Rheological and proteolytic properties of Monterey Jack goat's milk cheese during aging

To enhance the understanding of the quality traits of goat's milk cheeses, rheological and proteolytic properties of Monterey Jack goat's milk cheese were evaluated during 26 weeks of 4 degrees C storage. As expected with aging, beta-casein levels decreased with concomitant increases in peptide levels and were correlated with changes in rheological properties of the cheese. Hydrolysis of the protein matrix resulted in more flexible (increased viscoelastic properties) and softer (decreased hardness, shear stress, and shear rigidity) cheeses. During the first 4-8 weeks of storage, cheese texture changed significantly (P < 0.05) and then stabilized. Characterization of rheological and proteolytic properties of the goat's milk semihard cheese during aging provided insight into the changes occurring in the protein matrix, the relationship to structure, and a shift in cheese quality.     

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.     

Detection of pH 4.6 insoluble beta-lactoglobulin in heat-treated milk and Mozzarella cheese

Different protein aggregates including beta-lactoglobulin (beta lg) were detected in the pH 4.6 insoluble fraction recovered from actual heat-treated milk samples by gel electrophoresis and immunoblotting. A competitive enzyme-linked immunosorbent assay (ELISA) using anti-beta lg polyclonal antibodies was developed to analyze the beta lg partition in the protein fractions obtained upon acidification of both milk and Mozzarella cheese at pH 4.6. According to ELISA determinations, nearly 90% of the pH 4.6 soluble beta lg included in raw milk was found in the pH 4.6 insoluble fraction of ultrahigh temperature (UHT)-treated milk. As concerns Mozzarella cheese analysis, ELISA results indicated that about 36% of the total beta lg milk content was transferred from pasteurized milk to Mozzarella cheese, whereas less than 0.5% was transferred from raw milk. The pH 4.6 insoluble beta lg proved to be a suitable indicator of the intensity of the heat treatment applied to milk. The ELISA-based detection of this parameter was suggested for quality control of both drinking milk and raw milk cheese.     

Competitive adsorption between beta-casein or beta-lactoglobulin and model milk membrane lipids at oil-water interfaces

This study investigated the competitive adsorption between milk proteins and model milk membrane lipids at the oil-water interface and its dependence on the state of the lipid dispersion and the formation of emulsions. Both protein and membrane lipid surface load were determined using a serum depletion technique. The membrane lipid mixture used was a model milk membrane lipid system, containing dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, milk sphingomyelin, dioleoylphosphatidylserine, and soybean phosphatidylinositol. The model composition mimics the lipid composition of natural milk fat globule membranes. The interactions were studied for two proteins, beta-lactoglobulin and beta-casein. The mixing order was varied to allow for differentiation between equilibrium structures and nonequilibrium structures. The results showed more than monolayer adsorption for most combinations. Proteins dominated at the oil-water interface in the protein-emulsified emulsion even after 48 h of exposure to a vesicular dispersion of membrane lipids. The membrane lipids dominated the oil-water interface in the case of the membrane lipid emulsified emulsion even after equilibration with a protein solution. Protein displacement with time was observed only for emulsions in which both membrane lipids and beta-casein were included during the emulsification. This study shows that kinetics controls the structures rather than the thermodynamic equilibrium, possibly resulting in structures more complex than an adsorbed monolayer. Thus, it can be expected that procedures such as the mixing order during emulsion preparation are of crucial importance to the emulsification performance.     

Ethyl ester formation is enhanced by ethanol addition in mini Swiss cheese with and without added propionibacteria

Esters are important contributors to cheese flavor, but their mechanisms of synthesis in cheese are largely unknown. This study aimed to determine whether ethanol concentration limits the formation of ethyl esters in cheese. Mini Swiss cheeses were manufactured with (E) or without (C) the addition of ethanol to cheese milk. Ethanol concentrations (enzymatic analysis) were 64 +/- 17 and 330 +/- 82 microg g(-1), respectively, in C and E cheeses. E cheeses also contained 5.4 +/- 2.3 times more of the five ethyl esters quantified than C cheeses, regardless of the concentrations of esters in C cheeses (range 1-128 ng g(-1)). Furthermore, the presence of propionibacteria added as acid-producing secondary starters was associated with greater concentrations of esters, due to the increase in acid concentrations that propionibacteria induced and/or to an involvement of propionibacteria enzymes in ester synthesis. This study demonstrates that ethanol is the limiting factor of ethyl ester synthesis in Swiss cheese.     

Milk excretion of ivermectin and moxidectin in dairy sheep: assessment of drug residues during cheese elaboration and ripening period

Ivermectin (IVM) and moxidectin (MXD) are broad-spectrum endectocide antiparasitic drugs extensively used in food-producing animals. The patterns of IVM and MXD excretion in milk were comparatively characterized following their subcutaneous administration (200 microg.kg(-1) of body weight) to lactating dairy sheep. The relationship between milk excretion and plasma disposition kinetics of both compounds was characterized. A pool of milk collected from all of the animals in each experimental group was used for cheese elaboration. IVM and MXD residual concentrations were assessed during the cheese-making process and ripening period. IVM and MXD concentrations were measured in plasma, milk, and milk product (whey, curd, and cheese) samples using an HPLC-based methodology with fluorescence detection. IVM and MXD were extensively distributed from the bloodstream to the mammary gland, and large quantities, particularly of MXD, were excreted in milk. Residual concentrations of both compounds were recovered in milk up to 30 (IVM) and 35 (MXD) days post-treatment. The total fraction of the administered dose excreted in milk for MXD was significantly higher than that of IVM. During cheese production, the highest residual concentrations of both molecules were measured in the curd. Thirty-four percent of the total drug residue measured in the pooled milk collected from treated sheep was lost during the cheese-making process. The lowest residual concentrations were measured in the whey. IVM and MXD concentrations in the elaborated cheese tended to increase during the ripening period, reaching the highest residual level at 40 days of cheese maturation. The long persistence of milk residual concentrations of MXD and IVM in lactating dairy sheep and the high concentrations found in cheese and other milk-related products should be seriously considered before recommendation of the extralabel use of these antiparasitic drugs in dairy animals.     

Evaluation of APHA and AOAC methods for phosphatase in cheese

Varieties of market cheese were analyzed for alkaline phosphatase by the modified rapid colorimetric method of the American Public Health Association (APHA) and the official AOAC method, 16.304-16.306. In the APHA method, 5 g cheese (pH less than 7.0) is macerated with 2 mL 1:1 carbonate buffer, or 2 mL water (for cheese with pH greater than 7.0). Addition of 0.1 mL magnesium acetate (1 mg magnesium) to test portions of cheese extracts yielded reproducible and quantitative recovery of added phosphatase. In the AOAC method, macerating 0.5 g cheese with 1 mL borate buffer before adding milk phosphatase improved recovery among cheeses. Addition of magnesium ion increased phosphatase activity in some cheeses. Phosphatases in blue mold-ripened and Swiss cheeses were inactivated by heat faster than was milk phosphatase, yet milk phosphatase added to various soft cheeses was completely inactivated at 60 degrees C for 10 min. The lability of phosphatase was due to the heat-denaturing effect of NaCl present in finished cheeses. Some Mexican style soft cheeses contained both heat-labile and heat-stable phosphatases. These data suggest that the phosphatase test to differentiate milk and microbial phosphatases on the basis of repasteurization and analysis of cheese is no longer valid.     

Interactions of vitamin D3 with bovine beta-lactoglobulin A and beta-casein

It is of nutritional significance to fortify processed dairy products (e.g., cheese, yogurt, and ice cream) with vitamin D3; however, the inherent complexity of these foods may influence the stability and bioavailability of this nutrient. In the present study, the interactions of vitamin D3 with beta-lactoglobulin A and beta-casein were investigated under various environmental conditions (i.e., pH and ionic strength) using fluorescence and circular dichroism spectroscopic techniques. The results indicated that vitamin D3 was bound to both beta-lactoglobulin A and beta-casein depending on the solution conditions. The apparent dissociation constants ranged from 0.02 to 0.29 microM for beta-lactoglobulin A, whereas the beta-casein apparent dissociation constants ranged from 0.06 to 0.26 microM. The apparent mole ratios were also comparable, i.e., 0.51-2.04 and 1.16-2.05 mol of vitamin D3 were bound per mole of beta-lactoglobulin A and beta-casein, respectively. It was concluded that these interactions may strongly influence vitamin D3 stability and, hence, bioavailability in processed dairy products.     

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.     

Real-time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples

Mozzarella cheese obtained from buffalo (Bubalus bubalis) milk is a typical Italian product certificated by means of the European Protected Designation of Origin (PDO). Mozzarella cheese can also be obtained from bovine milk or bovine/buffalo milk mixtures, but in this case, it cannot be sold as PDO product, and its label must report the actual ingredients. However, bovine milk in PDO products was frequently detected in the past, suggesting fraudulent addition or accidental contamination. Several methods based on end-point polymerase chain reaction (PCR) have been profitably applied in a large number of tests to detect the presence of undeclared ingredients, also in dairy products. In the present study we report a real-time PCR method able to quantify bovine milk addition to pure buffalo cheese products. We validated a normalized procedure based on two targets: bovine mitochondrial cytochrome b (cyt b) to detect and quantify the bovine DNA and nuclear growth hormone (GH) gene used as a universal reference marker. With the use of this real-time PCR assay, 64 commercial mozzarella di bufala cheese samples purchased at local supermarkets, dairy shops, or directly from cheese manufacturers were analyzed. The results obtained demonstrate that most of the commercial samples were contaminated with bovine milk. Therefore, this assay could be conveniently employed to carry out routine and accurate controls aimed not only to discourage any fraudulent behavior but also to reduce risks for consumer health.     

Key odorants of Oscypek, a traditional Polish ewe's milk cheese

The unique flavor of Oscypek, a Polish ewe's milk smoked cheese, is described as slightly sour, piquant, salted, and smoked. In this paper with the application of gas chromatography-olfactometry (GC-O) and combination of aroma extract dilution analysis (AEDA) 20 potent odorants of this cheese have been identified within the flavor dilution factor (FD) range of 4-2048. Among them, 2-methoxyphenol, 2-methoxy-4-methylphenol, 4-methylphenol, and butanoic acid showed the highest FD factors. Quantification results based on labeled standard addition followed by calculation of odor activity values (OAV) of 13 compounds with the highest FD factors revealed that 11 compounds were present at concentrations above their odor threshold values and therefore mostly contribute to the overall aroma of smoked ewe's milk cheese. Six of those compounds were represented by phenolic derivatives, with the highest OAV for 2-methoxyphenol (1280). Analysis of key odorants of an unsmoked cheese sample showed that the smoking process had a fundamental influence on Oscypek aroma formation.     

Use of recombinant cyprosin in the manufacture of ewe's milk cheese

A recombinant cyprosin from the cardoon (Cynara cardunculus L.) was assayed and compared with calf rennet in batches of ewes' milk cheese by determining different chemical, biochemical, and microbiological parameters over 4 months of ripening. There were no differences between the two types of coagulants in most chemical parameters, a(w), and pH. Proteolysis was more marked and rapid in cheese containing recombinant cyprosin as coagulant, the soluble nitrogen content of which was significantly higher (p < 0.01) than that of the cheese obtained with animal rennet; at the end of ripening the nonprotein nitrogen of cheese produced with recombinant cyprosin was slightly higher (p > 0.05) as compared with that in cheeses obtained with animal rennet. Microbial counts in the milk used for making cheese were high in most of the groups analyzed. Despite slight differences in counts, the main microbial groups analyzed were similar in cheese produced with both types of coagulants throughout ripening.     

Identification and enumeration of beta-hemolytic Listeria monocytogenes in naturally contaminated dairy products

A DNA probe was used to identify hemolytic Listeria monocytogenes in naturally contaminated dairy products: unpasteurized milk, ricotta cheese, and imported semisoft cheeses. Of 34 milk samples, 12 were suspected to contain hemolytic L. monocytogenes; 1 contained greater than 6000 viable organisms/g. The ricotta cheese, although temperature-abused, had a titer of 3.6 x 10(6) beta-hemolytic L. monocytogenes cells/g, whereas the semisoft cheeses reached a maximum of 5.6 x 10(6) cells/g. Pure cultures of L. monocytogenes isolated from both types of cheese were found positive by the CAMP test and the DNA probe.     

Producibility and digestibility of antihypertensive beta-casein tripeptides, Val-Pro-Pro and Ile-Pro-Pro, in the gastrointestinal tract: analyses using an in vitro model of mammalian gastrointestinal digestion

Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) are antihypertensive tripeptides isolated from milk fermented with Lactobacillus helveticus and inhibit angiotensin-converting enzyme (ACE). We investigated whether these peptides were generated from beta-casein by digestive enzymes and whether they were resistant to enzymatic hydrolysis, using an in vitro model. VPP and IPP were not generated from beta-casein by gastrointestinal enzymes; instead, a number of longer peptides including VPP and IPP sequences were detected. The fermentation step would therefore be necessary to produce these antihypertensive tripeptides. VPP and IPP themselves were hardly digested by digestive enzymes, suggesting that orally administered VPP and IPP remain intact in the intestine, retaining their activity until adsorption. The present study also demonstrated that various functional peptide sequences in beta-casein were resistant to gastrointestinal enzymes. There may be a strong correlation between the resistance of peptides to gastrointestinal digestion and their real physiological effects after oral administration.     

Residue study of ivermectin in plasma,milk, and mozzarella cheese following subcutaneous administration to buffalo (Bubalus bubalis)

The distribution of ivermectin in buffalo plasma and milk after administration of a single subcutaneous dose (0.2 mg kg(-)(1) b.w.) was studied. Ivermectin reached the maximal concentration in plasma (28.5 +/- 1.7 ng mL(-)(1)) and milk (23.6 +/- 2.6 ng mL(-)(1)) after 2.4 +/- 0.32 and 2.8 +/- 0.44 days, respectively. The drug showed a parallel disposition in milk and plasma, with a ratio of 1.12 +/- 0.16. Ivermectin concentrations were detected in mozzarella cheese obtained from milk collected on days 1, 3, 4, and 20 following administration. The highest values (81.4 +/- 3.26 ng g(-)(1)) were found in the cheese produced on day 3 and were 4-fold higher than those present in the milk.     

Factors affecting the retention of rennet in cheese curd

The coagulant retained in cheese curd is a major contributor to proteolysis during ripening. The objective of this study was to quantify the effects of several milk-related factors and parameters during cheese manufacture on the retention of coagulant in cheese curd. The amount of coagulant retained in curd was determined by its activity on a synthetic heptapeptide (Pro-Thr-Glu-Phe-[NO2-Phe]-Arg-Leu) using reversed-phase HPLC. The retention of chymosin in cheese curd increased significantly when the pH of milk was reduced at rennet addition below pH 6.1, the pH at whey drainage below pH 5.7, or the average casein micelle size in milk and when the ionic strength of milk was increased. The casein content of milk and the quantity of chymosin added to milk had no significant effect on the retention of chymosin in curd; the quantity of coagulant bound per gram of casein remained unchanged.     

Peptides isolated from in vitro digests of milk enhance iron uptake by caco-2 cells

Milk proteins, during digestion, produce a range of biologically active peptides. Among those are peptides that may enhance iron absorption. The objective of this project was to investigate the effect of isolated milk peptides on iron uptake. Cow's milk, 0% fat, was subjected to a modified in vitro digestion process. The milk digest was further fractionated by gel filtration. All eluted fractions as well as beta-casein synthetic peptides (a tripeptide and a hexapeptide) were subsequently tested for effects on iron uptake with Caco-2 cell monolayers. Fractions of milk digests obtained through Sephadex G-25 gel filtration had a significant enhancing effect on iron uptake in Caco-2 cells compared to nonfractionated milk digests. Two fractions (P = 0) and the hexapeptide (P < 0.0001) enhanced iron uptake by up to 3-fold, whereas others and the tripeptide had no effect. These results suggest that selected peptides produced during the in vitro digestion of milk may enhance iron absorption; however, it remains to be demonstrated whether this effect may be nutritionally significant.     

Temperature-independent pectin gel method for coliform determination in dairy products: collaborative study

Ten laboratories participated in a collaborative study to compare a pectin-based violet red bile (VRB) method with the VRB agar-based standard method for estimation of coliform bacterial counts in 7 different dairy food groups: cream, cheddar cheese, cottage cheese, homogenized milk, raw milk, sour cream, and yogurt. Each laboratory analyzed 8 samples of each food group as sample pairs prepared at high, medium, and low inoculum levels, and an uninoculated control pair. Overall mean log counts were higher for the pectin gel method in 18 of 21 cases (7 samples at 3 inoculum levels); 12 results were significantly higher (P less than 0.01) than those for the VRB agar method. Of the 3 higher VRB agar method means, 2 were not significant at P less than 0.10, and one was of borderline significance (0.05 less than P less than 0.06). Repeatability variation (sr) favored the pectin gel method in 14 of 21 cases; 7 were highly significant differences (P less than 0.01). None of the 7 results favoring the VRB agar method was statistically significant. Reproducibility variation (sR) favored the pectin gel method in 14 of 21 results. These data indicate that the pectin gel method gives higher recovery of coliforms with better precision than does the VRB agar method, and strongly support the suitability of the pectin gel method as an alternative to the agar-based VRB method for coliform counts in dairy products. The pectin gel method has been adopted official first action.