Purification and characterization of a novel aspartic protease from basidiomycetous yeast Cryptococcus sp. S-2

An aspartic protease (Cap1) was purified from basidiomycetous yeast Cryptococcus sp. S-2 (FERM ABP-10961) using HiTrap DEAE FF column and HiTrap Q HP column chromatography with azocasein as a substrate. Cap1 has a molecular mass of 34 kDa on SDS-PAGE. It was stable up to 50°C with maximum activity at 30°C. Maximum proteolytic activity was observed at pH 5.0. Cap1 was stable in the pH range 3.0-7.0. Its enzyme activity was strongly inhibited by pepstatin A, an inhibitor of aspartic proteases, indicating that Cap1 is an aspartic protease. Cap1 hydrolyzed protein substrates, including BSA, hemoglobin, α-casein, β-casein, and κ-casein. It showed activity on synthetic substrates, such as MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-Arg-NH? and MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH?. Hydrolysis of the oxidized insulin B chain followed by amino acid sequencing analysis of the cleavage products revealed that 9 of its 30 peptide bonds were hydrolyzed by Cap1. This result was similar to that observed with pig pepsin A and human pepsin A. Cap1 also exhibited milk-clotting activity. We cloned the cDNA of CAP1 gene, which contained a 1254 bp open reading frame encoding a protein of 417 amino acid residues. Homology search in the NCBI database revealed that the amino acid sequence of Cap1 showed less than 39% identity to other known proteins. Therefore, we proposed that Cap1 is a novel aspartic protease.     

Relative Proteolytic Action of Milk-Clotting Enzyme Preparations on Bovine α-, β- and β-casein

Concentrations of seven milk-clotting enzyme preparations were standardized to equal clot times. Portions of bovine α_s-, β- and κ-casein were treated with enzymes. Proteolytic activity of the coagulants on each casein fraction was determined using the TNBS (2,4,6-trinitrobenzene-sulfonic acid) procedure. Recombinant chymosin showed the lowest degree of proteolysis on α_s- and β-caseins. Excessive proteolysis of calf rennet appeared to be due to the pepsin fraction. M. miehei and M. pusillus var Lindt proteases showed similar degradation of caseins, but M. pusillus var Lindt was more proteolytic when β-casein was the substrate. C. parasitica protease showed the highest degree of proteolysis on α_s- and β-caseins but was the least proteolytic on κ-casein.     

Short communication: Effect of kefir grains on proteolysis of major milk proteins

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse phase-HPLC analysis. The reduction of κ-, α-, and β-caseins (CN), α-lactalbumin (α-LA), and β-lactoglobulin (β-LG) contents during 48 and 90 h of incubation of pasteurized milk (100 mL) and respective cheese whey with kefir grains (6 and 12 g) at 20°C was monitored. Significant proteolysis of α-LA and κ-, α-, and β-caseins was observed. The effect of kefir amount (6 and 12 g/100 mL) was significant for α-LA and α- and β-CN. α-Lactalbumin and β-CN were more easily hydrolyzed than α-CN. No significant reduction was observed with respect to β-LG concentration for 6 and 12 g of kefir in 100 mL of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of α-LA and β-LG: α-LA was hydrolyzed between 60 and 90% after 12 h (for 6 and 12 g of kefir) and no significant β-LG proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of α-LA and κ-, α-, and β-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. β-Lactoglobulin is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase β-LG digestibility in whey-based or whey-containing foods.     

Influence of rennet milk-clotting activity on the proteolytic and sensory characteristics of an ovine cheese

Roncal cheese (regulated by an Apellation of Origin) is a traditional hard cheese manufactured from raw ewe's milk in the region of Navarre in Spain. Roncal cheeses, manufactured using two lamb rennets with different milk-clotting activity levels, were evaluated to compare their chemical, proteolytic, and sensory characteristics. A preliminary study of samples of lamb rennets indicated that a large proportion of such rennets did not fulfil current microbiological requirements and likewise revealed considerable variation in the milk-clotting activity of the samples examined. Trends in the overall physicochemical parameter values (pH, dry matter, fat, and protein) were similar in both cheese batches. Proteolysis of the nitrogen fractions was observed to take place at a faster rate in the cheeses made using the rennet with the higher milk-clotting activity (soluble nitrogen, non-protein nitrogen, and amino acid nitrogen values around 13–20% higher than in the cheeses made using the rennet with the lower milk-clotting activity after 180 days of ripening). Urea-PAGE electrophoretic analysis of the caseins from the cheeses manufactured using both types of rennet showed that the β-caseins were less susceptible to proteolysis than the α_s-caseins. The effect of the different milk-clotting activity levels was most pronounced on the α_s-caseins, in which the rennet with the higher milk-clotting activity gave higher breakdown. Nevertheless, the differences in the proteolysis rates did not yield any appreciable sensory differences.     

Characterisation of a milk-clotting extract from Balanites aegyptiaca fruit pulp

Milk-clotting proteases were extracted and characterised from Balanites aegyptiaca fruit pulp traditionally used in sub-Saharan countries to thicken gruel. The aim was to improve the extraction conditions and milk-clotting in the context of future cheese making. B. aegyptiaca fruits were disinfected, husked and soaked in different buffers and saline solutions. The extracts obtained were diafiltered, concentrated and decoloured with active charcoal. The effects of extract concentration, milk pH and temperature on clotting-time were studied. The use of specific inhibitors showed that the B. aegyptiaca extract contains two types of proteases: an aspartic protease and a serine protease, with an optimum activity at pH 5.0 and pH 8.0, respectively, and an optimal temperature at 50 °C. The proteolytic activity is more thermostable than bovine chymosin at pH 5.0. Partial purification by anion exchange chromatography and sodium docecylsulphate polyacrylamide gel electrophoresis allowed separation of the two milk-clotting proteases.     

Purification, characterization, and milk coagulating properties of ginger proteases

Ginger proteases are used as milk coagulants in making a Chinese traditional milk product (Jiangzhinai or Jiangzhuangnai), suggesting their potential as a source of rennet substitute that might be applicable in the modern dairy industry. In this study, ginger proteases were extracted from fresh ginger rhizome by using phosphate buffer and subsequently purified by ion exchange chromatography. Ginger proteases, all with a molecular weight around 31 kDa, were found to exist in 3 forms with isoelectric point values around 5.58, 5.40, and 5.22, respectively. These enzymes had very similar biochemical behavior, exhibiting optimal proteolytic activity from 40 to 60°C and maximum milk clotting activity at 70°C. They were capable of hydrolyzing isolated α_S1-, β-, and κ-casein, of which α_S1-casein was most susceptible to the enzyme; κ-casein was hydrolyzed with a higher specificity than α_S1- and β-casein. In addition, the ginger proteases exhibited a similar affinity for κ-casein and higher specificity with increasing temperature. Gel electrophoresis and mass spectra indicated that Ala90-Glu91 and His102-Leu103 of κ-casein were the preferred target bonds of ginger proteases. The milk clotting activity, affinity, and specificity toward κ-casein showed that ginger protease is a promising rennet-like protease that could be used in manufacturing cheese and oriental-style dairy foods.     

Proteolysis of milk proteins by AprX,an extracellular protease identified in Pseudomonas LBSA1 isolated from bulk raw milk,and implications for the stability of UHT milk

UHT milk made from milk contaminated by Pseudomonas LBSA1 destabilised during storage. Sedimentation of UHT milk was observed; zeta potential of casein micelles decreased, while contents of non-casein nitrogen and non-protein nitrogen increased. Pseudomonas LBSA1 produced an extracellular protease that hydrolysed caseins but not whey proteins; this was identified as AprX, a thermoresistant protease belonging to the serralysin family. This protease showed a broad range of pH activity (pH 6 to pH 10) and an optimal temperature of activity of 40 °C. Peptides released from purified α_S1-, β- and κ-caseins were determined by tandem mass spectrometry. The identified cleavage sites did not reveal a strong specificity of the extracellular protease. However, the presence of basic or aromatic amino acid residues in the P1 position had a positive influence on cleavage in comparison with acidic amino acid residues or proline.     

Short communication: Bovine α-casein is a ferritin-binding protein and inhibitory factor of milk ferritin immunoassay

Commercial bovine milk α-casein, but not β- and κ-caseins, bound to bovine spleen ferritin, as determined by an immunoassay for ferritin. In contrast, α-casein did not bind to apoferritin. The binding of α-casein to bovine spleen ferritin was strongly inhibited by increasing ionic strength by the addition of 0.5 M (NH₄)₂SO₄. The addition of α-casein to a known amount of bovine spleen ferritin resulted in significantly lower recovery (78-80%) of added ferritin, although β- and κ-caseins showed little inhibitory effect in the ferritin immunoassay. These results indicate that bovine α-casein is a specific ferritin-binding protein that may inhibit milk ferritin immunoassay.     

Fluorescein thiocarbamoyl-kappa-casein assay for the specific testing of milk-clotting proteases

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using κ-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity.     

Influence of residual milk-clotting enzyme on alpha(s1) casein hydrolysis during ripening of Reggianito Argentino cheese

Milk-clotting enzyme is considered largely denatured after the cooking step in hard cheeses. Nevertheless, typical hydrolysis products derived from rennet action on alpha(s1)-casein have been detected during the ripening of hard cheeses. The aim of the present work was to investigate the influence of residual milk-clotting enzyme on alpha(s1)-casein hydrolysis in Reggianito cheeses. For that purpose, we studied the influence of cooking temperature (45, 52, and 60 degrees C) on milk-clotting enzyme residual activity and alpha(s1)-casein hydrolysis during ripening. Milk-clotting enzyme residual activity in cheeses was assessed using a chromatographic method, and the hydrolysis of alpha(s1)-casein was determined by electrophoresis and high performance liquid chromatography. Milk-clotting enzyme activity was very low or undetectable in 60 degrees C- and 52 degrees C-cooked cheeses at the beginning of the ripening, but it increased afterwards, particularly in 52 degrees C-cooked cheeses. Cheese curds that were cooked at 45 degrees C had higher initial milk clotting activity, but also in this case, there was a later increase. Hydrolysis of alpha(s1)-casein was detected early in cheeses made at 45 degrees C, and later in those made at higher temperatures. The peptide alpha(s1)-I was not detected in 60 degrees C-cooked cheeses. The results suggest that residual milk-clotting enzyme can contribute to proteolysis during ripening of hard cheeses, because it probably renatures partially after the cooking step. Consequently, the production of peptides derived from alpha(s1)-casein in hard cheeses may be at least, partially due to this proteolytic agent.     

Ex vivo digestion of proteins and fat in buffalo milk

Ex vivo digestion of proteins and fat in whole buffalo milk (WBM) and skimmed buffalo milk (SBM) was studied. Proteolysis, generation of peptides, lipolysis and generation of fatty acids were monitored. In both types of milk, β- and κ-caseins were hydrolysed almost completely after 20 min of gastric digestion. Residual α_S-caseins were digested completely on 5 min of duodenal digestion. Of the main whey proteins, α-lactalbumin was only partially hydrolysed and β-lactoglobulin seemed to be resistant during 40 min of gastric digestion. However, by 5 min of duodenal digestion, both α-lactalbumin and β-lactoglobulin were fully hydrolysed in both WBM and SBM. Only small variations between WBM and SBM were found for the release of peptides. In the WBM, 30% lipolysis was observed after 30 min of duodenal digestion. Different extents of lipolysis of fatty acids among/within different groups, based on carbon number and double bond(s), were observed.     

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.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

Hydrolysis of caprine and ovine milk proteins,brought about by aspartic peptidases from Silybum marianum flowers

The flowers of cardoon (Asteraceae) are a rich source of aspartic peptidases which possess milk clotting activity – and are thus used in traditional cheesemaking in the Iberian Peninsula. This study was aimed at characterizing the enzymatic action of the aspartic peptidases present in flowers of Silybum marianum (L.) Gaertn. (Asteraceae), specifically upon degradation of caseins. The proteolytic activities toward Na-caseinates previously prepared from caprine and ovine milks were studied, in a comparative fashion, using urea-PAGE, tricine-SDS-PAGE, densitometry, electroblotting and sequencing. Caprine α_s1- and β-caseins were degraded up to 68% and 40%, respectively, during 24 h of incubation. Only one important and well-defined band corresponding to a molecular weight of 14.4 kDa – i.e. a fragment of β-casein, was observed by 12 h of hydrolysis. By 24 h of incubation, ovine α_s- and β-caseins were degraded up to 76% and 19%, respectively. In what concerns specificity, the major cleavage site in ovine caseinate was Leu99-Arg100 in α_s1-casein.     

The Impact of NaCl Substitution with KCl on Proteinase Activities Cell-Free Extract and Cell-Free Supernatant at Different pH Levels and Salt Concentrations: Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

The effect of NaCl substitution with KCl at different pH levels (6.0, 5.5, and 5.0) and salt concentrations on proteinase activities of cell-free and supernatant of Lactobacillus delbrueckii ssp. bulgaricus 11824 (L. bulgaricus) and Streptococcus thermophilus MS (ST) was investigated. MRS broths were separately mixed with 4 salt treatments (NaCl only, 1NaCl:1KCl, 1NaCl:3KCl, and KCl only) at 2 different concentrations (5% and 10%) and incubated at 37 °C for 22 h. The cell pellets were used to prepare proteinase of cell-free extract and the cell-free supernatants were used as source of extracellular proteinases. The proteolytic activities and protein contents of both fractions were determined. The supernatants after incubation of both fractions with 3 milk caseins (α-, β-, κ-casein) were subjected to angiotensin-converting-enzyme inhibitory (ACE-inhibitory) activity and proteolytic activity by ortho-phthalaldehyde (OPA) method. Significant differences were observed in ACE-inhibitory activities and proteolytic (OPA) between salt treatments of cell-free extract and cell-free supernatant of L. bulgaricus and S. thermophilus at same salt concentration and same pH level. There was a significant effect of pH level and salt treatments interaction on ACE-inhibitory activity, OPA activity and azocasein activity. Practical Application: To reduce sodium concentration in cheese by substituting of NaCl with KCl, it was important to study the effect on starter culture proteinases which play a vital role in ripening and texture profile of cheese.     

Genetic analysis of detailed milk protein composition and coagulation properties in Simmental cattle

The objective of this study was to estimate genetic parameters for milk protein fraction contents, milk protein composition, and milk coagulation properties (MCP). Contents of α_S1-, α_S2-, β-, γ-, and κ-casein (CN), β-lactoglobulin (β-LG), and α-lactalbumin (α-LA) were measured by reversed-phase HPLC in individual milk samples of 2,167 Simmental cows. Milk protein composition was measured as percentage of each CN fraction in CN (α_S1-CN%, α_S2-CN%, β-CN%, γ-CN%, and κ-CN%) and as percentage of β-LG in whey protein (β-LG%). Rennet clotting time (RCT) and curd firmness (a₃₀) were measured by a computerized renneting meter. Heritabilities for contents of milk proteins ranged from 0.11 (α-LA) to 0.52 (κ-CN). Heritabilities for α_S1-CN%, κ-CN%, and β-CN% were similar and ranged from 0.63 to 0.69, whereas heritability of α_S2-CN%, γ-CN%, and β-LG% were 0.28, 0.18, and 0.34, respectively. Effects of CSN2-CSN3 haplotype and BLG genotype accounted for more than 80% of the genetic variance of α_S1-CN%, β-CN%, and κ-CN% and 50% of the genetic variance of β-LG%. The genetic correlations among the contents of CN fractions and between CN and whey protein fractions contents were generally low. When the data were adjusted for milk protein gene effects, the magnitude of the genetic correlations among the contents of milk protein fractions markedly increased, indicating that they undergo a common regulation. The proportion of β-CN in CN correlated negatively with κ-CN% (r = −0.44). The genetic relationships between CN and whey protein composition were trivial. Low milk pH correlated with favorable MCP. Genetically, contents and proportions of α_S1- and α_S2-CN in CN were positively correlated with RCT. The relative proportion of β-CN in CN exhibited a genetic correlation with RCT of −0.26. Both the content and the relative proportion of κ-CN in CN did not correlate with RCT. Weak curds were genetically associated with increased proportions in CN of α_S1- and α_S2-CN, decreased contents of β-CN and κ-CN, and decreased proportion of κ-CN in CN. Negligible effects on the estimated correlations between a₃₀ and κ-CN contents or proportion in CN were observed when the model accounted for milk protein gene effects. Increasing β-CN and κ-CN contents and relative proportions in CN and decreasing the content and proportions of α_S1-CN and α_S2-CN and milk pH through selective breeding exert favorable effects on MCP.     

Comparative study of the protein fraction of goat milk from the Indigenous Greek breed and from international breeds

Individual goat milk samples, taken from animals of the Indigenous Greek breed and from the international breeds Saanen and Alpine, were studied by RP-HPLC regarding the qualitative and quantitative characteristics of their proteins. Thirty-two samples from the Indigenous Greek breed and 17 from the international breeds were characterised further by RP-HPLC/ESI-MS. The mean total protein content of the milk samples from the Greek breed was higher 38.8 g/l, compared to 31.9 g/l of those from international breeds, due to the great difference between their mean αs1-Cn contents (6.90 and 3.02 g/l, respectively). In the milk samples of the Greek breed, the strong αs1-Cn variants B3, B4 and As/B1 predominated, whereas in the milk samples from international breeds the medium variant E and the defectives F and null predominated. Variant A of αs2-Cn followed by variant C and the κ-Cn D (former B) were the most abundant in both groups. αs2-Cn F and the rare κ-Cn variant C/B were observed in the milk samples from the Greek breed. The β-caseins A and C were present in both groups of samples. Finally, the level of phosphorylation of the different genotypes is showed.     

Production and characterization of milk-clotting enzyme from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> JNU002 by submerged fermentation

Milk-clotting enzymes produced by microorganisms have been developed to replace calf rennet, yet the enzymatic level and the ratio of milk-clotting activity to proteolytic activity still need further improvement. This work described a strain Bacillus amyloliquefaciens JNU002 that screened from wheat bran that has a promising characterization. After optimization, B. amyloliquefaciens JNU002 showed a high milk-clotting activity (4969 SU/mL) and low proteolytic activity (4.02 U/mL) at 48 h with an inoculum size of 0.2% (v/v) at initial pH 6.0 in 15-L bioreactor. After purification, the purified enzyme gave a single protein band on SDS–PAGE, corresponding to 28 kDa. The purified enzyme showed a high ratio (2,575) of milk-clotting activity to proteolytic activity at 35 °C, and the ratio would be even higher (22,992) at 70 °C. The milk-clotting reaction and proteolytic reaction were prevented at 75 °C. This enzyme was stable at pH 4–6 and below 40 °C, and this was convenient for storage and transportation.     

辣木凝乳酶水解酪蛋白位点及其酪蛋白磷酸肽、酪蛋白糖巨肽

基于十二烷基硫酸钠-聚丙烯酰胺凝胶电泳,液相色谱-串联质谱法研究辣木凝乳酶对酪蛋白的酶切位点和水解特性,并分析该凝乳酶水解产生的酪蛋白糖巨肽和酪蛋白磷酸肽。结果表明,辣木凝乳酶酶切κ-酪蛋白的Arg 93-His 94位点导致凝乳,区别于其他已报道的凝乳酶,具有明显特异性。酶作用下κ-酪蛋白的米氏常数Km=0.49 mg/mL,最大反应速率Vm=45.2 U/min,有效促进酪蛋白胶束的聚集和凝乳。该酶对α-、β-、κ-酪蛋白产生不同程度的水解,对α-酪蛋白的水解速度最快,说明其适用于软质奶酪的加工。以酪蛋白为底物,通过钡-乙醇沉淀法得到酪蛋白磷酸肽（casein phosphopeptides,CPPs）,产率为15.87%,得到的CPPs可以使钙沉淀推迟23 min,有利于钙离子被肠道有效吸收。以水牛乳为底物时,乳凝固后析出的乳清中含酪蛋白糖巨肽6.61 mg/mL,糖基化程度477.527 μg/mg。研究为新型植物凝乳酶的研发,及其在奶酪、酪蛋白活性肽等产品的应用提供科学依据。     

Aggregation and Dissociation of Milk Protein Complexes in Heated Reconstituted Concentrated Skim Milks

Heating milk at 120°C at pH 6.55 or pH 6.85 caused the denaturation of whey proteins and increased their association with the casein micelles. The dissociation of K -, β-, and α_s-caseins (in that order by extent) from the casein micelles increased with severity of heat treatment. The effect was greater at higher pH. Gel filtration chromatography followed by gel electrophoresis of fractions showed the dissociated protein was composed of disulfide-linked k -casein/β-lactoglobulin complexes of varying composition, casein aggregates of varying sizes and some monomeric protein. When reconstituted concentrate was prepared from NFDM made from heated milk the non-sedimentable (88,000 ± g for 90 min) caseins or whey proteins/heating time profiles were altered and the rate of aggregation, as measured by turbidity of heated milks, was significantly reduced.