Stability of casein micelles cross-linked with genipin: a physicochemical study as a function of pH

Chemical or enzymatic cross-linking of casein micelles (CMs) increases their stability against dissociating agents. In this paper, a comparative study of stability between native CMs and CMs cross-linked with genipin (CMs-GP) as a function of pH is described. Stability to temperature and ethanol were investigated in the pH range 2.0-7.0. The size and the charge ($\zeta$-potential) of the particles were determined by dynamic light scattering. Native CMs precipitated below pH 5.5, CMs-GP precipitated from pH 3.5 to 4.5, whereas no precipitation was observed at pH 2.0-3.0 or pH 4.5-7.0. The isoelectric point of CMs-GP was determined to be pH 3.7. Highest stability against heat and ethanol was observed for CMs-GP at pH 2, where visible coagulation was determined only after 800 s at 140 $^\circ$C or 87.5% (v/v) of ethanol. These results confirmed the hypothesis that cross-linking by GP increased the stability of CMs.Comment: 5 pages, 2 figures, International Dairy Journal, 201

Simple and multilayers emulsion stabilized by lysozyme and β-lactoglobulin

β-lactoglobulina (BLG) é uma proteína presente no soro de leite, com propriedades emulsificantes, nutricionais e de transporte, amplamente utilizada como ingrediente. Esta proteína tem um ponto isoelétrico (PI) baixo, o que permite a sua interação eletrostática com proteínas de alto PI, como a lisozima (LIS), uma proteína da clara de ovo. Essas proteínas são usadas como emulsificantes, de forma a aumentar a estabilidade cinética de emulsões. A estabilidade das emulsões é afetada pela composição das fases contínua e dispersa. Emulsões óleo em água (O/A) de misturas de LIS e BLG foram avaliadas quanto ao efeito do pH e da adição de sal nas suas propriedades emulsificantes (tamanho, potencial ζ, floculação, concentração e composição interfacial proteica) e estabilidade cinética. Posteriormente, emulsões multicamadas formadas por uma emulsão primária de BLG ou LIS, seguida da adição de uma segunda camada de BLG, LIS ou pectina, com carga inversa, foram obtidas e avaliadas quanto a suas propriedades de emulsificação (floculação, diâmetro hidrodinâmico médio e potencial ζ) e estabilidade cinética. Emulsões formadas pelas proteínas puras apresentaram diâmetros menores. A BLG apresentou uma adsorção preferencial na interface em relação a LIS. O pH influenciou principalmente a carga superficial de emulsões, o que afetou a interação entre proteínas, o tamanho final e a estabilidade de emulsões. O aumento da força iônica influenciou negativamente a estabilidade das emulsões formadas pela mistura das proteínas, levando a um aumento dos tamanhos das gotículas, com posterior separação macroscópica de fases. As emulsões estabilizadas por múltiplas camadas, contendo pectina, apresentaram menores diâmetros e maior potencial ζ, em comparação com emulsões formadas apenas por proteínas. A adição de pectina, um polímero carregado e estabilizante, favoreceu a resistência de emulsões multicamadas ao armazenamento e à adição de NaCl. As emulsões podem ser usadas para dispersar compostos lipofílicos, como o β-caroteno (BC), que pode ainda interagir com as proteínas da interface, aumentando a capacidade de carreamento da emulsão. A interação de BLG e LIS com BC foi determinada por meio de espectroscopia de fluorescência, de forma a explorar a possibilidade de uso dessas proteínas em sistemas para a incorporação desse composto. A fluorescência da LIS e BLG foi suprimida pela presença do BC por um processo de supressão estático. As constantes de velocidade de supressão e as constantes de interação calculadas indicaram o mecanismo de supressão estática e a força de ligação média. A capacidade de ligação do BC a BLG diminui significativamente (p<0,05) com a desnaturação, indicando uma inativação parcial dos sítios de ligação. Esses resultados são importantes para embasar o uso dessas proteínas para incorporação do BC em alimentos, o que pode ser alcançado utilizando emulsões simples ou multicamadas de até duas camadas, utilizando pectina.β-lactoglobulin (BLG) is a protein present in whey, with emulsifying, nutritional and transport properties, widely used as an ingredient. It has a low isoelectric point (IEP), which allows its electrostatic interaction with high IEP proteins, such as lysozyme (LYS), an egg white protein with high IEP. These proteins can be used as emulsifiers in order to increase the kinetic stability of emulsions. The stability of the emulsions is affected by continuous and dispersed phase composition. Oil-in-water (O/W) emulsions of LYS and BLG mixtures were evaluated for the effect of pH and salt addition on their emulsifying properties (oil droplet size and potencial ζ, flocculation, protein concentration and interfacial composition) and kinetic stability. Subsequently, multilayer emulsions formed by a primary emulsion of BLG or LYS, followed by the addition of a second layer of BLG, LYS or pectin, with reverse charge, were obtained and also evaluated for their emulsification properties and kinetic stability. Emulsions formed by pure proteins showed smaller diameters. BLG showed a preferential adsorption at the interface comparing to LYS. The pH mainly influenced the surface charge of emulsions, which affected the interaction between proteins, final size and stability of emulsions. The increase in ionic strength negatively influenced the stability of the emulsions formed by the mixture of proteins, leading to an increase in droplet size, with subsequent macroscopic phase separation. The multilayer stabilized emulsions containing pectin had smaller diameters and higher potential ζ, compared to multilayer emulsions formed only by proteins. The addition of pectin, a charged polymer and stabilizer, favored the resistance of multilayer emulsions to the storage and addition of NaCl. Emulsions, composed of two slightly soluble phases, can be used to disperse lipophilic compounds, such as β-carotene (BC), which may further interact with interface proteins, increasing the emulsion load capacity. The interaction of BLG and LYS with BC was determined by fluorescence spectroscopy in order to explore the possibility of using emulsified systems containing those proteins in the incorporation of this compound. The quenching rate constants and binding constants calculated indicated the static quenching mechanism and medium binding force. The binding ability of the BC to both proteins decreased significantly (p<0.05) with denaturation, indicating partial inactivation of the binding sites. Those results are important to support the use of proteins for incorporation of BC in foods, which can be used using single or multilayer emulsions up to two layers, using pectin.Fundação de Amparo à Pesquisa do Estado de Minas Gerai

Development of a cation exchange solid supermacroporous adsorbent for lactoferrin recovery from whey

As proteínas do soro têm propriedades funcionais, nutricionais e tecnológicas que promovem a sua utilização como ingredientes e suplementos. Soro doce obtido a partir de produção de queijo Minas Frescal concentrado por ultrafiltração foi usado no processo de captura da lactoferrina em adsorvente contínuo supermacroporoso. O criogel foi preparado pela crio-copolimerização de acrilamida com comonômero funcional, éter alil glicidil e o monômero reticulante N, N-metileno-bis-acrilamida em colunas de vidro (Ø 5 mm). A polimerização do trocador ácido 2-acrilamido-2-metil-1-propanosulfônico na superfície do criogel foi usada para obtenção de grupos de ligação sulfônicos, projetado para troca catiônica. As colunas monolíticas apresentaram estrutura esponjosa e uniforme com supermacroporos interconectados de tamanho variando de 10-100 &#956;m. Os valores para a dispersão axial dentro do leito foram baixos para velocidades superficiais de 0,5 - 9 cm min-1. O coeficiente de dispersão axial aumentou exponencialmente com o aumento da velocidade superficial. Isotermas de adsorção da lactoferrina foram determinados para diferente valores de pH e força iônica. Como esperado, a capacidade da coluna diminuiu com o aumento da concentração de NaCl. A capacidade iônica foi determinada (2,24 ± 0,42 mmol SO3 g-1 criogel seco) e a capacidade média de ligação de lactoferrina do criogel foi de 136 mg mL-1 de criogel. A influência da razão de diluição, pH e vazão na adsorção de lactoferrina a partir do soro concentrado, em termos de recuperação e fator de purificação foi avaliada. Embora o eluído tenha obtido um alto fator de purificação (em média 11,3), a separação da lactoferrina do soro de leite concentrado não apresentou alta recuperação devido à presença de outras proteínas em maior quantidade. O fator de purificação foi influenciado positivamente pelo aumento do pH, sendo inversamente proporcional a adsorção de GMP. As melhores condições para a separação de lactoferrina do soro foram obtidas em pH 8,0 e razão de diluição igual a 0,33.Whey proteins have functional, nutritional and technological properties promoting their use as ingredients and supplements. Sweet whey obtained from Minas Frescal cheese production and concentrated by ultrafiltration was used in the lactoferrin capture in a supermacroporous adsorbent. Supermacroporous monolithic columns have been prepared by radical cryo-copolymerization of acrylamide with functional co-monomer, allyl glycidyl ether, and cross-linker N,N-methylene-bis-acrylamide in glass columns (Ø 5 mm). Graft polymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid was used to achieve a cryogel with sulfo binding groups, designed for cation-exchange. The monolithic columns have uniform sponge-like structure with interconnected supermacropores of pore size 10 100 &#956;m. The experimental results showed that axial liquid dispersion within the bed was weak in a wide water flow rate of 0.5 - 9 cm min-1. The axial dispersion coefficient was found to increased exponentially with the increase of liquid flow rate. Adsorption isotherms for lactoferrina were determined a different pH and ionic strength. The ionic capacity was 2.24 ± 0.42 (mmol SO3 g-1 dry cryogel) and the average binding capacity of lactoferrina was 136 (mg mL-1 cryogel). The influence of the dilution ratio, flow rate and pH on the adsorption of lactoferrin in terms of recovery and purification factor was evaluated. Although the eluate has reached a high purification factor (on average 11.3), the separation of lactoferrin from whey concentrate did not show high recovery due to the presence of other proteins in larger quantities. The purification factor was positively influenced by the increase in pH which is inversely proportional to the adsorption of GMP. The best conditions for separation of lactoferrin from whey were obtained at pH 8.0 and dilution ratio of 0.33

Foaming properties of suspensions composed by β-lactoglobulin and polysaccharides, in the presence of sucrose or polyols

This study compares the effect of different combinations between β-lactoglobulin (β-lact, 1.0–1.5% w/v), a polysaccharide (carrageenan gum, CG, or locust bean gum, LBG, 0.0–0.5% w/v) and a sweetener (sucrose, maltitol or sorbitol, 0.0–0.5% w/v) on the foaming capacity and half-life time. The experimental design was defined according to a simplex centroid mixture design with a restriction of at least 1.0% w/v of β-lact in each mixture. Measurements of particle size, zeta potential, surface tension, flow curve, foaming capacity and foam half-life time were obtained. All mixtures evaluated showed a wide particle size distribution with diameters between 5.35 and 4494 nm. Mixtures containing CG presented higher values for apparent viscosity and average particle size than mixtures containing LBG, according to the gum concentration in solution. All mixtures showed negative zeta potential values (−14.27 to −40.42 mV) due to the presence of negatively charged β-lact at the pH 7. However, CG exerted a dominant effect on the zeta potential when present in the mixtures. The surface tension and foaming capacity were affected by the combined effect of the gums and sweeteners used. These molecules seemed to concentrate the protein at the interface due to the exclusion volume effect. The foam half-life time was mainly improved by the CG due to the higher bulk apparent viscosity, regardless of the sweetener used. Replacing sucrose with maltitol or sorbitol lead to comparable foam characteristics, mainly the foam capacity and foam half-life time, which confirms the potential of these polyols for sucrose replacement in model foams

Foaming properties of suspensions composed by ?-lactoglobulin and polysaccharides, in the presence of sucrose or polyols.

This study compares the effect of different combinations between ?-lactoglobulin (?-lact, 1.0?1.5% w/v), a polysaccharide (carrageenan gum, CG, or locust bean gum, LBG, 0.0?0.5% w/v) and a sweetener (sucrose, maltitol or sorbitol, 0.0?0.5% w/v) on the foaming capacity and half-life time. The experimental design was defined according to a simplex centroid mixture design with a restriction of at least 1.0% w/v of ?-lact in each mixture. Measurements of particle size, zeta potential, surface tension, flow curve, foaming capacity and foam half-life time were obtained. All mixtures evaluated showed a wide particle size distribution with diameters between 5.35 and 4494?nm. Mixtures containing CG presented higher values for apparent viscosity and average particle size than mixtures containing LBG, according to the gum concentration in solution. All mixtures showed negative zeta potential values (?14.27 to ?40.42?mV) due to the presence of negatively charged ?-lact at the pH 7. However, CG exerted a dominant effect on the zeta potential when present in the mixtures. The surface tension and foaming capacity were affected by the combined effect of the gums and sweeteners used. These molecules seemed to concentrate the protein at the interface due to the exclusion volume effect. The foam half-life time was mainly improved by the CG due to the higher bulk apparent viscosity, regardless of the sweetener used. Replacing sucrose with maltitol or sorbitol lead to comparable foam characteristics, mainly the foam capacity and foam half-life time, which confirms the potential of these polyols for sucrose replacement in model foams

Stability of casein micelles cross-linked with genipin : a physicochemical study as a function of pH.

Chemical or enzymatic cross-linking of casein micelles (CMs) increases their stability against dissociating agents. In this paper, a comparative study of stability between native CMs and CMs cross-linked with genipin (CMs-GP) as a function of pH is described. Stability to temperature and ethanol were investigated in the pH range 2.0?7.0. The size and the charge (?-potential) of the particles were determined by dynamic light scattering. Native CMs precipitated below pH 5.5; CMs-GP precipitated from pH 3.5 to 4.5, whereas no precipitation was observed at pH 2.0?3.0 or pH 4.5?7.0. The isoelectric point of CMs-GP was determined to be pH 3.7. Highest stability against heat and ethanol was observed for CMs-GP at pH 2, where visible coagulation was determined only after 800 s at 140 ?C or 87.5% (v/v) of ethanol. These results confirmed the hypothesis that cross-linking by GP increased the stability of CMs