Obtaining Of β-Lactoglobulin By Gel Filtration Of Cow Milk Whey

Milk whey proteins carry out a number of important biological functions and also they are precursors of many biologically active peptides (antihypertensive peptides, antagonists of opioid receptors, regulators of intestinal motility, immunomodulatory, anti-microbial and anti-cancer peptides, appetite regulators and so on.). An important stage in natural bioactive peptides obtaining from milk whey proteins is the isolation of homogeneous proteins-precursors. Considering the significant difference in the molecular masses of whey proteins, a promising method for their selection is gel filtration. The purpose of the research was the fractionation of bioactive peptides precursors from milk whey using gel filtration on Sephadex G-150. The whey was obtained from fresh skimmed milk after isoelectric precipitation of casein. Gel filtration was carried out on the columns from a liquid chromatography kit by the “Reanal” company. The fractional composition and the degree of homogeneity of milk whey proteins were determined by disc-electrophoresis in the plates of a polyacrylamide gel. A repeated gel filtration of fractions from the chromatographic peaks, separated into sections, was performed to increase the fractionation efficiency. While choosing a dextran gel for gel filtration of precursors of biologically active peptides from milk whey proteins, we have taken into account the range of their molecular weights (from 10000 to 150000 Da), the ability to form supramolecular structures (β-LG), as well as the previously obtained results of gel filtration. As a result, it was shown that repeated gel filtration of milk whey on Sephadex G-150 allows efficiently fractionate the proteins-precursors of bioactive peptides. The range of peptides and proteins molecular weights that can be fractionated on this Sephadex is from 5000 to 300 000 Da. The usage of repeated gel filtration on Sephadex G-150 with the chromatogram separation into sectors allows to effectively fractionate proteins-precursors of bioactive peptides from milk whey. In particular, homogeneous β-lactoglobulin (degree of homogeneity > 95 %) and partially purified α-lactalbumin, as well as a group of immunoglobulins and a proteose-peptone fraction were obtained

Stability of whey protein derived peptides upon severe protein glycation

Cow’s milk and dairy products are major nutrients in the human diet, especially during infancy. Though at one time whey proteins were considered as by-product of the cheese making process, nowadays, due to their wide ranging nutritional, biological, and functional properties, whey proteins are often used in food technology as low-cost protein ingredients. However, whey-protein fractions, such as β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) represent the major allergens in cow’s milk. Therefore, the use of whey proteins in food might pose a serious threat to the milk allergic consumers. It is well reported that food processing may modify the allergenicity and detectability of proteins. This can be due to hydrolysis or chemical reactions with other food components (carbohydrates, fatty acids etc), leading to modification or destruction of the allergen’s structure. Therefore, the objective of this study was in the first stage to investigate the influence of glycation on the molecular changes induced in whey proteins. This was done with a special focus on the modifications induced on the lysine residues, free amino groups, the formation of protein bound carbonyls, formation of fluorescent compounds and brown polymers and on the protein aggregation. Matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) was used to get a better insight into the molecular changes that took place on the protein level. Unexpectedly, this study led to the identification of protein segments in the epitope region that remained unmodified during the experiments that mimic typical food processing conditions. The 57Val – Lys76 and 31Val – Arg56 from β–LG, remained unchanged disregarding the severe heating treatment in the presence of glucose and bulk proteins and they could be identified by either direct MALDI-TOF MS and MS/MS or after a more tedious separation using reversed phase chromatography. It is proposed that these peptide segments can be used as analytical targets for the development of more robust methods for the assessment of the presence of whey proteins in processed foodstuffs. Moreover, MALDI-TOF MS and MS/MS holds potential to be used as a screening tool for the identification of such stable peptides

Determination of processing conditions for industrial manufacture of pre-denatured WPC : a thesis presented in partial fulfilment of the requirements for the degree of Master in Food Technology

The purpose of this study was to establish processing conditions for manufacturing denatured whey protein concentrate (WPC) with the ability to form self-supporting gels upon addition of water with minimal or no heating. Such product could be used as thickening and gelling agents in various food applications. In the preliminary studies, fresh whey and ultrafiltrate (UF) retentate solutions were heated and analysed using polyacrylamide gel electrophoresis (PAGE). The results showed that heating 1% protein solution of retentate (pH 7.0) at 80°C for 20 min formed the desired "soluble" aggregates. Those aggregates were predominantly formed at lower protein concentration compared to that at higher protein concentration. Much larger aggregates were formed when acid whey was heated under similar conditions. The same heating conditions (1% retentate solution, pH 7.0, 80°C for 20 min) were used in two different pilot plant trials (Massey University and Anchor Products) to produce denatured WPC powders. The denatured WPC powders were capable of forming viscous solutions or gels at ~ 10% protein concentration upon re-hydration and addition of GDL, CaCl2 or NaCl at ambient temperatures. By contrast, the unheated WPC solutions did not gel under these conditions. The viscosity or gel strength of the denatured WPC solutions increased with protein concentration, incubation time and temperature in the presence of additives. The heat-denatured WPC powders produced in the pilot plants had desirable functional properties. The high viscosity and the ability to form a gel upon addition of GDL or salts at 5-40°C would enhance their application in food systems, such as in comminuted meat, pressed ham/bacon, mayonnaise and yoghurt products

Bioactivity in Whey Proteins Influencing Energy Balance

Peer-reviewedObesity develops due to energy (food) intake exceeding energy expenditure. Nutrients that reduce the positive energy balance are thus being considered as therapies to combat obesity. Here, we review the literature related to the physiological, cellular and endocrine effects of intake of whey proteins, namely α-lactalbumin, β-lactoglobulin, glycomacropeptide and lactoferrin. Moreover, we discuss how dietary composition and obesity may influence whey protein effects on the above parameters. Evidence suggests that intake of whey proteins causes a decrease in energy intake, increase in energy expenditure, influence insulin sensitivity and glucose homeostasis and alter lipid metabolism in the adipose, liver and muscle. These physiological changes are accompanied by alterations in the plasma levels of energy balance related hormones (cholecystokinin, ghrelin, insulin and glucagon-like peptide-1) and the expression of catabolic and anabolic genes in the above tissue in the direction to cause a negative energy balance

The Appetite-Suppressant and GLP-1-Stimulating Effects of Whey Proteins in Obese Subjects are Associated with Increased Circulating Levels of Specific Amino Acids

The satiating effect of whey proteins depends upon their unique amino acid composition because there is no difference when comparing whey proteins or a mix of amino acids mimicking the amino acid composition of whey proteins. The specific amino acids underlying the satiating effect of whey proteins have not been investigated to date. Aims and Methods. The aim of the present study was to evaluate the appetite-suppressant effect of an isocaloric drink containing whey proteins or maltodextrins on appetite (satiety/hunger measured by a visual analogue scale or VAS), anorexigenic gastrointestinal peptides (circulating levels of glucagon-like peptide 1 (GLP-1) and peptide tyrosine tyrosine (PYY)) and amino acids (circulating levels of single, total [TAA] and branched-chain amino acids [BCAA]) in a cohort of obese female subjects (n = 8; age: 18.4 \ub1 3.1 years; body mass index, BMI: 39.2 \ub1 4.6 kg/m2). Results. Each drink significantly increased satiety and decreased hunger, the effects being more evident with whey proteins than maltodextrins. Similarly, circulating levels of GLP-1, PYY and amino acids (TAA, BCAA and alanine, arginine, asparagine, citrulline, glutamine, hydroxyproline, isoleucine, histidine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tyrosine, and valine) were significantly higher with whey proteins than maltodextrins. In subjects administered whey proteins (but not maltodextrins), isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, and valine were significantly correlated with hunger (negatively), satiety, and GLP-1 (positively). Conclusions. Eight specific amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, and valine) were implicated in the appetite-suppressant and GLP-1-stimulating effects of whey proteins, which may be mediated by their binding with nutrient-sensing receptors expressed by L cells within the gastrointestinal wall. The long-term satiating effect of whey proteins and the effectiveness of a supplementation with these amino acids (i.e., as a nutraceutical intervention) administered during body weight reduction programs need to be further investigated

Study of the Different Ways of Proteins Extraction From Sheep and Cow Whey for “Urda” Cheese Production

The necessary stage of technological process of “Urda” albumin cheese production is whey proteins extraction from sheep whey (in classic technology of product) or from the whey mixtures, offered in the work. For whey proteins extraction from whey the following ways are used: thermal, acid, acid-alkaline and chlorine-calcium.There was established the equal dependence of the influence of the way of proteins extraction from both sheep and cow whey on the output of protein mass.The most output of protein mass (3,47±0,10 %) is at the chlorine-calcium way of proteins extraction, a bit less – 3,41±0,08 % – at the acid-alkaline way. But the acidity of such protein mass, received by the chlorine-calcium and acid-alkaline ways is not high – 37,2±1,8 and 45,6±1,4 % respectively that negatively influences the gustatory qualities of product. The least output is registered at the thermal way. Albumin cheese, received from such protein mass, was the best by its gustatory qualities. The output of protein mass from sheep whey is 1,60 times higher than from cow whey. The mass share of proteins in protein mass, received from sheep whey is by 6,9…8,0 % higher comparing with one, received from cow whey.Protein mass, received from sheep whey by the chlorine-calcium and acid-alkaline ways has the low titrated acidity. Cheese, received from protein mass, received of sheep whey by the acid method, has the extremely high acidity values (115,5±1,5 °Т), excessive sour-milk flavor and smell. These results don-t allow to provide the long storage term. That is why it is recommended to use the thermal way for proteins extraction from sheep whey and for proteins extraction from cow way in the technology of “Urda” albumin cheese.So the thermal way of proteins extraction from the mixture of sheep and cow whey in ratio 1:1 or 3:1 can be used for “Urda” albumin cheese production. The use of cow whey gives a possibility to cheapen the product because cow whey is cheaper than sheep one. Such cheese has the improved organoleptic parameters, namely homogenous consistence, tender sour-milk flavor and smell

Study of the interactions between milk proteins and hydroxyapatite particles : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Riddet Institute, Palmerston North, New Zealand

Hydroxyapatite (HA) and other insoluble calcium salts added to calcium-fortified milks are often described as inert, as they do not cause any protein aggregation and heat instability during heat treatment of the milk. However, it is well-known that proteins can interact with HA. The adsorption of milk proteins on HA has been demonstrated in many systems, for example in chromatography, bioceramic and dentistry applications, and has been shown to have consequence on the colloidal stability of HA, but has never been studied in food systems. The main objective of the present study was therefore to explore the adsorption of milk proteins onto HA particles under a range of physico-chemical conditions. The consequences of these interactions on the colloidal properties of the HA particles and on the stability of the milk proteins were investigated. It was shown that the five individual milk proteins aS-casein, ß -casein, ?-casein, ß- lactoglobulin and a-lactalbumin adsorbed onto the HA particles. A Langmuir model was used to fit the adsorption data and determine the affinity constant and maximum surface loads of the different proteins. The adsorption of the different milk proteins onto HA particles was found to be of competive nature. ß-casein and aS-casein were always preferred for adsorption over ?-casein, ß-lactoglobulin and a-lactalbumin. This was attributed to the presence of phosphoserine clusters in ß-casein and aS-casein, forming many anchor points capable of binding to the calcium sites of HA. ß-casein and aS-casein also adsorbed to higher maximum levels compared to ?-casein, ß-lactoglobulin and a-lactalbumin. Both ß-Casein and aS-casein were considered to self-associate or associate together in the adsorbed layer, therefore forming a thick layer onto the HA surface. Conversely, ?-casein, ß-lactoglobulin and a-lactalbumin adsorbed to lower maximum amounts and had lower affinities for HA, which was attributed to adsorption in a monolayer through their carboxyl groups binding to the calcium sites of HA. The amount of protein adsorbing to the HA surface was affected by the physico-chemical properties of the solution such as pH and ionic strength, for all proteins. Decreasing pH and increasing ionic strength decreased the electrostatic repulsive forces between HA and the proteins and the electrostatic repulsive forces within the protein molecules, which allowed more protein to adsorb onto the HA surface. Milk serum ions such as calcium, phosphate and citrate bound specifically onto HA particles, therefore competing with the milk proteins for adsorption. In milk, it was shown the addition of HA in milk disrupted the mineral equilibrium and the milk protein phase. When HA particles were added to milk, the milk serum ions bound to the HA surface. This caused the colloidal calcium phosphate to be released from the casein micelles and the casein micelles to dissociate. Therefore the casein micelles did not bind as intact micelles but as individual molecules or small aggregates onto the HA particles. The adsorption of milk proteins onto HA particles affected the colloidal properties of the HA particles in suspension. The adsorption of both caseins and whey proteins onto HA particles resulted in the particles becoming negatively charged, thus improving their suspension stability. Whey protein adsorption probably provided only electrostatic stabilisation, whereas casein adsorption also provided steric stabilisation. Overall, this work has provided a detailed understanding of the interactions between milk proteins and HA particles. Calcium fortification of milk using insoluble calcium salts such as HA should be approached using an awareness of these interactions, as they may have consequences on the stability of calcium fortified milks

Effect of isolation conditions on structural properties and surface behavior of soy-whey proteins

In this study, the impact of isolation conditions on structural and surface properties at the air/water interface of soy-whey proteins (SWP) was assessed. SWP were obtained by precipitation of soy-whey (at pH 4.5 or 8.0) with acetone or ammonium sulfate. Despite the fact that all SWP samples exhibited similar electrophoretic patterns, they showed different protein content (from 54.2 to 98.2% w/w). When precipitation was performed at pH 4.5, SWP samples evidenced a decrease of protein solubility (SP) and thermal stability, while the precipitation with acetone promoted the enrichment in polysaccharides and minerals. For all samples, intrinsic fluorescence, surface hydrophobicity and Fourier transform infrared (FTIR) studies revealed structural changes correlated to protein unfolding and aggregation processes. However, the surface behavior can be predicted from these studies mainly due to differences in surface hydrophobicity and the differential contribution of insoluble aggregates. The heating of SWP samples enhanced the surface activity, regardless of the pH of the raw material and the isolation method. These results can be useful as a reference research and as a starting point for industrial exploitation of proteins from soy wastewater.Fil: Ingrassia, Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Sobral, Pablo Antonio. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; ArgentinaFil: Risso, Patricia Hilda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Física de Rosario. Universidad Nacional de Rosario. Instituto de Física de Rosario; ArgentinaFil: Palazolo, Gonzalo Gastón. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Funcionalidad y Tecnología de Alimentos; ArgentinaFil: Wagner, Jorge Ricardo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Investigación en Funcionalidad y Tecnología de Alimentos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Development of methods for capillary isoelectric focusing of dairy proteins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Chemistry at Massey University

Capillary Isoelectric Focusing (CIEF) is a high-resolution technique which can be applied to the separation and characterisation of complex biological mixtures such as dairy proteins. Although dairy proteins are commonly analysed by traditional gel electrophoresis techniques including 2-Dimensional PAGE, CIEF offers the advantages of reduced analysis times, the ability to handle smaller sample volumes and increased sensitivity with improved separation efficiencies. Several methods for capillary isoelectric focusing of dairy proteins have been developed herein. For the analysis of soluble whey proteins methods that can be used with either UV or mass spectrometry (MS) detection have been set up. For MS detection a coaxial sheath flow interface in conjunction with electrospray ionisation has been utilised. For analysis of the inherently insoluble casein proteins with UV detection denaturing and reducing agents have been introduced into the system. Results have shown very close similarities to those obtained by IEF gels