Whey Protein Denaturation Research Articles

Effect of Micellar Casein on Heat Induced Denaturation of Whey Proteins in Milk

Denaturation of whey proteins by heat was higher in ultracentrifugal milk serum collected at 105,000×g for 30 minutes (no micellar casein) than in skimmilk with whole micellar casein. Denaturation decreased with increased micellar casein in milk serum. Denaturation further decreased when micellar casein was added to skimmilk. Buffalo milk whey proteins were more susceptible to heat denaturation than those of cow milk, with or without micellar casein. Micellar casein in the milk system partially stabilized whey protein against heat denaturation. Cow micellar casein stabilized more than buffalo micellar casein in their respective milks.

Hydrogen Peroxide Alteration of Whey Proteins in Whey and Concentrated Whey Systems

The extent of denaturation and physical aggregation of individual and total whey proteins, produced by treating whey and concentrated whey systems with hydrogen peroxide under experimental conditions that simulate pressure membrane and gel filtration processes for preparing an undenatured whey protein concentrate, was studied by preparative ultracentrifugation, Sephadex G-150 gel filtration chromatography and zonal electrophoresis. Proteose peptones were most susceptible; immunoglobulins, β-lactoglobulin and bovine serum albumin were intermediately susceptible; and α-lactalbumin was least susceptible to alteration by hydrogen peroxide. Variations in pH of the reaction mixture had only a minor influence upon whey protein alteration, whereas, all of the other variables, i.e., peroxide concentration, temperature, and reaction time, greatly affected the extent of total and individual whey protein alteration produced by the peroxide-catalase treatment.

Glucose-Residue Polymers as Protectants against Heat Denaturation of Whey Proteins

Thirty materials, 25 made up of varying numbers of glucose residue polymers (glucons) and 5 others, were tested for their potential to protect whey proteins during heat treatment and for their influence on fermentation media subsequently prepared from the wheys. Polyacrylamide gel electrophoresis of the proteins was made on unheated control, untreated, treated, pasteurized, and autoclaved wheys; and on supplemented whey media. Densitometric plots of the gels evaluated whey protein bands according to the Melachouris and Tuckey classification (14). Two chemically defined pectins and two chemically defined dextrins (40 to 60 glucose residues) were effective protectants; the effectiveness of hydrogen peroxide was proportional to protein denaturation by previous heat treatment of the wheys. Fermentation media prepared with the wheys were afforded similar whey protein protection.

Whey Protein Denaturation in Concentrated Skimmilks

The relative denaturation susceptibility of whey proteins in skimmilk and its concentrates was studied by heating milks of 9 to 44% total solids for 5, 10, 15 and 20min at 75 and 80C. Results indicated that solids are more critical than temperature variations. Denaturation at 80C for 20min decreased from 80 to 59 to 39% as the total solids increased from 9 to 28 to 44%. An equation relating percentage denaturation with time, temperature and solids was derived from the experimental data. Although denaturation susceptibility of whey proteins in skimmilk, expressed as percentage denatured, is reduced by concentration, the actual rate reaction is normal first order for heating in excess of five minutes.

Preparation and Properties of an Alcohol-Precipitated Whey Protein Concentrate

Aqueous methanol, ethanol, n-propanol, and n-butanol were investigated for precipitating whey proteins from liquid and dried whey to prepare a whey protein concentrate. Ethanol was more satisfactory than the other alcohols; methanol and n-propanol whey protein concentrates were slightly less soluble in water and neutral buffer than ethanol whey protein concentrate, and n-butanol whey protein concentrate contained gross amounts of lactose and was practically insoluble in water.The 72% ethanol treatment precipitated 45 to 60% of the whey proteins and produced a whey protein concentrate with ∼ a fourfold protein enrichment on a solids basis compared to whey. Minimum coprecipitation of minerals and lactose with the protein concentrate was produced by treating calcium-free acid whey with 72% (w/w) ethanol at 0 to 5C. Ethanol extraction of freeze-dried whey was less satisfactory than liquid whey treatments because of substantial coextraction of proteins with the lactose and minerals. Fifty to 70% of the freeze-dried whey protein concentrate was resolubilized at pH 6.6, whereas it was totally soluble at pH 8 to 9. The protein concentrate, which exhibited normal zonal electrophoretic properties at pH 8.6, contained 2 to 3 times as much bovine serum albumin and proportionately less α-lactal-bumin than control whey proteins. Pre-treatments to remove calcium and other minerals from whey and use of≥72% (w/w) ethanol during preparation improved the solubility of the protein concentrates. Possible mechanisms for ethanol-induced whey protein denaturation and precipitation are presented.

Studies on Denaturation of Whey Protein in Artificial Feeding—DEAE-Cellulose Chromatographic Analysis of Whey Protein in Different Milk Formulae—

Studies on Denaturation of Whey Protein in Artificial Feeding—DEAE-Cellulose Chromatographic Analysis of Whey Protein in Different Milk Formulae—

Protein Aggregation in Conventional and Ultra High-Temparature Heated Skimmilk

Ultracentrifugate supernatant fractions from raw and heated skimmilk were examined by Sephadex G-100 gel-filtration and zonal electorphoresis to investigate the protein aggregation produced by heating skimmilk at 90C for 10 or 30min, and at temperatures to 149C for as long as 16sec. Heating at 90C caused preferential formation of intermediate size ≥75 to 100 S ≤425 to 500 S protein particles; whereas, the ultra high-temperature heat treatments favored formation of larger size ≥425 to 500 S particles. Ultra high-temperature heat treatments produced only slight alteration of Sephadex gel-filtration patterns for the smallest size ≤75 to 100 S protein components; whereas, all of the ≤75 to 100 S protein components in 90C heated skimmilk were sufficiently aggregated as to be totally excluded from Sephadex G-100. Polyacrylamide gel electrophoresis data indicated that the rate and extent of heat-induced protein aggregation to particle sizes in excess of about 75 to 100 S closely parallel those for whey protein denaturation. The findings support the concept that ultra high-temperature heat treatments of 146 to 150C may produce small amounts of nonsedimenting casein components by disaggregation of casein micelles.

牛乳蛋白質の加熱変性におよぼすパラクロル水銀安息香酸(PCMB)の影響

The effect of PCMB on the interaction of milk proteins induced by heat-treatment was investigated.Skimmilk was made 0.2mM with PCMB and held at 4°C for 8 to 24hr before heating. PCMB treated milk was then heated at temperatures ranging from 65°C to 95°C and the nitrogen distribution, Sephadex gel filtration, PAE patterns and the stabilizing ability of κ-casein prepared from it were studied.The decrease of NCN by heating milk at 80°C for 10min and 95°C for 5min were 10.5% and 14.8%, respectively, in PCMB treated milk, while those of control (heated in the absence of PCMB)milk were 31.9% and 39.5%, respectively. This preventive effect of PCMB on the heat denaturation of whey protein was also found in Sephadex G-100 gel filtration patterns of heated milk, that is, the relative area of components corresponding to whey proteins was larger in PCMB treated milk than with control milk.The results of PAE showed that κ-casein interacted with other protein fractions in heated milk, this interaction being inhibited in PCMB treated milk.κ-Casein prepared from heated milk did not possess the αs-casein stabilizing ability, but, from heated PCMB treated milk, κ-casein with the full stabilizing ability was obtained.When skimmilk was heated soon after the addition of PCMB, the preventive effects of PCMB on whey protein denaturation were not found. In this case, PCMB might react non-specifically with the heat activated sites other than SH in heated milk.

Effect of Calcium, N-Ethylmaleimide and Casein upon Heat-Induced Whey Protein Aggregation

Low- and high-speed centrifugation, Sephadex gel-filtration and polyacrylamide gel electrophoresis were employed to further investigate the nature and extent of protein aggregation produced by heating whey and skimmilk systems.Results indicated that protein aggregation in heated whey systems is a multi-reaction process involving a) denaturation of whey proteins through thiol-disulfide group, hydrogen and hydrophobic bond reactions; b) aggregation to form intermediate-sized protein particles which were excluded from Sephadex G-100 and G-200 gels and sedimentable at 144,000g but not sedimentable at 1,000g, primarily through thiol-disulfide group reactions; and c) gross aggregation of the above protein particles in the presence of calcium ions to sizes sufficient to be sedimentable at 1,000g.n-Ethylmaleimide prevented heat-induced, irreversible whey protein denaturation and interaction of whey proteins with casein by thiol-disulfide group reactions, but did not interfere with the ability of whole casein to stabilize the whey proteins against gross aggregation. Therefore, it is proposed that in addition to the more specific thiol-disulfide group reaction between β-lactoglobulin and κ-casein, denatured whey proteins are stabilized against heat-induced gross aggregation in skimmilk by complexing with casein micelles through calcium-dependent linkages.

Denaturation of Whey Proteins in Milk Heated at High Temperatures for Short Times

Denaturation of Whey Proteins in Milk Heated at High Temperatures for Short Times

Effects of Direct Steam Heating and Vacuum Treatments on the Chemical Composition of Milk with Especial Reference to Substances Involved in Oxidized Flavor Development

Summary Milk was heated with live steam to from 190 to 240° F. and subjected to vacuum treatments of approximately 9 in. of mercury in one vacuum chamber and 20 in. of mercury in a second vacuum chamber. The direct steam heating and vacuum equipment were used in conjunction with a H.T.S.T. pasteurizing system and a homogenizer. The combined steam heating and vacuum treatment was found to produce a significant denaturation of whey proteins, as measured by the increase in the activity of sulfhydryl groups and the decrease in whey protein nitrogen. The decrease in whey protein nitrogen was shown to be approximately that which would be expected from published data on the effect of indirect heating. However, the extent of activation of sulfhydryl groups was lower than had been expected. The possibility that the vacuum treatment removed some of the volatile sulfhydryls was discussed. Increased activation of sulfhydryl groups was observed upon homogenization, particularly after direct steam heating. It was suggested that sulfhydryl activation may be a factor contributing to the high resistance of homogenized milk to oxidized flavor development. The lack of correlation between the nitroprusside test and cooked flavor is believed to indicate that the nitroprusside test is less sensitive than flavor perception, or that cooked flavor is not necessarily due to nitroprusside reactive substances. A high retention of ascorbic acid was observed in milk subjected to direct steam heating and vacuum treatment. This effect, along with resistance to oxidized flavor development, was noted even in milks to which copper was added. The removal of oxygen by the vacuum treatment and the activation of sulfhydryl groups by the heat treatment were considered to be contributing factors. Homogenization was also shown to contribute to the stability of ascorbic acid and flavor. However, homogenization alone was less effective than the combination of homogenization with steam heating-vacuum treatment.

Factors Affecting the pH of Skimmilk Coagulation by Lactic Culture

Summary The A-C (acid-coagulation) test measures the stage of gel formation in acidcoagulated skimmilk at which the gel will first shrink and expel whey when cut with a knife. This test was chosen to study certain factors influencing the pH of coagulation of skimmilk by action of lactic culture. The rate of acid development and the use of different commercial lactic cultures did not affect the pH at the A-C end-point. Increased amounts of rennet added early in the fermentation process caused the skimmilk to coagulate at progressively higher pH values, but very small additions had little or no effect. Different skimmilks did not respond alike to variations of rennet. Additions of chloride of calcium and sodium decreased the pH at the A-C end-point more than did monosodium phosphate or a mixture of salts when all were added to increase the ionic strength by 0.10 at pH 4.70. The pH at which the A-C end-point occurred decreased as the total solids in reconstituted nonfat dry milk increased, probably because of the greater ionic strength of milk in reconstituted nonfat dry milk with high total solids. Heat treatments of skimmilk which denatured whey proteins resulted in an increase in the pH at which the A-C end-point occurred.

Method for the Rapid Estimation of Undenatured Whey Proteins in Nonfat Dry Milk

Summary A simple spot-color test is described for use with nonfat dry milk, to estimate the amount of undenatured protein present and the baking quality. This method consists of precipitating casein and denatured whey proteins with 0.5% acetic acid and spotting the supernatant on filter paper. The paper is dried, stained with 0.1% bromphenol blue in ethanol saturated with mercuric chloride, washed with water, and dried. The intensity of the blue stain is used to estimate undenatured whey protein. The undenatured whey protein content of 17 samples was estimated by this technique. Samples of good baking quality and low undenatured whey-protein contents give pale spots, with characteristic diffuse edges. Results from 20 samples of nonfat dry milk show that this method is also satisfactory for estimating baking quality.

Sedimentation of Milk Proteins from Heated Milk

Summary Milk is a very sensitive system and the most marked changes produced by heat treatments can be measured only when the time and temperature of fractionation are carefully controlled. Some of the observations which have previously been made and that appear contradictory may be attributed to differences in one or both of these factors. Immediately following the heat treatment of skimmilk, sedimentation reveals such a marked drop in supernatant nitrogen that it must be concluded whey proteins are being sedimented. As pointed out by Edmondson and Tarassuk (1) , the measurement of acid-coagulable nitrogen in the supernatant will not reveal this if the whey proteins are sedimenting uniformly with the casein. During 24 hours of storage in the cold, rapid shifts in equilibria occur, which tend to restore the supernatant nitrogen to a degree dependent on the severity of the heat treatment. These results were confirmed, using skimmilk containing added S 35 -labeled whey proteins. Radioactive analyses demonstrated 52% of the whey proteins were present in the gels from a skimmilk which had received a strong heat treatment. In this case, considerable amounts of the denatured whey proteins remained centrifugable even after 24 hours' storage.