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     

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.     

The effect of gel structure on the kinetics of simulated gastrointestinal digestion of bovine β-lactoglobulin

The structure and properties of protein gels depend on the conditions under which they are formed. Here, we assessed the susceptibility of protein to simulated gastro-duodenal digestion of weak gels with contrasting structures, produced from either purified bovine β-lactoglobulin (β-Lg) or whey protein isolate (WPI) at pH ranging from 2.5 to 6.5 and using different heating regimes. Gels formed close to the isoelectric point proved to be very resistant to simulated gastric digestion, with more than 85% of β-Lg remaining and in the simulated duodenal phase of digestion. The sample heated to 85 °C was most resistant with over 40% remaining. In the WPI sample heated to 85 °C, more than 20% of the original β-Lg content remained undigested after simulated gastro-duodenal proteolysis. These results suggest that firm particulate gels can persist longer in the GI tract and may be useful in inducing satiety and thus provide another weapon in the fight against obesity.     

Effect of enzymatic hydrolysis and polysaccharide addition on the β-lactoglobulin adsorption at the air-water interface

The effect of enzymatic hydrolysis and polysaccharide addition on the interfacial adsorption of β-lactoglobulin (β-LG) was investigated in this work. The enzymatic treatment was performed in the hydrolysis degree (HD) range of 0.0-5.0% using bovine α-chymotrypsin II immobilized on agarose beads. Anionic non-surface active polysaccharides (PS), sodium alginate (SA) and λ-carrageenan (λ-C) were studied in the concentration range of 0.0-0.5 wt.%. The adsorption process at the air-water interface was evaluated by means of tensiometry and surface dilatational rheology. Biopolymer interactions in solution were analyzed by extrinsic fluorescence spectroscopy. The enzymatic hydrolysis improved β-LG interfacial properties. On the other hand, at low HD (1.0%), PS addition enhanced surface and elastic properties of β-LG hydrolysate films probably due to a higher repulsion between biopolymers in solution. However, at high HD (3.0-5.0%), SA addition caused a deterioration of surface and elastic properties of β-LG hydrolysate films probably due to the segregation and hydrolysate aggregation in solution, whereas λ-C addition could promote the formation of soluble complexes leading to a better control of elastic properties of β-LG hydrolysate films.     

Interactions between β-lactoglobulin and dextran sulfate at near neutral pH and their effect on thermal stability

The effect of interactions between β-lactoglobulin (β-LG) and dextran sulfate (DS) on thermal stability at near neutral pH was investigated. Samples containing 6% w/w β-LG and DS (M_w = 5–500 kDa) at different biopolymer weight ratios, pH (5.6–6.2), and NaCl concentrations (0–30 mM) were heated at 85 °C for 15 min. Turbidity results showed that the presence of DS at appropriate biopolymer weight ratio and pH significantly lowered the turbidity of heated β-LG. Solutions containing DS:β-LG weight ratios of 0.02 or less showed improved heat stability as indicated by decreased turbidity. Analysis of the unheated mixture by size exclusion chromatography coupled with multi-angle laser light scattering (SEC–MALLS) showed an interaction between β-LG and DS. The size of the aggregates increased as pH decreased. The β-LG–DS aggregates had a greater negative charge as seen from electrophoretic mobility measurement. Addition of 30 mM NaCl inhibited complex formation and the effect of DS on reducing the turbidity of heated β-LG, suggesting that the interaction was electrostatic in nature. Other than charge property, the amount and size of native aggregates appeared to be the major factor in determining how DS altered heat-induced aggregation. The presence of DS decreased denaturation temperature of β-LG, indicating that DS did not improve thermal stability of β-LG by stabilizing its native state but rather by altering its aggregation. The results provide information that will facilitate the application of whey proteins and polysaccharides as functional ingredients in foods and beverages.     

Effects of ultrasonic treatment on the gel properties of microbial transglutaminase crosslinked soy,whey and soy–whey proteins

Food Science and Biotechnology - This paper studied the influences of diverse ultrasonic power treatments on the physico-chemical properties of soy–whey mixed protein induced by microbial...     

Molecular dynamics simulation of the effect of heat on the conformation of bovine β-lactoglobulin A: A comparison of conventional and accelerated methods

Three molecular dynamics (MD) simulation methods are used to follow the thermal unfolding of bovine β-lactoglobulin (β-lac). The methods used, classical MD simulation at different temperatures in the range 300–500 K, essential dynamics and replica exchange MD, were chosen to give a range of conventional and accelerated methods. At 350 K, just above the experimentally determined denaturation temperature of β-lac only small changes to the tertiary fold and secondary structure were seen during a 110 ns simulation. At 400 K and 450 K more unfolding was observed, but it was not until the temperature was increased to 500 K that substantial disruption to the protein structure was seen. For the time that the heated simulated β-lac molecules occupy the same conformation space, they appear to follow similar unfolding pathways. ED simulations biased along the first 5 eigenvectors (determined at 350 K) give rise to conformations that have a more elongated tertiary fold compared to heated simulations, and do not follow similar unfolding pathways to the heated simulations. The REMD simulation, which is the equivalent of an approximately 0.7 μs simulation at 350 K, shows a small degree of tertiary structure unfolding, but very little secondary structure change. The results are discussed in terms of the structural changes that have been observed to occur experimentally in β-lac.     

Use of an electrodialytic reactor for the simultaneous β-lactoglobulin enzymatic hydrolysis and fractionation of generated bioactive peptides

The enzymatic hydrolysis of β-lactoglobulin and the fractionation of peptides were performed in one step in an electrodialysis cell with ultrafiltration membranes stacked. After 240 min of treatment, 15 anionic and 4 cationic peptides were detected in the anionic and cationic peptide recovery compartments. Amongst these 15 anionic peptides, 2 hypocholesterolemic, 3 antihypertensive and 1 antibacterial peptides were recovered and concentrated with migration rates ranging from 5.5% and 81.7%. Amongst the 4 cationic peptides, the peptide sequence ALPMHIR, identified as lactokinin and known to exert an important antihypertensive effect, was recovered with an estimated 66% migration rate. To our knowledge, it was the first attempt to perform hydrolysis under an electric field and to simultaneously separate anionic and cationic peptides produced.     

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.     

Effect of ascorbic acid and protein concentration on the molecular weight profile of bovine serum albumin and β-lactoglobulin by γ-irradiation

The effects of ascorbic acid and protein concentration on the molecular weight size distribution of BSA and β-lactoglobulin were examined after irradiation of proteins at various doses. Gamma-irradiation of protein solutions caused disruption of the ordered structure of protein molecules resulting in degradation, cross-linking, and aggregation of the polypeptide chains. SDS–PAGE and gel permeation chromatography study showed that ascorbic acid protected the aggregation and degradation of proteins by scavenging oxygen radicals produced by irradiation and the effect of irradiation on protein conformation was more significant at lower concentrations of proteins.     

One-step method for the isolation of α-lactalbumin and β-lactoglobulin from cow’s milk while preserving their antigenicity

Milk is a highly nutritional food, and separation of major allergens from milk has become important to people who are allergic. The aim of this study was to establish a simple and repeatable method for the isolation of α-lactalbumin and β-lactoglobulin from cow’s milk while preserving their antigenicity. Fractions of α-lactalbumin and β-lactoglobulin were salted-out using 50% ammonium sulfate from whey that was collected from cow’s milk after pH adjustment and then purified by anion-exchange chromatography with diethylaminoethyl-Sepharose Fast Flow. The antigenicity of the purified proteins was evaluated by indirect competitive enzyme-linked immunosorbent assay. The results showed that the purities of the α-lactalbumin and β-lactoglobulin collected were 84.85 and 94.91% and the cross-reactivities of the purified proteins were 93.2 and 95.4%, respectively. Therefore, this simple and efficient strategy consisting of a one-step process for α-lactalbumin and β-lactoglobulin is suitable for purifying the major allergens in cow’s milk. In addition, a scientific experimental basis for the preparation of non-allergenic milk was also offered in this study.     

Symposium review: The relevance of bovine milk phospholipids in human nutrition—Evidence of the effect on infant gut and brain development

This paper reflects the concepts reviewed during the presentation in the Joint MILK/Lactation Biology Symposium at the ADSA 2018 Annual Meeting. Our intention is to update the concepts and advances in the area of research regarding milk phospholipids or polar lipid fraction as part of a dairy ingredient used today in nutritional studies that focus on gut health as well as brain development of infants. Although processing advances have allowed the production of novel ingredients rich in milk fat globule membrane (MFGM) components, mostly monitored by phospholipid concentration and presence of membrane proteins, there is wide variability in their composition and structure. Furthermore, we aimed to include in the phospholipid fraction of milk nanovesicles designated as milk exosomes, which are secreted into milk by different secretion mechanisms than those of the fat globules but are also made up of a unique mixture of polar lipids. We consider imperative the study of polar lipid-derived structures from milk regarding composition and structure to gain insights into their biological effect in human health. Nevertheless, and tolerating the differences in composition and concentration of their components, studies supplementing the diet of infants with polar lipids (i.e., MFGM components) have shown significant advances in several areas of human health and well-being. Here we present a summary of the important components of MFGM and milk exosomes as well as an overview of the effects on gut health and brain and cognitive development when added to the diet of infants.     

The effect of different treatments for early-lactation hyperketonemia on blood β-hydroxybutyrate,plasma nonesterified fatty acids,glucose, insulin,and glucagon in dairy cattle

Despite increased efforts in preventing the occurrence of metabolic disorders in transition cows, hyperketonemia remains a frequent early-lactation metabolic disease affecting an average of 40% of cows in herds in the United States. Despite the demonstrated economic effect of this disorder, controlled clinical trials comparing different treatment strategies in affected cows are lacking. The objective of our study was to investigate the effect of treatment with intravenous glucose, oral propylene glycol, or a combination of both on the reduction in blood β-hydroxybutyrate (BHB) concentrations of early-lactation hyperketonemic dairy cows. Multiparous Holstein cows between 3 to 9 d in milk were screened for hyperketonemia using a handheld meter 3 times per week, and enrolled at whole blood BHB concentration ≥1.2 mmol/L to 1 of 4 treatment groups: (1) 500 mL of a 50% dextrose solution i.v. once daily for 3 d (GLU, n = 9), (2) 300 mL of propylene glycol as a drench once daily for 3 d (PG, n = 9), (3) a combination treatment of a 500 mL of 50% dextrose solution i.v. and 300 mL of propylene glycol orally once daily for 3 d (GLU+PG, n = 8), or (4) an untreated control group (CTRL, n = 8). Blood samples were collected immediately before as well as at 1, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h after administration of the first treatment through a jugular catheter and 3 times per week thereafter from coccygeal vessels. Concentrations of BHB were measured in whole blood, and plasma samples were analyzed for glucose, fatty acid (NEFA), insulin, glucagon, and electrolyte concentrations. The EDTA-anticoagulated blood samples were assessed for red blood cell indices, and smears were made for evaluation of red blood cell morphology. Outcomes were analyzed using repeated measures analysis. Overall least squares means (95% CI) of whole blood BHB concentrations between 1 h and d 11 relative to first treatment were 1.11 (0.95 to 1.30), 1.26 (1.07 to 1.47), 0.96 (0.81 to 1.13), and 1.53 (1.30 to 1.80) mmol/L for the GLU, PG, GLU+PG, and CTRL groups, respectively. Treatment with both glucose and propylene glycol led to a greater magnitude and more prolonged decrease in BHB concentrations compared with individual treatments. The NEFA and glucagon concentrations were lower immediately after treatment in GLU and GLU+PG groups compared with CTRL, and treatment with both glucose and propylene glycol was associated with a greater increase in glucose and insulin concentrations immediately after treatment compared with CTRL and GLU treatment alone. Treatments did not lead to differences in plasma mineral concentrations. We conclude that treatments varied in the magnitude of decreasing blood BHB concentrations in hyperketonemic postpartum cows, with the greatest decline after treatment with a combination of intravenous glucose and oral propylene glycol.     

The effect of new bovine viral diarrhea virus introduction on somatic cell count,calving interval,culling, and calf mortality of dairy herds in the Dutch bovine viral diarrhea virus–free program

Bovine viral diarrhea virus (BVDV) infection has a major effect on the health of cows and consequently on herd performance. Many countries have implemented control or eradication programs to mitigate BVDV infection and its negative effects. These negative effects of BVDV infection on dairy herds are well documented, but there is much less information about the effects of new introduction of BVDV on dairy herds already participating in a BVDV control program. The objective of our study was to investigate the effect of a new BVDV introduction in BVDV-free herds participating in the Dutch BVDV-free program on herd performance. Longitudinal herd-level surveillance data were combined with herd information data to create 4 unique data sets, including a monthly test-day somatic cell count (SCC) data set, annual calving interval (CIV) and culling risk (CR) data sets, and a quarterly calf mortality rate (CMR) data set. Each database contained 2 types of herds: herds that remained BVDV free during the whole study period (defined as free herds), and herds that lost their BVDV-free status during the study period (defined as breakdown herds). The date of losing the BVDV-free status was defined as breakdown date. To compare breakdown herds with free herds, a random breakdown date was artificially generated for free herds by simple random sampling from the distribution of the breakdown month of the breakdown herds. The SCC and CIV before and after a new introduction of BVDV were compared through linear mixed-effects models with a Gaussian distribution, and the CR and CMR were modeled using a negative binomial distribution in generalized linear mixed-effects models. The explanatory variables for all models included herd type, BVDV status, year, and a random herd effect. Herd size was included as an explanatory variable in the SCC, CIV, and CMR model. Season was included as an explanatory variable in the SCC and CMR model. Results showed that free herds have lower SCC, CR, CMR, and shorter CIV than the breakdown herds. Within the breakdown herds, the new BVDV introduction affected the SCC and CMR. In the year after BVDV introduction, the SCC was higher than that in the year before BVDV introduction, with a factor of 1.011 [2.5th to 97.5th percentile (95% PCTL): 1.002, 1.020]. Compared with the year before BVDV breakdown, the CMR in the year of breakdown and the year after breakdown was higher, with factors of 1.170 (95% PCTL: 1.120; 1.218) and 1.096 (95% PCTL: 1.048; 1.153), respectively. This study reveals that a new introduction of BVDV had a negative but on average relatively small effect on herd performance in herds participating in a BVDV control program.     

A new mechanistic growth model for simultaneous determination of lag phase duration and exponential growth rate and a new Bĕlehdrádek-type model for evaluating the effect of temperature on growth rate

A new mechanistic growth model was developed to describe microbial growth under isothermal conditions. The new mathematical model was derived from the basic observation of bacterial growth that may include lag, exponential, and stationary phases. With this model, the lag phase duration and exponential growth rate of a growth curve were simultaneously determined by nonlinear regression. The new model was validated using Listeria monocytogenes and Escherichia coli O157:H7 in broth or meat. Statistical results suggested that both bias factor (B_f) and accuracy factor (A_f) of the new model were very close to 1.0. A new B?lehdrádek-type rate model and the Ratkowsky square-root model were used to describe the temperature dependence of bacterial growth rate. It was observed that the maximum and minimum temperatures were more accurately estimated by a new B?lehdrádek-type rate model. Further, the inverse of square-roots of lag phases was found proportional to temperature, making it possible to estimate the lag phase duration from the growth temperature.     

Lifetimes of triplet dissolved natural organic matter (DOM) and the effect of NaBH₄ reduction on singlet oxygen quantum yields: implications for DOM photophysics

The natural lifetimes of triplet dissolved organic matter ((3)DOM) were determined by an O(2) saturation kinetics study of singlet oxygen quantum yields (Φ(1O2)) in buffered D(2)O. At least two distinct (3)DOM pools are present, and the observed lifetime range (～20 to 80 μs) leads to a dependence of Φ(1O2) on O(2) concentrations between 29 and 290 μM. Thus, steady-state (1)O(2) concentrations will depend on [O(2)] in natural waters. The lifetimes are essentially identical for DOM samples of different origins and do not vary with excitation wavelength. However, Φ(1O2) varies greatly between samples and decreases with excitation wavelength. These data strongly suggest that (3)DOM quantum yields decrease with excitation wavelength, which gives rise to the Φ(1O2) variation. Borohydride reduction of several samples in both D(2)O and H(2)O lowers the absorbance and (1)O(2) production rates, but it does not alter Φ(1O2). This is consistent with a model in which (1)O(2) sensitizing chromophores are borohydride reducible groups in DOM, such as aromatic ketones. Interpreted in the framework of a charge transfer (CT) model for DOM optical properties, the collective data suggest a model in which electron acceptor moieties are important (1)O(2) sensitizers and where CT interactions of these moieties disrupt their ability to produce (1)O(2).