The effect of glycation on foam and structural properties of β-lactoglobulin

The goal of the present work was to evaluate whether the Maillard reaction, with glucose and lactose as substrates, improves the foaming properties of β-lactoglobulin. Lactose led to the lowest degree of modification without significant differences by reaction time and by protein:sugar molar ratio. However, in the case of glucose, the degree of glycation increases with reaction time and molar ratio. The results obtained by UV fluorescence, surface hydrophobicity and differential scanning calorimetry clearly showed differences in the degree of folding of β-lactoglobulin upon modification with different sugars or thermal treatment, with changes in the foaming capacity of β-lactoglobulin. All the modified samples exhibited a significant increase (α ? 0.05) in draining stability (Kg) as compared to the non-thermally treated sample. In addition, foams formed by lactose-glycated samples were more stable than those formed by glucose-glycated ones. A significant increase (α ? 0.05) of foam stability with reaction time was also detected, particularly in glucose-glycated samples.     

Effects of heat-treated β-lactoglobulin and its aggregates on foaming properties

The effects on foaming properties of the aggregates formed by heating concentrate beta-lactoglobulin solutions (55 mg mL⁻¹, pH 6.8) at 85 °C from 1 to 15 min were investigated. Structural characteristics (size and molecular conformation), hydrophobicity and protein aggregates proportion were also studied. All tested methods pointed at 3 min of heating as a critical time, in terms of conformational changes and aggregation processes. At this time, the most significant conformational changes took place: non-native monomers were present and the greatest amount of dimers and trimers was produced, which was proved with the results of gel densitometry of SDS-PAGE, fluorescence quenching and circular dichroism tests. Foamability and foam stability were both improved by pre-heating the protein. A constant proportion among beta-lactoglobulin species (monomer 51%, dimer 33% and trimer 16%), regardless the protein concentration, led to the same results on foaming properties, confirming the link with structural changes. Aggregates formed by heating beta-lactoglobulin up to 10 min produced more stabilized foams, slowing down disproportionation, because of the formation of stiffer films. The increase in surface hydrophobicity was considered a decisive factor in the improved foamability and hydrophobic interactions improved the foam stability trough the rapid formation of a viscoelastic film.     

Interfacial and foaming properties of bovine β-lactoglobulin: Galactose Maillard conjugates

In this paper, the effect of the initial and advanced steps of glycosylation by Maillard reaction (MR) (glycation) of β-lactoglobulin (β-Lg) with galactose on the interfacial and foaming (foamability and foam stability) properties of this protein has been studied at both pH 7 and pH 5. Hardly any effect of glycation was observed at pH 7. However, a pH 5, due to its increased solubility, β-Lg glycated at 50 °C during 48 h (advanced steps of MR) presented the best dynamic of adsorption which lead to an increase of the surface dilatational modulus of adsorbed film. This resulted in a better foaming capacity, as well as higher stability of foams of β-Lg glycoconjugates with respect to native and control heated protein. These results could extend the applicability of β-Lg as a foaming agent, particularly in acid foods.     

Preparation and characterization of β-lactoglobulin hydrolysate-iron complexes

The purpose of this study was to determine the best preparation condition of β-lactoglobulin hydrolysate-iron complexes and characterize its structural transformation both before and after binding using the UV-visible absorption spectrum, Fluorescence spectrum, and Fourier transform infrared spectroscopy. Results showed that β-lactoglobulin hydrolysates obtained with alcalase after hydrolysis for 6h possessed the highest iron-binding capacity. The highest yield of complexes was obtained when the mass ratio between β-lactoglobulin hydrolysate and Fe(3+) reached 40:1, with the optimal pH value of 7.0. All of the spectra indicated that some sites such as amido bonds transformed during chelation, and nitrogen atoms could chelate with Fe(3+) to form coordinate bonds by offering electron pairs. Therefore, β-lactoglobulin hydrolysate-iron complexes may be good carriers for iron and possess great potential to be used as iron supplements.     

Rheological properties of patatin gels compared with β-lactoglobulin, ovalbumin, and glycinin

BACKGROUND: The thermal unfolding and rheological properties of patatin gels were compared with those of commonly used proteins (β‐lactoglobulin, ovalbumin, glycinin). RESULTS: A significant difference between these proteins was observed in both the denaturation temperature (59 °C for patatin; about 20 °C lower than the other proteins) and the onset temperature of gel formation (50–60 °C, compared to 70–85 °C for the other proteins). At low ionic strength the minimal concentration was only 6% (w/v) for patatin, compared to 8–11% for the other proteins. This effect was attributed to the relatively high exposed hydrophobicity of patatin as determined by hydrophobic interaction chromatography. For gels compared at ‘iso‐strength’, the frequency dependence was found to be close to identical, while small differences were observed in the strain at fracture. CONCLUSIONS: Patatin was found to form gels with comparable small‐deformational rheological properties as typical food proteins. In addition, at concentrations where the elastic modulus was similar for all proteins, the frequency and strain dependence were also comparable. From this it is concluded that patatin is a promising protein to be used in food applications as a gelling agent. Copyright © 2010 Society of Chemical Industry     

Allergenic and immunogenic potential of cow's milk β-lactoglobulin and caseins evidenced without adjuvant in germ-free mice

Scope: In most animal models of allergy, the development of an IgE response requires the use of an adjuvant. Germ‐free (GF) mice exhibit Th2‐polarized antibody responses combined with defective immunosuppressive mechanisms. The sensitizing potential of milk proteins was investigated in GF mice in the absence of adjuvant. Methods and results: β‐lactoglobulin (BLG) and whole casein (CAS) allergenicity was evaluated by means of intraperitoneal injections without adjuvant. Injections of BLG induced significant IgE and IgG1 responses in GF mice, while CAS injections provoked the production of IgG1 toward κ‐ and αS1‐caseins. No significant antibody response was evidenced in conventional (CV) mice. After in vitro BLG‐reactivation, IL‐4, IL‐5, IL‐13 and IFN‐γ productions by splenocytes were higher in GF mice than in CV mice. Heat‐treatment decreased BLG allergenicity as indicated by the absence of IgE production in GF mice. However, heat‐treatment increased protein immunogenicity and led to the production of anti‐BLG and anti‐κ‐casein IgG1 in both GF and CV mice. This correlated with enhanced productions of IL‐4, IL‐5 and IL‐13 in BLG‐reactivated splenocytes from CV mice. Conclusion: Gut colonization by commensal bacteria appeared then to significantly reduce the susceptibility of mice toward the intrinsic allergenic and immunogenic potential of milk proteins.     

Modulation of physicochemical properties of lipid droplets using β-lactoglobulin and/or lactoferrin interfacial coatings

There is considerable interest in the development of food-grade delivery systems to encapsulate, protect and release bioactive lipids. In this study, emulsion-based delivery systems were prepared consisting of lipid droplets covered by β-lactoglobulin (BLG) and/or lactoferrin (LF) coatings. BLG and LF are globular proteins with relatively low (pI ∼ 5) and high (pI ∼ 8) isoelectric points, respectively. Mixed systems were prepared by adding LF to BLG-stabilized corn oil-in-water emulsions (BLG/LF-coated droplets) or by adding BLG to LF-stabilized corn oil-in-water emulsions (LF/BLG-coated droplets) at pH 7, where there is an electrostatic attraction between the proteins. The influence of pH (3–7), ionic strength (0–60 mM CaCl₂ or 0–200 mM NaCl, pH 7), and thermal treatment (21–90 °C, pH 7, 20 min) on the physical stability of the resulting emulsions was examined. Emulsions with good stability to pH, salt, and thermal processing could be created using mixed interfacial coatings. In addition, we found that the lipids in these emulsions could still be digested using an in vitro digestion model to simulate small intestine conditions. This work may lead to the formation of emulsion-based delivery systems with improved physicochemical and functional performance.     

Lubricating properties of human whole saliva as affected by β-lactoglobulin

The effect of β-lactoglobulin (β-LG) at pH 3.5 and 7.0 on lubricating property of saliva as related to astringency perception was investigated using tribology. Saliva was adsorbed onto surfaces of a rotating poly dimethylsiloxane (PDMS) ball and disc to form a film under conditions that mimic the rubbing contacts in the oral cavity (Bongaerts, Rossetti, & Stokes, 2007) and the lubricity of saliva films upon exposure to astringent compounds was measured. While addition of non-astringent β-LG at pH 7.0 slowly increased friction of saliva film between tribopair surfaces, β-LG at pH 3.5 rapidly increased the friction coefficients of saliva, similar to other astringent compounds (epigallocatechin gallate and alum). This supports the hypothesis that astringency of β-LG arises from the loss of lubrication of saliva which is in agreement with the well-accepted astringency model of polyphenols. Increasing β-LG concentration at pH 3.5 (0.5–10% w/w) caused a rapid increase in friction coefficient; however, at the highest protein concentration, the friction coefficient, although higher than observed for water, was below the values observed for the lower protein concentrations. This suggests that static tribology testing is different from the dynamic in-mouth system such that a simple relationship between friction and sensory astringency cannot be found for all conditions.     

One-step method for isolation and purification of native β-lactoglobulin from bovine whey

BACKGROUND: The major whey protein β‐lactoglobulin (BLG) has been widely studied for its functional properties. The aim of this study was to develop an efficient, inexpensive and rapid one‐step method for the isolation and purification of BLG while preserving its native structure. RESULTS: BLG was purified from defatted whey obtained from raw cow's milk by anion exchange chromatography. Protein purity and identity were determined using reverse phase high‐performance liquid chromatography and mass spectrometry. Total BLG yield was 80% with protein purity from 97 to 99%. BLG isoforms A and B were separated into fractions of 91 and 99% purity respectively. The structure and native conformation of the isolated BLG were compared with those of standard commercial BLG by circular dichroism spectrometry, susceptibility to various crosslinking enzymes and enzyme‐linked immunosorbent assay inhibition. CONCLUSION: The proposed method is very useful for the rapid preparation of BLG suitable for studying antigenic and molecular characteristics of this protein, as well as the effect of food processing on these properties. The procedure requires only 1 day for the purification of about 300 mg of BLG from a single run using a small column (2.5 cm × 20 cm) of diethylaminoethyl Sephadex and has potential for scaling up. Copyright © 2011 Society of Chemical Industry     

Identification and characterization of yak α-lactalbumin and β-lactoglobulin

α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) were isolated from yak milk and identified by mass spectrometry. The variant of α-LA (L8IIC8) in yak milk had 123 amino acids, and the sequence differed from α-LA from bovine milk. The amino acid at site 71 was Asn (N) in domestic yak milk, but Asp (D) in bovine and wild yak milk sequences. Yak β-LG had 2 variants, β-LG A (P02754) and β-LG E (L8J1Z0). Both domestic yak and wild yak milk contained β-LG E, but it was absent in bovine milk. The amino acid at site 158 of β-Lg E was Gly (G) in yak but Glu (E) in bovine. The yak α-LA and β-LG secondary structures were slightly different from those in bovine milk. The denaturation temperatures of yak α-LA and β-LG were 52.1°C and 80.9°C, respectively. This study provides insights relevant to food functionality, food safety control, and the biological properties of yak milk products.     

Effect of underwater high-current discharge on the properties of low-concentration β-lactoglobulin solutions

Solutions of 0.5–2 mg/ml β-lactoglobulin (β-LG) at pH 7.0 were treated with underwater high-current discharges (UHCD) using a bench top system constructed in our laboratory. The short time duration of the UHCD (less than 3 μs) and the high energy delivered to the discharge (∼800 J) produced strong shock waves, in a microsecond time scale, as well as a powerful flash of light with a broad spectrum, ranging from soft X-rays to IR. Structural changes in β-LG were evaluated using hydrophobicity fluorescence of the β-LG molecule, differential scanning calorimetry, reactivity of the free thiol group to Ellman's reagent, small angle X-ray scattering (SAXS) and circular dichroism (CD). The results indicated that the application of the UHCD to β-LG leads to the increase of its surface hydrophobicity by approximately 40%. The enthalpy (ΔH_UHCD) of UHCD treated samples decreased as much as 40% compared to the enthalpy (ΔH_native) of native untreated proteins, indicating extensive but incomplete unfolding of the protein structure with no aggregation. It was found that no refolding occurred after the UHCD treatment. Also, it was shown that the reactivity of the free thiol group of UHCD treated samples increased 10-fold. Tryptophan residues in β-LG were markedly modified, as observed by UV absorption, indicating a change in its position. CD spectra indicated slight modifications in both secondary and tertiary structure.     

The effect of glycation on oil–water emulsion properties of β-lactoglobulin

The creaming, flocculation and coalescence processes of destabilization of emulsions prepared with glycated β-lactoglobulin was analyzed. The glycation process was carried out with glucose and lactose in different reaction conditions (reaction time and protein:carbohydrate molar ratio) The glycation of β-lactoglobulin with both with glucose and lactose causes an increase in the stability of oil–water emulsions. It was found that the process of creaming had a sigmoid behavior which fit to an equation with two parameters, one with hyperbolic and other with sigmoidal kinetics and is directly related to particle size of the dispersed phase of the emulsion. β-lactoglobulin glycated with lactose emulsions showed greater stability to creaming than those prepared with β-lactoglobulin glycated with glucose, which was related to the decrease in the particle size of the dispersed phase and the increased concentration of protein at the interface of the emulsions. Flocculation and coalescence were not influenced by the glycation.     

Genomic architecture of bovine κ-casein and β-lactoglobulin

The objective of this study was to characterize the genetic architecture underlying the absolute concentrations of 2 important milk proteins, κ-casein (κ-CN) and β-lactoglobulin (β-LG), in a backcross population of (Holstein × Jersey) × Holstein cattle. A genome-wide association analysis was performed using a selective DNA pooling strategy and the Illumina BovineHD BeadChip assay [777,000 (777K) SNP markers; Illumina Inc., San Diego, CA]. After correction for multiple testing, 25 single nucleotide polymorphisms were found to be associated with κ-CN and 36 single nucleotide polymorphisms were associated with β-LG. A pathway association analysis revealed 15 Gene Ontology (GO) terms associated with the κ-CN trait and 28 GO terms associated with β-LG. In addition, several GO terms were associated with both milk proteins. Further analysis revealed that κ-CN and β-LG production is regulated by both kinase and phosphatase activity, including mechanisms regulating the extracellular matrix. These results are in concordance with the complex multihormonal process controlling the expression of milk proteins and interactions between mammary epithelial cells and extracellular matrix components. Although κ-CN and β-LG milk proteins are expressed by single genes, the results from this study showed that many loci are involved in the regulation of the concentration of these 2 proteins.     

A large-scale isolation of native β-lactoglobulin: characterization of physicochemical properties and comparison with other methods

A novel method for β-lactoglobulin (β-lg) isolation from whey was investigated. The method comprised a peptic treatment of whey and membrane filtration under gentle conditions for isolation of native β-lg. The method was applied to 10,000 L batches of processed whey and the product quality obtained was compared with pilot-scale results of three other procedures: 3% trichloroacetic acid (TCA) precipitation, a salting-out procedure and a selective thermal precipitation. The calculated yields of native β-lg were 67.3, 44.9, 46.7 and 49.6% of the β-lg present in whey using the four methods, respectively. The composition of the different β-lg preparations was found to be comparable, however, analysis of purity revealed slight differences as indicated by electrophoresis and fast protein liquid chromatography (FPLC). The isolated β-lg retained a high degree of purity and native properties. The new method for isolation of native β-lg was found to be reproducible, selective and robust as processing of three 10,000 L batches did not indicate any technical problems or variations in the purity of the isolated β-lg.     

Effect of β-lactoglobulin A and B whey protein variants on cheese yield potential of a model milk system

Cheese yield mainly depends on the amount and proportion of milk constituents; however, genetic variants of the proteins present in milk may also have an important effect. The objective of this research was to study the effect of the variants A and B of β-lactoglobulin (LG) on cheese yield using a model system consisting of skim milk powder fortified with different levels of a mixture containing α-lactalbumin and β-LG genetic variants (A, B, or A-B) in a 1:2 ratio. Fortified milk samples were subjected to pasteurization at 65°C for 30 min. Miniature cheeses were made by acidifying (pH = 5.9) fortified milk and incubating with rennet for 1 h at 32°C. The clot formed was cut, centrifuged at 2,600 × g for 30 min at 20°C and drained for determining cheese yield. Cheese-yielding capacity was expressed as actual yield (grams of cheese curd per 100 g of milk) and dry weight yield (grams of dried cheese curd per 100 g of milk). Free-zone capillary electrophoresis was used for determining β-LG A or B recovery in the curd during rennet-induced coagulation. The presence of β-LG variant B resulted in a significantly higher actual and dried weight cheese yield than when A or A-B were present at levels ≤0.675% of whey protein (WP) addition. Results of free-zone capillary electrophoresis allowed us to infer that β-LG B associates with the casein micelles during renneting, as shown by an increase in the recovery of this variant in the curd when β-LG B was added up to a maximum at 0.45% (equivalent to 0.675% WP). In general, actual or dried weight cheese yield increased as WP addition was increased from 0.225 to 0.675%. However, when WP addition ranged from 0.675 to 0.90%, a drastic drop in cheese yield was observed. This behavior may be because an increase in the aggregation of casein micelles with a concomitant inclusion of whey protein in the gel occurs at low levels of WP addition, whereas once the association of WP with the casein micelles reach a saturation point at addition levels higher than 0.675%, rearrangements of the gel network result in larger whey expulsion and syneresis. This knowledge is expected to be useful to maximize cheese yield and optimize processing conditions during cheese and cheese analogs manufacturing.     

Rheology and microstructure of basil seed gum and β-lactoglobulin mixed gels

Gelling profiles and interaction between basil seed gum (BSG) and β-lactoglobulin (BLG) were characterized using dynamic rheology and scanning electron microscopy. Sequence of experimental sweeps of time–temperature, frequency, and strain were applied for BSG, BLG and BLG–BSG mixtures in the ratio of 10:1, 5:1, 2:1 & 1:2. Rheological behavior of BLG was found to be different from that of BSG and gelling point was raised during the heating period. By increasing BSG concentration, storage and loss moduli were increased and gel point was determined at about 80 °C, which was dependent of concentration and occurred during cooling period. Upon heating–cooling period (temperature sweep) of BLG–BSG mixture from 20 °C to 90 °C and coming back to original temperature, mixed gels presented a biphasic profile: the first phase, characterized by a sharp increase in the storage modulus in which gelation of BLG happened and the second phase exhibiting an increase in the storage modulus, corresponded to the build-up of a BSG network. Strain sweep results obtained for mixed gels at the end of the temperature sweep tests supported the biphasic hypothesis. The SEM results showed that BLG has a globular structure in comparison with BSG which has fibril and globular structure and the mean diameter of its globular structure was higher than that of BLG. Increasing the ratio of BSG to BLG resulted in a finer microstructure, which could retain higher water to form gel. In addition, microstructural study showed that BLG–BSG mixed is a bicontinuous network in which BSG dispersed in BLG continuous phase.     

Microstructure and rheology of β-lactoglobulin–galactomannan aqueous mixtures

The effect of the addition of a galactomannan (locust bean gum, LBG, or tara gum, TG) on the microstructure and rheological properties of a globular protein (β-lactoglobulin, β-Lg) solution was studied at pH 7.0, when the protein bears a net negative charge. Confocal laser scanning microscopy was used to explore the microstructure. Steady shear and dynamic oscillatory measurements were performed with a controlled stress rheometer AR2000 (TA Instruments) fitted with a cone-and-plate geometry. Mixtures were prepared with 6.5 wt% β-lactoglobulin concentration and 0.31–0.82 wt% LBG or 0.23–0.71 wt% TG concentration. All mixed systems were two-phase. The microstructure was clearly dependent on the concentration of the galactomannan in the mixture: the systems evolved from a continuous matrix of β-lactoglobulin enriched phase containing some small inclusions of the galactomannan, to a matrix of galactomannan-enriched continuous phase containing aggregates of β-lactoglobulin. Modifications of the flow and viscoelastic properties with respect to the individual components were clearly evidenced for the mixed systems. Phase inversion detected by microscopy could also be detected by rheology as a modification in the flow/viscoelastic behaviour.     

Synthesis and characterization of β-cyclodextrin inclusion complexes of thymol and thyme oil for antimicrobial delivery applications

Microencapsulation with β-cyclodextrin (β-CD) as a delivery platform offers a promising application for natural antimicrobial delivery for food safety. Hence, this study aimed to characterize inclusion complexes of thymol and thyme essential oil with β-CD and their resulting antimicrobial activities. Inclusion complexes were prepared by kneading (KN) and freeze-drying (FD) methods. The entrapment efficiencies ranged from 71 to 83 g/100 g with higher (P < 0.05) values for thymol and FD particles. All particles showed irregular shapes and sharp edges with significant differences in size distribution and strong tendency to agglomerate. FD produced higher (P < 0.05) particle sizes than KN method. Differential Scanning Calorimetry (DSC) analysis confirmed inclusion complexes formation between β-CD and both compounds. Phase solubility tests indicated B_S-type complexes formation between thymol and β-CD. All compounds effectively inhibited bacterial growth within the concentration range tested. Free thyme oil showed higher (P < 0.05) antimicrobial activity than thymol, and FD β-CD inclusion complexes were able to inhibit Escherichia coli K12 at lower (P < 0.05) active compound concentrations than corresponding free oils. Antimicrobial activities of β-CD inclusion complexes against total viable counts in pork meat system throughout storage were observed. The FD method showed to be the best encapsulation method with enhanced antimicrobial activity for both compounds.