Reduction of BetaLactoglobulin Content of Cheese Whey by Polyphosphate Precipitation

When cheddar cheese whey was treated under the optimized conditions, i.e., 1.33 mg/mL sodium hexametaphosphate (SHMP) at pH 4.07 and 22°C for 1 hr, more than 80% of β-lactoglobulin was removed by precipitation. In the supernatant, almost all of the immunoglobulins and the major portion of α-lactalbumin were retained, as indicated by SDS gel electrophoresis. Immunochemical assays showed that approximately 90% of immunoglobulin G activity was found in the supernatant. By dialysis against water for 48 hr, 72.2% and 45.3% phosphorous was removed from the supernatant and precipitate, respectively.     

Elimination of β-Lactoglobulin from Whey to Simulate Human Milk Protein

When the pH of cottage cheese whey was adjusted to 4.5 in the presence of 6.7 mM FeCI³, β-lactoglobulin was eliminated from the whey as a precipitate. However, the majority of immunoglobuhns were also coprecipitated. To recover immunoglobulins together with α-lactalbumin, the whey pH was adjusted to 3.0 in the presence of 4.0 mM FeCI³. After centrifugation of the whey, the supernatant contained exclusively β-lactoglobulin; other whey proteins were found in the precipitate. Excess Fe₊₊₊ in the precipitate was removed by ion exchange or by ultrafiltration. This protein concentrate had a protein composition much more similar to that of human milk whey than that of ultrafiltered whey protein concentrate.     

Degradation of α-Lactalbumin and β-Lactoglobulin by Actinidin

Susceptibility of the two major whey proteins, β-lactoglobulin and α-lactalbumin, to enzymatic degradation by actinidin as a function of pH and temperature was examined by a response surface methodology in order to elucidate the enzymatic action of the protease for controlled modification of these whey proteins. Pure whey protein fractions and commercial spray-dried whey were degraded by actinidin. The simultaneous effects of pH and temperature, in a range of 2.3 to 5 and 41 to 58°C respectively, on whey proteins degradation were studied, demonstrating a clear interrelationship between these two variables. With commercial whey, extended proteolysis of both β-lactoglobulin and α-lactalbumin was observed at pH 4.0 and temperature of 41.6°C; after an incubation time of 120 min, a degradation of 43.6% was obtained for the former, and 89.1% for the latter. Assays on pure proteins showed a complete degradation of α-lactalbumin and a 65.3% of degradation for β-lactoglobulin; therefore, the former appeared to be more susceptible to actinidin proteolysis.     

Effects of In Vitro Proteolysis on the Allergenicity of Major Whey Proteins

The effects of in vitro proteolysis on the allergenicity of major whey proteins (α-lactalbumin and β-lactoglobulin) by simulating the human gastrointestinal conditions were evaluated. The proteolysis of demineralized whey with pepsin (pH 2.0 for 30 min) and/or various pancreatic enzymes (pH 7.5 for 60 and 240 min) was performed by the pH-stat technique at 37°C. The enzyme inactivation was performed by heating at 80°C for 20 min. Allergenicity of the hydrolyzates was evaluated by RAST inhibition using sera obtained from children allergic to whey proteins. Selective proteolysis of whey by pepsin and α-chymotrypsin was the most efficient combination of enzymes to reduce the allergenicity of both α-lactalbumin and β-lactoglobulin. The above hydrolyzate could be used to develop an ingredient for infant milk formula with a lower allergenicity.     

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.     

Changes in the soluble nitrogen fraction of milk throughout PDO Grana Padano cheese-making

The behaviour of soluble nitrogen compounds during Grana Padano cheese-making was studied at eight dairies. Raw milk, skimmed milk, sweet whey and the derived natural whey culture, collected from 24 processes, were analysed for soluble whey proteins (α-lactalbumin and β-lactoglobulin), proteose-peptones (PP), small peptides (SP), caseinomacropeptides (CMPs), and free amino acids (FAAs). The PP fraction increased during milk natural creaming, then part of it was selectively retained in the curd and the rest degraded in the first few hours of whey fermentation, together with α-lactalbumin, CMPs and part of SP. Features outlined for the whey culture were confirmed on 30 samples collected at six different dairies. A time course study of the whey fermentation showed that degradation of α-lactalbumin began when the pH dropped below 4, whereas β-lactoglobulin content did not change. Uptake of specific FAAs was shown to support the initial growth of lactic acid bacteria in whey.     

Use of acid whey protein concentrate as an ingredient in nonfat cup set-style yogurt

Acid whey resulting from the production of soft cheeses is a disposal problem for the dairy industry. Few uses have been found for acid whey because of its high ash content, low pH, and high organic acid content. The objective of this study was to explore the potential of recovery of whey protein from cottage cheese acid whey for use in yogurt. Cottage cheese acid whey and Cheddar cheese whey were produced from standard cottage cheese and Cheddar cheese-making procedures, respectively. The whey was separated and pasteurized by high temperature, short time pasteurization and stored at 4°C. Food-grade ammonium hydroxide was used to neutralize the acid whey to a pH of 6.4. The whey was heated to 50°C and concentrated using ultrafiltration and diafiltration with 11 polyethersulfone cartridge membrane filters (10,000-kDa cutoff) to 25% total solids and 80% protein. Skim milk was concentrated to 6% total protein. Nonfat, unflavored set-style yogurts (6.0 ± 0.1% protein, 15 ± 1.0% solids) were made from skim milk with added acid whey protein concentrate, skim milk with added sweet whey protein concentrate, or skim milk concentrate. Yogurt mixes were standardized to lactose and fat of 6.50% and 0.10%, respectively. Yogurt was fermented at 43°C to pH 4.6 and stored at 4°C. The experiment was replicated in triplicate. Titratable acidity, pH, whey separation, color, and gel strength were measured weekly in yogurts through 8 wk. Trained panel profiling was conducted on 0, 14, 28, and 56 d. Fat-free yogurts produced with added neutralized fresh liquid acid whey protein concentrate had flavor attributes similar those with added fresh liquid sweet whey protein but had lower gel strength attributes, which translated to differences in trained panel texture attributes and lower consumer liking scores for fat-free yogurt made with added acid whey protein ingredient. Difference in pH was the main contributor to texture differences, as higher pH in acid whey protein yogurts changed gel structure formation and water-holding capacity of the yogurt gel. In a second part of the study, the yogurt mix was reformulated to address texture differences. The reformulated yogurt mix at 2% milkfat and using a lower level of sweet and acid whey ingredient performed at parity with control yogurts in consumer sensory trials. Fresh liquid acid whey protein concentrates from cottage cheese manufacture can be used as a liquid protein ingredient source for manufacture of yogurt in the same factory.     

OPTIMIZING STABILITY OF ORANGE JUICE FORTIFIED WITH WHEY PROTEIN AT LOW pH VALUES

Abstract The influence of temperature, heating time and pH on the stability of whey protein-fortified Valencia orange juice was determined by uronic acid content, degree of esterification (DE), % transmission measurements (%T) and capillary electrophoretic analysis of the juice-protein supernatants. Uronic acid content and charge of pectins showed no significant change in heat-treated samples with added proteins. The %T decreased with decreasing pH and increasing temperature and heating time for α-lactalbumin (α-lac), β-lactoglobulin (β-lg) and whey protein isolate (WPI). The lowest transmission values were shown at pH 3.0 and 85C. Capillary electropherograms confirmed more extensive juice-protein interactions in WPI and β-lg added juices than in those containing α-lac, especially at low pH, resulting in more stable juice-protein mixtures.     

Preparation of β-Lactoglobulin and p-Lactoglobulin-Free Proteins from Whey Retentate by NaCI Salting Out at Low pH

Solubility of the main proteins in 10 x acid and rennet whey retentates was studied in the pH range 2.0 to 4.0 in the presence of NaCI from 2 to 15% (w/v) final concentration, at 20°C to find fractionation conditions suitable for preparing pure β-lactoglobulin and β-lactoglobu-lin-free whey proteins and scaling up. At pH 2.0, 7% NaCI, 20 min holding time, nearly all (3-lactoglobulin remained soluble while a precipitate (PI) containing all other proteins was formed. Pure p-lacto-globulin was quantitatively recovered by salting-out the centrifugation supernatant at 30% NaCI (w/v) final concentration. PI, insoluble at pHs lower than 4.0, was made soluble at any pH by dissolving at pH 9.0, dialyzing against 50 mM formic acid (pH 3.0) and freeze-drying.     

Effects of pH and Calcium on Thermal Behavior of Isolated Whey Proteins

Heat stability of 0.2%α-lactalbumin (α-la) was studied up to pH 5.0 in normal and pH 7.0 in decalcified permeates. Heating 0.4%β-lactoglobulin (β-lg) resulted in rapid flocculation before reaching 93 °C at all pH levels, except 6.5–7.0, for permeates with varying Ca content. When both isolated whey protein fractions were heated together (0.2%α-la and 0.4%β-lg) in regular or decalcified permeates, precipitation characteristics of β-lg did not change. However, some α-la appeared to co-precipitate with β-lg at pH 6.0 and below regardless of Ca.     

Yak milk whey protein denaturation and casein micelle disaggregation/aggregation at different pH and temperature

At the natural pH of yak milk (pH 6.6), a low level (<30%) of κ-casein (κ-CN) was found in the serum phase after heating at 95 °C for 30 min, indicating that as much as 70% of the β-lactoglobulin (β-Lg) and κ-CN complexes is associated with the micelle colloidal particles. The β-Lg and κ-CN levels increased from 13.2% and 2.6% at pH 6.0 to 35.2% and 60.1% at pH 7.0, respectively, when yak milk was heated at 95 °C for 30 min. At pH 6.0–6.4, the denatured whey proteins were associated with the caseins in the colloidal phase, resulting in milk gelation upon heating. The distribution of β-Lg and κ-CN complexes increased in the serum phase, demonstrated by the increasing levels of both β-Lg and κ-CN with increasing pH; at high pH (6.6–7.0), large proportions of β-Lg and α-lactalbumin were lost, presumably forming complexes in the colloidal phase.     

The effect of heat treatment of caprine milk on the composition of cheese whey

In three independent trials, caprine milk from the same batch was divided into three lots, which were heated at 65 °C for 30 min, 80 °C for 5 min or 90 °C for 5 min. Representative whey samples collected during the whole cheese making process were analysed for fat, protein and dry matter contents, which decreased as the heating temperature of milk increased. Percentages of serum albumin and β-lactoglobulin in the total proteins of whey decreased as the heating temperature of milk increased, while α-lactalbumin and glycomacropeptide increased, particularly in the 90 °C whey. Lactoferrin and the immunoglobulin-heavy chain were only detected in the 65 °C whey.     

Heat-Induced Changes in the Proteins of Whey Protein Concentrate

Three-level fractional factorial experiments were used to study effects of heating conditions (pH, time, temperature, solids content, calcium addition) on whey protein concentrate. Increasing pH and temperature led to lower solubility at pH 4.6 and 7.0, lower sulfhydryl content, higher hydroxymethylfurfural, generally darker color, lower DNBS-available lysine and altered pepsin pancreatin digestion profiles. Mercaptoethanol and SDS demonstrated relative importance of disulfide and hydrophobic bonds on solubility loss. Polyacrylamide gel electrophoresis indicated heat stability of proteose peptones; susceptibility was greatest at pH 8.0, 95°C for β-lactoglobulin and α-lactalbumin, and pH 4.6, 95°C for bovine serum albumin. HPLC gel filtration showed that heating rendered a high molecular weight fraction undissociable by mercaptoethanol.     

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.     

Antioxidative ability of native and thermized sour whey in oxidation‐catalysed model systems†

Antioxidative properties of sour (cottage) were evaluated with (thermized) and without (native) heat treatment (80°C for 30 min). A model system comprising a Tween 20 stabilized peanut oil in phosphate buffer (pH 7.0) emulsion containing lipid oxidation catalysts, FeCl ₃, H₂O₂ and ascorbate was used. Native sour whey powder (SWP) was significantly better than thermized whey in terms of limiting the formation of thiobarbituric acid‐reactive substances and peroxide value. Antioxidative ability was best at pH 3.0 and decreased with increasing pH. SWP (20%, w/v) was significantly better than all commonly used antioxidants tested after 96 h of incubation at 40°C.     

Reduction of Mycobacterium avium ssp. paratuberculosis in colostrum: Development and validation of 2 methods,one based on curdling and one based on centrifugation

The aim of this study was to develop and validate 2 protocols (for use on-farm and at a central location) for the reduction of Mycobacterium avium ssp. paratuberculosis (MAP) in colostrum while preserving beneficial immunoglobulins (IgG). The on-farm protocol was based on curdling of the colostrum, where the IgG remain in the whey and the MAP bacteria are trapped in the curd. First, the colostrum was diluted with water (2 volumes colostrum to 1 volume water) and 2% rennet was added. After incubation (1 h at 32°C), the curd was cut and incubated again, after which whey and curd were separated using a cheesecloth. The curd was removed and milk powder was added to the whey. Approximately 1 log reduction in MAP counts was achieved. A reduction in total proteins and IgG was observed due to initial dilution of the colostrum. After curd formation, more than 95% of the immunoglobulins remained in the whey fraction. The semi-industrial protocol was based on centrifugation, which causes MAP to precipitate, while the IgG remain in the supernatant. This protocol was first developed in the laboratory. The colostrum was diluted with skimmed colostrum (2 volumes colostrum to 1 volume skimmed colostrum), then skimmed and centrifuged (at 15,600 × g for 30 min at room temperature). We observed on average 1.5 log reduction in the MAP counts and a limited reduction in proteins and IgG in the supernatant. To obtain a semi-industrial protocol, dairy pilot appliances were evaluated and the following changes were applied to the protocol: after 2:1 dilution as above, the colostrum was skimmed and subsequently clarified, after which the cream was heat treated and added to the supernatant. To investigate the effect of the colostrum treatment on the nutritional value and palatability of the colostrum and the IgG transfer, an animal experiment was conducted with 24 calves. Six received the dam's colostrum, 6 were given untreated purchased colostrum (control), and 2 groups of 6 calves received colostrum treated according to both of the above-mentioned methods. No significant differences were found between the test groups and the dam's colostrum group in terms of animal health, IgG uptake in the blood serum, milk, or forage uptake. Two protocols to reduce MAP in colostrum (for use on-farm or at a central location) were developed. Both methods preserve the vital IgG.     

Proteins recovered from acid whey/skim milk mixtures heated at alkaline pH values

Skim milk and mixtures prepared by combining acid whey with skim milk at volume ratios of 2:1, 1:1, 1:2, 1:3 and 1:4 were adjusted to pH 7.5 and heated at 90°C × 15 min. Protein was isolated from these heated samples by precipitation at pH 4.6 and it was found that 65% of the whey protein was recovered in each case. Non-recovered proteins included the proteose peptones and small quantities of β-lactoglobulin, α-lactalbumin and bovine serum albumin. The solubility of these isolates, which contained from 10–25% whey protein, decreased to > 95% when the whey protein exceeded ˜16%. Further characterization of the isolate, prepared from the 1:1 volume ratio of acid whey and skim milk, showed that ˜50% of the whey protein was insoluble, bound to casein and non-functional while the other ˜50% was complexed with casein and was soluble. The addition of a reducing agent suggests that sulphydryl bonding alone is not responsible for complex formation.     

Effect of pH and heat treatment conditions on physicochemical and acid gelation properties of liquid milk protein concentrate

Milk protein concentrates (MPC) are typically dried high-protein powders with functional and nutritional properties that can be tailored through modification of processing conditions, including temperature, pH, filtration, and drying. However, the effects of processing conditions on the structure-function properties of liquid MPC (fluid ultrafiltered milk), specifically, are understudied. In this report, the pH of liquid MPC [13% protein (70% protein DM basis), pH 6.7] was adjusted to 6.5 or 6.9, and samples at pH 6.5, 6.7, and 6.9 were subjected to heat treatment at either 85°C for 5 min or 125°C for 15 s. Sodium dodecyl sulfate PAGE was used to determine the distribution of caseins and denatured whey proteins in the soluble and micellar phases, and HPLC was used to quantify native whey proteins as a measure of denaturation, based on the processing conditions. Both heat treatments resulted in substantial whey protein denaturation at each pH, with β-lactoglobulin denatured more extensively than α-lactalbumin. Changes in liquid MPC physicochemical properties were monitored at d 1, 5, and 8 during storage at 4°C. Viscosity increased after heat treatment and also over time, regardless of pH and heating conditions, suggesting the role of whey protein denaturation and aggregation, and their interactions with casein micelles. The MPC samples processed at pH 6.9 had a significantly higher viscosity than those heated at pH 6.5 or 6.7, for both temperature and time conditions; and samples processed at 85°C for 5 min had higher viscosity than those heated at 125°C for 15 s. Particle size analysis indicated the presence of larger particles after 5 and 8 d of MPC storage after heating at pH 6.9. Acid-induced gelation of the liquid MPC led to significantly higher gel firmness after processing at 85°C for 5 min, compared with 125°C for 15 s. Also, gels made from MPC adjusted to pH 6.5 had higher storage moduli, with both time and temperature combinations, demonstrating the role of pH-dependent association of denatured whey proteins with casein micelles in gel network formation. These findings enable a better understanding of the processing factors contributing to structural and functional properties of liquid MPC and can be helpful in tailoring milk protein ingredient functionality for a variety of food products.     

Effect of Processing Temperature and Storage Time on Nonfat Dry Milk Proteins

Changes in physicochemical properties of pooled nonfat milk preheated to 63°C (I), 74°C (II), and 85°C (III), before spray-drying were examined. Insoluble material from III contained more protein (particularly at reduced pH) and more coagulated protein-lactose aggregates than either I or II. Soluble material from III was practically depleted of whey proteins which were utilized to form complexes stabilized through disulfide bonds. Milk protein micelles from III were heavier (ca 1 × 10¹¹ g/mole) than either I or II. An unsweetened milk-orange juice blend, which was pasteurized at 63°C for 30 min and stored at 4°C, developed a precipitate which contained more protein and pectin, but less sucrose than the supernatant.     

Foaming behaviour of EDTA-treated α-lactalbumin

The foaming properties of partially denatured α-lactalbumin was investigated. The partially denatured state was produced by removing bound Ca²⁺ by treatment with ethylenediaminetetraacetic acid (EDTA) at pH 8.0 and 25°C. Surface tension measurements showed that partially denatured α-lactalbumin unfolds easily at liquid interfaces compared with the native protein. The results of foam volume and stability measurements were consistent with the results of surface tension measurements. In the presence of EDTA a considerable amount of foam was obtained at low concentrations, such as 0.1 mg/ml, and the foam stability was improved. This indicates the importance of the protein structure on the adsorption of molecules at liquid interfaces. The presence of Ca²⁺ also resulted in an increase in the foamability and foam stability of α-lactalbumin compared with native protein, due to the saturation of the surface charges. This shows the binding affinity of protein to Ca²⁺. The investigation of the effect of Ca²⁺ on the surface behaviour of β-lactoglobulin, another whey protein, also showed an improvement in the foaming properties of protein.