharide are together with lipids the main ingredients of food and procure both nutrition and texture. A recent tendency in the food industry is to develop more healthy products that contain less fat and salt. The addition of polysaccharides is recognized as a good way to control the texture of food products. The texture of many food products is determined by gelation of either the proteins or the polysaccharides, or both. When both are present, gelation of the protein or the polysaccharide will be influenced by the presence of the other type. Understanding of the physical chemical properties of aqueous solutions and gels containing protein and polysaccharides by themselves and in mixtures is needed for a rational development of novel food products. This thesis describes an investigation of mixtures of the globular protein β-lactoglobulin (β-lg) and the polysaccharide κ-carrageenan (κ-car). κ-car is a polysaccharide isolated from algae that is often used as an additive in food industry. In solution it forms a gel below a critical temperature that depends on the amount and the type of salt. Addition of κ-car can improve the smoothness, creaminess, and body of food products and is often usedmodify the texture of dairy products. Therefore it is important to understand the interaction of κ-car with milk proteins such as β-lg, which is the main protein component of whey. The objective of the present investigation was to study the structure andthe mechanical properties of β-lg aggregates or gels when mixed with κ-car and to study the influence of the former on the gelation of κ-car. The focus was on the sensitivity of the system to calcium ions Protein particles were either formed separately and subsequently mixed with κ-car or formed directly in mixtures of κ-car and native β-lg by heating. The two different methods of preparation were compared with the same composition of polymers. The research presented in this thesis is essentially experimental using scattering techniques and confocal laser scanning microscopy to study the structure and shear rheology to study the dynamic mechanical properties.The size and morphology of protein aggregates formed by heating β-lg is strongly dependent on the concentration of Ca2+ that binds specifically to the proteins. It is shown that larger aggregates formed in the presence of Ca2+ micro-phase separate already at low κ-car concentrations. Therefore the structure of mixed systems is extremely sensitive to the amount of Ca2+ present in the system. The presence of protein aggregates was found to reinforce potassium induced κ-car gels, but it was also found that addition of CaCl2 strengthens potassium induced pure κ-car gels. We show that the reinforcement by addition of protein aggregates is caused by the transfer of a fraction of Ca2+ from β-lg to κ-car. It was shown that potassium induced gelation of κ-car also occurs during cooling heat-set β-lg gels formed in mixtures at higher protein concentrations leading to interpenetrated networks that are stronger than the sum of the individual networks. Themain conclusion of the investigation reported here is that the competition of κ-car and β-lg for calcium ions determines both the structure and the mechanical properties of the mixed systems., Les protéines et les polysaccharides constituent avec les lipides les principaux ingrédients de l’alimentation et lui confèrent à la fois ses propriétés de nutrition et de texture. Une tendance actuelle de l’industrie agroalimentaire est d’élaborer des aliments plus sains c'est-à-dire moins gras et moins salés. A ce titre, les polysaccharides sont des agents detexturation efficaces lorsqu’ils sont utilisés seuls ou en combinaison avec des protéines. Le développement de nouveaux produits alimentaires nécessite donc de rationaliser et mieux comprendre les propriétés physico-chimiques des solutions et des gels mixtes à base de protéines et de polysaccharides. Au cours de ce travail de thèse, nous avons étudié des mélanges de protéines globulaires (la β-lactoglobuline: β-lac) et de polysaccharide (le κ-carraghénane: κ-carr). Ce dernier provient d’algues et, en solution, il conduit à des gels au dessous d’une température critique qui dépend de la nature du sel ajouté. Le κ-carr est un additif important dans l’industrie alimentaire et plus particulièrement comme texturants des produits laitiers. Il est donc essentiel de comprendre les interactions qu’il développe avec les protéines du lait comme la β-lac.L’objectif de ce travail est d’étudier la structure et les propriétés mécaniques d’agrégats ou de gels de β-lac mélangés avec du κ-carr et d’étudier leur influence sur la gélification de ce dernier. Nous nous sommes plus particulièrement intéressés à la sensibilité des mélanges aux ions calcium. Des agrégats protéiques ont été formés soit indépendamment puis mélangés au κ-carr soit directement in situ en dénaturant thermiquement des mélanges κ-carr/β-lac native. Les deuxméthodes de préparation ont été comparées pour des compositions constantes des mélanges. La diffusion de la lumière, la rhéologie et la microscopie laser confocale ont été mises en oeuvre pour étudier la texture des mélanges.La taille et la morphologie des agrégats protéiques dépendent fortement de la concentration en ions calcium ajoutés qui se lient spécifiquement aux protéines. Nous avons montré que les très grands agrégats protéiques formés en présence de calcium conduisent à une microséparation de phase quand ils sont mélangés avec du κ-carr même à très faible concentration. Ainsi, la structure des systèmes mixtes est très sensible à la quantité de calcium en présence. Lesagrégats protéiques renforcent les gels de κ-carr formés en présence de potassium tout comme l’ajout de calcium. Ce renforcement dans le cas des agrégats protéiques est dû au transfert des ions calcium de la β-lac vers le κ-carr. De plus, nous avons montré que la gélification du β-carr induite par des ions potassium continuait à avoir lieu en refroidissantdes mélanges κ-carr/β-lac où cette dernière est dénaturée in situ. Cela conduit à des réseaux interpénétrés qui sont plus forts mécaniquement que la somme des deux réseaux pris individuellement. En conclusion, nous avons montré que la compétition entre la β-lac et le κ-carr pour les ions calcium était le paramètre de contrôle des propriétés texturales desgels mixtes.

Cross-linking of proteins has been exploited by the food industry to change food texture and functionality but the effects of these manipulations on food allergenicity still remain unclear. To model the safety assessment of these food biopolymers, we created cross-linked bovine beta-lactoglobulin (CL-BLG) by laccase treatment. The purpose of the present study was to compare the immunogenicity and allergenicity of CL-BLG with native BLG in a mouse model of food allergy. First, BALB/c mice were intragastrically sensitized and orally challenged with BLG or CL-BLG and BLG-specific serum antibodies and splenic leukocyte cytokine production and cell proliferation were measured. Hereafter, epithelial protein uptake was monitored in vitro and in vivo and the effects of BLG cross-linking on interactions with dendritic cells were analyzed in vitro. Sensitization of mice with CL-BLG resulted in higher levels of IgE, IgG1, and IgG2a. In contrast, a subsequent oral challenge with CL-BLG resulted in lower mast cell degranulation. Cross-linking of BLG reduced its epithelial uptake but promoted sampling through Peyer's patches. Differences in endocytosis by dendritic cells (DCs) and in vitro endolysosomal processing were observed between BLG and CL-BLG. CL-BLG primed DCs induced higher Th2 response in vitro. Cross-linking of BLG increased its sensitizing capacity, implying that the assessment of highly polymerized food proteins is of clinical importance in food allergy. Moreover, manufacturers of foods or therapeutic proteins should pay considerate attention to the health risk of protein aggregation. Free full text: [https://doi.org/10.1093/toxsci/kfu062]

Green tea polyphenols have a good antioxidant capacity but poor interfacial activity. β-lactoglobulin (β-lg) was used as an emulsifier agent and also as a carrier molecule by spontaneous nanocomplexes formation with green tea polyphenols. Oil-in-water emulsions containing liver fish oil rich in ω-3 fatty acids were formulated using these nanocomplexes at pH 6. The interfacial behavior of these complexes showed that both surface pressure and dilatational properties decreased as compared with pure β-lg. However, the initial droplet size and stability of emulsions were improved in the presence of the nanocomplexes. Moreover, the oxidative stability of liver fish oil was improved by the presence of polyphenols. Fil: Von Staszewski, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pizones Ruiz Henestrosa, Víctor Manuel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pilosof, Ana Maria Renata. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

A method for the recovery and fractionation of whey proteins from a whey protein concentrate (80%, w/w) by hydrophobic interaction chromatography is proposed. Standard proteins and WPC 80 dissolved in phosphate buffer with ammonium sulfate 1 M were loaded in a HiPrep Octyl Sepharose FF column coupled to a fast protein liquid chromatography (FPLC) system and eluted by decreasing the ionic strength of the buffer using a salt gradient. The results showed that the most hydrophobic protein from whey is α-lactalbumin and the less hydrophobic is lactoferrin. It was possible to recover 45.2% of β-lactoglobulin using the HiPrep Octyl Sepharose FF column from the whey protein concentrate mixture with 99.6% purity on total protein basis.

Beta-Lactoglobulin (BLG) is the main allergenic protein in cow’s milk and can cause allergy even when present at very low concentration. The aim of this work is to develop an innovative sample prepara- tion method fully compatible with capillary electrophoresis and laser-induced fluorescence detection for improving the sensitivity when analyzing BLG. Different types of baby food were on purpose contaminated with diverse dairy desserts and submitted to thermal treatment to simulate potential contamination at production. Sample preparation prior to CE analysis was performed by the classical extraction method and by the innovative one, and the results were compared. Analysis was performed by capillary electrophoresis with laser-induced fluorescence detection. The innovative method permitted to detect contaminations as low as 1 part of yoghurt in 10 000 parts of baby food., Financial support from the Spanish Ministry of Education and Science (projects DEP2006-56207 C03-01 and CTQ2006-05214) is acknowledged.

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 °C for 24 h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3 log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24 h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 μg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, α-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than β-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 μg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 μg/ml). Regarding α-lactalbumin and β-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages. © 2008. Fil: Pescuma, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Mozzi, Fernanda Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Font, Graciela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina

Çalısmada Siyah-Alaca sıgırlarda sütün protein içerigi ve komposizyonuna mevsim, laktasyon sayısı, laktasyon dönemi ve somatik hücre sayısının (SHS) etkileri arastırılmıstır. Laktasyon boyunca toplanan süt örneklerinde protein fraksiyonları Poliakrilamid-Jel-Elektroforezi’nde ayrılmıs ve densitometre yardımıyla her bir protein fraksiyonunun içerigi belirlenmistir. Sütün protein içerigine mevsim, laktasyon dönemi ve SHS’nın etkileri önemli bulunmustur. aS1-kazein (-Cn) içerigine mevsim, laktasyon dönemi, laktasyon sayısı, SHS; b-Cn içerigine mevsim, laktasyon sayısı, SHS; k-Cn içerigine laktasyon sayısı, laktasyon dönemi, mevsim; a-Laktalbumin (-La) içerigine mevsim, laktasyon dönemi, SHS; b-Laktoglobulin (-Lg) içerigine laktasyon dönemi, laktasyon sayısı, mevsim ve SHS önemli etkiye sahiptir. Sütün protein, aS1-Cn ve b-Lg içerigi Eylül- Aralık döneminde maksimum seviyededir. Laktasyon sayısının artmasıyla birlikte aS1-Cn içerigi azalmakta, b-Cn içerigi artmaktadır. Besinci ve daha sonraki laktasyonlarda k-Cn ve b-Lg içerigi minimum seviyededir. Laktasyonun ikinci ayında minimum olan protein ve b-Lg içerigi laktasyon sonuna kadar sürekli artmaktadır. aS1-Cn içeriginde laktasyon süresince azalma belirlenmistir. SHS’nın artmasıyla a-La, b-Lg ve k-Cn içeriginin arttıgı; aS1-Cn ve b-Cn içeriginin azaldıgı tespit edilmistir. Effects of season, parity number, stage of lactation and somatic cell count (SCC) on protein content as well as protein composition were investigated in Holstein Friesian cattle. Milk protein fractions were quantified after polyacrylamide gel electrophoresis, using computer-assisted densitometer. Milk protein content was effected by season, stage of lactation and SCC. Season, stage of lactation, parity number and SCC affected aS1-casein (-Cn) content. b-Cn content was effected by season, parity number and SCC. Parity number, stage of lactation and season had significant effect on k-Cn content. a-Lactalbumin (-La) content was effected by season, stage of lactation and SCC, while stage of lactation, parity number, season as well as SCC affected b-Lactoglobulin (-Lg) content. Protein, aS1-Cn and b-Lg content was higher between September-December. Increased parity number was associated with decreased aS1-Cn and increased b-Cn content. Last parity subclass had minimum k-Cn and b-Lg content. Protein and b-Lg content was minimum in second lactation monat and then increased as lactation progressed, while aS1-Cn content was decreased. Increased SCC were associated with increased a-La, b-Lg, k-Cn as well as decreased aS1-Cn and b-Cn content.

The use of hydrolysed formulas for feeding babies is efficient for preventing allergic diseases. Several hypoallergenic formulas (HF) based on hydrolysed cows’ milk proteins have been commercialised. However, some children fed such formulas suffer allergic reactions. b-lactoglobulin (bLG) is the main allergen present in cows’ milk. In addition to the whole protein, bLG fragments have also shown allergenic character. Detection of bLG and its peptides can be a useful marker of the presence of milk in food. Enzyme linked immunoassays (ELISAs) are general methods used to analyse allergens in foods. These techniques are sensitive and selective but they have several drawbacks. Immunoassay methods performed in chromatographic systems keep the advantages of classic ELISA while avoiding the disadvantages. A sandwich enzyme linked immunoaffinity chromatography (ELIAC) method was applied to determine bLG and its peptides in several commercial HF based on different milk proteins. Samples were also fractionated by employing membranes with different molecular weight cut-offs, and the antigenicity of the peptide fractions was determined. Applying the ELIAC method, for which one analysis takes 30 min, bLG was determined at the pM level in formulas based only on caseins, and at the nM to mM level in formulas based on hydrolysed whey proteins. Peptides of molecular weight less than 3000 derived from bLG showed positive results in ELIAC assays, indicating antigenic character., A.P. acknowledges the Spanish Ministry of Science and Technology for a predoctoral grant. This work has been supported by CICYT (projects AGL2000-1480 and TIC2003-1906).

Beta-Lactoglobulin (Beta;LG) is one of the main components of whey proteins. Among other reasons, its allergenic character makes necessary its determination in hypoallergenic foods and biopharmaceutical products at very low concentration making use of very sensitive methods. In this paper, the development of an enzyme linked immunoaffinity chromatographic (ELIAC) method in sandwich format is described. The optimized method includes the use of a minicolumn (4.9 mm × 1 mm) packed with Nucleosil® 4000-7OH derivatized with high density of antibody coverage, the employment of rabbit serum and Zwittergent® 3–12 as mobile phase additives, and the incubation of the sample containing BetaLG with anti BetaLG labeled with horseradish peroxidase prior to their injection into the immunochromatographic system. The method allows determination of betaLG with a practical detection limit of 20.7 pM and a sensitivity of 1.05 × 109 AU mol−1 L. The application of this ELIAC method to determine BetaLG in a commercial hypoallergenic formula based on caseins is shown. The amount of allergen, 0.32 pmol BetaLG per gram of sample, is about 5 million times lower than that naturally occurring in cow’s milk.

Orientador: Carlos Alberto Gasparetto Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos Resumo: Este é um trabalho sobre a incrustação de proteínas de origem animal, b-lactoglobulina e ovoalbumina, sobre superfícies cilíndricas aquecidas, comuns nos equipamentos de tratamento térmico e de concentração por evaporação. A incrustação é um fenômeno de transferência de massa que é uma função da solubilidade dessas proteínas. Para descrever a cinética da incrustação foi desenvolvido um algoritmo de cálculo que teve como subsídio a solubilidade das proteínas. A solubilidade foi determinada experimentalmente na faixa de 40-60°C, incluindo a dependência do pH na faixa de 3,5 a 7,8 para a b-lactoglobulina e 6,0 a 9,0 para a ovoalbumina. O algoritmo foi desenvolvido para aplicação no interior de tubos cilíndricos permitindo um modelo matemático unidimensional, mais simples devido à simetria. O sistema de equações foi resolvido pelo método de diferenças finitas com a aplicação de um algoritmo desenvolvido na linguagem Fortran 77. Os resultados mostram que a solubilidade protéica depende da temperatura e do pH da solução e que a cinética de incrustação é dependente da temperatura do fluido e da sua vazão mássica. O tempo necessário para o decréscimo de 30% no raio interno do tubo foi menor para maiores valores de temperatura e vazão. A deposição das proteínas foi mais acentuada na região de entrada do tubo Abstract: This is a research work on b-1actoglobulin and ovalbumin fouling over cylindrical heated surfaces, present on heat transfer equipments and evaporators. Fouling is a mass transfer phenomenon which is a function of protein solubility. To describe fouling kinetics an algorithm was developed based on the protein solubility. The protein solubility was determined experimentally in the range of 40-60°C, including dependence with pH in the range of 3.5-7.8 for b-1actoglobulin and 6.0-9.0 for ovalbumin. The algorithm was developed for cylindrical tubes thus leading to a less complex one-dimensional mathematical model, due to symmetry. The system of equations was solved by the finite differences method with an algorithm developed in Fortran 77 language. Experimental results showed that protein solubility depends on the solution temperature and pH and the fouling kinetics was dependent on the fluid temperature and flow rate. The time needed for 30% decrease on the tube internal radius was smaller for higher values of both temperatures and flow rate. Protein deposition was more intense in the tube entrance Doutorado Doutor em Engenharia de Alimentos

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS-PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β-Lg, para-κ-casein was also found in the serum fraction. A high proteolytic activity against κ-casein could be responsible for the hydrolysis of serum-liberated κ-casein or could have enhanced the liberation of β-Lg–para-κ-casein complexes through proteolysis of micellar κ-casein., We acknowledge the finnancial support given by the CICYT through project ALI 95-0046-CO2-02.

The genotypes for x-casein (x-CN), p-lactoglobulin -LG) and growth hormone (GH) were determined by polymerase chain reaction (PCR) and restriction enzyme digestion in seven breeds of cattle (Nelore, Gyr, Guzera, Caracu, Charolais, Canchim and Santa Gertrudis). k-Casein had two alleles with the A allele occurring at a higher frequency in Bos indicus breeds (0.93, 0.92 and 0.91% for Gyr, Guzera and Nelore, respectively). The p-lactoglobulin locus had two alleles in all of the breeds. European breeds had a higher frequency of the p-LG A allele than Zebu breeds. The GH locus had two alleles (L and V) in Bos taurus and was monomorphic (L allele only) in all of the Bos indicus breeds evaluated. The highest frequency for the V allele was observed in Charolais cattle. The markers used revealed a considerable similarity among breeds, with two main groups being discernible. One group consisted of Zebu and Santa Gertrudis breeds and the other consisted of European and Canchim breeds. Made available in DSpace on 2021-09-16T17:00:24Z (GMT). No. of bitstreams: 1 PROCI-1999.00091.pdf: 19756 bytes, checksum: 785de72a62b1f2212664f5d8cae7daf8 (MD5) Previous issue date: 1999

The solution behavior of [beta]-lactoglobulin ([beta]-Lg) at pH 7, 5 and 3 as a function of temperature, protein and solute concentration was studied. All possible morphologies for [beta]-Lg in water and ethanol or (NH4 )2SO4 were represented in ternary phase diagrams in the full range of each component. The effect of solute addition and temperature on phase behavior was found to be different according to pH. The characteristics of the intrinsic species involved in the formation of the different morphologies were studied in dilute solutions. It was found that dimers tended to dissociate into monomers as protein concentration decreased. Ethanol or (NH4) 2SO4 induced a conformational change on dissociated monomers that was accompanied by protein swelling. Changes in protein association were correlated with the rheological and thermal behavior of [beta]-Lg. The formation of gel and paste morphologies was compared using rheological analysis. Ethanol addition decreased while (NH4)2SO4 increased the thermal stability of [beta]-Lg and this behavior could explain several observed morphology changes. The microstructure of heat-induced gels as determined by scanning electron microscopy, also showed differences according to pH and solute addition. The solution behavior of [beta]-Lg and the previously studied proteins, ovalbumin, bovine serum albumin and gelatin were compared. From this work, it can be concluded that protein solution behavior is a complex function of a wide range of structural parameters.

Small amounts of dietary proteins pass undegraded into the circulation, an event initiating antibody production. Antibodies to dietary antigens might be beneficial by immune elimination of antigen as well as potentially disease-provoking by eliciting harmful immune effector functions, such as immune complex-mediated complement activation or antibody-dependent cell-mediated cytotoxicity (ADCC). The biological activities of antibodies are related to their characteristics, such as isotope, epitope specificity and avidity.The aim of the study was to evaluate characteristics of serum antibodies to dietary anti-gens in children with food allergy/intolerance, focussing on ADCC-mediating capacity, isotype/subclass pattern and avidity.Sera were analysed with regard to ADCC-mediating capacity, i.e. lysis of target cells (erythrocytes coated with dietary antigen) in the presence of cytotoxic effector cells (NK cells or monocytes). Serum antibody levels of IgG, IgG subclasses and IgA against b-lactoglobulin or gliadin were determined with ELISA. Affinity chromatography was used for selective depletion of IgG subclasses. Antibody avidity was estimated using a thiocyanate elution enzyme immunoassay.Sera from children with cow´s milk protein intolerance (CMPI) and gastrointestinal symptomatology showed a substantial ADCC reactivity in a b-lactoglobulin-specific system with NK cells as effectors. Coeliac children exhibited moderate ADCC reactivity of their sera, despite high levels of antibodies against b-lactoglobulin. Absorption experiments indicated that primarily IgG1 antibodies were responsible for this b-lactoglobulin-specific ADCC reactivity. Accordingly, ADCC reactivity of indi-vidual sera correlated with IgG1 anti-b-lactoglobulin antibody levels. Sera from CMPI children with gastrointestinal symptoms, most of which had a high ADCC reactivity, also had a distinctive subclass pattern of their anti-b-lactoglobulin antibodies with high relative proportions of IgG1.Sera from coeliac children had poor capacity to mediate NK cell-induced ADCC against gliadin-coated target cells. However, using monocytes as ADCC effector cells, sera from children with active coeliac disease showed a substantial ADCC-mediating capacity. Depletion of IgG1 markedly diminished the ADCC, indicating that IgG1 anti-bodies against gliadin predominantly mediate this gliadin-specific monocyte ADCC. Further, IgG1 as well as IgG3 correlated positively to ADCC reactivity.The avidity of antibodies against dietary antigens seemed to increase progressively during early childhood in healthy as well as diseased children. Active coeliac disease was connected with an accelerated avidity maturation.It is conceivable that several immune effector mechanisms may coexist and synergize in the disease process of food allergy/intolerance. The results of the present study suggest that IgG antibodies against gliadin or b-lactoglobulin, predominantly of the IgG1 iso-type, may be involved in the disease process of coeliac disease or CMPI with gastroin-testinal symptoms, via their capacity to mediate an ADCC reaction

Variously lactosylated species of β-lactoglobulin AB were analysed by capillary electrophoresis at low pH and high pH with and without buffer additives. By simple capillary zone electrophoresis at either low or high pH the lactosylated forms had longer migration times than the native β-lactoglobulin, most likely due to their larger molecular size and lower pI. By micellar electrokinetic chromatography using SDS as detergent the lactosylated forms of β-lactoglobulin had shorter migration times than the native species, due to less interaction with the micelles. The methods used were also able to distinguish between more or less lactosylated forms of β-lactoglobulin. By all methods used, partial separation of at least five forms of β-lactoglobulin were obtained. However, baseline separation of β-lactoglobulin with varying number of lactose units attached was not obtained, probably due to the presence of both variants of β-lactoglobulin with various numbers of lactosyl groups at different sites.

Food emulsion stability is strongly influenced by the interactions in the adsorbed layer around emulsion droplets. Competitive adsorption of pure milk proteins (13-casein or 13 -lactoglobulin) with nonionic surfactants in oil-in-water emulsions is shown to depend on the age of the adsorbed protein layer. Evidence is presented for slow accumulation of protein at the oil-water interface and/or partition of protein into the oil phase of emulsion droplets containing oil-soluble surfactant associated in reversed micelles. Small amounts of added water-soluble surfactant or calcium ions are shown to have a pronounced effect on the droplet coalescence rate under turbulent shear flow conditions. It appears that orthokinelic emulsion stability is related to the composition and surface rheology of the adsorbed layer and to the degree of perikinetic flocculation of the emulsion droplets.

A proper understanding of the behavior of food products requires knowledge of its structure, i.e., the spatial arrangement of the various structural elements and their interactions. The structure can properly be studied by visual observation techniques. In products such as fat spreads, creams, dressings, cheese, bread, milk, yoghurt, whipped cream, and ice cream, different structural elements can be distinguished. A number of those elements are discussed, viz., water droplets, oil droplets, gas cells, particles, fat crystals and strands. In addition examples of interactions between structural elements are presented, viz., oil droplets/matrix, protein/protein, protein carbohydrate, and fat crystal/fat crystal interactions. Finally, it is indicated how these elements cooperate in the formation of structure and contribute to function and macroscopic behavior of food products. Particular attention is given to fat spreads, processed cheese, protein gelation, and examples of the mutual interaction of milk proteins and of carbohydrates with milk proteins. It is expected that a proper understanding of the relation between structure and function will help us to design new ways of structuring in our continuing efforts to manufacture high quality, healthy and tasty food products.

Macromolecular assemblies of proteins and hydrocolloids in low-fat dairy products contribute to the structure and texture of these foods. Immunolocalization techniques were used to identify B-lactoglobulin, casein, bovine whey proteins, and egg albumin in low-fat frozen desserts, reduced fat process cheese, and salad dressings. SimpJesse18 I 00 protein particles were examined and characterized by these methods and compared to naturally occurring protein structures in the low-fat foods. Hydrocolloid identification in low-fat salad dressing was accomplished by complexing cellulase and hemicellulase with colloidal gold and applying the probe as a pre-embedding step, on sections of embedded specimens or on material dried on grids. This enzyme-gold method may be generally applied for identification of cellulose and hemicellulose in complex food systems. Immunolocalization techniques employed in this study should prove useful in probing the fundamental structure-texture relationship in foods.

The influence of Tween 20 (polyoxyethylene sorbitan monolaurate) on the composition of adsorbed protein films in hydrocarbon oil-in-water emulsions containing B-lactoglobulin and a-lactalbumin has been investigated at neutral pH. The kinetics of pol ymerization of adsorbed 13-lactoglobulin via sulphydryl-disulphide interchange is little affected by Tween 20. Add ing emulsifier after homogenization leads to slightly greater competitive d i splacement of protein from the interface for emul sions made with pure 8-lactoglobulin than for equivalent emulsions made with 0.-lactalbumin . For emulsions made with the two proteins in a 1 : 1 molar ratio, adding emulsifier after homogeniza t ion leads to more displacement of 8-l actoglobulin than a-lactalbumin. I n a n emulsion containing both proteins a nd Tween 20 added before homogenization at an emulsifier -toprotein molar ratio of 2:1, there i s three times as much a-lactalbumin adsorbed at the droplet surface as S-lactoglobulin. It appears that the kinetics of compe t itive protein exchange between the bulk phase and the oil-water interface is facilitated by the presence of water-soluble surfactant.

Irreversible modifications in tertiary structure, surface hydrophobicity, and association state of β-lactoglobulin were studied after exposure to high pressure (600 and 900 MPa) of solutions of the protein at neutral pH and at different concentrations. Only minor irreversible structural modifications were evident even for treatments as intense as 15 min at 900 MPa. The occurrence of irreversible modifications was time-progressive at 600 MPa but was complete within 2 min at 900 MPa. The irreversibly modified protein was soluble, but some covalent aggregates were formed. Formation of aggregates increased with increasing protein concentration and was prevented by blocking the free thiol moiety in each β-lactoglobulin monomer. Results are discussed in light of their practical relevance, and a unifying denaturation mechanism is envisaged for β-lactoglobulin. In the proposed mechanism, release of monomers represents one of the earliest events, while association of transiently modified monomers stabilizes the de...

The avarone/avarol quinone/hydroquinone couple, as well as their derivatives show considerable antitumor activity. In this work, covalent modifications of b-lactoglobulin, isolated from cow milk, by avarone, its model compound 2-tert-butyl-1,4-benzoquinone, and several of their alkylthio derivatives were studied. The techniques applied for assaying the modifications were: UV/VIS spectrophotometry, SDS PAGE and isoelectrofocusing. The results of the SDS PAGE suggest that polymerisation of the protein occurs. The shift of the pI of the protein upon modification toward lower values indicates that lysine amino groups are the principal site of the reaction of b-lactoglobulin with the quinones.