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.     

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%.     

Influence of various phosphopeptides of caseins on iron absorption

The influence of the origin and kind of caseinophosphopeptide (CPP) on iron absorption was assessed by comparing a commercially available CPP mixture (CPPs) and derived chromatographic fractions with the purified, chemically phosphopeptide of beta-casein [beta-CN(1-25)] using a perfused rat duodenal loop system; gluconate iron was used as control. Only iron complexed to beta-CN(1-25) displayed a better bioavailability than gluconate iron. The results obtained with various chromatographic fractions indicated that phosphopeptides of different origins (alpha(s)- versus beta-caseins) display specific effects. These findings contribute to the explanation of the discrepancy about the role of caseinophosphopeptides on mineral bioavailability in vivo.     

Antipeptide antibodies recognizing plasmin sensitive sites in bovine beta-casein sequence

To investigate plasmin activity in cheese, we produced antibodies to bovine beta-casein with controlled specificity, suitable as markers of the integrity of the major bonds involved in its initial breakdown. Sixteen rabbits were immunized with synthetic substitutes for six plasmin-sensitive peptides. Antisera raised to the peptides (f20-39), (f40-56), (f94-113), (f184-202), and (f193-209) recognized beta-casein in ACP-ELISA, Western-blot, and biosensor assays. Casein in vitro hydrolysis by plasmin or chymosin reduced the detection of these determinants in ACP-ELISA, in agreement with the enzymatic sensitivity of bonds included within the binding sites, or in their neighborhood. Antiserum to (f20-39) in particular allowed the specific detection of plasmin cleavage at the bond generating gamma1-CN. Antisera to C-terminus preferentially detected the cleavage by chymosin. Immunoassays using these antibodies would allow in situ monitoring of significant proteolysis events without bias originated in the secondary degradation of the released peptides.     

Expression and purification of glycosylated bovine beta-casein (L70S/P71S) in Pichia pastoris

Post-translational glycosylation of bovine beta-casein (L70S/P71S) that results in Asn(68)-linked glycan on the protein was obtained in up to 30% of total beta-casein expressed in the methylotrophic yeast Pichia pastoris. Among the growth/induction media used, buffered minimal glycerol (BMG)/buffered minimal methanol (BMM) media were best for the production of glycosylated bovine beta-casein, indicating pH-dependent glycosylation. Glycosylated bovine beta-casein (L70S/P71S) expressed in P. pastoris was purified to homogeneity by the combination of ammonium sulfate fractionation, Concanavalin A--Sepharose affinity column, and Mono Q anion-exchange FPLC. The purified glycosylated bovine beta-casein was specific only to Concanavalin A, and the oligosaccharide structure of glycosylated beta-casein was of high-mannose type. Unlike the hyperglycosylation that occurred in yeast, the majority of bovine beta-casein was not hyperglycosylated in P. pastoris, and its molecular weight was estimated to be 33.6 kDa. Glycosylated bovine beta-casein was normally phosphorylated to the same degree as native bovine beta-casein.     

Caseins and casein hydrolysates. 1. Lipoxygenase inhibitory properties

Whole casein from bovine origin, the different casein subtypes alpha, beta, and kappa, and the related dephosphorylated proteins were assayed as modulators of soybean lipoxygenase 1 activity and were found to inhibit it. To define the lipoxygenase inhibitory domain, whole casein and beta-casein were digested by proteases (trypsin, clostripain, and subtilisin). The beta-casein tryptic digest and the tryptic and subtilisin digests of whole casein retained their inhibitory properties. The tryptic beta-casein digest was the most potent inhibitor of lipoxygenase activity and was further fractionated by FPLC or HPLC. The collected peptides inhibited the lipoxygenase-catalyzed reaction to different extents. The active fractions were analyzed by ESI-MS, and the sequences of several lipoxygenase inhibitory peptides, corresponding mainly to the C-terminal moiety of beta-casein, were identified.     

Micellization of bovine beta-casein studied by isothermal titration microcalorimetry and cryogenic transmission electron microscopy

The association behavior, critical micellization concentration (CMC), and enthalpy of demicellization (DeltaHdemic) of bovine beta-casein were studied, for the first time by isothermal titration calorimetry, in a pH 7.0 phosphate buffer with 0.1 ionic strength and in pure water. In the buffer solutions, the CMC decreased asymptotically from 0.15 to 0.006 mM as the temperature was raised from 16 to 45 degrees C. DeltaHdemic decreased with increasing temperature between 16 and 28 degrees C but increased from 28 to 45 degrees C. Thermodynamic analysis below 30 degrees C is consistent with the Kegeles shell model, which suggests a stepwise association process. At higher temperatures, this model exhibits limitations, and the micellization becomes much more cooperative. The CMC values in water, measured between 17 and 28 degrees C, decreased with increasing temperature and, expectedly, were higher than those found in the buffer solutions. beta-Casein micelles were visualized and characterized, for the first time in their hydrated state, using advanced digital-imaging cryogenic transmission electron microscopy. The images revealed small, oblate micelles, about approximately 13 nm in diameter. The micelles shape and dimensions remained nearly constant in the temperature range of 24-35 degrees C.     

Iron tissue storage and hemoglobin levels of deficient rats repleted with iron bound to the caseinophosphopeptide 1-25 of beta-casein.

Caseinophosphopeptides (CPP) issued from enzyme digestion of caseins bind cations and keep them soluble in the digestive tract. They could be used as ligands to improve iron (Fe) bioavailability. Fe-deficient young rats were repleted with Fe (40 or 200 mg/kg of diet) bound either to the beta-CN (1-25) of beta-casein or to whole beta-casein or as FeSO(4). A control pair-fed group was given 200 mg of Fe (FeSO(4))/kg of diet for 6 weeks. After repletion, hemoglobin concentration of the control group was reached only by the ) animals fed 200 mg of Fe/kg; beta-CN (1-25) bound Fe (40 and 200 mg) produced higher Fe liver and spleen stores than FeSO(4). Binding Fe to the whole, nonhydrolyzed beta-casein gave results intermediate between the other experimental groups. Binding Fe to phosphoserine residues of low molecular weight CPP improved its ability to cure anemia and to restore iron tissue stores, as compared to Fe bound to the whole casein and to inorganic salts.     

Simultaneous liquid chromatographic determination of some tetracyclines in serum

A sensitive liquid chromatographic method has been developed for the simultaneous determination of oxytetracycline, minocycline, tetracycline, and doxycycline in serum. A serum sample is vortex-mixed with a solution of mobile phase for tetracyclines and 2% (v/v) phosphoric acid. The mixture is filtered using a 30,000 molecular weight cutoff microseparation tube which separates high-molecular-weight solutes following low-speed centrifugation. Tetracyclines are separated from other serum components by reverse phase liquid chromatography (LC) with buffered methanol mobile phase. Ultraviolet absorbance of the column effluent is monitored at 267 nm. Concentrations as low as 0.2 micrograms/mL of tetracyclines in serum are quantitatable, with recoveries from 76.2 to 102.6% and coefficients of variation from 2.69 to 5.36%. The method has been tested in bovine, porcine, equine, caprine, ovine, canine, feline, and avian (turkey) serum.     

基于骨蛋白拉曼光谱特异性的肉骨粉种属鉴别方法

为了快速检测肉骨粉的种属来源,该研究开发了一种简便、可靠、科学、高效的肉骨粉种属鉴别方法。以87个肉骨粉(猪,鸡,牛和羊肉骨粉)为研究对象,利用拉曼光谱技术,结合化学计量学方法,建立了基于骨蛋白拉曼光谱特性的肉骨粉种属鉴别分析方法与模型。研究结果表明:根据偏最小二乘判别分析(PartialLeastSquaresDiscriminant Analysis,PLS-DA)模型,发现鸡和哺乳动物(猪,牛和羊)肉骨粉主要在1 659、2 930、2 852、1 246和1 455 cm-1附近的特征谱带具有差异性;猪和反刍动物(牛和羊)肉骨粉主要是在2 852、1 659和1 246 cm-1附近的特征谱带具有差异性;牛和羊肉骨粉主要是在878、853、2 930、2 852、1 246、1 455和1 659 cm-1附近的特征谱带具有差异性,并且PLS-DA模型鉴别肉骨粉的灵敏度和特异度均大于93.8%。研究结果可以丰富肉骨粉种属鉴别方法体系以及为开发便携式拉曼光谱仪提供参考。     

Determination of grayanotoxins in biological samples by LC-MS/MS

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.     

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.     

Quantification of beta casein in milk and cheese using an optical immunosensor

beta-Casein was quantified in milk and cheese, using an optical immunosensor, based on surface plasmon resonance (SPR) measurement. The assay consists of a two-step sandwich strategy, with two anti-beta-casein antibodies directed against each extremity of the casein. This strategy permits only native beta-casein to be quantified and not its degradation products. The calibration curve was obtained with a reference milk powder of known beta-casein concentration. The analysis time per sample was less than 10 minutes. The antibody-coated surface could be used for more than 250 determinations. The detection limit was established at 85 ng x mL(-)(1) and the intra- and inter-assay variation coefficients were 2.6 and 6.2% respectively. The method was applied to raw milk to quantify intact beta-casein, with no pretreatment of the sample. A second application was realized with cheese, to follow the proteolysis of beta-casein during ripening.     

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.     

Impact of whey pH at drainage on the physicochemical, sensory, and functional properties of mozzarella cheese made from buffalo milk

In this study, the effects of whey pH at drainage on the physicochemical, sensory, and functional properties of mozzarella cheese made from buffalo milk during storage were investigated. Four cheese samples were manufactured using starter culture at different whey pH values [(A) 6.2, (B) 5.9, (C) 5.6, and (D) 5.3] and analyzed on the 1st, 28th, and 56th day. Ash, calcium, and phosphorus concentrations decreased as the whey pH at drainage was lowered. Cheese yield and calcium recovery were the highest in D cheeses. During storage, expressible serum levels decreased and nonexpressible serum levels increased, indicating an increase in the water holding capacity of the cheeses. Reducing the calcium content of cheeses increased meltability values, but an overly low calcium level (D cheeses) had an adverse effect on the meltability. The melting properties of cheese samples, except D cheeses, were improved with aging. A cheeses were the hardest and D cheeses the softest throughout storage. The 1st day sensory evaluations revealed that C and D cheeses were preferred and that A cheeses were not. All sensory properties of A cheeses were improved with storage. D cheeses were rated inferior to the others at the end of the storage time.     

反刍动物饲料中乳源性成分与牛羊肉骨粉的区分

利用奶粉的溶解性,通过水和0.5%次氯酸钠溶液洗涤混有乳源性成分和牛、羊肉骨粉的饲料样品,去除饲料中的乳粉成分后,再使用16S rDNA PCR方法进行动物源性检测。结果表明,实验所建立的方法能够完全区分饲料中的乳粉与肉骨粉。当乳粉含量分别为25%、50%和75%时,混合饲料中牛、羊肉骨粉的检出限分别为2%、0.5%和0.1%。此方法操作简单,容易掌握,可用于鉴别反刍动物饲料中非乳源性成分的牛、羊源性成分。     

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.     

Sensomics mapping and identification of the key bitter metabolites in Gouda cheese

Application of a sensomics approach on the water-soluble extract of a matured Gouda cheese including gel permeation chromatography, ultrafiltration, solid phase extraction, preparative RP-HPLC, and HILIC combined with analytical sensory tools enabled the comprehensive mapping of bitter-tasting metabolites. LC-MS-TOF and LC-MS/MS, independent synthesis, and sensory analysis revealed the identification of a total of 16 bitter peptides formed by proteolysis of caseins. Eleven previously unreported bitter peptides were aligned to beta-casein, among which 6 peptides were released from the sequence beta-CN(57-69) of the N terminus of beta-casein and 2 peptides originated from the C-terminal sequence beta-CN(198-206). The other peptides were liberated from miscellaneous regions of beta-casein, namely, beta-CN(22-28), beta-CN(74-86), beta-CN(74-77), and beta-CN(135-138), respectively. Six peptides were found to originate from alpha(s1)-casein and were shown to have the sequences alpha(s1)-CN(11-14), alpha(s1)-CN(56-60), alpha(s1)-CN(70/71-74), alpha(s1)-CN(110/111-114), and alpha(s1)-CN(135-136). Sensory evaluation of the purified, synthesized peptides revealed that 12 of these peptides showed pronounced bitter taste with recognition thresholds between 0.05 and 6.0 mmol/L. Among these peptides, the decapeptide YPFPGPIHNS exhibited a caffeine-like bitter taste quality at the lowest threshold concentration of 0.05 mmol/L.