Minor Whey Proteins Research Articles

Equine lactoferrin: Antioxidant properties related to divalent metal chelation

Lactoferrin is a minor whey protein known for its multifunctionalities, particularly for its rule in regulation of immune system and for its antimicrobial activities. Although bovine lactoferrin has been extensively studied, very few information exists on lactoferrin from mare's milk. This article studies the antioxidant properties via metal chelation of purified lactoferrin from Kazakhstan mare's milk. Equine lactoferrin shows free radical-scavenger activity when tested with radical DPPH and ABTS and has reducing capacities testing by FRAP. Moreover, the ferrous(II) and copper(II) ion-chelating power is highlighted by chemical colorimetric tests. Finally, the newly introduced biophysics switchSENSE® technology allowed to measure in real time the molecular interactions between equine lactoferrin and divalent cations. The order of magnitude of the KD values was tens or so μM for the four metal ions tested: Zn2+ (KD = 23.9 ± 4.0 μM), Ca2+ (KD = 28.2 ± 4.4 μM), Cu2+(KD = 43.5 ± 5.1 μM) and Fe2+ (KD = 54.1 ± 8.9 μM). According to these results, we can conclude that equine lactoferrin shows antioxidant activity by radical-scavenging mechanism, reducing capacities and divalent pro-oxidant metals chelation ability.

Effect of Malabari goat lactoferrin on tumour growth kinetics in experimentally induced tumours in mice

Lactoferrin, a minor whey protein, possesses a multitude of biological functions including antimicrobial, immunomodulatory and antitumour activities. The present study focuses on the antitumour activity of lactoferrin isolated from Malabari goats. Lactoferrin was isolated from Malabari goat colostrum by Cation exchange chromatography. The purity of the isolated protein was confirmed by SDS-PAGE and its identity was ascertained by Western Blotting. Solid tumours were induced in hind limbs of Swiss Albino mice using Daltons lymphoma ascites cells. Animals which developed the tumours were divided into groups and administered different doses of Malabari goat lactoferrin (MgLf) intratumourally as a single dose. The tumour positive animals were sacrificed on day 10 post treatment to study the effects of MgLf on tumour growth kinetics by assessing the tumour weight: body weight ratio and tumour volume inhibition of the slaughtered animals. It was found that MgLf @150μg significantly lowered the tumour weight: body weight ratio and tumour volume of the treated animals.

Development and validation of an enzyme-linked immunosorbent assay for the determination of bovine lactoferrin in various milk products

For the detection of the minor whey protein lactoferrin, a competitive enzyme-linked immunosorbent assay (ELISA) was developed. The method enables the quantification of low lactoferrin concentrations in different matrices. As the good recovery values indicate, the method can be applied for various milk-based products, e.g., milk (104% recovery), cream (106% recovery), curd (96% recovery) and whey (107% recovery). In the validation runs, an accuracy at different concentration levels between 93% and 106% was confirmed, while the precision within a day (91% intra-assay) and between different days (84% inter-assay) was in an acceptable range. The limit of quantification (LOQ) was determined at 62 μg L−1. The applicability was demonstrated by monitoring the lactoferrin content during curd production. An enrichment of lactoferrin in the curd phase (266 mg kg−1) was observed while a large percentage (>39%) of the initial lactoferrin was found in the whey stream.

Denaturation of selected bioactive whey proteins during pasteurization and their ability to modulate milk immunogenicity.

This research communication relates to the hypothesis that the consumption of raw or unprocessed cow's milk contributes to lowered prevalence of allergies. Thermal pasteurization of bovine milk can result in denaturation of minor whey proteins and loss of their bioactivity. Denaturation of bovine serum albumin (BSA), immunoglobulin G (IgG) and lactoferrin (LF) in skim milk was studied under different temperature (72, 75 or 78°C) and time (0-300 s) combinations. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that denaturation of all 3 proteins occurred at 72°C and progressed with increase in temperature and holding time. About 59% of LF and 12% of IgG denatured under high-temperature short-time (72°C/ 15 s) pasteurization, while BSA was least impacted. To assess modulation of milk immunogenicity, secretion of selected T helper (Th)-type cytokines by human peripheral blood mononuclear cells (PBMCs) was studied in vitro in response to different concentrations of BSA (0.4-1.0 mg/ml) and IgG (0.8-1.6 mg/ml) in unheated skim milk. Addition of IgG at 1.6 mg/ml induced a prominent Th1-skewed cytokine profile that may not trigger a Th2-skewed allergic reaction. BSA did not appear to modulate milk immunogenicity to any significant extent.

Analysis of ionic and nonionic surfactants blends used for the reverse micellar extraction of Lactoperoxidase from whey

AbstractBovine Lactoperoxidase (LP), a minor whey protein, is used as an antimicrobial in cosmetic, food, and pharmaceutical preparations. Industries are in pursuit of reliable, cheap, and scalable purification methods as the conventional techniques for LP purification like chromatography and membrane separation suffer from several drawbacks. The present work investigates the selective reverse micellar extraction of LP using the reverse micellar system formed by mixing food grade nonionic (Tween, Span, and Triton series) and ionic (AOT) surfactant blends. The analysis of LP extraction efficiency was performed by varying the concentration of nonionic surfactants with a constant AOT concentration of 100 mM and the initial pH of the system. Complete LP solubilization was achieved with reverse micelles formed by 100 mM AOT and 20 mM Tween 80 at pH 8. It was found that the extraction efficiency was dependent on the chain length or the number of ethylene oxide units in the Triton surfactant tail and the carbon–carbon double bond in Tween 80 tail, that is, on oleic acid. Span series however showed poor extraction in the organic phase substantiating the lesser water content. The forward extracted LP was successfully back‐extracted into a fresh aqueous phase containing 1 M KCl at pH 10.5. The aqueous phase (whey) from the forward transfer can be further used to fractionate other whey proteins.

Shearing accelerates denaturation of β-lactoglobulin and α-lactalbumin in skim milk during heating

Shearing is an embedded step during thermal processing of milk that can add additional impact on native proteins. Behaviour of native proteins in bovine skim milk was studied at different temperatures (70–100 °C) for 5 min with or without applying a constant shear rate at 1000 s−1. Shear stress generated generally decreased with increase in temperature due to decrease in milk viscosity which displayed a Newtonian fluid behaviour. β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La) denatured rapidly even at low temperature (70 °C) under the shearing and about 25% more of them denatured at 85 °C than those of the non-sheared counterparts. Shearing predominantly induced formation of thiol/disulphide aggregates mainly consisting of β-Lg and α-La, as well other minor whey proteins and caseins. These results highlighted the necessity of assessing impact of shearing on milk proteins when optimising thermal processing conditions.

Cheese Whey Fermentation by Its Native Microbiota: Proteolysis and Bioactive Peptides Release with ACE-Inhibitory Activity

Cheese whey contains about 20% of the total milk protein and has high nutritional and technological value, as well as attractive biological properties. Whey protein represents an important source of bioactive peptides with beneficial effects on health (e.g., antioxidant, antidiabetic, antihypertensive, etc.). Microbiota in cheese whey can hydrolyze proteins and generate bioactive peptides through a fermentation process. The objective of this study was to evaluate the effect of temperature on the fermentation of cheese whey by its native microbiota, and the action of microbial proteolytic activity on whey proteins to release peptides with inhibitory activity of the angiotensin-converting enzyme (ACE). Whey proteins hydrolysis occurred at all incubation temperatures evaluated (32–50 °C), with the major proteolytic effect within the range of 35–42 °C. Minor whey proteins (i.e., Lf, bovine serum albumin (BSA), and IgG) were more susceptible to degradation, while β-lactoglobulin and α-lactalbumin showed major resistance to microbial proteolytic action. Alfa-amino groups increased from 36 to 360–456 µg Gly/mL after 120 h of fermentation. A higher lactic acid production (11.32–13.55 g/L) and lower pH (3.3–3.5) were also observed in the same temperature range (32–42 °C). In addition, ACE-inhibitory activity increased from 22% (unfermented whey) to 60–70% after 120 h of fermentation. These results suggest that the fermentation of cheese whey by its native microbiota represents an attractive process to give value to whey for the production of whey-based beverages or functional foods with potential antihypertensive properties.

Fractionation of casein micelles and minor proteins by microfiltration in diafiltration mode. Study of the transmission and yield of the immunoglobulins IgG, IgA and IgM

There is little information concerning the fractionation by microfiltration (MF) of casein micelles and immunoglobulins plus other minor whey proteins with ceramic gradient membranes. The order of transmission was α-lactalbumin (α-La, 2.3 nm)> β-lactoglobulin (β-Lg; 4.2 nm)>IgG (10.7 nm)> lactoperoxidase (8.2 nm)> IgA (18.1 nm)> IgM (23.8 nm)> lactoferrine > blood serum albumin (7.8 nm), irrespective of the applied transmembrane pressure (0.6–3 bar) and equal to 55% > 50% > 47% > 41%>39% > 32% > 22% > 19%. Including preconcentration, it was possible to obtain 90% of the initial IgG, IgA and IgM within 85, 119, and 160 min, based on 1 m2 of membrane area and 50 L of skim milk volume. The long-term process exposure at 50 °C did not affect α-La and IgG but β-Lg (3–5% denaturation), which, however, was selectively retained by the MF. In conclusion, MF is not only suitable for fractionation of the major whey proteins and caseins, but also for the minor and far bigger immunoglobulins.

Optimization of Lactoperoxidase and Lactoferrin Separation on an Ion-Exchange Chromatography Step

Lactoperoxidase (LP), which is a high-value minor whey protein, has recently drawn extensive attention from research scientists and industry due to its multiplicity of function and potential therapeutic applications. In this study, the separation and optimization of two similar-sized proteins, LP and lactoferrin (LF) were investigated using strong cation exchange column chromatography. A two-step optimization strategy was developed for the separation of LP and LF. Optimization was started with central composite design-based experiments to characterize the influences of different decision variables, namely, flow rate, length of gradient, and final salt concentration in the linear elution gradient step on the yield of LP. This was followed by a more accurate optimization of ion-exchange chromatography (IEC) separation of LP and LF based on an experimentally verified chromatographic model. The optimal operating points were found and the results were compared with validation experiments. Predictions respecting yield confirmed a very good agreement with experimental results with improved product purity.

Impact of operating conditions on chromatographic column performance: experimental studies on adsorption of high-value minor whey proteins

Over the last decade, ion-exchange chromatography (IEC) has been extensively explored for protein purification at both small and large scales. Despite several IEC columns are commercialized, the physical phenomena underlying the adsorption of proteins on ion-exchange columns performance has not been thoroughly investigated. In this work, the influence of operating conditions on the adsorption of lactoperoxidase (LP) and lactoferrin (LF) on cation exchange chromatography adsorbent is experimentally studied in order to understand fundamental pertaining to underlying mechanism. Analysis was carried out in columns with different IDs (7.7 and 16 mm), packed for 100 mm with 90 μm particle size polymer-grafted cation exchanger. The flow distribution was measured using acetone as a non-binding tracer. An evaluation of van Deemter plots was done as well as LP breakthrough curves at different flow rates and LP loading concentrations. The results were compared with two columns in terms of efficiency and the LP binding capacity. The dynamic binding capacity at 10% breakthrough was found to be independent of the applied flow rate. Surprisingly for both systems, LP breakthrough takes place later at higher loading concentrations, which is in contrast to IEC. The results propose a major presence of non-ideal effects as steric shielding and charge repulsion of protein in the adsorption. In addition, the accessibility of binding sites for protein at higher concentrations seems more available than sodium counter-ions in buffer.

Determination of adsorption isotherm parameters for minor whey proteins by gradient elution preparative liquid chromatography

Ion-Exchange Chromatography (IEC) techniques have been extensively investigated in protein purification processes, due to the more selective and milder separation steps. To date, existing studies of minor whey proteins fractionation in IEC have primarily been conducted as batch uptake studies, which require more experimental search space, time and materials. In this work, the selected resin's (SP Sepharose FF) equilibrium and dynamic binding capacity were first investigated. Next, adsorption of the pure binary mixture of lactoperoxidase and lactoferrin was studied to calibrate steric mass action (SMA) model using a simplified approach with data from single column experiments. The calibrated model was then verified by performing factorial-design based experiments for various process operating conditions assessing process performance on a larger bed height column. The model predicted results demonstrated a realistic agreement with the experiments providing reproducible column elution profile and reduced experimental work. Finally, whey protein isolate was used to evaluate model parameters in real conditions. Results obtained herein are suitable for future large scale applications.

Optimization of lactoferrin and bovine serum albumin separation using ion-exchange membrane chromatography

Lactoferrin (LF), which is a high value minor whey protein, has recently received extensive attention from research scientists and industry due to its multifunction and potential therapeutic applications. In this study, the separation of two similar-sized proteins: bovine serum albumin (BSA) and LF was investigated using strong cation and anion exchange membrane chromatography (MC). Single protein and BSA–LF mixture adsorption were performed on Sartobind Q75 and S75 at pH between the LF and BSA isoelectric points. Identical breakthrough curves were obtained for both single protein and binary protein mixture, which suggests that there is no protein adsorption competition at the binding sites. The process optimization was further studied to yield optimum buffer and operating conditions. The highest BSA flux per membrane area (728.00gm−2h−1) was obtained using 100mM phosphate buffer solution at pH 6.0 on the cation exchange membrane, whereas LF was bound to the membrane with the dynamic binding capacity at 10% breakthrough (DBC10%) of about 60mg. On the anion exchange membrane, LF was collected in the effluent at the flux of 287.46gm−2h−1 using 5mM phosphate buffer at pH 6.0, while BSA was retained on the membrane with DBC10% equal to 60.96mg. The combination effect between pH change and hydrophobic interaction improved the eluted protein mass for both anion and cation exchangers. Furthermore, the completed separation cycle was operated with the Sartobind S75 device with a short process time of 34.19min and optimal LF productivity over 2628.84mgmL−1h−1. This study confirms the advantage of MC for the separation of biomolecules with similar molecular weight and different isoelectric points, such as BSA–LF mixture separation. This fast and effective protein separation method could be applied at an industry scale.

Skim milk protein distribution as a result of very high hydrostatic pressure

This work studies the micellar size and the distribution of caseins, major and minor whey proteins in different fractions of skim milk treated up to 900MPa for 5min. Transmission electron microscopy showed that the smallest casein micelles were formed around 450MPa with no variations at higher pressures. The changes found in micellar size correlated with the concentration of soluble casein, because treatments at 250MPa significantly enhanced the level of non-sedimentable casein while, between 700 and 900MPa, there were no further increases with respect to lower pressures. There was a severe β-lactoglobulin (β-Lg) denaturation at pressures ≥700MPa, which reached 77–87%. α-Lactalbumin (α-La) was stable up to 550MPa, but it denatured at higher pressures. The content of soluble lactoferrin (Lf) decreased with pressure, particularly from 550 to 800MPa, while that of secretory IgA (sIgA) progressively decreased from 250 up to 700MPa. Our results indicated that treatment of milk at very high pressures, from 700 to 900MPa, did not reduce micellar size nor released more soluble casein with respect to treatments at lower pressures (250–550MPa). However, these treatments led to a severe denaturation of the whey proteins, in particular of β-Lg and the minor proteins Lf and sIgA. The possibility of using high hydrostatic pressure to obtain a soluble milk fraction with a casein and whey protein composition similar to that of human milk is discussed.

Processing and protein-fractionation characteristics of different polymeric membranes during filtration of skim milk at refrigeration temperatures

Serum protein concentrates (SPCs) were generated from reconstituted skim milk (3.2% protein) using lab-scale tangential-flow filtration at 3–4 °C. The influence of membrane type on process performance (e.g., permeate flux) and protein-enrichment (e.g., protein profile) was assessed with polyvinylidene-difluoride membranes (0.1 μm and 0.45 μm pore-size), and a polyethersulfone membrane (1000 kDa cut-off). The 1000 kDa membrane exhibited the highest starting flux (6.7 L m−2 h−1), followed by the 0.1 μm (5.4 L m−2 h−1) and 0.45 μm (4.8 L m−2 h−1) membranes. Flux decreased by >40% during filtration with the 1000 kDa and 0.1 μm membranes, while the decrease was lower (<20%) with the 0.45 μm membrane. β-Casein comprised >97% of casein in SPCs from the 0.1 μm and 1000 kDa membranes. SPCs from the 0.45 μm membrane had higher β-casein:αs-casein ratios than the feed and higher levels of minor whey proteins (e.g., lactoferrin) relative to the other SPCs.

Fractionation of all major and minor whey proteins with radial flow membrane adsorption chromatography at lab and pilot scale

A radial flow membrane adsorber system with improved fluid distribution, thereby avoiding high back pressures, was investigated for the complete fractionation of whey proteins. A 50-fold scale-up of the chromatographic separation of all major and minor proteins in acid whey was achieved. The process with anion and cation exchanger membranes consists of two steps. In the first step β-lactoglobulin (β-Lg) and bovine serum albumin are removed. In the second step the minor proteins lactoferrin, lactoperoxidase and immunoglobulin G are isolated and α-lactalbumin remains in the serum phase. The membrane performance over five repeated cycles without cleaning was stable at both scales. The loaded β-Lg equalled the dynamic binding capacity, which was 0.5 mg cm−2 and depletion of β-Lg was 96–99.8%. The yield and purity of the minor whey proteins were consistent at both scales in a range between 80 and 97%, with a concentration factor between 6 and 14.

Enrichment and Purification of Casein Glycomacropeptide from Whey Protein Isolate Using Supercritical Carbon Dioxide Processing and Membrane Ultrafiltration.

Whey protein concentrates (WPC) and isolates (WPI), comprised mainly of β-lactoglobulin (β-LG), α-lactalbumin (α-LA) and casein glycomacropeptide (GMP), are added to foods to boost nutritional and functional properties. Supercritical carbon dioxide (SCO2) has been shown to effectively fractionate WPC and WPI to obtain enriched fractions of α-LA and β-LG, thus creating new whey ingredients that exploit the properties of the individual component proteins. In this study, we used SCO2 to further fractionate WPI via acid precipitation of α-LA, β-LG and the minor whey proteins to obtain GMP-enriched solutions. The process was optimized and α-LA precipitation maximized at low pH and a temperature (T) ≥65 °C, where β-LG with 84% purity and GMP with 58% purity were obtained, after ultrafiltration and diafiltration to separate β-LG from the GMP solution. At 70 °C, β-LG also precipitated with α-LA, leaving a GMP-rich solution with up to 94% purity after ultrafiltration. The different protein fractions produced with the SCO2 process will permit the design of new foods and beverages to target specific nutritional needs.

Effect of milk thermal history on the recovery of TGF-β2 by acid precipitation of whey protein concentrates

The objective of the study was to characterize the effects of preheating of milk and of pasteurization steps during whey processing on the separation of transforming growth factor-beta 2 (TGF-β2) from whey protein concentrates (WPCs). Three heating conditions were applied to skimmed milk: no heating, 63 °C for 20 s and 68 °C for 20 s. The effect of pasteurizing liquid acid whey and liquid WPC was also assessed. Heating milk at 63 or 68 °C decreased TGF-β2 content of whey. A pasteurization treatment of whey at 72 °C for 15 s further decreased their TGF-β2 content. However, an additional pasteurization of WPCs significantly increased TGF-β2 only in samples from unheated milk. Acid precipitation of WPCs showed that cumulative heat treatments increased the amount of precipitate obtained, and that pH 4.5 was found to be optimal in terms of TGF-β2 content of precipitates. The highest TGF-β2 concentrations in acid precipitates (>10,000 pg.mg−1 solids) were obtained with a combination of heating milk at 63 °C for 20 s and pasteurization of whey at 72 °C for 15 s. It was found that, although whey obtained from raw milk represents the best source of TGF-β2, an appropriate selection of heat treatments of liquid whey and WPC improves the recovery of TGF-β2. The presence of casein and minor whey proteins in TGF-β2-enriched fractions underlines the occurrence of complex heat-induced interactions between TGF-β2 and other milk constituents.

Changes in the bovine whey proteome during the early lactation period

To investigate time-dependent change in the bovine whey proteome during the early lactation period, a two-dimensional gel-based approach was used in this study. Milk samples were collected from five healthy Friesian-Holstein dairy cows up to 10 days postpartum. Spot patterns of whey proteins varied drastically from immediately after parturition to 48 h, but no significant changes occurred thereafter. Protein identification by mass spectrometry revealed that the ratios of caseins and immunoglobulins drastically decreased during 48 h postpartum, while those of lower molecular mass proteins such as α-lactalbumin and β-lactoglobulin increased. More than 100 spots were detected, being much more abundant in colostral whey than in mature milk whey. Of a total of 25 proteins identified, four, viz. zinc-α-2-glycoprotein, vitamin D-binding protein, immunoglobulin G2 chain C and β2-microglobulin, were detectable only in colostrum. Our results indicate that most of the minor whey proteins in colostrum relate to the passive immunity of newborn calves, but some of them play significant roles in nutritional supplementation of the neonate. The characteristics of whey proteins in transition imply that enhancement of innate immunity becomes more important than protection of the neonate against pathogens via passive immunity after 48 h postpartum.

Fractionation of bovine whey proteins and characterisation by proteomic techniques

To further elucidate and understand bovine milk protein composition and its relation to bioactivity, we have investigated the bovine whey proteome by gel-based proteomic methods, after first fractionating whey from late-lactation milk into acidic, basic and non-bound fractions by semi-coupled anion and cation exchange chromatography. Characteristic two-dimensional gel fingerprints were obtained for each fraction, with protein isoform patterns clearly apparent. A large number of minor whey proteins were identified, several of which have not been reported previously in bovine milk. Notably, a cluster of osteopontin peptides not priorly described in milk was consistently observed in the acidic protein fraction, presupposing novel bioactivities.

In vitro studies of the digestion of caprine whey proteins by human gastric and duodenal juice and the effects on selected microorganisms.

The in vitro digestion of caprine whey proteins was investigated by a two-step degradation assay, using human gastric juice (HGJ) at pH 2.5 and human duodenal juice (HDJ) at pH 7.5. Different protein and peptide profiles were observed after the first (HGJ) and second (HDJ) enzymatic degradation. The minor whey proteins serum albumin, lactoferrin and Ig were rapidly degraded by HGJ, while alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) were more resistant and survived both 30 and 45 min of the enzymatic treatment. Further digestion with HDJ still showed intact beta-LG, and the main part of alpha-LA also remained unchanged. The protein degradation by HGJ and HDJ was also compared with treatment by commercial enzymes, by using pepsin at pH 2.5, and a mixture of trypsin and chymotrypsin at pH 7.5. The two methods resulted in different caprine protein and peptide profiles. The digests after treatment with HGJ and HDJ were screened for antibacterial effects on some selected microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus rhamnosus GG and Streptococcus mutans. Active growing cells of E. coli were inhibited by the digestion products from caprine whey obtained after treatment with HGJ and HDJ. Cells of B. cereus were inhibited only by whey proteins obtained after reaction with HGJ, while the products after further degradation with HDJ demonstrated no significant effect. Screenings performed on cells of Lb. rhamnosus GG and S. mutans all showed no signs of inhibition.