Prediction of the ripening times of ewe's milk cheese by multivariate regression analysis of capillary electrophoresis casein fractions

The effect of the ripening time on the proteolytic process in cheeses made from ewe's milk during a 139-day ripening period was monitored by the use of capillary electrophoresis of pH 4.6 insoluble fraction. Totals of 18 and 21 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks correspond to intact ovine caseins and their hydrolysis products (alpha(s1)-casein I, alpha(s1)-casein II, alpha(s1)-casein III, alpha(s2)-casein, beta(1)-casein, beta(2)-casein, p-kappa-casein, alpha(s1)-I-casein, gamma(1)-casein, gamma(2)-casein, and gamma(3)-casein). The alpha(s)-caseins (alpha(s1)- and alpha(s2)-casein) displayed similar degradation pattern to one another, but different from those of beta-caseins (beta(1)- and beta(2)-casein). beta-Caseins were very much undergoing lesser degradation during the ripening time than alpha(s)-casein. Finally, partial least-squares regression and principal components regression were used to predict the ripening time in cheeses. The models obtained yielded good results since the root-mean-square error in prediction by cross validation was <8.6 days in all cases.     

Uncommonly thorough hydrolysis of peptides during ripening of Ragusano cheese revealed by tandem mass spectrometry

Ragusano is a pasta filata cheese produced from raw milk in Sicily. The proteolysis was extensively analyzed after stretching (day 0), at 4 and 7 months of ripening through soluble nitrogen, urea-PAGE, and peptide identification by tandem mass spectrometry. After stretching, 123 peptides were identified: 72 arising from β-casein, 34 from α(s1)-casein, and 17 from α(s2)-casein. The main protein splitting corresponded to the action of plasmin, chymosin, cathepsin D, cell envelope proteinase, and peptidase activities of lactic acid bacteria. Unlike other types of cheeses, <10% residual β- and α(s)-caseins remained intact at 7 months, indicating original network organization based on large casein fragments. The number of identified soluble peptides also dramatically decreased after 4 and 7 months of ripening, to 47 and 25, respectively. Among them, bioactive peptides were found, that is, mineral carrier, antihypertensive, and immunomodulating peptides and phosphopeptides.     

Peptides identified during Emmental cheese ripening: origin and proteolytic systems involved

To determine the proteolytic changes occurring during Emmental cheese ripening, peptides released in cheese aqueous phase were analyzed by reversed-phase HPLC and identified by tandem mass spectrometry sequencing, for which different strategies were illustrated by some examples. Among the 91 peptides identified, most of them arose from alpha(s1)- (51) and beta-caseins (28), and a few arose from alpha(s2)- (9) and kappa-caseins (1). An attempt was made to correlate the released peptides with the proteolytic systems potentially involved during Emmental cheese manufacture. Besides the well-known action of plasmin on beta- and alpha(s2)-caseins, and in the absence of residual fungal coagulant from Endothia parasitica, two other proteinases seem to be involved in the hydrolysis of alpha(s1)-casein in Emmental cheese: cathepsin D originated from milk and cell-envelope proteinase from thermophilic starters. Moreover, peptidases from starters were also active throughout ripening, presumably like those from nonstarter lactic acid bacteria, in contrast to those from propionic acid bacteria.     

Immunochemical evaluation of bovine beta-casein and its 1-28 phosphopeptide in cheese during ripening

Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.     

Dynamics of competitive adsorption of alphas-casein and beta-casein at planar triolein-water interface: evidence for incompatibility of mixing in the interfacial film

Competitive adsorption of alpha(s)-casein and beta-casein from a bulk solution mixture to the triolein-water interface has been studied. Although the binding affinity of alpha(s)-casein to the triolein-water interface was lower than that of beta-casein in single-component systems, in a 1:1 mixture of alpha(s)-casein and beta-casein in the bulk solution the ratio of interfacial concentrations of alpha(s)-casein to beta-casein at equilibrium was about 2:1, indicating that alpha(s)-casein was preferentially adsorbed to the triolein-water interface. Furthermore, the equilibrium composition of alpha(s)-casein and beta-casein in the interfacial film at various bulk concentration ratios did not follow a simple Langmuir adsorption model. This deviation from ideal behavior was mainly due to thermodynamic incompatibility of mixing of these caseins in the interfacial region. The value of the incompatibility parameter, X(12), for these caseins at the triolein-water interface was much greater than that at the air-water interface. Displacement experiments showed that while alpha(s)-casein could dynamically displace beta-casein when the latter was in an unsaturated monolayer state at the interface, it could not do so when beta-casein was in a saturated monolayer film state. It is hypothesized that, because of thermodynamic incompatibility of mixing, the alpha(s)-casein and beta-casein mixed film at the oil-water interface may undergo two-dimensional phase separation.     

Variability of hydrolysis of β-, αs1-, and αs2-caseins by 10 strains of Streptococcus thermophilus and resulting bioactive peptides

Milk proteins contain numerous potential bioactive peptides, which may be released by digestive proteases or by the proteolytic system of lactic acid bacteria during food processing. The capacity of Streptococcus thermophilus to generate peptides, especially bioactive peptides, from bovine caseins was investigated. Strains expressing various levels of the cell envelope proteinase, PrtS, were incubated with α(s1)-, α(s2)-, or β-casein. Analysis of the supernatants by LC-ESI-MS/MS showed that the β-casein was preferentially hydrolyzed, followed by α(s2)-casein and then α(s1)-casein. Numbers and types of peptides released were strain-dependent. Hydrolysis appeared to be linked with the accessibility of different casein regions by protease. Analysis of bonds hydrolyzed in the region 1-23 of α(s1)-casein suggests that PrtS is at least in part responsible for the peptide production. Finally, among the generated peptides, 13 peptides from β-casein, 5 from α(s2)-casein, and 2 from α(s1)-casein have been reported as bioactive, 15 of them being angiotensin-converting enzyme inhibitors.     

Sugar-casein interaction in deuterated solutions of bovine and caprine casein as determined by oxygen-17 and carbon-13 nuclear magnetic resonance: a case of preferential interactions

17O NMR spectroscopy and (13)C NMR spectroscopy have been used to study the mechanism of interaction of sugars with bovine and caprine caseins in D(2)O. The (17)O NMR relaxation results showed in all cases an increase in water of hydration, as a result of added sugar; this was predominantly associated with "trapped" water in the caseins. Analysis of the vir al coefficients, obtained from the (17)O relaxation data, suggested that preferential interactions occur in the sugar-protein solutions. This could be the result of either sugar binding or a solute-solute thermodynamic effect, preferential hydration. The addition of sugars to deuterated solutions of bovine casein and caprine casein high in alpha(s1)-casein had little or no effect on either line width or chemical shifts of the (13)C NMR spectra of these milk proteins. (13)C NMR studies of sucrose, at various concentrations (100-300 mM) in the presence of caprine casein high in alpha(s1)-casein, showed no changes in either chemical shifts or T(1) values. This indicates that the sugar molecules tumble isotropically and therefore neither bind to the protein nor affect viscosity in the protein-sugar studies. All of these data suggest that the preferential exclusion of the sugar from the domain of the caseins results in preferential hydration of the caseins.     

Changes in the volatile composition of a semihard ewe milk cheese induced by high-pressure treatment of 300 MPa

The effect of high-pressure (HP) treatment (300 MPa, 10 min) on the volatile profile of semihard ewe milk cheeses was investigated. The HP treatment was applied at two different stages of ripening (1 and 15 days; 3P1 and 3P15) and microbiota, proteolysis indexes (soluble nitrogen and total free amino acid content), and volatile compounds were assayed at 15, 60, 90, and 150 days of ripening. The intensity of odor and aroma of cheeses was also assayed. 3P1 cheeses presented the highest content of free amino acids and were characterized by the lowest amounts of aldehydes, ketones, short-chain free fatty acids, and terpenes and higher levels of ethanol and ethyl esters. 3P15 cheeses were characterized by the highest content of short-chain free fatty acids and pyruvaldehyde and the lowest abundance of secondary alcohols and were more similar to control cheeses than those HP-treated on the first day. Intensities of odor and aroma were not significantly influenced by the HP treatment. However, the panellists found some differences in 3P1 as compared with control and 3P15 cheeses in what they perceived as lower odor and aroma quality.     

Peptides from Lactobacillus hydrolysates of bovine milk caseins inhibit prolyl-peptidases of human colon cells

Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from α(S1)-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-amino-dipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects.     

A novel approach to the quantification of bovine milk in ovine cheeses using a duplex polymerase chain reaction method

A duplex Polymerase Chain Reaction (PCR) method able to detect bovine milk in ovine cheeses was developed. This method is based on the mitochondrial 12S and 16S rRNA genes to generate fragments of different lengths. The proposed methodology presents an alternative DNA extraction procedure faster and more economical than the kits commercially available. A linear normalized calibration curve was obtained between the log of the ratio of the bovine band intensity and the sum of bovine and ovine band intensities versus the log of cow's milk percentage. The method was applied successfully to the detection and quantification of raw, pasteurized, and powdered bovine milk in different cheeses. The proposed duplex PCR provides a simple, sensitive, and accurate approach to detect as low as 0.1% bovine milk in cheeses and to quantify bovine milk in ovine cheeses in the range of 1-50%.     

Binding of 2-nonanone and milk proteins in aqueous model systems

Interactions of the model flavor compound 2-nonanone with individual milk proteins, whey protein isolate (WPI), and sodium caseinate in aqueous solutions were investigated. A method to quantify the free 2-nonanone was developed using headspace solid-phase microextraction followed by gas chromatography with flame ionization detection. Binding constants (K) and numbers of binding sites (n) for 2-nonanone on the individual proteins were calculated. The 2-nonanone binding capacities decreased in the order bovine serum albumin > beta-lactoglobulin > alpha-lactalbumin > alpha s1-casein > beta-casein, and the binding to WPI was stronger than the binding to sodium caseinate. All proteins appeared to have one binding site for 2-nonanone per molecule of protein at the flavor concentrations investigated, except for bovine serum albumin, which possessed two classes of binding sites. The binding mechanism is believed to involve predominantly hydrophobic interactions.     

Topography of the casein micelle surface by surface plasmon resonance (SPR) using a selection of specific monoclonal antibodies

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), β, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, β-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.     

Production of hard-type cheese using free or immobilized freeze-dried kefir cells as a starter culture

This study provides a contribution to hard-type cheese starter culture production through the use of a freeze-dried culture in the ripening of hard-type cheeses. The effect of initial cell concentration, ripening temperature, and cell immobilization of kefir on the degree of openness, mold spoilage, microbial associations, physicochemical characteristics, and aroma-related compounds was studied. Use of kefir starter cultures resulted in cheese with an increased shelf life and resistance to spoilage as compared to control cheeses without kefir inoculants. Furthermore, the freeze-dried kefir culture improved aroma, taste, and texture characteristics while increasing the degree of openness in comparison to traditional hard-type cheese products. The kefir culture resulted in an increase in counts of total aerobic bacteria, yeasts and molds, lactococci, and lactobacilli until the 15th day of ripening. From then on, only lactobacilli counts increased, reaching levels up to 9.17 log CFU/g in cheeses ripened at 5 degrees C using freeze-dried kefir cells immobilized on casein. SPME-GC/MS analysis revealed major differences in volatile composition, especially with regard to alcohols (up to 75%), carbonyl compounds (up to 75%), and esters (up to 64%) between cheeses made with kefir cells and cheeses made without kefir inoculants.     

Removal of cholesterol from Cheddar cheese by beta-cyclodextrin

This study was carried out to determine the cholesterol removal rate and resulting changes in flavor, fatty acid and bitter amino acid production in reduced-cholesterol Cheddar cheese, made by cream separation followed by 10% beta-cyclodextrin (beta-CD) treatment. The cholesterol removal from the cheese was 92.1%. The production of short-chain free fatty acids (FFAs) increased the ripening time in control and cream-treated cheeses. The quantity of short-chain FFAs released between treatments during ripening was different, while not much difference was found in the production of neutral volatile compounds in the samples. Reduced-cholesterol cheese produced much higher levels of bitter amino acids than the control. In sensory analysis, the texture score of control Cheddar cheese increased significantly with ripening time; however, that of the cream treatment group decreased dramatically with ripening time. On the basis of our results, we conclude that the cheese made from beta-CD-treated cream had a higher rate of cholesterol removal and ripened rapidly.     

Biogenic amines and polyamines in milks and cheeses by ion-pair high performance liquid chromatography

The proposed chromatographic method provides a complete resolution of twelve amines in a single run in milks and unripened cheeses, avoiding the losses of resolution linked to fluctuations in working temperature. We also propose an alternative chromatographic gradient, which can be useful for samples that have undergone long ripening periods, like ripened cheeses. According to the results of the reliability study, the method described was precise, accurate, and sensitive. The method was applied to several samples of milks and cheeses and the results showed that the biogenic amine profiles varied greatly, not only between different types of samples but also among the samples from the same kind of products. In unripened cheeses, milks, and yogurts, spermidine and spermine were the prevailing amines, but in ripened cheeses the major amine was tyramine, followed by putrescine and cadaverine.     

Isolation and characterization of three novel peptides from casein hydrolysates that stimulate the growth of mixed cultures of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

In this study, sodium caseinate hydrolysates produced by papain with strong growth-stimulating activity for Streptococcus thermophilus (St) and Lactobacillus delbrueckii subsp. bulgaricus (Lb) were obtained. A series of separation methods including ultrafiltration, macroporous adsorption resin chromatography, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC) were applied to isolate and purify the peptide(s), which were mainly responsible for the activity. Finally, three novel growth-stimulating peptides, H-2-A, F2-c, and F2-b, corresponding to amino acid residues 29-35 and 103-108 of bovine α(S2)-casein and 181-186 of bovine α(S1)-casein, respectively, were obtained. With supplementation of H-2-A, F2-b, or F2-c at a protein concentration of 0.3%, the biomass yield of these two lactic acid bacteria (LAB) was enhanced by 193.3, 166.7, or 151.7%, respectively. In addition, there were significant (p < 0.05) increases in viable counts of St and lactic acid production of LAB in the presence of the purified peptides.     

Chemometric analysis of Ragusano cheese flavor

Ragusano cheeses were produced in duplicate from milk collected from pasture-fed and total mixed ration (TMR)-fed cattle at four time intervals. The cheeses were subjected to chemical analysis, conventional sensory testing, and gas chromatography-olfactometry (GCO). Data from each type of analysis were examined by principal component and factor analysis and by pattern recognition (SIMCA) to see if sufficient information for classification into pasture-fed and TMR-fed groups was contained therein. The results clearly indicate that there are significant differences in sensory panel and chemical analysis results between the two cheeses. The data were also examined to see if models of sensory responses as a function of analytical or GCO results or both could be constructed with the modeling technique partial least-squares regression (PLS). Strong PLS models of some sensory responses (green and toasted odor; salt, pungent, bitter, and butyric sensations; and smooth consistency) were obtained.     

Metabolic profiling of potential probiotic or synbiotic cheeses by nuclear magnetic resonance (NMR) spectroscopy

To assess ripening of potential probiotic cheeses (containing either Lactobacillus casei -01 or Bifidobacterium lactis B94) or synbiotic cheeses with fructooligosaccharides (FOS) or a 50:50 mix of FOS/inulin, metabolic profiles have been obtained via classical biochemical analyses and by NMR spectroscopy. The addition of prebiotics to the cheeses resulted in lower proteolysis indices, especially in those synbiotic cheeses inoculated with B. lactis B94. Among synbiotic cheeses the combination of FOS and inulin resulted in an increase in lipolytic activity. The metabolic profiles of the cheeses analyzed by NMR spectroscopy, combined with multivariate statistics, allowed profiles to be distinguished by maturation time, added probiotic bacteria, or, in the case of B. lactis B94 cheese, added prebiotic. The NMR results are in agreement with the biochemical analyses and demonstrate the potential of NMR for the study of metabolic processes in probiotic/synbiotic food matrices.     

Formation of early and advanced Maillard reaction products correlates to the ripening of cheese

The present study deals with the characterization of the ripening of cheese. A traditional German acid curd cheese was ripened under defined conditions at elevated temperature, and protein and amino acid modifications were investigated. Degree of proteolysis and analysis of early [Amadori compound furosine (6)] and advanced [N(ε)-carboxymethyllysine (4), N(ε)-carboxyethyllysine (5)] Maillard reaction products confirmed the maturation to proceed from the rind to the core of the cheese. Whereas 6 was decreased, 4 and 5 increased over time. Deeper insight into the Maillard reaction during the ripening of cheese was achieved by the determination of selected α-dicarbonyl compounds. Especially methylglyoxal (2) showed a characteristic behavior during storage of the acid curd cheese. Decrease of this reactive structure was directly correlated to the formation of 5. To extend the results of experimental ripening to commercial cheeses, different aged Gouda types were investigated. Maturation times of the samples ranged from 6 to 8 weeks (young) to more than 1 year (aged). Again, increase of 5 and decrease of 2 were able to describe the ripening of this rennet coagulated cheese. Therefore, both chemical parameters are potent markers to characterize the degree of maturation, independent of coagulation.     

A rapid method for the quantitative determination of short-chain free volatile fatty acids from cheese

The determination of free volatile fatty acids (FVFA) is of interest in the analysis of cheeses. As these compounds are components of taste and flavor, they give indications on metabolic reactions taking place during cheese ripening and can provide an evaluation of cheese defects and their causes. One of the most widely used methods for the determination of FVFA in cheese involves preliminary recovery from the matrix by steam distillation, followed by gas chromatography separation. Relatively high distillate volumes must be collected to achieve a quantitative yield of all the compounds of interest, so that, as a result, the solution is too diluted to achieve good instrumental sensitivity. In this paper, an alternative method for the determination of C2-C6 free carboxylic acids in cheeses involving the use of a Nukol capillary column and crotonic acid as internal standard is described. This method is quick and cheap, as the sample preparation is a simple extraction with water. The underivatized FVFA are then directly separated by gas chromatography. Using this method, all FVFA in cheeses can be quantified with good repeatability and excellent recovery.