Effect of ripening time on peptide dynamics and bioactive peptide composition in Tulum cheese

Skin bag Tulum cheeses traditionally produced in the Central Taurus region of Turkey were studied to identify peptide profiles by liquid chromatography–tandem mass spectrometry over 180 d of ripening. After mass spectrometry analysis, 203 peptides were identified: 59 from α_S1-casein (CN), 11 from α_S2-CN, 129 from β-CN, and 4 from κ-CN. Numbers of α_S1- and β-CN-derived peptides increased with increasing number of ripening days due to the dependence of newly formed peptides on proteolysis. However, similar increases were not observed for α_S2- and κ-CN-derived peptides. Most identified peptides consisted of β-CN-derived peptides, followed by α_S1-, α_S2-, and κ-CN-derived peptides. Among these, bioactive peptides were found, including antihypertensive, antibacterial, antioxidant, dipeptidyl peptidase-4 inhibitory, metal chelating, skin regenerating, glucagon-like peptide-1 secretion enhancing, opioid, cathepsin B inhibitory, prolyl endopeptidase inhibitory, immunomodulatory, brain function improving, antiamnesic, antihypercholesterolemic, anti-inflammatory, and anticarcinogenic peptides.     

Ripening of extra-hard cheese made with mesophilic DL-starter

Extra hard cheese is commonly made with thermophilic starters using high temperatures to stimulate expulsion of whey. In this work, microflora, proteolysis and volatiles were investigated in an extra-hard cheese made with mesophilic DL-starter, produced using challenging cooking temperatures for the starter bacteria over several hours. Cheese from six commercially produced vats was investigated over 56 weeks. The number of starter bacteria decreased after three weeks of ripening. Casein breakdown was characterised by chymosin and plasmin activity on α_s1- and β-caseins, respectively. Peptide profiles showed accumulation of Lactococcus derived peptides from α_s1-CN f1–23, and the peptide β-CN 29–93 as a result of joint plasmin and chymosin activity and absence of highly proteolytic thermophilic Lactobacillus, commonly present in extra-hard cheese. The composition of amino acids depended mainly on starter during the first 26 weeks of ripening. The content of volatiles depended both on ripening time and the starter used.     

Qualitative and Quantitative Determination of Caseins with Reverse-Phase and Anion-Exchange HPLC

Whole native caseinate (WNC) and casein (CN) fractions from preparative DE-52 cellulose urea columns were chromatographed using C-8 reverse-phase (RP) and DEAE-type anion-exchange (AEx) HPLC systems. With RP, α_S2-CN and κ-CN eluted first as several small peaks; α_S1-CN eluted later as two peaks, followed by β-CN peaks. With AEx, κ-CN eluted early as a group of peaks, β-CN eluted next, and α_S1-CN and α_S2-CN coeluted last. Standard curves were prepared for α_S1-CN and β-CN using RP-PHLC and showed correlation coefficients of 0.99 and 0.98, respectively. The caseins in WNC, nonfat dry milk casein, commercial casein(ates) and caseins from milks of individual cows were determined.     

Lipolysis and proteolysis profiles of fresh artisanal goat cheese made with raw milk with 3 different fat contents

The objective of this study was to describe the proteolysis and lipolysis profiles in goat cheese made in the Canary Islands (Spain) using raw milk with 3 different fat contents (0.5, 1.5, and 5%) and ripened for 1, 7, 14, and 28 d. β-Casein was the most abundant protein in all cheeses and at all ripening times. Quantitative analysis showed a general decrease in caseins as ripening progressed, and degradation rates were higher for α_S1-casein than for β-casein and α_S2-casein. Furthermore, the degradation rate during the experimental time decreased with lower fat contents. The α_S2-casein and α_S1-casein levels that remained in full-fat and reduced-fat cheeses were less than those in low-fat cheese. In contrast, β-casein also showed degradation along with ripening, but differences in degradation among the 3 cheese types were not significant at 28 d. The degradation products increased with the ripening time in all cheeses, but they were higher in full-fat cheese than in reduced-fat and low-fat cheeses. The free fatty acid concentration per 100 g of cheese was higher in full-fat cheese than in reduced- and low-fat cheese; however, when the results were expressed as milligrams of free fatty acids per gram of fat in cheese, then lipolysis occurred more rapidly in low-fat cheese than in reduced- and full-fat cheeses. These results may explain the atypical texture and off-flavors found in low-fat goat cheeses, likely the main causes of non-acceptance.     

Identification of casein peptides in plasma of subjects after a cheese-enriched diet

Despite several studies support the functionality of casein peptides in vitro, a gap of knowledge still exist about their bioavailability.In this pilot study the bioavailability of phosphopeptides (CPPs) was investigated in four healthy subjects who consumed for one week 100 g/day of Parmigiano Reggiano cheese after one week of a dairy products-free diet. CPPs were detected in plasma samples from fasting subjects after the cheese-enriched diet and peptides were detected variously in almost all the 4 samples. Some α_s1- and α_s2-CN-derived CPPs, such as α_s1-CN (f43–52 and f43–50) and α_s2-CN (f8–12), (f7–12) and (f6–12) as well as four non-phosphorylated peptides belonging to the C-terminal end of β-CN (f193–209, f194–209, f200–209) were detected in plasma samples submitted to extraction and enrichment by hydroxyapatite (HA) chromatography followed by MALDI-TOF and nano LC-ESI/MS/MS analysis.Data indicated that casein oligopeptides are bioavailable after a continued intake of cheese. Future studies are warranted to ascertain this finding on a wider population and to clarify the mechanisms behind CPPs bioaccessibility and absorption.     

Phosptiopeptides from Comté Cheese: Nature and Origin

Thirteen low-molecular-weight phosphopeptides were isolated from the water-soluble fraction of Comté cheese. The sample was fractionated and purified by gel permeation chromatography and reverse-phase HPLC. The peptide sequences were identified by Edman degradation and primary molecular structure was confirmed by mass spectrometry. The different peptides purified correspond to fragments of the sequence Val13-Lys 28 of β-casein and of the sequence Glu S-Lys 21 of α₃₂ casein. These fragments probably originated from an initial proteolysis of the two caseins by plasmin, followed by further endopeptidase aminopeptidase and, possibly, carboxypeptidase digestions. Partial dephosphorylation of some β-casein fragments was observed. These peptides probably influence the flavor profile of comté cheese.     

Chymosin-mediated proteolysis, calcium solubilization, and texture development during the ripening of cheddar cheese

Full fat, milled-curd Cheddar cheeses (2 kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0 μmol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8°C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0 μmol/L of pepstatin showed complete inhibition of hydrolysis of α_S1-casein (CN) at Phe₂₃-Phe₂₄ at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact α_S1-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of α_S1-CN at Phe₂₃-Phe₂₄ is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.     

Milk-clotting properties on bovine caseins of a novel cysteine peptidase from germinated Moringa oleifera seeds

A cysteine peptidase was previously identified from germinated Moringa oleifera seeds, but its milk-clotting properties on bovine caseins was still unclear. In this study, this novel cysteine peptidase (MoCP) showed preferential activity on κ-casein (κ-CN), with greater hydrolytic activity compared with calf rennet, whereas weak hydrolysis of α-casein and β-casein made MoCP suitable for application in cheesemaking and may yield various functional peptides. All 3 evaluated caseins were hydrolyzed to form relatively stable peptide bands within 3 h of proteolysis with MoCP. Cleavage sites were determined by gel electrophoresis, liquid chromatography mass spectrometry/mass spectrometry, and peptide sequencing, which revealed that cleavage of κ-CN by MoCP occurred at residue Ile129-Pro130 and generated a 14,895.37-Da peptide. The flocculation reaction between MoCP and κ-CN determined by 3-dimensional microscopy with super-depth of field revealed that the initial 30 min of reaction were key for milk coagulation, which may affect curd yield. Overall, the findings presented herein suggest that the cysteine peptidase from germinated M. oleifera seeds can be considered a promising plant-derived rennet alternative for use in cheese manufacture.     

Edam牦牛干酪成熟过程中品质变化及蛋白质降解

为研究Edam牦牛半硬质干酪成熟机理，分别测定成熟0、20、40、60、80 d Edam牦牛干酪的感官、理化、物性、蛋白质和脂肪分解指标，采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和傅里叶变换红外光谱等方法研究酪蛋白降解情况，通过Pearson相关性分析成熟时间与各指标间的相关性。结果表明：随着成熟时间延长，感官评分先下降后上升；水分含量、pH值下降；亮度值（L*）下降，红度值（a*）和黄度值（b*）值上升；硬度、弹性、胶黏性均上升，凝聚性逐渐下降；储能模量和损耗模量升高，损耗角正切值始终小于1；成熟时间与总氮含量、pH 4.6和12%三氯乙酸条件下干酪中可溶性氮含量等呈极显著正相关（P<0.01）；脂肪含量先升高后降低，游离脂肪酸含量和硫代巴比妥酸值逐渐增加。酪蛋白（casein,CN）降解研究结果表明：αs1-CN、αs2-CN、β-CN及κ-CN均随成熟时间延长而不断降解，成熟80 d时大分子蛋白降解明显；成熟过程中β-折叠、α-螺旋逐渐向无规卷曲转化；成熟时间与羰基含量、表面疏水性呈极显著正相关（P<0.01），与总巯基含量呈显著负相关（P<0.05）。     

地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响

利用地衣芽孢杆菌凝乳酶制作切达干酪和切达干酪类似物,分析干酪成熟过程中各蛋白水解指标的变化规律,以揭示地衣芽孢杆菌凝乳酶对切达干酪成熟过程中蛋白水解的影响。结果表明,CDF组（添加地衣芽孢杆菌D3.11凝乳酶所制切达干酪）、CD3组（添加地衣芽孢杆菌D3.11凝乳酶但未添加发酵剂制成的干酪类似物）和CCF组（添加商品凝乳酶所制切达干酪）干酪蛋白含量、pH 4.6-可溶性氮、12%三氯乙酸-可溶性氮、5%磷钨酸-可溶性氮、总游离氨基酸含量均随着成熟时间延长呈显著增加趋势,并且成熟期间CDF组干酪均显著高于CCF组干酪（P＜0.05）;十二烷基硫酸钠-聚丙烯酰氨凝胶电泳分析表明,CDF组干酪α-酪蛋白水解程度较大;pH 4.6-可溶性肽段分析表明,随着干酪的成熟,总肽含量呈先增加后下降趋势,但疏水性肽与亲水性肽的比值呈持续下降趋势,在成熟第6个月时,CDF组、CD3组和CCF组干酪疏水性肽与亲水性肽比值分别为2.668、2.822、3.788。主成分分析表明,3 组干酪的蛋白水解程度与成熟度呈正相关,与疏水性肽和亲水性肽的比值呈负相关。以上结果表明,利用地衣芽孢杆菌凝乳酶制作的干酪蛋白水解度更高,但其疏水性肽比例较小,研究结果可为地衣芽孢杆菌凝乳酶在干酪生产中的应用提供理论依据。     

Structural equation modeling for unraveling the multivariate genomic architecture of milk proteins in dairy cattle

The aims of this study were to investigate potential functional relationships among milk protein fractions in dairy cattle and to carry out a structural equation model (SEM) GWAS to provide a decomposition of total SNP effects into direct effects and effects mediated by traits that are upstream in a phenotypic network. To achieve these aims, we first fitted a mixed Bayesian multitrait genomic model to infer the genomic correlations among 6 milk nitrogen fractions [4 caseins (CN), namely κ-, β-, α_S1-, and α_S2-CN, and 2 whey proteins, namely β-lactoglobulin (β-LG) and α-lactalbumin (α-LA)], in a population of 989 Italian Brown Swiss cows. Animals were genotyped with the Illumina BovineSNP50 Bead Chip v.2 (Illumina Inc.). A Bayesian network approach using the max-min hill-climbing (MMHC) algorithm was implemented to model the dependencies or independence among traits. Strong and negative genomic correlations were found between β-CN and α_S1-CN (?0.706) and between β-CN and κ-CN (?0.735). The application of the MMHC algorithm revealed that κ-CN and β-CN seemed to directly or indirectly influence all other milk protein fractions. By integrating multitrait model GWAS and SEM-GWAS, we identified a total of 127 significant SNP for κ-CN, 89 SNP for β-CN, 30 SNP for α_S1-CN, and 14 SNP for α_S2-CN (mostly shared among CN and located on Bos taurus autosome 6) and 15 SNP for β-LG (mostly located on Bos taurus autosome 11), whereas no SNP passed the significance threshold for α-LA. For the significant SNP, we assessed and quantified the contribution of direct and indirect paths to total marker effect. Pathway analyses confirmed that common regulatory mechanisms (e.g., energy metabolism and hormonal and neural signals) are involved in the control of milk protein synthesis and metabolism. The information acquired might be leveraged for setting up optimal management and selection strategies aimed at improving milk quality and technological characteristics in dairy cattle.     

Antibacterial activities of peptides from the water-soluble extracts of Italian cheese varieties

Water-soluble extracts of 9 Italian cheese varieties that differed mainly for type of cheese milk, starter, technology, and time of ripening were fractionated by reversed-phase fast protein liquid chromatography, and the antimicrobial activity of each fraction was first assayed toward Lactobacillus sakei A15 by well-diffusion assay. Active fractions were further analyzed by HPLC coupled to electrospray ionization-ion trap mass spectrometry, and peptide sequences were identified by comparison with a proteomic database. Parmigiano Reggiano, Fossa, and Gorgonzola water-soluble extracts did not show antibacterial peptides. Fractions of Pecorino Romano, Canestrato Pugliese, Crescenza, and Caprino del Piemonte contained a mixture of peptides with a high degree of homology. Pasta filata cheeses (Caciocavallo and Mozzarella) also had antibacterial peptides. Peptides showed high levels of homology with N-terminal, C-terminal, or whole fragments of well known antimicrobial or multifunctional peptides reported in the literature: α_S1-casokinin (e.g., sheep α_S1-casein (CN) f22-30 of Pecorino Romano and cow α_S1-CN f24-33 of Canestrato Pugliese); isracidin (e.g., sheep α_S1-CN f10-21 of Pecorino Romano); kappacin and casoplatelin (e.g., cow κ-CN f106-115 of Canestrato Pugliese and Crescenza); and β-casomorphin-11 (e.g., goat β-CN f60-68 of Caprino del Piemonte). As shown by the broth microdilution technique, most of the water-soluble fractions had a large spectrum of inhibition (minimal inhibitory concentration of 20 to 200 μg/mL) toward gram-positive and gram-negative bacterial species, including potentially pathogenic bacteria of clinical interest. Cheeses manufactured from different types of cheese milk (cow, sheep, and goat) have the potential to generate similar peptides with antimicrobial activity.     

A Survey on the Some Chemical and Biochemical Properties of Civil Cheese,a Traditional Turkish Cheese

In this article, 15 randomly selected samples of Civil cheese, were purchased from different retail markets in the Erzurum province, Turkey and were investigated for some chemical and biochemical analyses. All cheese samples were analyzed for dry matter, fat, salt, ash, titrable acidity, total nitrogen, soluble nitrogen, ripening index, α_s-and β-casein degradation, γ-casein, and peptides. Dry matter, fat, fat in dry matter, salt, salt in dry matter, ash, and acidity values in samples analyzed were found to be as found between 31.33 and 40.12 g/100 g cheese; 1.00 and 7.00 g/100 g cheese; 2.49 and 18.98 g/100 g cheese; 0.11 and 0.34 g/100 g cheese; 0.27 and 1.04 g/100 g cheese; 1.42 and 5.14 g/100 g cheese and, 0.63 and 2.16%, respectively. TN, WSN/TN, TCA-SN/TN, and PTA-SN/ TN values, expressed as TN%, were found between 3.01 and 5.57 g/100 g cheese, 4.25 and 8.80 g/100 g cheese, 3.23 and 6.12 g/100 g cheese, 1.03, and 5.53 g/100 g cheese in Civil cheese samples analyzed, respectively. SDS-PAGE showed that both α_s-CN and β-CN ratios were not high compared with similar cheeses, and are not completely hydrolyzed in all Civil cheese samples. A broad range of values from chemical and biochemical analysis indicated that Civil cheeses collected from retail markets lacked standardization. Consequently, it was decided that Civil cheese samples do not undergo an excessive proteolysis.     

Reconstituted skim milk concentration affects partitioning of some individual caseins between rennet curd and whey

Milk composition is a major factor determining cheesemaking efficiency where caseins (CNs) play key roles, but little is known about individual partitioning during coagulation. This study evaluated the impact of reconstituted skim milk concentration (8%–25% solids) on the partitioning of individual CNs between rennet curd and whey produced with or without slight acidification. α_s2-CN and intact κ-CN fractions partitioned in rennet whey were less than 10% and decreased with rise in milk concentration, whereas α_s1-CN and β-CN appeared 100% retained into the curds. Loss of some α_s2- and κ-CN into rennet whey is attributed to their soluble complexes with whey proteins.     

Proteolysis and biogenic amine buildup in high-pressure treated ovine milk blue-veined cheese

Penicillium roqueforti plays an important role in the ripening of blue-veined cheeses, mostly due to lactic acid consumption and to its extracellular enzymes. The strong activity of P. roqueforti proteinases may bring about cheese over-ripening. Also, free amino acids at high concentrations serve as substrates for biogenic amine formation. Both facts result in shorter product shelf-life. To prevent over-ripening and buildup of biogenic amines, blue-veined cheeses made from pasteurized ovine milk were high-pressure treated at 400 or 600 MPa after 3, 6, or 9 wk of ripening. Primary and secondary proteolysis, biogenic amines, and sensory characteristics of pressurized and control cheeses were monitored for a 90-d ripening period, followed by a 270-d refrigerated storage period. On d 90, treatments at 400 MPa had lowered counts of lactic acid bacteria and P. roqueforti by less than 2 log units, whereas treatments at 600 MPa had reduced lactic acid bacteria counts by more than 4 log units and P. roqueforti counts by more than 6 log units. No residual α-casein (CN) or κ-CN were detected in control cheese on d 90. Concentrations of β-CN, para-κ-CN, and γ-CN were generally higher in 600 MPa cheeses than in the rest. From d 90 onwards, hydrophilic peptides were at similar levels in pressurized and control cheeses, but hydrophobic peptides and the hydrophobic-to-hydrophilic peptide ratio were at higher levels in pressurized cheeses than in control cheese. Aminopeptidase activity, overall proteolysis, and free amino acid contents were generally higher in control cheese than in pressurized cheeses, particularly if treated at 600 MPa. Tyramine concentration was lower in pressurized cheeses, but tryptamine, phenylethylamine, and putrescine contents were higher in some of the pressurized cheeses than in control cheese. Differences in sensory characteristics between pressurized and control cheeses were generally negligible, with the only exception of treatment at high pressure level (600 MPa) at an early ripening stage (3 wk), which affected biochemical changes and sensory characteristics.     

Comparison of milk protein composition and rennet coagulation properties in native Swedish dairy cow breeds and high-yielding Swedish Red cows

Recent studies have reported a very high frequency of noncoagulating milk in Swedish Red cows. The underlying factors are not fully understood. In this study, we explored rennet-induced coagulation properties and relative protein profiles in milk from native Swedish Mountain and Swedish Red Polled cows and compared them with a subset of noncoagulating (NC) and well-coagulating (WC) milk samples from modern Swedish Red cows. The native breeds displayed a very low prevalence of NC milk and superior milk coagulation properties compared with Swedish Red cows. The predominant variants in both native breeds were α_S1-casein (α_S1-CN) B, β-CN A² and β-lactoglobulin (β-LG) B. For κ-CN, the B variant was predominant in the Swedish Mountain cows, whereas the A variant was the most frequent in the Swedish Red Polled. The native breeds displayed similar protein composition, but varied in content of α_S1-CN with 9 phosphorylated serines (9P) form. Within the Swedish Mountain cows, we observed a strong inverse correlation between the relative concentration of κ-CN and micelle size and a positive correlation between ionic calcium and gel firmness. For comparison, we investigated a subset of 29 NC and 28 WC milk samples, representing the extremes with regard to coagulation properties based on an initial screening of 395 Swedish Red cows. In Swedish Red, NC milk properties were found to be related to higher frequencies of β-CN A², κ-CN E and A variants, as well as β-LG B, and the predominant composite genotype of β- and κ-CN in the NC group was A²A²/AA. Generally, the A²A²/AA composite genotype was related to lower relative concentrations of κ-CN isoforms and higher relative concentrations of α_S1-, α_S2-, and β-CN. Compared with the group of WC milk samples, NC milk contained a higher fraction of α_S2-CN and α-lactalbumin (α-LA) but a lower fraction of α_S1-CN 9P. In conclusion, milk from native Swedish breeds has good characteristics for cheese milk, which could be exploited in niche dairy products. In milk from Swedish Mountain cows, levels of ionic calcium seemed to be more important for rennet-induced gel firmness than variation in the relative protein profile. In Swedish Red, lower protein content as well as higher fraction of α_S2-CN and lower fraction of α_S1-CN 9P were related to NC milk. Further, a decrease in the frequency of the composite β-κ-CN genotype A²A²/AA through selective breeding could have a positive effect on milk coagulation properties.     

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.     

In vitro gastrointestinal digestion of bovine αS1- and αS2-casein variants gives rise to different IgE-binding epitopes

The occurrence and differences of resistant regions containing IgE-binding epitopes of α_S1-casein (α_S1-CN) variants B and C, as well as α_S2-CN A and B, after in vitro gastrointestinal digestion was investigated using mass spectrometry. The amino acid substitutions characterising the genetic variants affected the peptide pattern arising from the caseins and thus modifications in their allergenic epitopes occurred. Peptides f174–193 in α_S1-CN B and f179–198 in α_S1-CN C correspond to the IgE-binding epitope f173–194, which has been reported as one of the major epitopes in α_S1-CN B. Within α_S2-CN, the two variant-specific peptides, f7–29 from variant A and f1–22 from variant B, contain the previously identified IgE-binding epitope f1–20. These peptides, and in consequence the protein variants, may exhibit different immunoreactions, which could be significant in the production of milk with improved nutritional properties, such as hypoallergenic quality, by selection and breeding of cows with particular milk protein genotypes.     

Occurrence,origin and fate of pyroglutamyl-γ3-casein in cheese

During plasmin-catalysed proteolysis of β-casein (β-CN) in cheese, pyroglutamyl-γ₃-casein (pγ₃-CN), originates from the cyclisation of the N-terminal Glu of γ₃-CN (β-CN f108–209). This peptide progressively increased in content during ripening of Grana Padano and Parmigiano-Reggiano Protected Designation of Origin cheeses. A preliminary survey revealed pγ₃-CN in both hard and extra-hard cheese varieties; in semi-hard cheeses it was present only in mature samples. To understand the mechanisms of pγ₃-CN formation/degradation, in vitro trials (real cheese, model cheeses and buffered γ₃-CN solutions) were performed. Both the effect of cheese pH and the capacity of certain microorganisms to cyclise the N-terminal Glu residue of γ₃-CN were studied. The results suggested that pγ₃-CN formed spontaneously during cheese ripening, mainly at the acid pH characteristic of hard and extra-hard cheeses; the role of cyclase enzymes was negligible. Differences in pγ₃-CN degradation were observed due to the proteolytic activity of bacteria involved in the cheese ripening.     

Evolution of chemico-physical characteristics during manufacture and ripening of Castelmagno PDO cheese in wintertime

Biochemical, volatile and textural profiles during manufacture and ripening were determined in samples of Castelmagno PDO cheese obtained from three different batches in the main artisan cheese plant of Castelmagno PDO production area. At the end of manufacture, samples were characterised by a pH of 6.57% and 52.4% moisture content. The HPLC analysis of organic acids and sugars showed the exhaustion of lactose content, while Urea-PAGE indicated extensive primary proteolysis of both β-casein and α_s1-casein. During ripening, cheeses were characterised by high degradation of β-casein and α_s1-casein, due to bacterial action. RP-HPLC profiles showed a high production of peptides eluted between 20 and 30 min. In total, 92 volatile compounds were identified in cheese headspace. Texture profiles showed an increase in hardness, gumminess, chewiness and adhesiveness values, as well as a decrease in cohesiveness during ripening.