Complexation between milk proteins and polysaccharides via electrostatic interaction: principles and applications – a review

This article details recent research conducted on the complexation between milk proteins and polysaccharides and the properties of the complexes, and the application of such relationships to the food industry. Complexation between proteins and polysaccharides through electrostatic interactions gives either soluble complexes in a stable solution or insoluble complexes, leading to phase separation. The formation and the stability of these complexes are influenced by pH, ionic strength, ratio of protein to polysaccharide, charge density of protein and polysaccharide as well as processing conditions (temperature, shearing and time). The functional properties of milk proteins, such as solubility, surface activity, conformational stability, gel‐forming ability, emulsifying properties and foaming properties, are improved through the formation of complexes with polysaccharides. These changes in the functional properties provide opportunities to create new ingredients for the food industry.     

蛋白聚糖的结构和功能特性

蛋白质与多糖之间通过静电相互作用发生的复合物凝集或结合是形成蛋白聚糖的主要原因，象pH值、离子强度、蛋白质和多糖的浓度及比例、蛋白质和多糖的带电情况以及分子大小等理化条件会影响蛋白聚糖的形成及稳定性。同时，温度及一些物理因素（压力、剪切速率和时间）对此也有一定的影响。蛋白聚糖具有较好的水合作用（溶解性、粘度），结构特性和表面特性（起泡性、乳化性），可用于大分子物质的纯化，微胶囊材料，食用成分（脂肪替代物等）和生物材料的合成等（可食用薄膜，人造皮肤）。     

Polysaccharide–protein interactions in dairy matrices,control and design of structures

Protein–polysaccharide interactions are of great importance in the design of dairy formulations, as they play a key role in the formation of structure and texture in dairy products. With a detailed understanding of the factors affecting the interactions, the ability of charged polysaccharides to associate with the milk proteins is continuously exploited to create functional complexes, novel ingredients and delivery systems. In addition, formulations containing non-interacting polysaccharides also need to be carefully controlled, as these biopolymers may give rise to segregative phase separation, with important consequences to the stability and quality of the final matrix. As casein micelles play a major role in imparting structure to dairy products, emphasis in this review will be given to the molecular details of the interactions between polysaccharides with these protein particles. Some of the most researched polysaccharides will be highlighted in this context, and the progress made in understanding their role during structure formation of dairy matrices will be discussed. The opportunity of creating novel microstructures provided by association or/and incompatibility of milk proteins and different polysaccharides will be assessed.     

Mixed biopolymers at interfaces: Competitive adsorption and multilayer structures

Mixed biopolymer layers are commonly involved in the stabilization of food emulsions and foams. The interfacial composition and structure of mixed layers are predominantly determined by two mechanistic phenomena—competitive adsorption from mixed solution and cooperative adsorption into multilayers. The surface-active protein components typically dominate primary layers around droplets and bubbles, and the interacting polysaccharides form outer secondary stabilizing layers. This article reviews progress in understanding the factors controlling the nanoscale structure and physico-chemical properties of adsorbed layers in colloidal systems containing mixtures of biopolymers. Contributions from different experimental techniques are described, with particular attention directed towards the role of surface shear rheology in providing information on competitive adsorption of proteins and macromolecular interactions at fluid interfaces. We also consider here the phenomenon of phase separation in mixed protein monolayers, the balance of thermodynamic and kinetic factors in determining biopolymer layer properties, and the involvement of electrostatic interactions in the stabilization of emulsions by protein–polysaccharide complexes.     

Metal-polysaccharide complexes—Part I

Many factors, including linear charge density, effective nuclear charge of the cation, basicity of the donor group, chelation, intermolecular binding of two or more donor groups by a single cation and steric fit, contribute—or possibly contribute—to the formation and stability of metal ion-polysaccharide complexes. In order to form a proper foundation for studying and interpreting metal ion-polysaccharide interactions, the author introduces into his treatise on polysaccharide complexes much current information concerning the interaction of metal ions with simple unidonor and simple multidonor ligands. For convenience of discussion, the subject of polysaccharide complexes is divided into two categories: interactions of neutral polysaccharides, such as cellulose and starch, and interactions of anionic polysaccharides, such as alginate, pectate, mucopolysaccharides and carrageenan. On the one hand, neutral polysaccharides form weak complexes with cations in neutral or non-alkaline media. Only in alkaline solution is there any great affinity between cation and donor. On the other hand, anionic polysaccharides have a strong affinity for metal counterions, even at low concentrations. Attempts are made to provide reasonable explanations for the formation of these complexes and for the often large differences in affinity between metals for a particular polysaccharide or between polysaccharides for a particular metal. In some instances, rheological behaviour of polysaccharide systems can be related to structural features of macromolecular complexes.     

Depletion flocculation and thermodynamic incompatibility in whey protein stabilised O/W emulsions

The stability of whey protein stabilised emulsions, containing methylcellulose added after emulsification in their bulk phase, was investigated. The phase diagram of the ternary system whey proteins/methylcellulose/water was first established and used to identify the conditions permitting polymer phase separation within the emulsion bulk phase. Emulsions containing a whey protein and methylcellulose concentration in the bulk phase below and above the phase separation threshold could therefore be prepared. Below the phase separation threshold, the creaming rate of the oil droplets was faster than the one predicted by the Stokes equation, due to methylcellulose-induced depletion flocculation. Above the phase separation threshold, the destabilisation of the emulsion involved different mechanisms, depending on the emulsifier adsorbed at the O/W interface. In the case of Tween 40 stabilised droplets, depletion flocculation led to a complete creaming of the fat globules while phase separation led to the formation of two polymer-rich phases, namely a protein-rich phase at the bottom of the tube and a methylcellulose-rich phase above. In the case of whey protein stabilised droplets, phase separation between bulk whey proteins and methylcellulose occurred, and the fat globules were entrapped in the protein-rich phase. These results permitted to describe the destabilisation mechanisms of both Tween 40 and whey protein stabilised emulsions in the presence of unadsorbed polysaccharide. They could be used to better understand the destabilisation processes arising in food emulsions, especially in those emulsions containing whey proteins, small surfactant molecules and polysaccharides.     

Utilizing whey protein isolate and polysaccharide complexes to stabilize aerated dairy gels

Heated soluble complexes of whey protein isolate (WPI) with polysaccharides may be used to modify the properties of aerated dairy gels, which could be formulated into novel-textured high-protein desserts. The objective of this study was to determine the effect of polysaccharide charge density and concentration within a WPI-polysaccharide complex on the physical properties of aerated gels. Three polysaccharides having different degrees of charge density were chosen: low-methoxyl pectin, high-methoxyl type D pectin, and guar gum. Heated complexes were prepared by heating the mixed dispersions (8% protein, 0 to 1% polysaccharide) at pH 7. To form aerated gels, 2% glucono-δ-lactone was added to the dispersions of skim milk powder and heated complex and foam was generated by whipping with a handheld frother. The foam set into a gel as the glucono-δ-lactone acidified to a final pH of 4.5. The aerated gels were evaluated for overrun, drainage, gel strength, and viscoelastic properties. Without heated complexes, stable aerated gels could not be formed. Overrun of aerated gel decreased (up to 73%) as polysaccharide concentration increased from 0.105 to 0.315% due to increased viscosity, which limited air incorporation. A negative relationship was found between percent drainage and dispersion viscosity. However, plotting of drainage against dispersion viscosity separated by polysaccharide type revealed that drainage decreased most in samples with high-charge-density, low-methoxyl pectin followed by those with low-charge-density, high-methoxyl type D pectin. Aerated gels with guar gum (no charge) did not show improvement to stability. Rheological results showed no significant difference in gelation time among samples; therefore, stronger interactions between WPI and high-charge-density polysaccharide were likely responsible for increased stability. Stable dairy aerated gels can be created from WPI-polysaccharide complexes. High-charge-density polysaccharides, at concentrations that provide adequate viscosity, are needed to achieve stability while also maintaining dispersion overrun capabilities.     

多糖-蛋白质复合水凝胶研究进展

多糖和蛋白质是食品中最重要的2种功能大分子,可以通过相互作用形成复合水凝胶。与单一组分相比,多糖和蛋白质形成的复合水凝胶不仅具有优异的物理结构和化学性质,而且具有提高复合体系机械性能的潜在优势。该文对部分多糖-蛋白质复合水凝胶的研究进展进行总结,综述了形成复合水凝胶的多糖及蛋白质的类型和条件、二者主要相互作用及影响二者相互作用的内部和外部因素,阐述了多糖对其与蛋白质形成复合水凝胶机械性能的影响,并概述了多糖-蛋白质复合水凝胶在食品工业及生物医药等领域的应用现状。该文将为多糖-蛋白质基创新凝胶的设计、开发及应用提供理论参考依据。     

Polysaccharide protein interactions

The interaction between proteins and polysaccharides, as can be observed in food related systems, is systematically discussed by separating biopolymer interactions into respectively enthalpy- and entropy-dominated types. We present examples of typically enthalpy driven phase separations, such as biopolymer incompatibility, described by the classical Flory-Huggins theory. This behavior is for instance observed for the system gelatin–dextran. In mixed systems where excluded volume or depletion interaction plays an important role the phase separation is mainly driven by entropy. Here the interaction of (random coil) polysaccharides and protein (covered) particles such as casein micelles and emulsion droplets in dairy systems may lead to phase separation as well.     

Interactions between cell wall polysaccharides and polyphenols

In plant-based food systems such as fruits, vegetables, and cereals, cell wall polysaccharides and polyphenols co-exist and commonly interact during processing and digestion. The noncovalent interactions between cell wall polysaccharides and polyphenols may greatly influence the physicochemical and nutritional properties of foods. The affinity of cell wall polysaccharides with polyphenols depends on both endogenous and exogenous factors. The endogenous factors include the structures, compositions, and concentrations of both polysaccharides and polyphenols, and the exogenous factors are the environmental conditions such as pH, temperature, ionic strength, and the presence of other components (e.g., protein). Diverse methods used to directly characterize the interactions include NMR spectroscopy, size-exclusion chromatography, confocal microscopy, isothermal titration calorimetry, molecular dynamics simulation, and so on. The un-bound polyphenols are quantified by liquid chromatography or spectrophotometry after dialysis or centrifugation. The adsorption of polyphenols by polysaccharides is mostly driven by hydrophobic interactions and hydrogen bonding, and can be described by various isothermal models such as Langmuir and Freundlich equations. Quality attributes of various food and beverage products (e.g., wine) can be significantly affected by polysaccharide–polyphenol interactions. Nutritionally, the interactions play an important role in the digestive tract of humans for the metabolism of both polyphenols and polysaccharides.     

Effect of limited hydrolysis of soy protein on the interactions with polysaccharides at the air–water interface

The objective of the work was to study the effect of limited hydrolysis of soy protein on the interactions with polysaccharides with and without surface activity at the air–water interface at neutral pH where a limited incompatibility between macromolecules can occur. The surface pressure and phase angle as a function of time were evaluated with a drop tensiometer at 20 °C, pH 7 and ionic strength 0.05 M.Hydrolysates of 2% (H1) and 5.4% (H2) degree of hydrolysis (DH) with neutral protease from Aspergillus oryzae were obtained from a commercial soy protein isolate. The polysaccharides used were: hydroxypropylmethylcellulose (HPMC) as surface active polysaccharide; lambda carrageenan (λC) and locust bean gum (LB) as non-surface active polysaccharides.It was found that increasing DH decreased the surface pressure and increased film viscoelasticity (determined as the phase angle, θ) of soy protein hydrolysates and the nature of protein–polysaccharide interactions was strongly affected by DH. The presence of polysaccharides led to an increase of surface pressure of H1 but when added to H2, HPMC and λC decreased the surface pressure.The less hydrolyzed protein H1 gave rise to a higher surface pressure and film viscoelasticity in combination with the polysaccharides. This result points out that a limited protein hydrolysis was sufficient to improve the surface properties of soy proteins if used in combination with polysaccharides.Polysaccharides used in admixture with hydrolyzed soy proteins could control and improve the stability of foams and emulsions not only by increasing bulk viscosity but also by improving film viscoelasticity.     

Phase transitions of dairy proteins,dextrans and their mixtures as a function of water interactions

The water sorption behaviour and phase transitions of dairy proteins (β-casein and β-lactoglobulin), dextrans (dextran6 and dextran500) and their mixtures were studied at low water content. Freeze-dried polysaccharide samples containing between 20 and 80% dairy protein were equilibrated at different water activities (a_w) between 0.11 and 0.75, at 25 °C. Water sorption isotherms of pure compounds and mixtures, as well as glass transition at different water activities were determined. Crystallization of polysaccharides was also investigated. BET and Gordon and Taylor equations were used to model water adsorption isotherms and glass transition temperature behaviour, respectively. Polysaccharides showed a higher water adsorption capacity than dairy proteins in the range of a_w studied, which decreased with the addition of protein. The addition of β-casein decreased the Tg values of dextran systems. This effect was attributed to water migration from β-casein to the polysaccharide fraction following the formation of β-casein hydrophobic interactions. Likewise, dairy proteins provoked an increase in the temperature of dextran crystallization and a decrease in the enthalpy. This effect did not reflect the increase of dextran molecular mobility in the presence of β-casein but could be masked by other factors, like steric hindrance. The effect of dairy proteins, especially β-casein, on the phase transitions of polysaccharides should be considered for controlling the Maillard reaction, as well as physical and chemical changes that occur during processing and storage of food systems.     

Interpolymeric Complexes Formed Between Whey Proteins and Biopolymers: Delivery Systems of Bioactive Ingredients

Whey proteins are obtained from dairy industry waste. Studies involving the analysis of the bioactive compounds in whey show health benefits, as it is an excellent source of indispensable amino acids. Milk whey contains principally β‐lactoglobulin, α‐lactoglobulin, bovine serum albumin, and lactoferrin, proteins with innumerable functional and technological properties. One application of these proteins in food is the formation of interpolymer complexes, along with other proteins or anionic polysaccharides. The formation of complexes occurs mainly through electrostatic interactions between a negatively charged biopolymer and a positively charged biopolymer. This formation is influenced by factors such as pH, ionic strength, and biopolymer ratio. Because they do not use high temperatures and chemical reagents and have additional nutritional and functional value, these complexes have been used as encapsulating agents for bioactive ingredients. Recent studies on their training and applications are addressed in this review to boost new research and applications in the food industry, thus increasing opportunities for utilizing whey proteins.     

Effect of pH,biopolymer mixing ratio and salts on the formation and stability of electrostatic complexes formed within mixtures of lentil protein isolate and anionic polysaccharides (κ‐carrageenan and gellan gum)

Associative phase separation within lentil protein isolate (LPI), polysaccharide (κ‐carrageenan (κ‐CG) and gellan gum (GG)) mixtures was investigated as a function of pH (1.50–8.00) and mixing ratio (1:1–30:1; LPI/polysaccharide) by turbidity and electrophoretic mobility. Effects of salts (NaCl, KCl and CaCl₂) on complex stability were also studied as a function of ionic strength. Coacervation typically follows two pH‐dependent forming events associated with the formation of soluble and insoluble complexes. The addition of polysaccharides to a LPI system (at all ratios) resulted in a significant drop in turbidity over the entire pH range and a shift in net neutrality to lower pH relative to LPI alone; where LPI aggregation was inhibited by repulsive forces between neighbouring polysaccharide chains. As the biopolymer mixing ratio increased, structure formation was less inhibited. The addition of salts resulted in the disruption of formed LPI/polysaccharide complexes.     

Effect of polysaccharide charge on formation and properties of biopolymer nanoparticles created by heat treatment of β-lactoglobulin–pectin complexes

Biopolymer nanoparticles can be formed by heating globular protein/polysaccharide mixtures above the thermal denaturation temperature of the protein under pH conditions where the two biopolymers are weakly electrically attracted to each other. In this study, the influence of polysaccharide linear charge density on the formation and properties of these biopolymer nanoparticles was examined. Mixed solutions of globular proteins (β-lactoglobulin) and anionic polysaccharides (high and low methoxyl pectin) were prepared. Micro-electrophoresis, dynamic light scattering, turbidity and atomic force microscopy (AFM) measurements were used to determine the influence of protein-to-polysaccharide mass ratio (r), solution pH, and heat treatment on biopolymer particle formation. Biopolymer nanoparticles (d < 500 nm) could be formed by heating protein–polysaccharide complexes at 83 °C for 15 min at pH 4.75 and r = 2:1 in the absence of added salt. The biopolymer particles formed were then subjected to pH and salt adjustment to determine their stability. The pH stability was greater for β-lactoglobulin-HMP complexes than for β-lactoglobulin-LMP complexes. The addition of 200 mM sodium chloride to heated complexes greatly improved the pH stability of HMP complexes, but decreased the pH stability of LMP complexes. The biopolymer particles formed consisted primarily of β-lactoglobulin, which was probably surrounded by a pectin coating at low pH values. AFM measurements indicated that the biopolymer nanoparticles formed were spheroid in shape. These biopolymer particles may be useful as delivery systems or fat mimetics.     

Improved thermal gelation of oat protein with the formation of controlled phase-separated networks using dextrin and carrageenan polysaccharides

The thermal gelation of oat protein (OP) was investigated in the presence of polysaccharides at different pHs. The compressive stress dramatically increased in these phase-separated protein–polysaccharides gels due to an apparent increase in protein concentration. The polysaccharide structure significantly affected the degree of phase-separation and gel mechanical properties. The observed two-fold increase in gel compressive stress can be attributed to strong repulsive forces caused by carrageenan molecules. These resulted in a greater degree of phase-separation with the formation of carrageenan rich domains embedded in the protein phase, and a highly ordered protein network, stabilized by hydrogen and hydrophobic interactions. In the case of OP-dextrin gels, the rate of phase separation was slower than the rate of protein aggregation, thus the dextrin particles were uniformly distributed within the protein network. This research contributes to the basic understanding required for designing textures for novel plant-based protein products.     

基于蛋白质、多糖混合体系的食品凝胶结构设计策略

随着现代食品工业的快速发展与人们健康饮食意识的普遍增强,凝胶食品因高含水量、低能量、口感宜人、质构特性独特等优点逐渐受到青睐。多糖和蛋白质是食品体系中广为存在的天然大分子,是食品凝胶结构设计的良好原料。有研究表明,多糖和蛋白质混合体系的相行为是决定两者混合凝胶微观结构及物理性质的主要因素。本文首先阐述了多糖和蛋白质双相混合体系的相行为及影响因素,随后总结了混合体系在构建食品凝胶结构中的设计原则。最后,以乳清蛋白和几种常见多糖的混合体系为例,证明蛋白质和多糖在食品凝胶结构设计方面的巨大潜力。     

Influence of pH and biopolymer ratio on sodium caseinate—guar gum interactions in aqueous solutions and in O/W emulsions

Many colloidal food systems contain both proteins and polysaccharides. In the present study, the phase behaviour of mixed sodium caseinate—guar gum aqueous solutions was investigated: segregative phase separation was observed in solutions containing at least 0.04% of guar gum and 1.6% of sodium caseinate, thus indicating the limited compatibility of the polysaccharide and the protein.In addition, the functionality of guar gum as gravitational stabilizer in sodium caseinate stabilized 25% O/W emulsions was checked. At pH conditions significantly larger than the iso-electric point (IEP) of sodium caseinate, addition of small amounts of guar gum (0.1–0.2%) gave raise to fast serum separation, which was thought to be due to depletion flocculation. Increasing the polysaccharide concentration and/or the oil volume fraction limited the degree of phase separation, since depletion flocculation induced a sufficiently strong three-dimensional network to withstand gravity effects.Considering different guar gum concentrations at pH 5.0, 5.5, 6.0 and 6.5, it became obvious that the phase separation behaviour in the absence of guar gum was largely affected by the pH, whereas in the presence of at least 0.1% of guar gum it became mainly affected by the guar gum concentration. Hereby, higher guar gum concentrations introduced a longer delay time before separation could effectively be detected. As laser diffraction particle size analysis results were not significantly affected by guar gum addition, it was concluded that the guar gum-induced flocculation was weak in nature and largely reversible.Combining all results, it was concluded that guar gum could effectively be used to prevent phase separation problems that could occur due to flocculation around the protein's IEP, provided that at least 1.0% of guar gum is added to ensure depletion stabilization by formation of a sufficiently strong three-dimensional network to overcome separation effects. Increasing the ionic strength through addition of salt further reinforces the network in order to prevent its collapse due to gravity.     

大豆蛋白凝胶制备及其影响因素的研究进展

大豆作为我国重要的粮食作物之一,具有较高的营养价值。凝胶性作为大豆分离蛋白重要的功能特性备受关注。大豆蛋白在产品中多用作多种配合物如水分子、糖类、脂质以及不稳定小分子活性物质的包埋载体,但大豆蛋白天然凝胶制品存在结构松散、成品率低等问题,极大地限制了其凝胶制品的应用与发展。本文从大豆分离蛋白凝胶形成机理进行解析,并对大豆蛋白构象及组成、多糖、脂质间的相互作用、离子强度等内在影响因素,以及物理、化学、生物等外部因素对凝胶形成产生的影响进行了深入探讨和系统分析,以期对今后大豆蛋白凝胶制品加工与利用提供理论依据。     

蛋白质-多糖多尺度复合物结构的形成机制及其应用前景

蛋白质-多糖复合物结构的形成基础是分子间相互作用，包括共价和非共价相互作用。在特定加工条件下，由于分子间作用力的驱动，蛋白质和多糖可组装形成分子水平-微观水平-宏观水平上的多尺度复合物。而调控蛋白质-多糖复合物多尺度结构的形成，发挥不同组分的协同增效作用，对于设计具有特定或新功能的蛋白质-多糖复合物具有重要意义。本文以蛋白质与多糖分子间的相互作用为出发点，综述了蛋白质-多糖复合物多尺度结构的形成及其对功能特性的影响，同时介绍了蛋白质-多糖多尺度复合体系的应用前景，以期为设计和开发功能优异的蛋白质-多糖复合体系产品提供参考。