Rheological properties of acid gels prepared from heated milk fortified with whey protein mixtures containing the A,B and C variants of β-lactoglobulin

The effect of fortification of reconstituted skim milk with different levels of a whey protein mixture containing a 1:2 ratio of α-lactalbumin (α-la) and different genetic variants of β-lactoglobulin (β-LG) on the rheological properties of acid milk gels, formed by acidification with glucono-δ-lactone, was investigated. Milk samples were either unheated or heated at 80°C for 30 min before acidification. Acid gels prepared from unheated skim milk had very low G′ values, long gelation times and low gelation pH. Samples prepared from heated milk had markedly higher G′ values, a reduced gelation time and an increased gelation pH. The addition of increasing levels of whey protein mixtures containing β-LG B or β-LG C to the milk prior to heating and acidification caused an almost linear increase in the G′. In contrast, whey protein mixtures containing β-LG A caused a progressive increase in the G′ with added protein levels up to about 0.7% (w/w) but little further change at higher addition levels. A mixture of the A and B variants of β-LG gave an intermediate behaviour between those of the A and B variants. In all samples, the G′ value at 5°C was approximately twice that at 30°C so that the relative differences as a result of the β-LG genetic variants were similar for the two temperatures.     

Effect of prolactin, β-lactoglobulin, and κ-casein genotype on milk yield in East Friesian sheep

The effect of prolactin (PRL), β-lactoglobulin (β-LG), and κ-casein (CSN3) on milk yield was estimated in an East Friesian dairy sheep population from Old Chatham Sheepherding Company, New York. Genotypes were determined by PCR amplification followed by digestion with HaeIII and RsaI for PRL and β-LG, respectively, and by PCR amplification for CSN3. Monthly milking records and pedigree information were used to evaluate the effect of each polymorphism on milk yield. Results indicated that PRL genotype had a significant effect on milk yield. Ewes carrying one A allele produced 110.6 g more milk per day than ewes with no A alleles. There was no statistical difference between ewes with only one A allele and ewes with 2 A alleles. No association among polymorphisms at the β-LG and CSN3 loci and milk yield was found. The results presented in this study indicate that the PRL gene is a potential marker that could be used in selection programs for improving milk yield in dairy sheep.     

Effect of κ-casein B relative content in bulk milk κ-casein on Montasio, Asiago, and Caciotta cheese yield using milk of similar protein composition

The aim of this study was to investigate the effect exerted by the relative content of κ-casein (κ-CN) B in bulk milk κ-CN on coagulation properties and cheese yield of 3 Italian cheese varieties (Montasio, Asiago, and Caciotta). Twenty-four cheese-making experiments were carried out in 2 industrial and 1 small-scale dairy plant. Detailed protein composition of bulk milk of 380 herds providing milk to these dairies was analyzed by reversed-phase HPLC. To obtain 2 experimental milks differing in the relative content of κ-CN B in κ-CN, herds were selected on the basis of bulk milk protein composition and relative content of κ-CN genetic variants. Milk was collected and processed separately for the 2 groups of selected herds. A difference of 20% in the relative content of κ-CN B in κ-CN was obtained for the 2 experimental milks for Montasio and a difference of 15% for Asiago and Caciotta. The 2 experimental milks were of similar protein and CN content, casein number, pH, CN composition, and β-CN genetic composition. For each cheese-making trial, amounts of milk, ranging from 2,000 to 6,000 kg, were manufactured. Each vat contained milk collected at least from 4 dairy herds. Cheese yield after brining and at the end of the aging was recorded. Milk with a greater proportion of κ-CN B in κ-CN (HIGHB) exhibited similar coagulation properties and greater cheese yield compared with milk with a lower proportion of κ-CN B in κ-CN (LOWB). The increased cheese yield observed for HIGHB when manufacturing Montasio cheese was ascribed to a greater fat content compared with LOWB. The probability of HIGHB giving a cheese yield 5% greater than that of LOWB ranged from 51 to 67% for Montasio cheese, but was less than 21% for Asiago and Caciotta cheeses. Variation in relative content of κ-CN B in κ-CN content did not relevantly affect industrial cheese yield when milks of similar CN composition were processed. An indirect effect due to the increased κ-CN content of κ-CN B milk is thought to explain the favorable effects of κ-CN B on cheese yield reported in the literature.     

Effects of lipid supplementation on the yield and composition of milk from cows with different β-lactoglobulin phenotypes

Responses to lipid supplementation differ between dairy breeds and genetic lines suggesting nutrition by genotype interactions. β-Lactoglobulin phenotype is associated with changes in yield and composition of milk. The response of cows with different β-lactoglobulin phenotypes to lipid supplementation has not been examined. Furthermore, we examined whether lipid supplementation alters milk protein composition. By using a randomized block design, we fed Holstein cows for 3 wk either a control diet containing 2.8% crude fat (n = 19) or an experimental diet that was supplemented with 4.2% tallow (n = 20). Before randomization, all cows were fed the supplemental tallow diet for at least 2 wk. Dry matter intake, body weight, milk yield, and milk composition were measured in the last week before and during the experimental period. Feeding supplemental tallow increased dry matter intake and yields of milk and milk components, including casein content, without decreasing milk component content or altering milk protein composition. On the low-fat control diet, cows with the β-lactoglobulin allele B had a greater milk and milk component yield than cows with the A allele, whereas no differences by β-lactoglobulin phenotype were observed in cows on the tallow supplement diet. Our results suggest that cows that differ in β-lactoglobulin phenotype respond differently to a low-fat diet and that feeding cows 4.2% of additional tallow increases milk yield without affecting milk component content and milk protein composition.     

Antigenic response of whey proteins and genetic variants of β-lactoglobulin — the effect of proteolysis and processing

Limited enzymatic hydrolysis trials of WPC-80 and β-lactoglobulin AA, AB and BB were performed using specific enzymes (trypsin, Neutrase, Corolase PP, Corolase PS) in a pH-stat under controlled conditions. The hydrolysates of WPC-80 and β-lactoglobulin were fractionated into high and low molecular fractions. Residual antibody binding activity of the peptides was dependent on the degree of hydrolysis (DH 2-20), but also on the enzyme used. Heat treatment affected the solubility and thereby the antigenic response. Dialysis influenced the antibody binding activity of the peptides. Tryptic hydrolysis of β-lactoglobulin AA was slower than for β-lactoglobulin BB and AB. Antigenic responses of the hydrolysates and fractions were measured by SLOT-BLOT and ELISA. SLOT-BLOT, a rapid screening method, was not able to differentiate the hydrolysates. The ELISA, being a more sensitive method, differentiated between the genetic variants, but was more time consuming. The lowest antigenicity was observed in the 1000–5000 Da fraction and β-lactoglobulin AA showed the lowest response.     

Ultrasonic generation of aerated gelatin gels stabilized by whey protein β-lactoglobulin

Dispersed air provides an additional phase within gel-type foods may accommodate new textural and functional demands. This paper addresses the effect of using whey protein β-lactoglobulin (β-lg), with different degrees of denaturation, as stabilizing agent in the formation of aerated gelatin gels using ultrasound as a novel method to incorporate bubbles in model foods. The heat denaturation, aggregate formation and surface properties of β-lg dispersions were studied at three pHs (6.0, 6.4 and 6.8) and at a heating temperature of 80 °C. β-Lg dispersions with four degrees of denaturation (0%, 20%, 40% and 60%) were used to stabilize bubbles generated by high intensity ultrasound in aerated gelatin gels. Experimental methods to determine gas hold-up, bubble size distributions and fracture properties of aerated gelatin gels stabilized by β-lg (AG), as well as control gels (CG), aerated gelatin gels without β-lg, are presented. Gas hold-up of AG peaked at a degree of denaturation of 40% when AG were fabricated using β-lg heated at pH 6.4 and 6.8, whereas using β-lg heated at pH 6.0 gas hold-up decreased constantly with increasing degree of denaturation. The use of β-lg as surfactant at pH 6.8 and 6.4 reduced the bubble sizes of AG compared with CG, but no effect was observed at pH 6.0. AG showed values of stress and strain at fracture lower than CG (5.86 kPa and 0.62), probably because of the lower gas hold-up of CG. However, both type of aerated gels were weaker and less ductile than non-aerated gels, with a decrease in stress and strain at fracture for AG between 56–71% and 33–43%, respectively. This study shows that the presence of bubbles in gel-based food products results in unique rheological properties conferred by the additional gaseous phase.     

Effect of 3 lactobacilli on immunoregulation and intestinal microbiota in a β-lactoglobulin–induced allergic mouse model

Milk is one of the earliest and most common allergen sources in the world, with β-lactoglobulin representing a major allergen protein. Numerous studies have reported that probiotics exert antiallergic and anti-inflammatory effects. Here, we examined the effects of 3 strains of Lactobacillus on immunomodulatory functions, intestinal barrier functions, and intestinal microbiota through a β-lactoglobulin–induced allergic mouse model. We found that the oral administration of Lactobacillus plantarum ZDY2013 and Lactobacillus rhamnosus GG suppressed allergic response, attenuating serum IgE and relieving anaphylaxis symptoms. The 3 strains of Lactobacillus could induce T helper (Th) 1 or T regulatory cells to differentiate to inhibit the Th2-biased response for regulating Th1/Th2 immune balance. Furthermore, L. plantarum ZDY2013 and L. rhamnosus GG enhanced intestinal barrier function through the regulation of tight junction. We also found that L. plantarum ZDY2013 and L. plantarum WLPL04 could regulate alterations in intestinal microbiota caused by allergies. In particular, Rikenella, Ruminiclostridium, and Lachnospiraceae UCG-006 were considerably reduced after treatment with L. plantarum ZDY2013 and L. plantarum WLPL04. These results suggested that 3 Lactobacillus strains may serve as an effective tool for the treatment of food allergies by regulating immune and gut microbiota.     

Influence of milk pre-heating conditions on casein–whey protein interactions and skim milk concentrate viscosity

The viscosity of concentrates (50–55% total solids) prepared from skim milk heated (5 min at 80 or 90 °C) at pH 6.5 and 6.7 was examined. The extent of heat-induced whey protein denaturation increased with increasing temperature and pH. More denatured whey protein and κ-casein were found in the serum phase of milk heated at higher pH. The viscosity of milk concentrates increased considerably with increasing pH at concentration and increasing heating temperature, whereas the distribution of denatured whey proteins and κ-casein between the serum and micellar phase only marginally influenced concentrate viscosity. Skim milk concentrate viscosity thus appears to be governed primarily by volume fraction and interactions of particles, which are governed primarily by concentration factor, the extent of whey protein denaturation and pH. Control and optimization of these factors can facilitate control over skim milk concentrate viscosity and energy efficiency in spray-drying.     

Effects of Maillard reaction conditions on the antigenicity of α-lactalbumin and β-lactoglobulin in whey protein conjugated with maltose

The effects of Maillard reaction conditions (weight ratio of protein to sugar, temperature and time) on the antigenicity of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG) in conjugates of whey protein isolate (WPI) with maltose were investigated. Response surface methodology was used to establish models to predict the antigenicity of α-LA and β-LG and find an optimal reaction condition under which the antigenicity of α-LA and β-LG reduces to minimum value. Conjugating WPI with maltose was an effective way to reduce the antigenicity of α-LA and β-LG. The antigenicity of α-LA decreased from 32.25 μg mL⁻¹ to 10.91 μg mL⁻¹. And the antigenicity of β-LG decreased from 272.4 μg mL⁻¹ to 38.17 μg mL⁻¹. Temperature had the greatest effect on the antigenicity of α-LA, while weight ratio of WPI to maltose was the most significant factor on the antigenicity of β-LG.     

Partitioning of α-lactalbumin and β-lactoglobulin in whey protein concentrate/hydroxypropylmethylcellulose aqueous two-phase systems

Whey protein concentrate (WPC) was fractionated by using hydroxypropylmethylcellulose (HPMC) at pH 6.5. Incompatible mixtures with different proportions of HPMC and WPC were prepared. After phase separation, the protein concentration in both phases was determined by the Kjeldahl method and the proportion of each protein by SDS-PAGE combined with image analysis. The results show that the low molecular weight proteins α-lactalbumin (α-lac) and β-lactoglobulin (β-lg) were retained in high proportion in the upper phase (about 90% compared to 64% of WPC). The most efficient condition to fractionate β-lg and α-lac was the phase separation of an incompatible mixed system with a high initial concentration of WPC and a low initial concentration of HPMC i.e., WPC 20%, wt/wt/HPMC 0.5%, w/w. It can be concluded that the thermodynamic incompatibility which arises from mixing WPC with HPMC could be used as a method for fractionation of whey proteins.     

Effect of high-pressure treatment on in-vitro digestibility of β-lactoglobulin

The effect of high-pressure (HP)-treatment on β-lactoglobulin (β-Lg) was investigated using in-vitro pepsin digestion under simulated gastric conditions. HP-treatment of β-Lg at 400 MPa for 10 min only slightly increased its subsequent hydrolysis by pepsin. However, higher pressure treatments (600 and 800 MPa) resulted in rapid digestion of β-Lg. After these higher pressure treatments, β-Lg disappeared in less than 1 min of pepsin incubation as determined by SDS-PAGE analysis. Mass spectrometry analysis of the digestion products at corresponding incubation times revealed rapid and progressive degradation of β-Lg. Most (> 90%) of the peptide products following pepsin digestion of HP-treated β-Lg were less than 1500 Da in size. Peptide products from pepsin digestion were identified and mapped to β-strand regions (Leu₃₂–Leu₅₄ and Phe₈₂–Leu₁₀₄) and to the N- and C-terminals regions (Leu₁–Leu₁₀ and Ser₁₅₀–Leu₁₅₆) of β-Lg. While these regions corresponded to known IgE epitopes of β-Lg, the predominant peptides resulting from 60 s of incubation were short (7–10 residues) in length. These results demonstrate that HP-treatment increased the digestibility of β-Lg and represents a promising processing technology for reducing the allergenicity of known allergens in a wide variety of food materials.Industrial relevanceHigh-pressure treatment is widely used to enhance the functional attributes of food proteins. The potential for enhanced nutritional value of β-Lg was also demonstrated here by its increased digestibility. High-pressure treatment followed by incubation with proteases may represent a method for the commercial production of bioactive peptides such as inhibitors of angiotensin converting enzyme. More importantly, high-pressure-induced unfolding of milk proteins may reduce their allergenicity. Unfolded proteins are less likely to become agents of immunological sensitization because they are more readily hydrolyzed. Thus high-pressure treatment applied to food ingredients such as whey protein isolate may contribute to the development of hypoallergenic foods.     

Effect of pulsed-light processing on the surface and foaming properties of β-lactoglobulin

Pulsed-light processing was used to treat β-lactoglobulin (BLG) solutions. The impact of pulsed light (PL) on the structural properties of this protein was explored through far-UV, CD spectral analysis, size exclusion chromatography, surface hydrophobicity and NMR spectroscopy. Changes on these physicochemical properties were related to surface rheology, surface tension, foam stability and foam capacity of the non-treated and treated BLG to elucidate adsorption mechanism and consequences on foaming capacity. Conformational modification of BLG was related with PL total fluence as important conformational changes increased when total fluence was higher. Consequently, adsorption rate of treated BLG at the air/water interface was faster than native BLG. Additionally, treated BLG formed highly elastic interfaces. This was found to have an impact on the foam stability. Pulsed-light treatment seemed to enhance the overall strength of the interface, resulting in more stable foams.     

Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin

Immunoreactivity of bovine β-lactoglobulin (β-Lg) hydrolysates obtained after a simulated gastrointestinal digestion and previously glycated via Maillard reaction with galactose, tagatose, and dextran of 10 or 20 kDa has been determined, with a view to study the effect of glycation and aggregation degree of β-Lg on its residual immunoreactivity. High levels of glycation impaired β-Lg proteolysis and, consequently, increased the IgG- and IgE-reactivities of hydrolysates, regardless of the carbohydrate used. Protein aggregation during the advanced stages of Maillard reaction had a masking effect on β-Lg epitopes, counteracting the negative effect of the lower digestibility of glycated protein on its allergenicity. Finally, the use of polysaccharides as glycation agents did not contribute to enhancement of the masking effect of the attached carbohydrate on β-Lg epitopes. These findings stress the importance of evaluating the impact of glycation on protein gastrointestinal digestibility prior to investigation of the immunoreactivity of protein Maillard complexes.     

Effect of high-pressure treatment on denaturation of bovine β-lactoglobulin and α-lactalbumin

The effect of high-pressure treatment on denaturation of β-lactoglobulin and α-lactalbumin in skimmed milk, whey, and phosphate buffer was studied over a pressure range of 450–700 MPa at 20 °C. The degree of protein denaturation was measured by the loss of reactivity with their specific antibodies using radial immunodiffusion. The denaturation of β-lactoglobulin increased with the increase of pressure and holding time. Denaturation rate constants of β-lactoglobulin were higher when the protein was treated in skimmed milk than in whey, and in both media higher than in buffer, indicating that the stability of the protein depends on the treatment media. α-Lactalbumin is much more baroresistant than β-lactoglobulin as a low level of denaturation was obtained at all treatments assayed. Denaturation of β-lactoglobulin in the three media was found to follow a reaction order of n = 1.5. A linear relationship was obtained between the logarithm of the rate constants and pressure over the pressure range studied. Activation volumes obtained for the protein treated in milk, whey, and buffer were −17.7 ± 0.5, −24.8 ± 0.4, and −18.9 ± 0.8 mL/mol, respectively, which indicate that under pressure, reactions of volume decrease of β-lactoglobulin are favoured. Kinetic parameters obtained in this work allow calculating the pressure-induced denaturation of β-lactoglobulin on the basis of pressure and holding times applied.     

Effect of κ-carrageenan on milk protein polysaccharide mixtures

The effect of κ-carrageenan (0, 0.025, 0.05%) on phase separation between polysaccharides (0.36% of locust bean gum (LBG), guar gum, or xanthan gum) and milk proteins (from 10.5% skim milk powder) in solution was studied. Xanthan gum was seen to be the most incompatible with milk proteins, followed by guar gum and LBG. Casein micelles were more incompatible with all polysaccharides than whey proteins. Whereas at either concentration κ-carrageenan inhibited visual phase separation, it was seen by transmission electron microscopy that samples with κ-carrageenan showed microscopic phase separation. Samples with 0.05% κ-carrageenan and either LBG or guar gum and all samples with xanthan gum could be described rheologically as weak gels, while those with no or 0.025% κ-carrageenan and either LBG or guar gum could be described as concentrated solutions. Thus, no correlation was seen between the inhibition of macroscopic phase separation by κ-carrageenan and the formation of a weak gel in solution.     

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.     

Dipeptidyl peptidase-IV inhibitory peptides generated by tryptic hydrolysis of a whey protein concentrate rich in β-lactoglobulin

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease involved in the degradation and inactivation of incretin hormones that act by stimulating glucose-dependent insulin secretion after meal ingestion. DPP-IV inhibitors have emerged as new and promising oral agents for the treatment of type 2 diabetes. The purpose of this study was to investigate the potential of β-lactoglobulin as natural source of DPP-IV inhibitory peptides. A whey protein concentrate rich in β-lactoglobulin was hydrolysed with trypsin and fractionated using a chromatographic separation at semipreparative scale. Two of the six collected fractions showed notable DPP-IV inhibitory activity. These fractions were analysed by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) to identify peptides responsible for the observed activity. The most potent fragment (IPAVF) corresponded to β-lactoglobulin f(78–82) which IC₅₀ value was 44.7 μM. The results suggest that peptides derived from β-lactoglobulin would be beneficial ingredients of foods against type 2 diabetes.     

The β-lactoglobulin content of bovine milk: Development and application of a biosensor immunoassay

An optical biosensor immunoassay exploiting surface plasmon resonance is described for the quantification of β-lactoglobulin in milk. Samples were diluted with buffer, and the protein estimated from binding with a polyclonal antibody immobilised on the sensor surface. Analytical method performance characteristics including range, detection limit, precision and accuracy were determined and reported. The temporal variability in the β-lactoglobulin content of milk from pasture-fed cows during early lactation and across a production season was investigated. The content of β-lactoglobulin decreased from >10 mg mL⁻¹ in early colostrum to <5 mg mL⁻¹ in mature milk, and the β-lactoglobulin content of skim milk powder trended from 25 to 60 mg g⁻¹ across a season. In view of its allergenicity, these data will improve understanding of the expression of innate β-lactoglobulin in the milk of pasture-grazed dairy herds, thereby providing information that is applicable to the formulation of bovine milk-based products.     

Pilot-scale formation of whey protein aggregates determine the stability of heat-treated whey protein solutions—Effect of pH and protein concentration

Denaturation and consequent aggregation in whey protein solutions is critical to product functionality during processing. Solutions of whey protein isolate (WPI) prepared at 1, 4, 8, and 12% (wt/wt) and pH 6.2, 6.7, or 7.2 were subjected to heat treatment (85°C × 30 s) using a pilot-scale heat exchanger. The effects of heat treatment on whey protein denaturation and aggregation were determined by chromatography, particle size, turbidity, and rheological analyses. The influence of pH and WPI concentration during heat treatment on the thermal stability of the resulting dispersions was also investigated. Whey protein isolate solutions heated at pH 6.2 were more extensively denatured, had a greater proportion of insoluble aggregates, higher particle size and turbidity, and were significantly less heat-stable than equivalent samples prepared at pH 6.7 and 7.2. The effects of WPI concentration on denaturation/aggregation behavior were more apparent at higher pH where the stabilizing effects of charge repulsion became increasingly influential. Solutions containing 12% (wt/wt) WPI had significantly higher apparent viscosities, at each pH, compared with lower protein concentrations, with solutions prepared at pH 6.2 forming a gel. Smaller average particle size and a higher proportion of soluble aggregates in WPI solutions, pre-heated at pH 6.7 and 7.2, resulted in improved thermal stability on subsequent heating. Higher pH during secondary heating also increased thermal stability. This study offers insight into the interactive effects of pH and whey protein concentration during pilot-scale processing and demonstrates how protein functionality can be controlled through manipulation of these factors.     

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.