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.     

Proteolysis texture and microstructure of low‐fat Tulum cheese affected by exopolysaccharide‐producing cultures during ripening

The objective of the study was to determine the effects of exopolysaccharide (EPS)‐producing or non‐EPS‐producing starters on proteolysis, physical and microstructural characteristics of full‐fat or low‐fat Tulum cheeses during ripening. For this purpose, Tulum cheese was manufactured using full‐ or low‐fat milk with EPS‐producing and non‐EPS‐producing starter cultures. Chemical composition, proteolysis, texture profiles and microstructure of the cheeses were studied during 90 days of ripening. Urea‐PAGE of water‐insoluble and RP‐HPLC peptide profiles of water‐soluble fractions of the cheeses showed that the use of starters resulted in different degradation patterns in all cheeses during ripening. Although β‐casein exhibited similar degradation patterns in all cheeses, small differences are present in α_s1‐casein degradation during ripening. Reducing fat in Tulum cheese changed the RP‐HPLC peptide profile of the cheeses. The use of EPS‐producing cultures improved the textural characteristics and changed the microstructure and proteolysis of low‐fat Tulum cheese.     

TORSION ANALYSIS OF FRESH AND AGED CHEESES1

Six cheese varieties encompassing a wide compositional and age range were analyzed fresh and after aging to determine correlations between casein proteolysis products, fat content, moisture content, and torsion results. Shear stress, a measure of the strength of the casein matrix, was dependent on the amount of intact α_sl‐casein. Shear strain, a measure of the cohesiveness of a structure, exhibited a negative correlation with fat content and a positive correlation with moisture content. Shear strain was also correlated with the ratio of moisture to protein, an indication of the interaction between casein particles. Torsion tests provide information on the level of degradation of the casein network in cheese, which can be used by manufacturers to alter storage conditions to optimize the quality of their aged cheese., April 10, 2003     

Proteolytic properties of Turkish white‐brined cheese (Beyaz peynir) made by using wild‐type Lactococcal strains

The development of proteolysis in white‐brined Turkish cheese made by using wild strains of Lactococcus lactis subsp. lactis (namely MBLL9, MBLL23 and MBL27) was monitored for 90 days. Proteolysis in cheeses was investigated using urea‐PAGE gel electrophoresis of pH 4.6‐insoluble and RP‐HPLC of both 70% ethanol‐insoluble and 70% ethanol‐soluble nitrogen fractions. Results indicated that developments of proteolysis in the experimental cheeses were strain dependent. The degradation of casein fractions was more evident in the cheeses made using strain MBLL23. The lowest levels of proteolysis and development of acidity were obtained in the cheese made using strain MBLL9.     

A Survey of the Peptide Profile in Prato Cheese as Measured by MALDI‐MS and Capillary Electrophoresis

In this study, we describe the characterization of the peptide profile in commercial Prato cheese by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and capillary electrophoresis (CE). Ten commercial Prato cheese brands were characterized via their physicochemical composition and subjected to fractionation according to solubility at pH 4.6. The pH 4.6 insoluble fraction was evaluated by CE, whereas MALDI‐MS was applied to the fraction soluble at pH 4.6 and in 70% ethanol. CE revealed a characteristic pattern of hydrolysis, with formation of para‐κ‐casein, hydrolysis of α_s1‐casein at the Phe₂₃ ‐ Phe₂₄ bond, and hydrolysis of β‐casein. For the MALDI‐MS data, a complex peptide profile was observed, with the identification of 44 peptides previously reported (24 peptides from α_s1‐casein, 14 from β‐casein, 3 from κ‐casein, and 3 from α_s2‐casein). It was also observed that cheeses with salt‐in‐moisture content greater than 5% showed an accumulation of a bitter‐tasting peptide (m/z 1536, α_s1‐CN f1‐13), suggesting a relationship between the higher salt concentration and the abundance of this peptide. In conclusion, the results showed that even commercial cheeses produced with different raw material and processing conditions showed very similar peptide profiles when assessed at the molecular level, and only 9 peptides were responsible for discrimination of cheeses.     

Characteristics of Miniature Cheddar‐Type Cheese Made by Microbial Rennet from Bacillus amyloliquefaciens: A Comparison with Commercial Calf Rennet

Miniature Cheddar‐type cheeses were produced using microbial rennet from Bacillus amyloliquefaciens (milk‐clotting enzyme [MCE]) or calf rennet (CAR). With the exception of pH, there were no significant differences in gross composition between MCE‐cheese (MCE‐C) and CAR‐cheese (CAR‐C). The pH value of CAR‐C was significantly higher than that of MCE‐C at 40 and 60 d of ripening. The total nitrogen content of the pH 4.6‐soluble fraction obtained from MCE‐C was higher than that obtained from CAR‐C. However, nitrogen content of the 12% TCA‐soluble fraction was similar between CAR‐C and MCE‐C. The extent of α_s1‐casein and β‐casein hydrolysis, measured by urea‐PAGE, was similar in both cheese samples. The hydrolysis of β‐casein was lower than that of α_s1‐casein. Different reverse phase‐high‐performance liquid chromatography peptide profiles of ethanol‐soluble and ethanol‐insoluble fractions were obtained from CAR‐C and MCE‐C. The peptide content in the 2 cheese samples increased throughout ripening; the ratio of hydrophobic to hydrophilic peptides was lower in MCE‐C than in CAR‐C. Compared with CAR‐C, MCE‐C was softer as a result of higher protein hydrolysis. Microbial rennet from B. amyloliquefaciens contributed to higher proteolytic rates, which reduced ripening time.     

Casein Degradation of Fynbo Cheese Salted with NaCl/KCl Brine and Ripened at Various Temperatures

ABSTRACT: Effect of temperature and salt substitution on casein degradation of Fynbo cheese was studied. Fynbo cheeses, salted in solutions of 190 g NaCl/L and of 100 g NaCl/L and 100 g KCl/L and ripened at 5, 12, and 16 °C, were sampled at 1, 5, 10, 20, 30, 60, and 90 d of ripening, at central and external zones. Samples were analyzed for moisture and chloride contents, maturation index, and casein degradation by urea‐polyacrylamide gel electrophoresis. NaCl replacement by KCl did not affect any of the parameters studied. Total salt concentration and ripening temperature affected proteolysis significantly. First‐order kinetics constants for α_s1‐casein degradation were in the range of 0.002 to 0.016 day^‐1 and the activation energy of the reaction was approximately 26 kcal/gmol.     

Characterisation of the casein fraction of Ibérico cheese by electrophoretic techniques

The physicochemical characteristics of Ibérico cheese, a semi‐hard Spanish variety manufactured from mixtures of cow's, ewe's and goat's milk, were studied. The casein fraction and breakdown products of 6‐month‐old cheeses were characterised by various electrophoretic techniques: urea polyacrylamide gel electrophoresis (urea‐PAGE) at alkaline pH, isoelectric focusing (IEF) and two‐dimensional polyacrylamide gel electrophoresis (2DE). Proteins were separated in 2DE according to their charge/mass ratio by urea‐PAGE at alkaline pH in the first dimension and according to their isoelectric point by IEF in the second dimension. Some individual bands considered homogeneous by urea‐PAGE at alkaline pH (ie different grades of phosphorylation of α_s1‐casein and α_s2‐casein) or by IEF (ie overlapping of several bands of α_s2‐casein with γ‐casein bands) were found to be complex mixtures of casein components by 2DE. The two‐dimensional electrophoretic pattern was characteristic of the milk of each animal species included in Ibérico cheese. Capillary electrophoresis (CE) was also used to study the Ibérico cheeses. The high resolution of this technique allowed the identification of the main caseins of the different species (ie para‐κ‐casein, β‐casein, γ₂‐casein and γ₃‐casein). © 2002 Society of Chemical Industry     

Viability of the Lactobacillus rhamnosus HN001 Probiotic Strain in Swiss‐ and Dutch‐Type Cheese and Cheese‐Like Products

The objective of this study was to determine the viability of the probiotic Lactobacillus rhamnosus HN001 in Swiss‐type and Dutch‐type cheese and cheese‐like products (milk fat is substituted by stearin fraction of palm fat) during manufacture, ripening, and storage. The use of the probiotic L. rhamnosus HN001 in Dutch‐type cheese and cheese‐like products significantly (P = 0.1) changed their chemical composition (protein and fat content) and an insignificant increase (approximately 1.6% in cheese‐like products and approximately 0.3% in cheese) in yield. L. rhamnosus HN001 did not affect the rate of changes in the pH of ripened cheese and cheese‐like products. A minor increase in probiotic counts was observed in initial stages of production and were partially removed with whey. Ripened cheese and cheese‐like products were characterized by high survival rates of probiotic bacteria which exceeded 8 log CFU/g after ripening. An insignificant reduction in the number of viable probiotic cells was noted during storage of Swiss‐type and Dutch‐type cheese, whereas a significant increase in probiotic cell counts was observed in cheese‐like products during storage.     

Kinetics of Casein Degradation during Ripening of Fynbo Cheese Salted with NaCl/KCl Brine

First-order kinetics with respect to the α_s1-casein concentration was used to study casein degradation during low-fat Fynbo cheese ripening. Effects of partial NaCl replacement by KCI during cheese salting were studied by statistical treatment of the casein degradation results. Four zones from cheeses at 1, 5, 10, 20, and 30 ripening days were analyzed by a polyacrylamide gel electrophoresis method. Similar kinetic parameters were obtained for a cheese salted with a NaCl/KCl brine and for a control cheese during ripening. Results were more affected by salt concentration than by salt substitution. KCl did not strongly influence kinetics of Fynbo cheese proteolysis.     

Discrimination of Swiss Cheese from 5 Different Factories by High Impact Volatile Organic Compound Profiles Determined by Odor Activity Value Using Selected Ion Flow Tube Mass Spectrometry and Odor Threshold

Swiss cheese contains more than 200 volatile organic compounds (VOCs). Gas chromatography‐mass spectrometry has been utilized for the analysis of volatile compounds in food products; however, it is not sensitive enough to measure VOCs directly in the headspace of a food at low concentrations. Selected ion flow tube mass spectrometry (SIFT‐MS) provides a basis for determining the concentrations of VOCs in the head space of the sample in real time at low concentration levels of parts per billion/trillion by volume. Of the Swiss cheese VOCs, relatively few have a major impact on flavor quality. VOCs with odor activity values (OAVs) (concentration/odor threshold) greater than one are considered high‐impact flavor compounds. The objective of this study was to utilize SIFT‐MS concentrations in conjunction with odor threshold values to determine OAVs thereby identifying high‐impact VOCs to use for differentiating Swiss cheese from five factories and identify the factory variability. Seventeen high‐impact VOCs were identified for Swiss cheese based on an OAV greater than one in at least 1 of the 5 Swiss cheese factories. Of these, 2,3‐butanedione was the only compound with significantly different OAVs in all factories; however, cheese from any pair of factories had multiple statistically different compounds based on OAV. Principal component analysis using soft independent modeling of class analogy statistical differentiation plots, with all of the OAVs, showed differentiation between the 5 factories. Overall, Swiss cheese from different factories was determined to have different OAV profiles utilizing SIFT‐MS to determine OAVs of high impact compounds.     

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.     

Casein breakdown during ripening of Idiazabal cheese: influence of starter and rennet type

The objective of this work was to determine the effect of starter and rennet type on casein breakdown during Idiazabal cheese ripening. Four batches of cheeses were manufactured with two rennets, commercial calf rennet and artisanal lamb rennet, and the use of natural flora or a commercial starter. Electrophoretic analysis of cheese samples showed six bands identified as α_s1‐, α_s2 + β‐, α_s1‐I‐, γ₁‐, β‐I‐ and para‐κ‐casein. As expected, the casein breakdown during cheese ripening was considerably affected by rennet type and the use of a commercial starter. The artisanal lamb rennet produced a higher hydrolysis of casein fractions than the commercial calf rennet, probably owing to its high percentage of chymosin (around 78%). The effect of addition of starter on proteolysis was dependent on the casein fractions generated by artisanal lamb rennet or commercial calf rennet. © 2000 Society of Chemical Industry     

The effect of refrigerated storage of raw milk on the physicochemical and microbiological quality of Tunisian semihard Gouda‐type cheese during ripening

A Tunisian semihard Gouda‐type cheese made from milk kept at 4 °C for 24, 48, 72 and 96 h was monitored during 45 days of ripening. The effect of milk refrigeration on the evolution of physicochemical parameters in relation to the quantitative variation of the microbial population during ripening of Gouda‐type cheese was investigated. Microbiological and physicochemical analyses were performed on raw milk and cheese samples after curding, 2, 9, 16, 23, 30, 37 and 45 days of ripening time. The raw milk kept under refrigeration at 4 °C for 96 h showed the highest microbial count and proteolysis level. The duration of storage significantly reduced the cheese yield as a result of important solubilisation casein in proteoses‐peptones. Results of different nitrogenous fractions by Kjeldahl method showed enzymatic hydrolysis products of casein whose intensity depended on the maturing stage as well as the refrigeration time. Besides the evident action of the plasmin, original milk protease, on the hydrolysis of casein in soluble fractions, the proteolysis of cheese caseins is also initiated by proteolytic action of the chymosin and extracellular heat‐resistant proteases notably produced by the same psychrotrophic microflora. Lactic acid bacteria starters that constitute the dominant microflora of this type of cheese are also considered as aroma precursors.     

Effect of sesame protein isolate in partial replacement of milk protein on the rheological,textural and microstructural characteristics of fresh cheese

Soft cheeses were manufactured from bovine milk with the addition of 0–12% sesame protein isolate (SPI) were utilised to investigate rheology, texture and microstructure at different stages of cheese making. SPI addition reduced the speed of milk fermentation, kappa‐casein proteolysis of rennet and elongated the time of cheese curd formation. Renneted milk storage modulus G′_60min was decreased and coagulation time increased with increasing SPI content. Low SPI supplements (4% and 8%) enhanced the hardness, cohesiveness, adhesiveness and gumminess of the soft cheese, while high SPI addition (12%) deteriorated the texture. In the cheese curd gel matrix, SPI distributed as specific SPI‐gel clusters on the surface of curd fractures, stacked or fused with ball‐shaped casein micelles and wrapped up to casein gel strands. In summary, SPI actively interacted with casein colloid throughout the cheese making process.     

Formation of bitter peptides in cheese and from casein]

Whereas a slightly bitter taste is desirable in certain foods, it is an off-flavour in cheese which may even lead to unfitness for consumption. Bitter principles from cheese have been found to be peptides with molecular weights ranging from 2000 to 3000. For the purpose of further characterization, bitter peptides were isolated from enzymatic casein hydrolysates as well as from bitter cheese and purified. 30 proteases from different origins proved to be able to form peptides with bitter taste of varying intensity from casein. Present experience shows that the formation of bitter peptides during casein hydrolysis can be inhibited only to a very small measure. Bitter peptides are extrmely resistant to proteases, which is probably attributable to their high contents of hydrophobic amino acids and hydrophobic bonds. The detection of only N- or C-terminal amino acid in each of 11 different bitter peptides shows that peptide chains are present and not cyclic peptides as repeatedly assumed. It must be aimed at avoiding the cheese defect "bitter" by using appropriate starter cultures and rennet substitutes as little disposed as possible to produce bitter peptides.     

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from α_S1-CN, 813 from α_S2-CN, and 234 from κ-CN. The majority of β-CN- and α_S1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from α_S2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, α_S2-CN, as well as α_S1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and α_S1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of α_S2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.     

Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of CaCl₂ and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight lower than 2509 Da and 2254 Da respectively as determined using size exclusion HPLC, particularly when a degree of hydrolysis of 20% for the casein hydrolyzates was achieved. At the end, the recovery of casein phosphorylated peptides reached 24%. Phosphorus component of casein phosphorylated peptides was found to be 3.08%. The nitrogen recovery of casein non-phosphorylated peptides was about 76%.     

酪蛋白降解对牦牛乳硬质干酪苦味的影响

针对牦牛乳硬质干酪的苦味缺陷,分别以小牛皱胃酶、微生物凝乳酶和木瓜蛋白酶制作的牦牛乳硬质干酪为研究对象,利用尿素聚丙烯酰胺凝胶电泳,研究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白的降解程度,且对成熟过程中的牦牛乳硬质干酪苦味进行感官评价,探究牦牛乳硬质干酪pH 4.6水不溶性酪蛋白降解对其苦味的影响。结果表明：牦牛乳硬质干酪在成熟期间酪蛋白发生了明显的降解,且αs-酪蛋白均比β-酪蛋白降解速率快。经尿素聚丙烯酰胺凝胶电泳分离后,发现木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白在Pre-αs-酪蛋白区域有较强的蛋白带。木瓜蛋白酶制作的牦牛乳硬质干酪pH 4.6水不溶性酪蛋白中αs-酪蛋白和β-酪蛋白降解程度均显著或极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪（P＜0.05或P＜0.01）,木瓜蛋白酶制作的牦牛乳硬质干酪的苦味值极显著高于微生物凝乳酶和小牛皱胃酶制作的牦牛乳硬质干酪的苦味值（P＜0.01）,通过主成分分析得出3 种凝乳酶制作牦牛乳硬质干酪的苦味值和未降解β-酪蛋白和αs-酪蛋白含量成极显著负相关。这为控制牦牛乳硬质干酪品质提供了理论参考。