Chemometrical analysis of capillary electrophoresis casein fractions for predicting ripening times of milk mixture cheese

The effect of the ripening time on the proteolytic process in cheeses manufactured from mixtures of cow's and ewe's milk during a 167-day ripening period was monitored by capillary electrophoresis of the pH 4.6-insoluble fraction. Totals of 21 and 16 peaks were recognized and matched in the electropherograms obtained with a fused-silica capillary and a neutral capillary (hydrophilically coated), respectively. These peaks corresponded to intact bovine and ovine caseins and their hydrolysis products (e.g., alpha(s1)-casein, gamma-caseins). In 167-day-old cheeses, bovine alpha(s0)-casein (alpha(s1)-casein 9P) had been completely degraded and 6% of the residual bovine alpha(s1)-casein remained intact. Breakdown of the beta-casein fraction was lower than that of the alpha(s)-casein fraction. Finally, partial least-squares regression and principal component regression were used to predict the ripening time in cheeses. The root-mean-square errors in prediction by cross-validation were <7.8 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.     

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.     

Immunochemical detection of formylated gamma(2)-casein in cheese

An immunochemical approach has been developed to detect the use of formaldehyde as a bacteriostatic agent in dairy products. A synthetic peptide, reproducing the first five amino acid residues of the gamma(2)-casein sequence, was formylated to generate the novel haptenic structure, already well-recognized in formaldehyde-treated milk and arising out of molecular rearrangement after the addition of formaldehyde to the alpha-amino group of the histidine residue at the N terminus of gamma(2)-casein. A polyclonal antibodies preparation produced against the formylated peptide adduct proved to be a highly specific analytical tool for detecting the formylated adduct of gamma(2)-casein in formaldehyde-treated milk. Polyclonal antibodies obtained against the unmodified peptide were able to detect selectively residual native gamma(2)-casein in ripened cheese.     

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.     

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.     

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.     

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.     

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.     

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.     

Enrichment of cheeses manufactured from cow's and sheep's milk blends with sheep-like species-related alkylphenols

Enhancement of concentrations of species-related sheep-like alkylphenols, p- and m-cresols and 3- and 4-ethylphenols, in experimental Manchego-type cheeses manufactured from cow's and sheep's milk blends (80:20) by using arylsulfatases was investigated. A food-grade arylsulfatase from Aspergillus oryzae (ATCC 20719) was produced using a stimulatory medium, and crude dried cells were used as the enzyme source. Exogenous arylsulfatases from Helix pomatia and A. oryzae were added to cheese curd, and the amounts of species-related alkylphenols were measured. Arylsulfatase from H. pomatia released limited amounts of alkylphenols in the cheese only when used at a high level. Arylsulfatase from A. oryzae released substantial amounts of alkylphenols during 2 months of ripening. The concentrations of alkylphenols in A. oryzae arylsulfatase-treated cheese were comparable to the previously reported levels present in aged Manchego-type cheeses manufactured from pure sheep's milk.     

Chemical and microbiological characteristics of ewes' milk cheese manufactured with extracts from flowers of Cynara cardunculus and Cynara humilis as coagulants

The chemical and microbial characteristics as well as the flavor and aroma of Los Pedroches cheese made using aqueous extracts of Cynara cardunculus L. flowers were compared with those of cheeses manufactured with extracts of Cynara humilis L. throughout ripening. The two thistle species assayed were found to have no appreciable effect on the moisture, fat, protein, and NaCl contents of the cheese or on its water activity, flavor, and aroma; however, the use of C. humilis resulted in reduced lactic acid content (p < 0.001) and higher pH values (p < 0.05) relative to those of cheese specimens produced with C. cardunculus. The protein breakdown of the cheeses was assessed in terms of soluble nitrogen (SN), nonprotein nitrogen (NPN), and amino acid nitrogen (AAN). Proteolysis was more marked and rapid in cheese containing C. cardunculus as coagulant, the SN and NPN contents of which were significantly higher (p < 0. 01) than those of the cheese obtained with the species C. humilis; AAN contents were similar in both species of Cynara throughout ripening. Although total viable, coliform, and lactobacilli counts were similar in cheeses produced with both types of plant coagulant throughout ripening, enterobacteria and yeasts counts (p < 0.01) and molds counts (p < 0.05) were higher in cheese produced with C. humilis than in cheese obtained with C. cardunculus.     

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.     

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.     

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.     

Predicting alpha, beta and gamma plant diversity from physiognomic and physical indicators as a tool for ecosystem monitoring

We searched for predictive models for alpha, beta and gamma plant diversity based in easy to measure field indicators. The study was conducted on the upper belt of the Córdoba mountains (Argentina). We established 222 permanent plots of 4 × 4 m distributed on sites with different physiognomy, topography and management. At each plot we measured physical and physiognomic indicators and recorded the presence of all vascular plants. We estimated alpha diversity as the number of species detected in a plot, beta diversity as the floristic dissimilarity between two plots, and gamma diversity as the number of species detected in a landscape. Through linear regression we found predictive models for alpha and pair-wise beta diversity. Then we analysed if predicted average alpha and beta diversity were good estimators of gamma diversity. We recorded a total of 288 species (5-74 species per plot). Alpha diversity was highest in sites on shallow soils with high structural richness (i.e. high number of cover categories), half covered by lawns, at sunny slopes and rough landscapes (r² = 0.66). For beta diversity, the difference between plots in structural richness and in cover of thick tussocks grasses and lawns were the best predictors (r² = 0.45). For different sets of simulated landscapes, gamma diversity was well explained by predicted average alpha and beta diversity, plus the sampling effort (r² = 0.92). We concluded that using easy to measure field indicators it is possible to estimate plant diversity at different levels with a good accuracy.     

Time course and specificity of lipolysis in Swiss cheese

Controlling lipolysis in cheese is necessary to ensure the formation of desirable flavor. To get a better understanding of the mechanism of lipolysis in Swiss cheese, cheeses were manufactured with and without (control) the addition of Propionibacterium freudenreichii. Products of lipolysis were quantified throughout ripening. Half of the free fatty acids (FFA) released in milk (3.66 mg/g fat), in particular the short-chain FFA, were lost in the whey during curd drainage, whereas diglycerides and monoglycerides were retained within the curd. P. freudenreichii was responsible for the release of most FFA during ripening (10.84 and 0.39 mg/g fat in propionibacteria-containing and control cheeses, respectively). Indices of lipolysis displayed low specificity. All types of FFA were released, but butyric and palmitic acids more significantly, which could be due to a low sn-1,3 regioselectivity. All glycerides were hydrolyzed in the following order: monoglycerides>diglycerides>triglycerides. The results of this study show the quantitative and qualitative contributions of the different lipolytic agents to Swiss cheese lipolysis.     

Influence of a probiotic adjunct culture of Enterococcus faecium on the quality of cheddar cheese

Cheddar cheese has previously been shown to be an effective vehicle for delivery of viable cells of a probiotic Enterococcus faecium strain to the gastrointestinal tract. The particular strain, E. faecium PR88, has proven efficacy in the treatment of irritable bowel syndrome, and in this study it was evaluated for suitability as a starter adjunct for Cheddar cheese manufacture. When added to cheesemilk at an inoculum of 2 x 10(7) cfu/mL, the enterococcal adjunct maintained viability in Cheddar cheese at levels of up to 3 x 10(8) cfu/g during 9 months of ripening. Increased proteolysis and higher levels of some odor-active volatile compounds were observed in Cheddar cheeses containing the PR88 adjunct compared with the control throughout the ripening period. In addition, the enterococcal adjunct strain did not affect cheese composition. Although sensory evaluation showed no significant difference in flavor/aroma and body/texture scores between control and experimental cheeses, repeated comments by the commercial grader consistently described the cheeses containing PR88 as 'more advanced than the control' and as having 'better flavor'. These findings indicate that the presence of the PR88 adjunct strain in Cheddar cheese at levels of >/=10(8) cfu/g may positively influence Cheddar flavor.     

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.