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.     

Compositional analysis of nonfat dry milk by using near infrared diffuse reflectance spectroscopy

Proximate data from 82 nonfat dry milk (NFDM) samples were correlated with near infrared reflectance (NIR) measurements. The best wavelengths for determining constituent concentrations were chosen from 19 preselected filters by using linear regression analysis. The correlation coefficient (r) was 0.971 and the standard error of prediction (SEP) was 0.274 when the predicted values (from NIR measurements) using the 3 wavelengths selected for determining moisture content were compared with laboratory values; r and SEP were 0.961 and 0.099, respectively, when the predicted values using the 4 wavelengths selected for fat content were compared with laboratory results; 0.887 and 0.594, respectively, using the 4 wavelengths selected for lactose content; 0.905 and 0.438 using the 8 wavelengths selected for protein (micro-Kjeldahl) content; and 0.911 and 0.509 using the 7 wavelengths selected for protein (dye binding). These data indicate that NIR can be used to estimate moisture, fat, lactose, and protein content of NFDM.     

Disruption and reassociation of casein micelles under high pressure: influence of milk serum composition and casein micelle concentration

In this study, factors influencing the disruption and aggregation of casein micelles during high-pressure (HP) treatment at 250 MPa for 40 min were studied in situ in serum protein-free casein micelle suspensions. In control milk, light transmission increased with treatment time for approximately 15 min, after which a progressive partial reversal of the HP-induced increase in light transmission occurred, indicating initial HP-induced disruption of casein micelles, followed by reformation of casein aggregates from micellar fragments. The extent of HP-induced micellar disruption was negatively correlated with the concentration of casein micelles, milk pH, and levels of added ethanol, calcium chloride, or sodium chloride and positively correlated with the level of added sodium phosphate. The reformation of casein aggregates during prolonged HP treatment did not occur when HP-induced disruption of casein micelles was limited (<60%) or very extensive (>95%) and was promoted by a low initial milk pH or added sodium phosphate, sodium chloride, or ethanol. On the basis of these findings, a mechanism for HP-induced disruption of casein micelles and subsequent aggregation of micellar fragments is proposed, in which the main element appears to be HP-induced solubilization of micellar calcium phosphate.     

Effect of mean velocity gradient and mixing time on particle removal in seawater induced flocculation

Various combinations of mean velocity gradient (G) and mixing time (t _d ) in the seawater flocculation of industrial wastewater and oxidation pond effluent were investigated. The optimum product of these parameters (Gt _d ) lies within the range of 16,000 to 160,000. Inclusion of a slow mixing phase improved particle removal over that achieved by rapid mixing alone at equal gSGt_d. The minimum Gt _d at which peak particle removal was achieved decreased as seawater dosage increased. Similar trends were demonstrated with increasing dosages of the flocculant chitosan. An empirical model of flocculation kinetics, having the same form as the partice aggregation-floe breakup model, was found to portray accurately the effects of G and t _d .     

Influence of competitive adsorption on flocculation and rheology of high-pressure-treated milk protein-stabilized emulsions

The effect of high-pressure treatment (HPT) on the droplet-size distribution and small-deformation rheology of oil-in-water emulsions containing beta-lactoglobulin and a nonionic surfactant or sodium caseinate has been investigated at neutral pH. Addition of Tween 20 (polyoxyethylenesorbitan monolaurate) to a beta-lactoglobulin-stabilized emulsion results in competitive displacement of the adsorbed globular protein film and, following HPT, the formation of a less flocculated emulsion. The age of the beta-lactoglobulin-stabilized emulsion prior to addition of sodium caseinate influences the competitive adsorption behavior. The strengthening of the beta-lactoglobulin layer with time makes it more resistant to disruption by sodium caseinate. The level of pressure-induced flocculation of beta-lactoglobulin-coated oil droplets depends on the intensity of processing conditions and on the degree of interfacial displacement. In contrast, beta-lactoglobulin added after emulsification appears to show little evidence of competitive adsorption behavior at the caseinate oil-water interface. Changes in the rheological properties of these latter systems following HPT can be attributed to pressure-induced denaturation and gelation of beta-lactoglobulin in the continuous phase of the emulsion.     

Estimation of whey protein in casein coprecipitate and milk powder by high-performance liquid chromatography quantification of cysteine

An analytical high-performance liquid chromatography (HPLC)-fluorescence method for indirect measuring of whey protein in casein coprecipitate and milk powder was developed. Samples were hydrolyzed with HCl, and cysteyl residues were derivatized with 3,3'-dithiodipropionic acid and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The cysteine content was used to calculate the percentage of whey protein in commercial samples with use of European Union Regulation cysteine reference values in both casein and whey protein. Method validation studies were performed for caseinates and milk powder, and results indicate that the present HPLC approach can be applied as a fast method with a standard deviation of repeatability between 3.3 and 9.5%. Applicability was studied by analysis of 40 commercial caseinate samples, and all complied to European legislation with a content of whey protein not exceeding 5%. Finally, an approach used to estimate the cysteine amount in pure casein by comparison of calculated and experimental values questions the generally accepted cysteine reference value in casein, which is most likely an overestimation.     

Stabilization of ascorbic acid and its measurement by liquid chromatography in nonfat dry milk

The determination of ascorbic acid by liquid chromatography (LC) was improved by performing the analysis in the presence of solvents that had been purged with argon to reduce the concentration of oxygen. This methodological modification eliminated the oxidation of ascorbic acid during the chromatographic procedure and reduced the minimum detection level to 1 microgram. Solutions of ascorbic acid have been successfully stabilized for 67 days by addition of dithiothreitol to a deaerated solution of water-acetonitrile (25 + 75 v/v), sealed under argon in amber vials and stored at -20 degrees C. In a second independent study, a procedure for the extraction of ascorbic acid from nonfat dry milk in a single step was developed. The ascorbic acid content of Nonfat Dry Milk (SRM 1549) was determined by LC, using the method of standard additions. The mean ascorbic acid content was 54 +/- 5 micrograms/g of sample. Analysis of variance of the analytical results indicates that there is a significant continual increase in the content of the ascorbic acid in each bottle from first to last sample.     

Near-infrared analysis of fat, protein, and casein in cow's milk.

Fat, crude protein, true protein, and casein were determined in cow milks by near-infrared transmission spectroscopy (NIR). Partial and overall PLS calibrations were performed on two sets of samples: partial calibration included 76 unhomogenized samples, whereas overall calibration used 96 homogenized and unhomogenized samples. Standard errors of calibration were 0.12% for fat, 0.06% for crude protein, 0.04% for true protein, and 0.05% for casein in the overall calibration. Validation of the overall calibration with an independent set of samples gave standard errors of prediction of 0. 07% for fat, 0.06% for crude protein and casein, and 0.05% for true protein. Except for fat, all of the statistical parameters were better with overall than with partial calibrations, which indicates that homogenization has an effect on NIR fat determination. Despite the relatively small number of samples included in the calibration model, NIR transmission was found to be a reliable method for the determination of fat and nitrogenous constituents in milk.     

Oxygen permeability of films made from CO2-precipitated casein and modified casein

Oxygen permeabilities (OP) of CO(2)-casein (CO(2)CN), calcium caseinate (CaCN), and acylated casein (AcCN) films were determined as functions of % relative humidity (% RH), temperature, and plasticizer type. Tensile properties and water vapor permeabilities (WVP) were also measured. Plasticizers were glycerol (GLY) or a 3:1 ratio of GLY:poly(propylene glycol) (PPG), a hydrophobic plasticizer. OP of the CO(2)CN:GLY film was almost twice that of films containing either plasticizer at 35% RH, but its OP approached that of the other films at 70% RH. OP and WVP of films plasticized with GLY were greater than that for films plasticized with PPG. Plasticizer type had little impact on the tensile strength of CO(2)CN films while tensile strength of CaCN-GLY:PPG (3:1) films approximately doubled. Results show that structural dissimilarities in the films contribute to differences in OP only under conditions of low RH where the plasticizing effects of water are not significant.     

Heat-induced covalent complex between casein micelles and beta-lactoglobulin from goat's milk: identification of an involved disulfide bond.

Goat milk is characterized by a very low heat stability that could be attributed, in part, to the covalent interaction between whey proteins and casein micelles. However, the formation of such a complex in goat milk has never been evidenced. This study was designed to assess whether heat-induced covalent interaction occurs between purified casein micelles and beta-lactoglobulin. We used a multiple approach of ultracentrifugation of heated mixture, chromatographic fractionation of resuspended pellets, sequential enzyme digestion of disulfide-linked oligomers, and identification of disulfide-linked peptides by on-line liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS), and tandem MS. We identified three different types of disulfide links: (1) expected intermolecular bridges between beta-Lg molecules; (2) disulfide bond involving two kappa-casein molecules; and (3) a disulfide bond between two peptides, one from beta-Lg and the other from kappa-casein. The involved sites in this last bond were Cys(160) of beta-Lg and Cys(88) of kappa-casein. Although the identified heterolinkage is possibly only one of several different types, the results of this study constitute the first direct evidence of the formation of a covalent complex between casein micelles and beta-lactoglobulin derived from goat milk.     

有机奶奶源基地建设的研究与实现

奶源生产是发展有机奶的首要环节,也是最重要的环节之一。建设有机奶奶源基地是保持正常奶源生产的有力保障。有机奶及其生产已成为发达国家的奶品消费及生产主流。虽然我国已制定了《OFDC有机认证标准》,但真正的有机奶生产还没有开始。人们需要更多的高品质的奶品。根据我国《OFDC有机认证标准》中提出的要求,结合多年从事奶源基地建设的实际经验,提出了在国内有机奶奶源基地建设理念,并在养殖工艺(包括引中、饲料、疾病防治等)、设施结构、环境控制(包括消毒隔离、粪污处理等)3个方面进行了研究与探讨;并就我国发展有机奶奶源基地建设中可能出现的问题以及解决办法进行了讨论,为我国发展有机奶奶源基地建设、走奶业可持续发展之路、生产优质安全的有机奶提供了一些切实可行的方法。     

有机奶奶源基地建设的研究与实现

奶源生产是发展有机奶的首要环节，也是最重要的环节之一。建设有机奶奶源基地是保持正常奶源生产的有力保障。有机奶及其生产已成为发达国家的奶品消费及生产主流。虽然我国已制定了《OFDC有机认证标准》，但真正的有机奶生产还没有开始。人们需要更多的高品质的奶品。根据我国《OFDC有机认证标准》中提出的要求，结合多年从事奶源基地建设的实际经验，提出了在国内有机奶奶源基地建设理念，并在养殖工艺（包括引中、饲料、疾病防治等）、设施结构、环境控制（包括消毒隔离、粪污处理等）3个方面进行了研究与探讨；并就我国发展有机奶奶源基地建设中可能出现的问题以及解决办法进行了讨论，为我国发展有机奶奶源基地建设、走奶业可持续发展之路、生产优质安全的有机奶提供了一些切实可行的方法。     

Rapid high pressure liquid chromatographic determination of aflatoxin M1 in milk and nonfat dry milk

A modification of the current revised AOAC method, 26.A10-26.A15, is described for the rapid analysis of aflatoxin M1 in milk and nonfat dry milk. The method incorporates chloroform extraction and eliminates the need for column chromatography by using liquid-liquid partition for sample extract cleanup. Quantitation is carried out by using fluorescence detection combined with high pressure liquid chromatography (HPLC) of aflatoxin M1 which has been converted to aflatoxin M2a with trifluoroacetic acid. The method has a detection limit of 0.014 micrograms/L (2 X signal/noise) for whole milk. For 6 samples of naturally contaminated nonfat dry and freeze-dried milk, the modified method gave an average result of 0.698 micrograms/L; the AOAC method gave an average result of 0.386 micrograms/L.     

Addition of pectin and soy soluble polysaccharide affects the particle size distribution of casein suspensions prepared from acidified skim milk

Pectins are negatively charged polysaccharides employed as stabilizers in acidified milk dispersions, where caseins aggregate because of the low pH and serum separation needs to be prevented. The objective of this research was to study the effect of charge on the stabilizing functionality of the polysaccharide in acid milk drinks. Unstandardized pectins with various charges (as degree of esterification, DE) as well as soybean soluble polysaccharide (SSPS) were tested for their stabilizing behavior as a function of pH and concentration. Skim milk was acidified by glucono-delta-lactone and then homogenized in the presence of polysaccharide at different pH values (in the range from 4.2 to 3.0). Measurements of particle size distribution demonstrated that pectins with a DE of 71.4, 68.6, and 67.4 stabilized milk at pH > 4.0. Pectins with a lower DE (63.9%) needed a higher concentration (0.4%) at the same pH to show a monomodal distribution of particle sizes. Pectins with lower DE (<50%) did not stabilize the dispersions. Although this difference in behavior was attributed mainly to the pectin charge, the efficiency in stabilizing the casein dispersion decreased with decreasing pectin size. For example, the high methoxyl pectin (HMP) with 63.9 DE was smaller in size than the HMPs with a higher charge. Pectins showed a pH-dependent stabilization effect, as at pH < 4.0 the dispersions contained aggregates. When SSPS was used to stabilize acid milk, at pH < 4.0, it showed a better stabilization behavior than HMP. When SSPS and pectin were used in combination, the particle size distribution of the acid milk dispersion was pH-dependent, and results were similar to those for samples containing pectin alone. This suggested that in the mixture, pectin dominated the behavior over SSPS, even when an excess of SSPS was added to the dispersions before homogenization.     

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.     

旱育缺锰水稻秧苗在大田生长发育特征比较研究

对田间条件下旱育缺Mn水稻苗和正常苗移栽水作田和旱作田后生长发育及产量性状研究结果表明，缺Mn苗除株高与正常苗无明显差异外，其他性状包括分蘖、成穗和产量均低于正常苗，穗粒数、千粒重也表现同样的规律。若生产上采用缺Mn苗，不论采用传统的淹水方式、地膜覆盖旱作或麦秸覆盖旱作以及采用其他任何补救措施，均不能达到正常苗所达产量。     

Enumeration of total bacteria and coliforms in milk by dry rehydratable film methods: collaborative study

Eleven laboratories participated in a collaborative study to compare the dry rehydratable film (Petrifilm SM and Petrifilm VRB) methods, respectively, to the standard plate count (SPC) and violet red bile agar (VRBA) standard methods for estimation of total bacteria and coliform counts in raw and homogenized pasteurized milk. Each laboratory analyzed 16 samples (8 different samples in blind duplicate) for total count by both the SPC and Petrifilm SM methods. A second set of 16 samples was analyzed by the VRBA and Petrifilm VRB methods. The repeatability standard deviations (the square root of the between-replicates variance) of the SPC, Petrifilm SM, VRBA, and Petrifilm VRB methods were 0.05104, 0.0444, 0.14606, and 0.13806, respectively; the reproducibility standard deviations were 0.7197, 0.06380, 0.15326, and 0.13806, respectively. The difference between the mean log10 SPC and the mean log10 Petrifilm SM results was 0.027. For the VRBA and Petrifilm VRB methods, the mean log10 difference was 0.013. These results generally indicate the suitability of the dry rehydratable film methods as alternatives to the SPC and VRBA methods for milk samples. The methods have been adopted official first action.     

Recovery of Salmonella species from nonfat dry milk rehydrated under rapid and reduced pre-enrichment conditions: collaborative study

A collaborative study was conducted to compare the relative efficiency of the AOAC rapid rehydration method with the reduced rehydration soak method for the recovery of Salmonella species from nonfat dry milk (NFDM). In the AOAC method, a 25 g sample of NFDM is rapidly rehydrated at a 1:9 sample/water ratio and mixed by swirling. After 60 min, the flask contents are adjusted to a pH of 6.8, and 0.45 mL of 1% aqueous brilliant green dye solution is added. The flasks are then incubated at 35 degrees C. In the soak method, a 25 g sample of NFDM is gently added to the sterile brilliant green (BG) water at a 1:9 sample/BG water ratio and allowed to soak undisturbed for 60 min at room temperature before incubation. Twelve collaborators analyzed 3 shipments of samples with the following results for the AOAC and soak methods: shipment 1-31 and 46 positive samples, respectively, with a 48% increase in detection by the soak method; shipment 3-45 and 66 positive samples, respectively, with a 47% increase in detection by the soak method; shipment 2--no significant difference in recovery of Salmonella species by the 2 methods. It is recommended that the official final action method for the detection of Salmonella species, 46.054-46.067, be revised to use the soak method for the analysis of nonfat dry milk.