Relationship between MALDI-TOF analysis of beta-CN f193-209 concentration and sensory evaluation of bitterness intensity of aged cheddar cheese

An internal standard method was previously developed to measure the concentration of a synthetic bitter peptide, beta-CN f193-209, by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The objective of this study was to evaluate the relationship between beta-CN f193-209 concentration in an aqueous extract of aged Cheddar cheese and bitterness intensity of the cheese. Concentrations of beta-CN f193-209 in cheese extracts were determined by MALDI-TOF at 0, 120, 180, and 270 days. Trained panels evaluated the bitterness intensity of the cheeses at 180 and 270 days. Correlation coefficients between MALDI and sensory data at 180 and 270 days were 0.803 and 0.554, respectively. The decreased correlation may be due to the presence of other bitter peptides more responsible for bitterness at longer aging or the production of compounds that mask bitterness intensity.     

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.     

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.     

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.     

Proteolytic activities of suparen and rennilase on buffalo, cow, and goat whole casein and beta-casein.

The proteolytic specificity and activity of Mucor miehei protease (Rennilase) and Endothia parasitica protease (Suparen) on buffalo, cow, and goat whole casein and beta-casein (CN) were studied by analyzing the degradation products. The results suggest that Rennilase hydrolyzes casein of the three species in a manner similar to that of chymosin, resulting in the formation of alpha(s1)-I and beta-I, -II, -III as initial degradation fragments of alpha(s1)- and beta-CN. alpha(s1)-I was also the initial breakdown product of alpha(s1)-CN by Suparen. Contrary to Rennilase, Suparen showed a higher affinity toward beta-CN and hydrolyzes beta-CN, giving rise to degradation products characterized by mobility lower than that of beta-CN. Increasing NaCl concentration (>3%) reduced the proteolysis of beta-CN of the three species by Rennilase but not by Suparen. The hydrolysis of alpha(s1)-CN and alpha(s1)-I by the two enzymes was enhanced in the presence of NaCl.     

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.     

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.     

Effects of caseins on thermal stability of bovine beta-lactoglobulin

Casein fractions have been shown to act as molecular chaperones and inhibit aggregation of whey proteins in dilute solutions (< or =1% w/v). We evaluated if this approach would stabilize protein solutions at higher concentration and thermal processing temperatures desired for beverage applications. Mixtures of beta-lactoglobulin (BLG) (6% w/v) with either beta-casein (BCN) (0.01-2% w/v) or alpha s-casein (ACN) (2% w/v) were adjusted to pH 6.0 and heated (70-90 degrees C) for 20 min, cooled, and then analyzed to determine the degree of aggregation. Aggregation was determined by solution turbidity as optical density (OD) at 400 or 600 nm. The addition of 0.05% (w/v) BCN or greater caused a drop in turbidity for solutions heated at 70-90 degrees C. In contrast, inhibition was observed in BLG-ACN mixtures at 70 degrees C but not at > or =75 degrees C. Moreover, prolonged heating (90 min) of BLG with 2% (w/v) BCN (pH 6.0) at 90 degrees C produced a clear solution while BLG-ACN solutions formed translucent gels after heating for 15 min. The weight-averaged molar mass and root-mean-square (rms) radius of soluble aggregates were determined by size exclusion chromatography in conjunction with multiangle laser light scattering (SEC-MALS). SEC-MALS confirmed the turbidity results by showing that the BLG-BCN mixture (8% w/v protein) produced aggregates with lower molar mass and smaller rms radius (majority 20-40 nm). These results showed that BCN is a feasible component to stabilize higher concentrations of whey proteins in beverages.     

Functionality of beta-casein peptides: importance of amphipathicity for emulsion-stabilizing properties.

To investigate structure-function relationships with regard to emulsion-stabilizing properties, peptides from bovine beta-casein (betaCN), obtained by plasmin hydrolysis and fractionation of the hydrolysate, were isolated and identified on the basis of their masses determined by electrospray ionization mass spectrometry, the primary structure of the intact protein, and the known specificity of the enzyme. An amphipathic peptide fraction was fractionated further by ion-exchange chromatography and subsequent hydrophobic interaction chromatography resulting in the components betaCN[f 1-105/107] and betaCN[f 29-105/107]. The latter peptides had poor emulsion-stabilizing properties compared to the former ones, and the stability of an emulsion formed with betaCN[f 29-105/107] was also more sensitive to hydrophobic impurities than that of an emulsion formed with betaCN[f 1-105/107]. The highly charged N-terminal part appeared to be important for the emulsion-stabilizing properties of these peptides. A hypothesis for the structure-function relationship is given.     

Quantitative studies and taste re-engineering experiments toward the decoding of the nonvolatile sensometabolome of Gouda cheese

The first comprehensive quantitative determination of 49 putative taste-active metabolites and mineral salts in 4- and 44-week-ripened Gouda cheese, respectively, has been performed; the ranking of these compounds in their sensory impact based on dose-over-threshold (DoT) factors, followed by the confirmation of their sensory relevance by taste reconstruction and omission experiments enabled the decoding of the nonvolatile sensometabolome of Gouda cheese. The bitterness of the cheese matured for 44 weeks was found to be induced by CaCl2 and MgCl2, as well as various bitter-tasting free amino acids, whereas bitter peptides were found to influence more the bitterness quality rather than the bitter intensity of the cheese. The DoT factors determined for the individual bitter peptides gave strong evidence that their sensory contribution is mainly due to the decapeptide YPFPGPIHNS and the nonapeptides YPFPGPIPN and YPFPGPIHN, assigned to the casein sequences beta-CN(60-69) and beta-CN(60-68), respectively, as well as the tetrapeptide LPQE released from alphas1-CN(11-14). Lactic acid and hydrogen phosphate were identified to play the key role for the sourness of Gouda cheese, whereas umami taste was found to be due to monosodium L-glutamate and sodium lactate. Moreover, saltiness was induced by sodium chloride and sodium phosphate and was demonstrated to be significantly enhanced by L-arginine.     

Control of heat-induced aggregation of whey proteins using casein

The ability of alphas1/beta-casein and micellar casein to protect whey proteins from heat-induced aggregation/precipitation reactions and therefore control their functional behavior was examined. Complete suppression (>99%) of heat-induced aggregation of 0.5% (w/w) whey protein isolate (pH 6.0, 85 degrees C, 10 min) was achieved at a ratio of 1:0.1 (w/w) of whey protein isolate (WPI) to alphas1/beta-casein, giving an effective molar ratio of 1:0.15, at 50% whey protein denaturation. However, in the presence of 100 mM NaCl, heating of the WPI/alphas1/beta-casein dispersions to 85 degrees C for 10 min resulted in precipitation between pH 6 and 5.35. WPI heated with micellar casein in simulated milk ultrafiltrate was stable to precipitation at pH>5.4. Protein particle size and turbidity significantly (P相似文献   

Flavor release measurement from gum model system

Flavor release from a mint-flavored chewing gum model system was measured by atmospheric pressure chemical ionization mass spectroscopy (APCI-MS) and sensory time-intensity (TI). A data analysis method for handling the individual curves from both methods is presented. The APCI-MS data are ratio-scaled using the signal from acetone in the breath of subjects. Next, APCI-MS and sensory TI curves are smoothed by low-pass filtering. Principal component analysis of the individual curves is used to display graphically the product differentiation by APCI-MS or TI signals. It is shown that differences in gum composition can be measured by both instrumental and sensory techniques, providing comparable information. The peppermint oil level (0.5-2% w/w) in the gum influenced both the retronasal concentration and the perceived peppermint flavor. The sweeteners' (sorbitol or xylitol) effect is less apparent. Sensory adaptation and sensitivity differences of human perception versus APCI-MS detection might explain the divergence between the two dynamic measurement methods.     

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.     

Chemical characterization and anti-inflammatory effect of polysaccharides fractionated from submerge-cultured Antrodia camphorata mycelia

Five polysaccharide fractions (AC-1, AC-2, AC-3, AC-4, and AC-5) were obtained after systemic solvent extractions and precipitations from Antrodia camphorata mycelia with yields of 2.92, 10.38, 1.65, 0.34, and 1.64%, respectively. Gel permeation chromatography (GPC) analysis showed that the distribution of mean molecular mass of the fractionated polysaccharides was in the range of 394-940 kDa. The proximate compositions from each polysaccharide fraction revealed that all fractions belonged to the category of glycoprotein, having ratios of carbohydrate/protein ranging from 0.29 to 10.79 (w/w). Glucose or galactose was the major monosaccharide in all fractions except fraction AC-2, which has a mean molecular mass of 394 kDa with lyxose as the most prominent constituent. In the evaluation of the DPPH* radical scavenging capability, fraction AC-1 and AC-2 polysaccharides showed the better capabilities, around 74.5 and 50.5%, respectively, compared to the reference control of Trolox (87.5%) at a concentration of 1 microM. In testing with macrophage RAW264.7 cells, fraction AC-2 demonstrated a rather potent anti-inflammatory capability. Furthermore, the lipopolysaccharide-induced NO production and the protein expression by the inducible nitric oxide synthase (iNOS) gene were inhibited, respectively, in a dose-dependent (50-200 microg/mL) manner by fraction AC-2 polysaccharide.     

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.     

Ethanol-dependent heat-induced dissociation of casein micelles

The dissociation of casein micelles when heated to approximately 65 degrees C in the presence of ethanol [1:1 mixture (v/v) of milk and 65% (w/w) aqueous ethanol] was investigated using L* values and transmission measurements. Mixtures of milk and ethanol became transparent on heating, which suggests dissociation of casein micelles. Results of experiments using confocal laser scanning microscopy, light scattering (static and dynamic), and dialysis to examine the changes of milk during heating in the presence of ethanol supported the assertion that such treatments result in dissociation of casein micelles, as did studies of model beta-casein micellar systems.     

Isolation of Small Starch Granules and Determination of Their Fat Mimic Characteristics

Small starch granules from wheat and amaranth were isolated and examined by light microscopy (wheat), scanning electron microscopy (wheat and amaranth), and rapid viscosity (RVA) analysis. The isolated wheat and amaranth starches were incorporated into reduced‐fat frozen dessert mixes (2%, w/w) and the frozen desserts were characterized by sensory analysis for smoothness, creaminess, and preference. The results of the sensory analysis suggest that up to 50% of the fat in a frozen dessert may be replaced when substituted by these starches at the 2% level (w/w).     

Effect of transglutaminase-catalyzed polymerization of beta-casein on its emulsifying properties

beta-Casein was polymerized to various extents by the transglutaminase-catalyzed cross-linking reaction. Under the reaction conditions used, dimer, trimer, and tetramer of beta-casein were produced as predominant species at short reaction times (20-90 min) and large polymers at longer reaction times (3-24 h). Examination of the emulsifying properties of these cross-linked beta-casein polymers showed that although the emulsifying activity index decreased, the storage stability of the emulsions increased with increasing degree of polymerization. Experiments with mixtures of native and polymerized beta-casein also showed that increasing the fraction of polymerized beta-casein in the mixture increased the emulsion stability. This enhancement of emulsion stability might be due either to an enhancement in steric stabilization, attributable to the branched nature of the trasgutaminase-catalyzed polymers, or to stronger cohesive interactions between polymerized beta-casein molecules than between monomeric beta-casein molecules in the film.