Unheated Skim Milk Research Articles

Acid gelation of heat-treated recombined milk: Fat globule membrane composition and gelation functionality

Recombined whole milk was prepared by heating skim milk before homogenising with milkfat (HEHO milk) or by homogenising milkfat with unheated skim milk and heating the recombined milk (HOHE milk). Heat treatment caused whey protein denaturation, more at higher temperatures and slightly more in HEHO milk than in HOHE milk. Less protein was adsorbed to the fat globules of the HEHO milk than the HOHE milk; the former had a higher proportion of αS-casein and β-casein and a lower proportion of κ-casein and denatured whey proteins at the interface, indicating a different interaction mechanism. Gelation pH and gelation time were similar for acidified HEHO and HOHE samples and correlated to the denaturation of whey proteins. The final G’ and yield stresses were lower for the HEHO than the HOHE milk. The differences in gelation behaviour were related to the compositions and interactions of the proteins adsorbed to the fat globules.

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.

Physico-chemical properties of casein micelles in unheated skim milk concentrated by osmotic stressing: Interactions and changes in the composition of the serum phase

The changes in processing functionality of concentrated milk are caused by a number of factors, amongst the most important, the ionic equilibrium and the increase in the interactions between the casein micelles because of their increased volume fraction. The objective of this work was to characterize the physico-chemical properties of casein micelles as a function of their volume fraction, by using osmotic stressing as a non-invasive method to obtain concentrated milk, in the attempt to preserve the ionic balance during concentration. Osmotic concentration was carried out for 18 h at 4 °C, using different concentrations of polyethylene glycol dissolved in permeate as the stressing polymer. The viscosity of the concentrated milk could be predicted using established rheological models, when the changes occurring to the viscosity of the serum phase were taken into account. Both Eilers and Mendoza equations predicted a maximum packing volume fraction of 0.8 for the casein micelles. After concentration up to 20% protein, the casein micelles did not show a change in their size upon redilution. Light scattering measurements carried out using diffusing wave spectroscopy without dilution suggested that casein micelles behave as hard spheres with the characteristic of free diffusing Brownian particles up to a volume fraction of 0.3, and restricted motion at higher concentrations. Results of total and soluble calcium suggested release of colloidal calcium phosphate from the micelles at volume fractions >0.35. This research brings new insights on the changes occurring in skim milk during concentration.

Rheological properties of acid gels prepared from pressure- and transglutaminase-treated skim milk

The effect of heat treatment, transglutaminase treatment, and high hydrostatic pressure treatment of fresh skim milk on the rheological properties of acid milk gels, formed by acidification with glucono-δ-lactone, was investigated. Acid gels prepared from unheated skim milk had long gelation times, low gelation pH, and very low final G′ values. Heat treatment and pressure treatment of milk had similar effects in that both treatments decreased the gelation time, increased the gelation pH, and markedly increased the final G′ of the resultant acid gels. Transglutaminase treatment of milk prior to acid gel formation also markedly increased the final G′ of the acid gels; however, when transglutaminase treatment was performed during pressure treatment, a markedly higher final G′ was obtained than when only pressure or transglutaminase treatment was performed. It is proposed that there is increased cross-linking of the whey proteins, and an increased cross-linking between the whey proteins and casein when transglutaminase treatment is performed under high pressure. The introduction of a higher number of intermolecular cross-links will result in increased acid gel firmness and therefore an increased final G′.

Studies of the acid gelation of milk using ultrasonic spectroscopy and diffusing wave spectroscopy

Processes involved in the coagulation of heated and unheated skim milk during acidification by glucono-δ-lactone have been studied by a combination of ultrasonic spectroscopy and forward-scattering diffusing wave spectrometry (DWS). Ultrasonic velocity and attenuation seem to be governed by the changes in the casein micellar structure as the acid dissolves the micellar calcium phosphate. Both parameters show only small effects of gelation. The DWS shows that there are large differences between heated and unheated milks, especially in respect of the photon mean free path in the milks before coagulation has occurred. Measurement of the particle sizes by this technique shows that slow aggregation occurs at pH values considerably higher than those at which the full gelation of the casein micelles is seen to occur. The general conclusion is that the process of acidification, especially in heated milks, may be less simple than had previously been supposed.

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

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

Protein composition of micellar casein obtained by cross-flow micro-filtration of skimmed milk

In this study of the products of cross-flow micro-filtration of unheated skim milk, it was found that some lactoferrin, serum albumin and immunoglobulins were retained with the casein fraction. An acid-soluble casein, identified as proteose peptone component 5, also remained associated with the micellar fraction during micro-filtration.

Enterotoxin-Binding Glycoproteins in a Proteose-Peptone Fraction of Heated Bovine Milk

The binding of Escherichia coli heat-labile enterotoxin to caseins, whey proteins, milk fat globule membrane, and proteose-peptone fraction from bovine milk was studied by using the Western blot technique. Two toxin-binding glycoproteins, ppl6k and pp20k, with molecular weights of 15,500 and 20,000, respectively, were detected only in a proteose-peptone fraction. These glycoproteins were partially purified by ammonium sulfate precipitation and Toyopearl HW 55 gel filtration chromatography. The binding ability to the toxin was destroyed by periodate treatment or β-galactosidase treatment, indicating that a carbohydrate moiety, particularly a terminal galactosyl residue, was essential for the binding of the toxin. In contrast, the binding ability was not changed by mild acid treatment, and these glycoproteins did not bind cholera toxin, which can bind to ganglioside GM1, suggesting that the carbohydrate structure of the glycoproteins is different from that of GM1. The N-terminal amino acid sequence and immunoblot analysis indicated that the protein moieties of ppl6k and pp20k are identical to α-lactalbumin and β-lactoglobulin, respectively. These toxin-binding glycoproteins were not detected in whey proteins isolated from unheated skim milk, suggesting that they are newly generated during heat treatment of skim milk before the preparation of a proteosepeptone fraction.

Effect of Blood and High Density Lipoprotein Preparations upon Lipase Distribution and Spontaneous Lipolysis in Bovine Milk

The mechanism by which high density lipoprotein promotes spontaneous lipolysis in milk was investigated. Spontaneous milk samples were characterized by an elevated cream-associated lipoprotein lipase activity compared with nonspontaneous milk samples. This cream lipase activity was correlated (.70) with cold storage acid degree value. Blood preparations (serum, plasma) added to warm milk, upon milk cooling, caused correlated (.89) increases in cream lipase activity and acid degree values. Total milk lipase activity was not correlated with level of cold storage lipolysis; however, skim milk lipase activity was negatively correlated (–.38). Addition of high density lipoprotein to recombined milk caused transfer of active lipoprotein lipase from unheated skim milk onto fat globules, which had been recovered from milk heated to inactivate cream-associated lipoprotein lipase. Transfer of lipoprotein lipase to fat globules by high density lipoprotein occurred in a dose-response manner with a correlation of .68 between high density lipoprotein added and cream lipase activity. These observations supported the concept that biological variation in high density lipoprotein content of milk can contribute to the variability in spontaneous lipolysis observed among milk samples. This contribution by high density lipoprotein is mediated primarily through its ability to redistribute lipoprotein lipase toward fat globules rather than activation of the lipoprotein lipase that was initially associated with fat globules.

Enhancement of proteolysis in bovine skim milk by heat and chemical treatments

SummaryEffect of heating and of some chemical treatments on the proteolysis of skim milk caused by its major proteinase system, plasmin, was investigated. Degree of proteolysis was expressed as the increase in γ-casein content of skim milk (mg/ml) after incubation with 0·02% (w/v) NaN3 at 37 °C for 20 h. Apart from reduced proteolysis in skim milk heated at > 70 °C for 10 min or at 75 °C for ∼ 5 min, some enhancement (10–80%) was observed on heating at 55–63 °C for 10 min or at 60–65 °C for 15 s. Heating at pasteurization conditions (63 °C/30 min or 72 °C/15 s) had no apparent effect on the amount of proteolysis. Addition of ascorbic acid (0·2 mg/100 ml) or H2O2 (1/100 ml) to unheated skim milk also increased the amount of proteolysis by 38 and 28%, respectively. However, the extent of this increase diminished with increasing amounts of both chemicals. Furthermore, there was a steady increase (∼ 33%) in proteolysis with time of exposure to light for up to 3 h. These results suggest that oxidative conditions lead skim milk to enhanced proteolysis by inactivating an inhibitor(s) operating in the plasmin system in milk.

Inhibition of the Antibacterial Lactoperoxidase-Thiocyanate-Hydrogen Peroxide System by Heat-Treated Milk

The antibacterial system, lactoperoxidase-H2O2-SCN− was affected by the presence of heated milk or skim milk reconstituted from powders having received severe heat treatment. This inhibitory effect was related to the increase in exposed sulfhydryl groups and to the redistribution of protein between micellar and whey phases. Chromatographic analyses of heat-treated milk showed that the inhibitory factor was associated with the casein micelle fraction. The inhibition, however, was overcome by addition of unheated skim milk.

Effect of extender, number of Spermatozoa and HCG on equine fertility

One ejaculate from each of 10 stallions was used in a facrorially-designed experiment to study the effects of two extender treatments and three incubation temperatures on spermatozoal livability. The percentage of progressively motile spermatozoa in fresh unheated and heated skimmilk was compared at incubation temperatures of 5, 15 and 38°C. Spermatozoal motility was higher (P<0.05) in heated slcimmilk than in unheated skimmilk at all incubation temperatures. Thus, for maximum spermatozoal survival of equine spermatozoa in a skimmilk extender it is essential that the skimmilk be heated. As incubation temperature increased, spermatozoal motility in heated skimmilk decreased (P<0.05); whereas survival was similar at 5 and 15°C in unheated skimmilk, but declined (P<0.05) when exposed to an incubation temperature of 38°C. Two stallions and 48 nonlactating mares were used in a factorially-designed experiment to study the effects of three extenders, two spermatozoal numbers and human chorionic gonadotropin (HCG) on fertility. Each mare was inseminated with 50 or 500 × 10 6 progressively motile spermatozoa extended in skimmilk, skimmilk-gelatin (gel) or cream-gel. One-half of the mares were injected (IM) with 3,300 IU of HCG 24 hours prior to their first insemination on cycle 1. Pregnancy rates for mares inseminated with spermatozoa extended in skimmilk, skimmilk-gel and cream-gel were identical (18.8%) after the first cycle, and 62.5, 62.5 and 43.8%, respectively, after three cycles (P > 0.10). The pregnancy rate of mares inseminated with 50 × 10 6 spermatozoa were 20.8, 15.8, 6.2 and 37.5% for cycles 1, 2, 3 and overall, respectively, compared to 16.7, 55.0, 33.3 and 75.0% for mares receiving 500 × 10 6 progressively motile spermatozoa per insemination. There was a difference (P <0.05) in pregnancy rates obtained during cycle 2 (15.8 vs 55.0%) and overall (37.5 vs 75.0%) in favor of mares inseminated with 500 × 106 spermatozoa. Duration of estrus was shorter (P <0.001) during cycle 1 for the HCG-treated mares than for the controls (4.8 vs 8.6 days). There was no difference (P > 0.10) in pregnancy rates after three cycles betwen HCG-treated and control mares (66.7 vs 45.8%).

Proteose-Peptone Fraction of Bovine Milk: Lacteal Serum Components 5 and 8—Casein-Associated Glycoproteins

Lacteal serum Components 5 and 8 were isolated from both heated and unheated skimmilk. At pH 8.6 Component 5 migrated as a double zone in starch-urea-gels (discontinuous buffer) and as a single zone in poly aery lamide gels (continuous buffer). Component 8 migrated in the ion front on starch gels but resolved into two principal zones in the polyacrylamide gel system, designated as 8-f ast and 8-slow in ascending order of relative mobility. Components 8-fast and 8-slow were fractionated by gelpermeation chromatography on Bio-Gel P-10 polyacrylamide beads.Component 5 is characterized by its high proline (≈ 10.5%) and relatively low carbohydrate contents (≈1.5%). Components 8-fast and 8-slow contain relatively high concentrations of total phosphorus (≈7.4 and 5.5% as H2P03) and intermediate amounts of carbohydrate (≈2.07 and 9.0%). All three components are void of cysteine and cystine and contain only low concentrations of methionine.In veronal buffer at pH 8.6, r/2 = 0.1, a sedimentation coefficient (S°20,w) of 1.22 and a molecular weight (M¯W) of ≈ 14,300 were observed for Component 5. Similar parameters for Components 8-fast and 8-slow were Sdegrees =°2o,w = 4,100 and 9,900. An isoelectric pH of 3.3 is reported for Component 8-fast and partial specific volumes (V¯) of 0.736, 0.700, and 0.719 (cc/g) for Component 5, 8-slow, and 8-fast were calculated from composition data.

Proteose-Peptone Fraction of Bovine Milk: Lacteum Serum Component 3—A Whey Glycprotein

Lacteal serum Component 3 was isolated from both heated and unheated skimmilk. Unlike lacteal serum Components 5 and 8 which exist both in whey and in association with the casein components, Component 3 is found only in the whey protein fraction of skimmilk.Component 3 is characterized by a high content of carbohydrates (≈17.3%) and a low content of aromatic and sulfur-containing amino acids. In veronal buffer, the following physical parameters were determined: D°20,w = 1.8, S°20,w = 3.9, and M¯°W=163,000. In veronal-GU · HCl, they were D°20,w = 1.1, S°20,w = 1.6, and M¯°W=40,800, respectively. A minimum molecular weight, calculated from the limiting amino acid residues, was estimated at 22,000. An isoelectric pH of —3.7 and a partial specific volume of 0.73 were reported for Component 3.

Activity of Bacillus cereus Proteinases in Milk

The selective activity of the proteinase enzyme system of a strain of Bacillus cereus was determined in sterile nonheated and pasteurized skimmilks. They were inoculated with B. cereus spores, incubated at 35C for 12 hours, and changes in the protein system were followed by polyacrylamide gel disc electrophoresis. For the proteins soluble in acid whey, two major changes were noted in the electrophoretic patterns: The proteose-peptone fraction disappeared completely within 12 hours and a new fraction appeared with a mobility similar to that of the dye marker. The caseins were degraded, being more pronounced for β- than for αs-casein and more rapid in pasteurized than in raw unheated skimmilk.

Proteose-Peptone Fraction of Bovine Milk: Distribution in the Protein System

Proteose-peptone preparations were isolated from both unheated (95C for 30min) skimmilk and from thrice-washed casein micelles. Preparations obtained from both the heated and unheated skimmilk contained three principal components, i.e., serum Components 3, 5, and 8. The preparations obtained from micellar casein did not contain Components 3. Thus, Component 3 appears to be a serum component, possibly of blood origin; whereas, Components 5 and 8, which seem to exist in equilibria between micellar casein and the serum, might be minor caseins.In starch-urea gels, Components 3 and 8 move as single electrophoretic zones, whereas Component 5 migrates as a doublet.

Protein Changes Associated with Extended Storage of Sterile Unheated Skimmilk

Changes in the protein system of sterile unheated skimmilk during extended storage were investigated with polyacrylamide gel electrophoresis. The results reveal that decreases in the concentration of immune globulins, blood serum albumin, β-casein, and αs-casein occurred during a 15-day storage period at 30C, with the effect being greater and occurring more rapidly for the casein components. No changes in the proteins were observed during 15 days’ storage at 4C. Storage of the skimmilk beyond 15 days at 30C resulted in coagulation and serum separation.

Effect of Heat upon Electrophoresis and Ultracentrifugal Sedimentation Properties of Skimmilk Protein Fractions

Skimmilk was centrifuged at 11,700×g, 46,900×g, and 92,000×g and the supernatant and sediment fractions were incorporated into phosphate buffer or treated with potassium oxalate to remove calcium and disperse the proteins. They were examined by free-boundary electrophoresis and ultracentrifugal sedimentation.Heating skimmilk (88 C-10min) had little effect upon electrophoretic properties of the three protein sediment fractions, but reduced the magnitude of the differences in electrophoretic composition of the supernatant fractions. These results indicate that interaction of α-casein with β-lactoglobulin is favored among the smaller skimmilk protein particles.Skimmilk and its sediment and supernatant fractions all exhibited two ultra-centrifugal sedimentation components at pH 6.98 and 25C. Oxalate treatment of unheated skimmilk increased Component I from 28 to 57% of the pattern area and reduced its sedimentation constant from 12.7 to 6.9 S. Heating skimmilk increased Component I from 28 to 83% of the pattern area and reduced its sedimentation constant to 6.7 S. The sedimentation patterns of heated skimmilk protein fractions were relatively unaffected by the oxalate treatment. No evidence of 25-48 S casein-serum protein complex particles was obtained in any of the heated skimmilk fractions.

Fractionation of Skimmilk Proteins by Sephadex Gel Filtration

Summary Sephadex G-75 and G-100 gel filtration was employed to determine the protein particle size distribution in skimmilk and ultracentrifugal serum as affected by heat, dialysis, and oxalate-solubilization. Sephadex G-75 failed to fractionate skimmilk proteins satisfactorily, but yielded two partially separated protein components. Sephadex G-100 fractionated unheated skimmilk into four heterogeneous components with about 52, 11, 10, and 26% of the total elution pattern area in each of these components in the order of elution. Oxalate solubilization altered the gel filtration properties of the proteins to a much greater extent than dialysis against phosphate buffer alone. Heating skimmilk (74–90.5 C—10 min) caused two major alterations in the Sephadex patterns: (a) an increase in the largest-sized protein component at the expense of the serum proteins and (b) an increase in the nonprotein nitrogen component. Heating prevented the alteration of the proteins by oxalate-solubilization, as was achieved with the unheated systems.

Inhibition of the Spermicidal Factor(s) in Unheated Skimmilk by the Addition of Egg Yolk

Fresh milk, skim or whole, raw or pasteurized, must be heated or chemically treated with compounds containing reactive sulfhydryl groups (SH) to support extended livability of bovine spermatozoa (1,3,8,9,12). Complete protection is apparently afforded by the addition of SH compounds and, in some cases, the motility of spermatozoa extended in chemically treated milk exhibited greater motility than split-samples extended in heated milk (3,8,9). The spermicidal factor(s) in milk and the possible role of SH in supporting motility have been investigated (6,8,10,12). In regard to fertility, Hayden et al. (7) and Bartoff (2) have reported favorable results with extenders containing added glutathione.While several studies have dealt with mixtures of heated milk plus egg yolk, with or without glycerol, as a diluent for bull semen, some of these indicated that extenders of this type are excellent for suporting spermatozoan motility, but inferior in maintaining fertility (4,11,14).In view of the observations that SH-treated milk and heated milk plus egg yolk provided an excellent environment for maintenance of motility, an experiment was conducted to study the effect on spermatozoal motility of adding various levels of cysteine hydrochloride (cysteine HCl) to an unheated skimmilk-egg yolk diluent.