Moisture-induced aggregation of alpha-lactalbumin: effects of temperature, cations, and pH

Abstract: Alpha‐lactalbumin is an important dairy protein ingredient, and has been widely used in high‐protein foods such as infant formula and nutritional bars for its nutritional and functional properties. The purpose of this study was to investigate the moisture‐induced aggregation of alpha‐lactalbumin in premixed protein dough model systems, and to illustrate the effects of temperature, cations, and pH on the progress of protein aggregation. Our results suggested that storage temperature was a critical factor for protein aggregation in model systems, and the formation of protein aggregates became faster with increases in storage temperature. Calcium significantly improved the thermal stability of alpha‐lactalbumin and slowed down the formation of protein aggregates. The increases in pH accelerated the aggregation of alpha‐lactalbumin. Our results also suggested that the formation of intermolecular disulfide bonds together with noncovalent interactions are the main mechanisms resulting in the moisture‐induced aggregation of alpha‐lactalbumin in model systems. Practical Application: Alpha‐lactalbumin is an important dairy protein ingredient, and has been widely used in high‐protein foods such as infant formula and nutritional bars for its nutritional and functional properties. Our results suggested low storage temperature, the presence of calcium and low pH condition can make high‐protein food products containing alpha‐lactalbumin more stable.     

Effect of protein–polysaccharide mixtures on the continuous manufacturing of foamed food products

The aim of this work is to understand the effects of protein and polysaccharide interactions on the physicochemical properties of highly viscous Newtonian model foods and their impact on continuous foaming operation in laminar flow conditions. Model foods consisted of modified glucose syrups. Foaming was carried out at constant gas-to-liquid flow rate ratio as a function of rotation speed. Overrun, mean bubble diameter d₃₂ and stability over time were used to characterize foams. Results showed that blow-by occurred during foaming of models including either 0.1% guar or xanthan without proteins, while 0.1% pectin allowed a total incorporation of the gas phase with large bubbles. For proteins, models with 2% whey protein isolate (WPI) were able to form foams with the desired overrun and small bubbles, while foaming was less effective with 2% Na-caseinates. With WPI, guar addition did not improve significantly foam properties. Overrun was reduced in WPI–xanthan mixtures, probably because the matrix exhibited viscoelastic trends even though xanthan decreased d₃₂. WPI–pectin mixtures provided abundant and stable foams with the smallest d₃₂ and the best stability because WPI reinforced the time-dependent behaviour of pectin recipes. However, blow-by was observed with 0.1% pectin when WPI was replaced by Na-caseinates, which demonstrates the key role of specific protein–polysaccharide interactions on overrun. Conversely, bubble diameters in foams were governed by process parameters and could be adequately described using a laminar Weber number based on foam viscosity measured during foaming for all model foods that provided stable foams.     

Physical and chemical stability of β-carotene-enriched nanoemulsions: Influence of pH, ionic strength, temperature, and emulsifier type

The enrichment of foods and beverages with carotenoids may reduce the incidences of certain chronic diseases. However, the use of carotenoids in foods is currently limited because of their poor water-solubility, high melting point, low bioavailability, and chemical instability. The potential of utilising oil-in-water (O/W) nanoemulsions stabilised by a globular protein (β-lactoglobulin) for encapsulating and protecting β-carotene was examined. The influence of temperature, pH, ionic strength, and emulsifier type on the physical and chemical stability of β-carotene enriched nanoemulsions was investigated. The rate of colour fading due to β-carotene degradation increased with increasing storage temperature (5-55 °C), was faster at pH 3 than pH 4-8, and was largely independent of ionic strength (0-500 mM of NaCl). β-Lactoglobulin-coated lipid droplets were unstable to aggregation at pH values close to the isoelectric point of the protein (pH 4 and 5), at high ionic strengths (NaCl >200 mM, pH 7), and at elevated storage temperatures (55 °C). β-Carotene degradation was considerably slower in β-lactoglobulin-stabilised nanoemulsions than in Tween 20-stabilised ones. These results provide useful information for facilitating the design of delivery systems to encapsulate and stabilise β-carotene for application within food, beverage, and pharmaceutical products.     

The effect of pH on gel structures produced using protein–polysaccharide phase separation and network inversion

Forming heat-induced gels through combined effects of micro-phase separation of whey protein isolate (WPI; 5%, w/v, 100 mm NaCl) by pH change (5.5, 6.0, and 6.5), and addition of κ-carrageenan (0–0.3%, w/w), were evaluated. The microstructure of WPI gels was homogeneous at pH 6.0 and 6.5 and micro-phase separated at pH 5.5. Addition of 0.075% κ-carrageenan to WPI solutions caused the microstructure of the gel to switch from homogeneous (pH 6.0 and 6.5) to micro-phase separated; and higher concentrations led to inversion of the continuous network from protein to κ-carrageenan. Protein solutions containing 0.075% (w/w) κ-carrageenan produced gels with increased storage modulus (G′) at pH 6.5 and decreased G′ at pH 5.5. All gels containing 0.3% (w/w) κ-carrageenan had κ-carrageenan-continuous networks. It was shown that microstructural and rheological changes were different in WPI and κ-carrageenan mixed gels when micro-phase separation was caused by pH rather than ionic strength.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Korean mothers’ food choice behavioral intent for children: An examination of the interaction effects of food type,household income,and healthism

This study identifies how economic factors, like household income, and psychological factors, like healthism, affect the food choice behavioral intent of mothers in Korea. We designed a 2 (type of food: sweet snack as hedonic food vs. milk as utilitarian food) × 2 (household income: low vs. high) × 2 (healthism: low vs. high) stimulus. The participants were Korean mothers raising children in Seoul, Korea. Participants were exposed to an advertisement for milk as the utilitarian food and a sweet snack as the hedonic food and then asked for favorability and purchase intention toward each type of food and about participants’ household income and concerns regarding health. Our study found high-income mothers were not influenced by food type, but low-income mothers were. Lower-income mothers were more willing to purchase utilitarian foods than hedonic foods. High-healthism mothers did not favor hedonic foods, regardless of household income, while low-healthism, high-income mothers favored hedonic foods more than low-healthism, low-income mothers. In contrast, low-healthism mothers did not favor utilitarian foods, regardless of their household income, while high-healthism, low-income mothers favored utilitarian foods more than high-healthism, high-income mothers. The results of our study may assist the government and marketers to understand how healthism and household income affect food choice behavioral intent.     

Deamidated wheat protein–dextran Maillard conjugates: Effect of size and location of polysaccharide conjugated on steric stabilization of emulsions at acidic pH

A soluble isolated wheat protein fraction (sIWP) prepared from commercially deamidated wheat protein (30–35% deamidation) was incubated with dextrans D10 or D65 (MW 6400 Da or 41,000 Da) at 60 °C and 75% relative humidity to form Maillard type complexes. After 72 h reaction, approximately 1–2 smaller dextran D10 molecules were attached to per protein molecule whereas only about 0.5 mol of the larger dextran D65 was attached to per protein molecule. Both sIWP–dextran complexes formed a thicker interfacial layer at the surface of the polystyrene particles (23.6 nm and 21.2 nm for the sIWP–D65 and sIWP–D10 respectively) than that adsorbed protein alone (17.7 nm). Both conjugates provided enhanced steric stabilization of the emulsions at acidic pH (∼pH 4), with the sIWP–D65 conjugate being more effective than the sIWP–D10 conjugate even though there was lower number of dextran D65 molecules conjugated to the protein. This is because that the conjugation of larger size dextran D65 was preferred at the N-terminal domain and thus provided an additional steric layer of ∼6 nm thickness in addition to the protein layer at the oil-in-water emulsion interface which is sufficient to provide steric repulsion for the oil droplets even in the absence of electrostatic repulsion. The number of dextrans conjugated to the protein and the location of conjugation was dependent on the size of the dextran. This, in turn, influenced the ability of conjugates to form effective interfacial steric layer and to maintain emulsion stability at acidic pH (∼pH 4).     

pH Influence on the stability of foams with protein–polysaccharide complexes at their interfaces

Food foams such as marshmallow, Chantilly and mousses have behavior and stability directly connected with their microstructure, bubble size distribution and interfacial properties. A high interfacial tension inherent to air/liquid foams interfaces affects its stability, and thus it has a direct impact on processing, storage and product handling. In this work, the interactions of egg albumin with various types of polysaccharides were investigated by drop tensiometry, interfacial rheology and foam stability. The progressive addition of egg albumin and polysaccharide in water induced a drop of the air–water surface tension which was dependent on the pH and polysaccharide type. At pH 4, that is below the isoeletric point of egg albumen (pI = 4.5) the surface tension was decreased from 70 mN/m to 42 mN/m by the presence of the protein, and from 70 mN/m to 43 mN/m, 40 mN/m and 38 mN/m by subsequent addition of xanthan, guar gum and κ-carrageenan, respectively. At pH 7.5 the surface tension was decreased from 70 mN/m to 43 mN/m by the simultaneous presence of the protein and κ-carrageenan. However, a higher surface tension of 48 and 50 mN/m was found when xanthan and guar gum were added, respectively, when compared with carrageenan addition. The main role on the stabilization of protein–polysaccharide stabilized interfaces was identified on the elasticity of the interface. Foam stability experiments confirmed that egg-albumin/κ-carrageenan at pH below the protein isoeletric point are the most efficient systems to stabilize air/water interfaces. These results clearly indicate that protein–polysaccharide coacervation at the air/water interface is an efficient process to increase foam stability.     

Structural,morphological and rheological characterisation of bovine serum albumin–cress seed gum complex coacervate

The interactions between the bovine serum albumin (BSA) and cress seed gum (CSG) were investigated by structural, morphological and rheological characterisation as a function of pH level (7.0–2.0) and biopolymers’ concentrations (BSA, 0.1%, 0.5% and 1%, w/w and CSG, 0.01%, 0.05% and 0.1%, w/w). The results showed that turbidity, zeta potential and coacervate yield values had correlations with the initial number of biopolymers, which are influenced by the level of the positive and negative charges of BSA and CSG. Furthermore, the optimal complexation conditions in terms of pH, CSG-BSA content and yield were 3.5%, 0.05–0.1% and 61.17% respectively. Rheological properties revealed the formation of a weakly gel-like structure with a shear-thinning behaviour. X-ray diffraction (XRD) and scanning electron microscopy (SEM) authenticated an amorphous and branch-like network structure in the coacervate phase respectively. These results reflect that CSG-BSA complex coacervate could be an appropriate biopolymer carrier for susceptible and bioactive compounds.     

Formation and stability of α-lactalbumin–lysozyme spherical particles: Involvement of electrostatic forces

The formation and the subsequent stability of spherical microparticles resulting from the self-assembly between two oppositely charged proteins, lysozyme (LYS) and apo α-lactalbumin (apo α-LA), at pH 7.5 and 45 °C were studied under different physico-chemical conditions—ionic strength, type of salts, type of buffer. Increasing the ionic strength reduced the ability of the two proteins to interact together and to form microspheres. The formation of such microparticles was completely abolished at an ionic strength of 100 mM. Increasing salt concentration also allowed destabilisation and dissociation of formed microspheres in salt-dependent manner as assessed by turbidity experiments and microscopic observations. Microparticles were destabilised with either NaCl, MgCl₂ or CaCl₂, but the latter showed the greatest destabilising effect. The higher efficiency of calcium ions in the destabilisation experiment could be attributed to the presence of specific calcium binding site on α-LA. Interestingly, complete disappearance of formed particles was not reached even after adding salt concentrations as high as 250 mM of NaCl or 7.5 mM of divalent cations. Our results suggest that electrostatic interactions are clearly involved in the first events of the two-protein assembly and spherical particles building. Moreover, non-electrostatic forces are also involved in maintaining the integrity and stability of formed microparticles.     

Preparation,characterisation and activity of the inclusion complex of paeonol with β-cyclodextrin

The inclusion complex of β-cyclodextrin (β-CD) and paeonol (2′-hydroxy-4′-methoxyacetophenone, PAE) was synthesised and characterised by thermal gravimetric analysis (TGA) and two-dimensional rotating frame spectroscopy (2D ROESY). The antioxidant activity and tyrosinase inhibition activity were also studied. The TGA results indicated that the thermal stability of PAE was improved when it was included with β-CD. Based on the 2D ROESY analysis, an inclusion structure of the PAE–β-CD complex was proposed, in which PAE penetrated β-CD in a tilted manner due to the interaction of intermolecular hydrogen bonds between PAE and β-CD. The complex of PAE with β-CD increased the antioxidant activity and tyrosinase-inhibiting activity of PAE.     

Use of ultrasounds in the food industry–Methods and effects on quality,safety, and organoleptic characteristics of foods: A review

The use of ultrasounds has recently gained significant interest in the food industry mainly due to the new trends of consumers toward functional foods. Offering several advantages, this form of energy can be applied for the improvement of qualitative characteristics of high-quality foods as well as for assuring safety of a vast variety of foodstuffs, and at the same time minimizing any negative effects of the sensory characteristics of foods. Furthermore, the non-destructive nature of this technology offers several opportunities for the compositional analysis of foods. However, further research is required for the improvement of related techniques and the reduction of application costs in order to render this technology efficient for industrial use. This review paper covers the main applications of ultrasounds as well as several advantages of the use of the technology in combination with conventional techniques. The effects of ultrasounds on the characteristics, microbial safety, and quality of several foods are also detailed     

Formation and functionality of whey protein isolate–(kappa-, iota-, and lambda-type) carrageenan electrostatic complexes

The formation of electrostatic complexes between whey protein isolate (WPI) and (κ-, ι-, λ-type) carrageenan (CG) was investigated by turbidimetric measurements as a function of pH (1.5–7.0), biopolymer weight-mixing ratio (1:1–75:1 WPI:CG) and NaCl addition (0–500 mM) to better elucidate underlying mechanisms of interaction. Emulsion stabilizing effects of formed complexes was also studied to assess their potential as emulsifiers. Complex formation followed two pH-dependent structure-forming events associated with the formation of soluble (pH_c) and insoluble (pH_?1) complexes. For both the WPI–κ-CG and WPI–ι-CG mixtures, pH_c and pH_?1 occurred at pH 5.5 and 5.3, respectively, whereas in the WPI–λ-CG mixture values were slightly higher (pH_c = 5.7; pH_?1 = 5.5). In all mixtures, maximum turbidity was found to occur near pH 4.5, before declining at lower pHs. Biopolymer mixing ratios corresponding to maximum OD was found to occur at the 12:1 ratio for both the WPI–κ-CG and WPI–λ-CG mixtures, and 20:1 ratio for WPI–ι-CG mixture. The addition of NaCl disrupted complexation within WPI–κ-CG mixtures as levels were raised, whereas when ι-CG and λ-CG was present, complexation was enhanced up to a critical Na⁺ concentration before declining. Adsorption of CG chains to the small WPI–WPI aggregates during complexation was proposed to be related to both the linear charge density and conformation of the CG molecules involved. Emulsion stability in the mixed systems (12:1 mixing ratio), regardless of the CG type (κ, ι, λ), was significantly higher than individual WPI solutions indicating enhanced ability to stabilize the oil-in-water interface.     

Preparation and characterisation of a novel agar oligosaccharide–iron (III) complex

This study aimed to synthesise a complex of agar oligosaccharides (AOS) with ferric iron and to evaluate its physicochemical and structural characteristics. Based on single factor, Plackett–Burman and response surface optimal experiments of three factors at three levels, the optimised chelate conditions of the AOS-iron (III) complex were mass ratio of AOS to iron ions of 4.5:1 g:g, reaction temperature of 74 °C and reaction time of 60 min. Under these conditions, the iron content and total sugar content were 14.03 ± 0.42% and 66.07 ± 0.96%, respectively. The physicochemical analysis showed that AOS–iron (III) was soluble and steady at physiological pH, and it was easily reduced to iron (II). The structural characteristics of AOS and AOS–iron (III) were analysed by Raman spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction and Nuclear magnetic resonance, and results showed that the iron (III) ions were chelated to AOS by –OH groups and –COOH groups and widely dispersed on the AOS backbone. Therefore, AOS–iron (III) could be a candidate for iron supplementation to treat iron deficiency anaemia.     

The effect of protein concentration and heat treatment temperature on micellar casein–soy protein mixtures

The objective of this study was to investigate the effect of concentration and temperature on the rheological properties of soy proteins (SP) and micellar casein (MCN) systems. Individual and mixed (1:1) protein systems of 2–15% concentration were prepared and heat treated for 5 min at 40–90 °C. After cooling to 20 °C, their rheological properties were determined using steady-shear rheology. Zeta potential and particle size measurements were also conducted. Both proteins were negatively charged under all experimental conditions, but the absolute values of zeta potential and thus the stability of the protein solutions decreased with temperature and concentration. For SP solutions, viscosity and apparent yield stress increased with concentration. Shear thinning behavior was prevalent, becoming more pronounced with increasing concentration. Heat treatments at T ≥ 80 °C induced glycinin denaturation, followed by aggregation and network formation when C ≥ 7.5%. Heat treatment did not significantly affect viscosity of MCN systems, while increasing concentration resulted in a significant increase in apparent viscosity and apparent yield stress. Most MCN systems exhibited Newtonian flow behavior, with the exception of systems with C ≥ 12.5% treated at T ≥ 80 °C, which became slightly shear thickening. Mixed SP–MCN systems mimicked the behavior of SP, with most values of rheological parameters intermediate between SP and MCN-only systems. Mixtures of 7.5–12.5% concentration treated at 90 °C displayed local phase separation, low viscosity and apparent yield stress, while 15% mixtures treated at 90 °C showed protein aggregation and incipient network formation. The data generated in this study can be used to develop a range of protein based products with unique flow characteristics and storage stability.     

Electric field effects on β-lactoglobulin thermal unfolding as a function of pH – Impact on protein functionality

The presence of moderate electric fields (MEF) during ohmic heating (OH) treatment of whey protein systems have demonstrated potential to change physicochemical and functional properties, like aggregation rate and extension or viscoelastic behaviour. However, the specific action of MEF upon the molecular structure of proteins, particularly during thermal processing has yet to be clarified. The effects of MEF in pure fractions of β-lactoglobulin (β-lg) under non-aggregating conditions (low concentration and ionic strength), were investigated in this work. Protein samples were identically heat-treated through conventional and OH methods and at different pH values. β-lg's structural features were characterized by evaluation of secondary structure distribution and local conformational changes using techniques such as circular dichroism, intrinsic and extrinsic fluorescence and free thiol groups reactivity. It was confirmed that MEF affects β-lg upon thermal unfolding, resulting in distinctive structural features, surface hydrophobicity and SH reactivity. The mechanism of action is probably related with the molecular motion induced by the oscillating electric field and is more pronounced at neutral pH, where β-lg is more susceptible to thermal structural changes. These results contribute to a better understanding of OH processing and its effects in food matrices reinforce the possibility of using MEF as a toll to change protein functionality.Industrial relevanceOhmic heating is an emerging technology and is being established as reference method for processing protein-rich food such as dairy and egg products. Non-thermal effects of the applied electric fields during ohmic heating have been addressed but few works deal with their real impact in structural and molecular properties of food proteins with high biological value.In this work demonstrated that the presence of an electric field during ohmic heating processing influences structural aspects of beta-lactoglobulin. This knowledge plays an important role on process design (i.e. pasteurization binomials and fouling control) and product quality because these proteins play an essential role in food's nutritional and organoleptic properties, as well as on functionality, allergenicity and stability aspects.     

Water–water and water–macromolecule interactions in food dehydration and the effects of the pore structures of food on the energetics of the interactions

A molecular dynamics (MD) modeling and simulations approach has been rationally built and developed to study porous food systems constructed with amylose and dextran chains. The findings from our MD studies indicate that the presence of food macromolecules decreases the energetics of the water–water interactions for the nearby water molecules in the pore space, but provides additional water–macromolecule interactions that can significantly outweigh the partial loss of water–water interactions to make the adjacent water molecules strongly bound to the food macromolecules so that the water activity and water removal rate are decreased as dehydration proceeds and, thus, the dehydration energy requirement would be increased. The effects of pore structures are greater in systems with higher densities of food macromolecules, smaller in size pores, and stronger water–macromolecule interactions. Dehydration of food materials can thus be reasonably expected to start from the largest pores and from the middle of the pores, and to have non-uniform water removal rates and non-planar water–vapor interfaces inside individual pores as well as across sections of the food materials. The food porous structures are found to have good pore connectivity for water molecules. As dehydration proceeds, water content and the support from water–water and water–macromolecule interactions both decrease, causing the food porous structures to adopt more compact conformations and their main body to decrease in size. Dehydration in general also reduces pore sizes and the number of pore openings, increases the water–macromolecule interactions, and leads to the reduction of the overall thermal conductivity of the system, so that more energy (heat), longer times, and/or greater temperature gradients are needed in order to further dehydrate the porous materials. Our thermodynamic analysis also shows that the average minimum entropy requirement for food dehydration is greater when the water–macromolecule interactions are stronger and the food macromolecular density is higher. The importance of the physicochemical affinity of food molecules for water and of the compatibility of the resultant porous structures with water configurational structures in determining food properties and food processing through the water–macromolecule interactions, is clearly and fundamentally verified by the results and discussion presented in this work.