Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6 h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12 h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α_S1-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α_S1-casein. Two-dimensional gel electrophoretograms of milk 6 h after infusion with the toxin confirmed hydrolysis of β- and α_S1-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C₁₈ columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α_s1- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously.     

Effect of micellar structure of casein and its modification on plasmin-induced hydrolysis

Plasmin-induced hydrolysis of casein in milk can lead to many defects including proteolysis, age gelation, and bitterness. The susceptibility of casein to plasmin can be affected by micellar structure and modification of the lysine residues on caseins. Different levels of casein modification and dissociation of the casein micelle structure were achieved through succinylation. Succinylation occurred at residues Lys7, Lys34, Lys36, Lys42, Lys83 and Lys124 in α_S1-casein; Lys80, Lys150, Lys152, Lys158 and Lys165 in α_S2-casein; Lys28, Lys29, Lys32, Lys99, Lys105, Lys107 and Lys113 in β-casein, as identified using liquid chromatography–tandem mass spectrometry. The dissociation of caseins from the casein micelle reduced steric hindrance and made the protein more readily susceptible to hydrolysis by plasmin. However, the formation of succinyl-lysine rendered β-casein unrecognisable to the substrate-binding pocket of plasmin, resulting in a non-linear decrease in level of hydrolysis because of the competitive effect of micelle dissociation.     

Effect of photo-oxidation of major milk proteins on protein structure and hydrolysis by chymosin

Changes in the protein structure of different major milk proteins in response to photo-oxidation and its effect on hydrolysis by chymosin were studied by determination of free amino-terminal groups and by peptide mapping using liquid chromatography coupled with mass spectrometry. Changes were seen in the formation of peptides by chymosin after photo-oxidation of all substrates studied, but the extent of changes varied between the different substrates. Oxidative changes involving tryptophan residues and formation of dityrosines were high in the casein, and conformational changes of both α_S- and β-casein were indicated by changes in the micro-environment of the tryptophan residues after oxidation. A decreased accessibility of chymosin to oxidized caseins was reflected in a lower overall level of free amino-terminal groups, and changed peptide maps. Although β-lactoglobulin and lactoferrin showed marginal conformational changes, a higher level of free amino-terminal groups was observed, and a range of peptides were found to increase for these two proteins.     

Impact of milk processing on the generation of peptides during digestion

Milk processing may induce changes in dairy product composition and influence digestibility and nutrient bioavailability. Differences in protein degradation and peptide generation were studied for β-lactoglobulin and α_S1-casein from commercially available dairy products before, during, and after in vitro digestion. All major milk proteins, except β-lactoglobulin, were degraded to smaller peptides during the gastric phase in all investigated products. After the gastric phase, a shortened fragment of β-lactoglobulin was identified in the non-fermented dairy products, underlining differences in protein conformation due to the fermentation process. During the gastric phase, greater numbers of small peptides were generated from α_S1-casein than from β-lactoglobulin. The monitoring of generation of specific β-lactoglobulin and α_S1-casein peptide profiles by liquid chromatography–mass spectrometry allowed the identification of potential bioactive peptides. Peptides with satiety-influencing DPP-4 inhibiting properties were monitored and quantities were compared between products to identify promising targets for the development of new health promoting products.     

Composition and Structure of Fat Globule Surface Layers in Recombined Milk

Protein coverage, composition and structure of surface layers of fat globules in recombined milk were determined. Average protein load was ～6 mg/m² fat surface. Both casein and whey proteins were present in the fat globule surface layer, with casein adsorbed in preference to whey proteins and α_s (α_sl+α_s2)-casein adsorbed in preference to β-casein. Transmission electron microscopy showed that the surface layer of fat globule was made up of casein micelles, fragments of casein micelles and a thin layer of protein, possibly whey proteins. Experiments with surface layers that had been dispersed in EDTA showed that the extent of dissociation of caseins followed the order: β-casein > α_s-casein ≦ K-casein, suggesting that most of the K-casein was probably associated directly with the fat surface.     

Differences in the peptide profile of raw and pasteurised ovine milk cheese and implications for its bioactive potential

Cheese produced using raw ovine milk (R) or pasteurised ovine milk (P) was subjected to peptide extraction, characterisation by mass spectrometry and bioinformatic analysis with the aim of assessing the impact of milk pasteurisation on the final peptide profile. In total, 187 peptides arising from β-casein, α_S1-casein, and α_S2-casein were identified. Upon label-free quantitation, 58 peptides were found to be significantly different in the two preparations; 38 were more abundant in R and 20 were more abundant in P. Of these, 27 were unique to R and 10 were unique to P. Bioinformatic analysis by EnzymePredictor provided insights into the influence of milk pasteurisation on susceptibility of cheese proteins to proteolytic enzymes during ripening. Finally, BIOPEP analysis predicted a biological activity for 37 of the 187 identified sequences (20%), with a significantly higher abundance of peptides with immunomodulating and ACE-inhibitor properties in R cheeses.     

Whole-genome association study for milk protein composition in dairy cattle

Our objective was to perform a genome-wide association study for content in bovine milk of α_S1-casein (α_S1-CN), α_S2-casein (α_S2-CN), β-casein (β-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), β-lactoglobulin (β-LG), casein index, protein percentage, and protein yield using a 50K single nucleotide polymorphism (SNP) chip. In total, 1,713 Dutch Holstein-Friesian cows were genotyped for 50,228 SNP and a 2-step association study was performed. The first step involved a general linear model and the second step used a mixed model accounting for all family relationships. Associations with milk protein content and composition were detected on 20 bovine autosomes. The main genomic regions associated with milk protein composition or protein percentage were found on chromosomes 5, 6, 11, and 14. The number of chromosomal regions showing significant (false discovery rate <0.01) effects ranged from 3 for β-CN and 3 for β-LG to 12 for α_S2-CN. A genomic region on Bos taurus autosome (BTA) 6 was significantly associated with all 6 major milk proteins, and a genomic region on BTA 11 was significantly associated with the 4 caseins and β-LG. In addition, regions were detected that only showed a significant effect on one of the milk protein fractions: regions on BTA 13 and 22 with effects on α_S1-CN; regions on BTA 1, 9, 10, 17, 19, and 28 with effects on α_S2-CN; a region on BTA 6 with an effect on β-CN; regions on BTA 13 and 21 with effects on κ-CN; regions on BTA 1, 5, 9, 16, 17, and 26 with effects on α-LA; and a region on BTA 24 with an effect on β-LG. The proportion of genetic variance explained by the SNP showing the strongest association in each of these genomic regions ranged from <1% for α_S1-CN on BTA 22 to almost 100% for casein index on BTA 11. Variation associated with regions on BTA 6, 11, and 14 could in large part but not completely be explained by known protein variants of β-CN (BTA 6), κ-CN (BTA 6), and β-LG (BTA 11) or DGAT1 variants (BTA 14). Our results indicate 3 regions with major effects on milk protein composition, in addition to several regions with smaller effects involved in the regulation of milk protein composition.     

Effect of minor milk proteins in chymosin separated whey and casein fractions on cheese yield as determined by proteomics and multivariate data analysis

The objective of this work was to find regressions between minor milk proteins or protein fragments in the casein or sweet whey fraction and cheese yield because the effect of major milk proteins was evaluated in a previous study. Proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry in combination with multivariate data analysis were used to study the effect of variations in milk protein composition in chymosin separated whey and casein fractions on cheese yield. By mass spectrometry, a range of proteins significant for the cheese yield was identified. Among others, a C-terminal fragment of β-casein had a positive effect on the cheese yield expressed as grams of cheese per 100 g of milk, whereas several other minor fragments of β-, α_s1-, and α_s2-casein had positive effects on the transfer of protein from milk to cheese. However, the individual effect of each identified protein was relatively low. Therefore, further studies of the relations between different proteins/peptides in the rennet casein or sweet whey fractions and cheese yield are needed for advanced understanding and prediction of cheese yield.     

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.     

Peptidome comparison following gastrointestinal digesta of bovine versus caprine milk serum

Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), β-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from α_S1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.     

Changes in proteins,physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.     

In vitro Digestibility of Whey Protein/K-Casein Complexes Isolated from Heated Concentrated Milk

The disulfide-linked complex of _K-casein and whey proteins that forms when concentrated milk is heated was isolated by centrifugation and column chromatography on Sephacryl S-1000. The rate of hydrolysis of β-lactoglobulin and _K-casein in the complex and the reduced and carboxymethylated components of the complex were measured by polyacrylamide gel electrophoresis. The rates of hydrolysis at pH 2.0 (pepsin) and pH 8.0 (trypsin and chymotrypsin) were similar for _k-casein in the complex and its reduced form. β-Lactoglobulin hydrolysis was faster for the reduced complex than for the complex which was much faster than for the native protein for all three enzymes. The results suggest that heating milk increases the digestibility of whey proteins, despite the formation of large protein complexes between the whey proteins and _K-casein.     

Short communication: Genetic parameters for milk protein composition predicted using mid-infrared spectroscopy in the French Montbéliarde,Normande, and Holstein dairy cattle breeds

Genetic parameters for the major milk proteins were estimated in the 3 main French dairy cattle breeds (i.e. Montbéliarde, Normande, and Holstein) as part of the PhénoFinlait program. The 6 major milk protein contents as well as the total protein content (PC) were estimated from mid-infrared spectrometry on 133,592 test-day milk samples from 20,434 cows in first lactation. Lactation means, expressed as a percentage of milk (protein contents) or of protein (protein fractions), were analyzed with an animal mixed model including fixed environmental effects (herd, year × month of calving, and spectrometer) and a random genetic effect. Genetic parameter estimates were very consistent across breeds. Heritability estimates (h²) were generally higher for protein fractions than for protein contents. They were moderate to high for α_S1-casein, α_S2-casein, β-casein, κ-casein, and α-lactalbumin (0.25 < h² < 0.72). In each breed, β-lactoglobulin was the most heritable trait (0.61 < h² < 0.86). Genetic correlations (r_g) varied depending on how the percentage was expressed. The PC was strongly positively correlated with protein contents but almost genetically independent from protein fractions. Protein fractions were generally in opposition, except between κ-casein and α-lactalbumin (0.39 < r_g < 0.46) and κ-casein and α_S2-casein (0.36 < r_g < 0.49). Between protein contents, r_g estimates were positive, with highest values found between caseins (0.83 < r_g < 0.98). In the 3 breeds, β-lactoglobulin was negatively correlated with caseins (?0.75 < r_g < ?0.08), in particular with κ-casein (?0.75 < r_g < ?0.55). These results, obtained from a large panel of cows of the 3 main French dairy cattle breeds, show that routinely collected mid-infrared spectra could be used to modify milk protein composition by selection.     

Effects of milk protein variants on the protein composition of bovine milk

The effects of β-lactoglobulin (β-LG), β-casein (β-CN), and κ-CN variants and β-κ-CN haplotypes on the relative concentrations of the major milk proteins α-lactalbumin (α-LA), β-LG, α_S1-CN, α_S2-CN, β-CN, and κ-CN and milk production traits were estimated in the milk of 1,912 Dutch Holstein-Friesian cows. We show that in the Dutch Holstein-Friesian population, the allele frequencies have changed in the past 16 years. In addition, genetic variants and casein haplotypes have a major impact on the protein composition of milk and explain a considerable part of the genetic variation in milk protein composition. The β-LG genotype was associated with the relative concentrations of β-LG (A » B) and of α-LA, α_S1-CN, α_S2-CN, β-CN, and κ-CN (B > A) but not with any milk production trait. The β-CN genotype was associated with the relative concentrations of β-CN and α_S2-CN (A² > A¹) and of α_S1-CN and κ-CN (A¹ > A²) and with protein yield (A² > A¹). The κ-CN genotype was associated with the relative concentrations of κ-CN (B > E > A), α_S2-CN (B > A), α-LA, and α_S1-CN (A > B) and with protein percentage (B > A). Comparing the effects of casein haplotypes with the effects of single casein variants can provide better insight into what really underlies the effect of a variant on protein composition. We conclude that selection for both the β-LG genotype B and the β-κ-CN haplotype A²B will result in cows that produce milk that is more suitable for cheese production.     

Pancreatic hydrolysis of bovine casein: Peptide release and time-dependent reaction behavior

Pancreatic hydrolysis of bovine casein was carried out at pH 8.0 in a batch stirred tank reactor. The resulting peptides were identified by liquid chromatography–tandem mass spectrometry and their release kinetics were tracked. During 24 h of digestion, a total of 297 peptides consisting of 109, 48, 126, and 14 peptides, from α_s1-, α_s2-, β-, and κ-caseins (α_s1-, α_s2-, β-, and κ-CN), respectively, were identified by LC–MS–MS. We found that pancreatin attacked different caseins with varying hydrolysis rates (β-CN > α_s1-CN > α_s2-CN > κ-CN). Based on our analysis of the peptide fragments produced, we determined that pancreatin preferentially cleaved the N-terminal phosphorylated region, the C-terminal hydrophobic region and the middle region of β-casein, the terminal regions of α_s1-casein, and both the terminals and the middle region of α_s2-casein. The hydrolysis of κ-casein occurred at the slowest rate, releasing peptides only after 30 min of hydrolysis.     

Genetic and nongenetic factors contributing to differences in αS-casein phosphorylation isoforms and other major milk proteins

Relative concentrations of α_S-casein (α_S-CN) phosphorylation isoforms vary considerably among milk of individual cows. We aimed to explore to what extent genetic and other factors contribute to the variation in relative concentrations of α_S-CN phosphorylation isoforms and the phosphorylation degree of α_S-CN defined as the proportion of isoforms with higher degrees of phosphorylation. We also investigated the associations of genetic variants of milk proteins and casein haplotypes with relative concentrations of α_S-CN phosphorylation isoforms and with the phosphorylation degree of α_S-CN in French Montbéliarde cattle from the cheese production area of Franche-Comté. Detailed milk protein composition was determined by liquid chromatography coupled with electrospray ionization mass spectrometry from 531 test-day morning milk samples. Parity, lactation stage, and genetic variation of cows contributed to the phenotypic variation in relative concentrations of individual α_S-CN phosphorylation isoforms and in the phosphorylation degree of α_S-CN. As lactation progressed, we observed a significant increase for relative concentrations of α_S-CN isoforms with higher degrees of phosphorylation (α_S1-CN-9P, α_S2-CN-13P, and α_S2-CN-14P) as well as for the phosphorylation degree of both α_S1-CN and α_S2-CN. Furthermore, the β-CN I variant was associated with a greater proportion of isoforms with lower degrees of phosphorylation (α_S1-CN-8P, α_S2-CN-10P, and α_S2-CN-11P); the β-CN B variant was associated with a greater proportion of isoforms with higher degrees of phosphorylation (α_S1-CN-9P, α_S2-CN-12P to α_S2-CN-14P). The heritability estimates were low to moderate for relative concentrations of α_S2-CN phosphorylation isoforms (0.07 to 0.32), high for relative concentrations of α_S1-CN-8P (0.84) and α_S1-CN-9P (0.56), and moderate for phosphorylation degrees of α_S1-CN (0.37) and α_S2-CN (0.23). Future studies investigating relations between the phosphorylation degree of α_S-CN and technological properties of milk will be beneficial for the dairy industry.     

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92–100, 125–135, 125–138, and 149–162) in β-LG, 2 peptides in α-LA (AA 80–93 and 63–79), 2 peptides in α_S1-casein (AA 84–90 and 125–132), and 1 peptide (AA 25–32) in α_S2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.     

Proteins and proteolysis in pre-term and term human milk and possible implications for infant formulae

Understanding the differences between the protein system of human milk and bovine milk is critical in the development of infant formulae. In this study, the proteins of bovine milk and a bovine-based whey-dominant infant formula were compared with those of human milk for infants born prematurely (pre-term) or at full term (term). The protein distribution of infant formula differed significantly from that of either type of human milk. A proteomic comparison between pre-term and term human milk showed a reduction of levels of β-casein and α_s-casein and appearance of additional products, corresponding to low molecular weight hydrolysis products of the caseins, in pre-term milk. Pre-term milk samples also had higher total nitrogen concentration and plasmin activity, consistent with the proteomic data. These results suggest the operation of a physiological mechanism that may adjust enzyme and/or protein expression to modify protein digestibility, and may facilitate design of infant formulae, closer to maternal milk, particularly for premature infants.     

Milk nutrition and childhood epilepsy: An ex vivo study on cytokines and oxidative stress in response to milk protein fractions

We present a pilot study on the effects of milk protein fractions [α_S1-casein (CN), α_S2-CN, κ-CN, β-CN, and a mix of α-lactalbumin (α-LA) and β-lactoglobulin (β-LG)] from different animal species (bovine, ovine, and caprine) on pro- and anti-inflammatory cytokines and oxidative status in cultured peripheral blood mononuclear cells from children with generalized epilepsy. Peripheral blood mononuclear cells (PBMC) were obtained by density gradient from blood of 10 children with generalized epilepsy (5 males; mean age 33.6 ± 5.4 mo) and 10 controls (5 males; mean age 35.6 ± 6.8 mo). Children with epilepsy were grouped according to cytokine levels as follows: children with epilepsy having low levels of cytokines not different from those of control children (LL-EC); children with epilepsy having cytokine levels at least 5-fold higher (medium levels) than those of control children (ML-EC); and children with epilepsy having cytokine levels at least 10-fold higher (high levels) than those of control children (HL-EC). The production of tumor necrosis factor-α (TNF-α), IL-10, IL-6, and IL-1β was studied in cultured PBMC incubated with α_S1-CN, α_S2-CN, κ-CN, β-CN, and a mix of α-LA and β-LG from bovine, caprine, and ovine milks. The levels of reactive oxygen and nitrogen species (ROS/RNS) and catalase activity were assessed in cultured supernatant. In the HL-EC group, β-CN from small ruminant species (ovine and caprine) induced the highest levels of TNF-α, whereas PBMC incubated with α_S2-CN from ovine milk and the mix of β-LG and α-LA from all tested milk species had the lowest levels of TNF-α. Within the HL-EC group, production of IL-1β was higher for bovine and ovine α_S2-CN fractions and lower for caprine and ovine β-CN and κ-CN. In the HL-EC group, IL-6 was higher in cultured PBMC incubated with α_S2-CN from bovine and ovine milk than from caprine milk. The cytokine IL-10 did not differ among milking species. The highest levels of ROS/RNS were found after incubation of PBMC with the β-CN fraction in bovine milk. Catalase activity was higher in PBMC cultured with β-CN isolated from bovine and caprine milk and with α_S1-CN from ovine milk.     

Effects of genetic type,stage of lactation,and ripening time on Caciocavallo cheese proteolysis

The objective of this experiment was to evaluate the effects of genetic type, stage of lactation, and ripening time on proteolysis in Caciocavallo cheese. One hundred twenty Caciocavallo cheeses made from the milk of 2 breeds, Italian Brown and Italian Holstein and characterized by different stages of lactation were obtained and ripened for 1, 30, 60, 90, and 150 d. Cheese proteolysis was investigated by ripening index (ratio of water-soluble N at pH 4.6 to total protein, %) and by the study of degradation of the protein fractions (α_S1-, β-, and para-κ-casein), which was determined by densitometric analysis of isoelectric focusing results. The statistical analysis showed a significant effect of the studied factors. Ripening index was higher in Italian Brown Caciocavallo cheese and in cheeses made with early lactation milk, whereas casein solubilization was greater in the first 2 mo of ripening. Isoelectric focusing analysis of cheese samples during ripening showed extensive hydrolysis of caseins. In particular, the protein fraction that underwent major degradation by proteolytic enzymes was α_S1-casein, followed by β-casein, whereas para-κ-casein was less degraded. Italian Brown cheese showed a lower residual quantity of β- and para-κ-casein, whereas Italian Holstein cheese showed a lower residual quantity of α_S1-casein. In addition, significant interactions of both first and second order were found on both ripening index and degradation of protein fractions. This study demonstrated that the analyzed factors influenced proteolysis of Caciocavallo cheese, which forms the basis of new knowledge that could lead to the production of a pasta filata cheese with specific characteristics.