Effect of heat treatment on denaturation of bovine alpha-lactalbumin: determination of kinetic and thermodynamic parameters

The effect of heat treatment on the denaturation of alpha-lactalbumin was studied, under different conditions, over a temperature range of 78-94 degrees C. The concentration of the residual immunoreactive protein after different treatments was determined by kinetic analysis, obtaining D and Z values. Thermodynamic parameters were also calculated. Denaturation of alpha-lactalbumin, measured by the loss of immunoreactivity, could be described as an order of reaction of n = 1.5. Results obtained indicated that alpha-lactalbumin was more heat-sensitive when treated in milk than in phosphate buffer. The protein was also denatured more rapidly in the apo form than in the calcium-saturated form. Besides, the thermal stability of apo-alpha-lactalbumin decreased with the binding of oleic acid.     

Direct optical biosensor analysis of folate-binding protein in milk

An automated, rapid, sensitive, and label-free biosensor-based assay for folate-binding protein (FBP) in bovine milk utilizing surface plasmon resonance optical detection is described. The active concentration of FBP is estimated from its specific interaction with a pteroyl-l-glutamic (folic) acid derivative immobilized on the sensor surface in a direct binding assay format. Milk, colostrum, and milk powders are prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration, have been defined, and nonspecific binding considerations were evaluated. Performance parameters include a working range for FBP in buffer of 0-200 ng/mL, a method detection limit of 0.13 microg/mL in fluid milk, overall instrument response RSD(R) of 0.64%, a mean interassay RSD(R) of 7.3% for skim milk powder, and surface stability over ca. 200 samples. The technique was applied to the measurement of active FBP content of consumer milks, the heat classification of skim milk powders manufactured under a wide range of thermal processing protocols, and change during early bovine lactation.     

Determination of taurine in infant formulas using ultrafiltration and cation-exchange chromatography

A fast and simple method for determination of taurine in infant formulas has been developed. The sample preparation uses disposable ultrafiltration cartridges to remove protein and clarify the sample. Hydrolysis is avoided, simplifying the procedure and increasing efficiency. One mL sample is centrifuged in a cartridge for 45 min. The filtrate is diluted with pH 2.2 citrate buffer and injected into a high performance amino acid analyzer. A cation-exchange column (sodium phase) is used with a single buffer eluant and an isocratic chromatographic program. Colorimetric detection is performed following post-column ninhydrin reaction. Chromatographic resolution from other ninhydrin-positive compounds is excellent. Average recoveries for 3 levels of spike for various products were 100-102%. Precision is 1-3% RSD, depending on product. Linearity, specificity, and ruggedness are excellent. The method is applicable to quality control testing of milk-based, soy-based, and prehydrolyzed protein-based infant formulas in the ready-to-use, concentrate, and powder forms. A variety of commercially available infant formulas from different manufacturers were analyzed and all were found to contain taurine levels comparable to human milk. Some human milk and cow's milk samples were also analyzed and results compare well with literature values.     

Determination of the heat treatment undergone by milk by following the denaturation of alpha-lactalbumin with a biosensor

Dairy industries are interested in knowing the heat treatment undergone by milk so as to control the quality of drinking milks or to control their heating systems. Among the different techniques available to characterize the heat treatment of milk, estimation of the denaturation of proteins has been widely used. However, because the concentration of the proteins in raw milk can fluctuate significantly, determining only the concentration of a native protein without knowing its concentration in the raw milk before undergoing heat treatment can lead to significant imprecision. The objective of this study was to develop, on Biacore 3000, a biosensor assay for determining the denaturation index of alpha-lactalbumin by quantifying separately the native and "heat-denatured" forms of alpha-lactalbumin with specific monoclonal antibodies. alpha-Lactalbumin denaturation index is independent of the concentration of alpha-lactalbumin in the original raw milk. The technique developed is discriminating, fast, repeatable, fully automated, and requires no pretreatment of the milk sample.     

Characterization of the heat treatment undergone by milk using two inhibition ELISAs for quantification of native and heat denatured alpha-lactalbumin

Dairy industries are interested to know the heat treatment undergone by milk for controlling the quality of drinking milks or to control their heating systems. The purpose of this work was to develop a specific and sensitive technique for classification of the heat treatment a milk has been submitted to, without disposing of the original raw milk. For this purpose, alpha-lactalbumin was chosen as a bioindicator of heat treatment, and monoclonal antibodies specific for its native or heat-denatured form were raised and used in two inhibition ELISAs. ELISA allowed differentiation among raw, pasteurized, ultrahigh-temperature-treated, and sterilized milks without even having to know the alpha-lactalbumin concentration of the original raw milk. However, this technique was more suitable for intense heat treatments such as UHT treatment and sterilization because of the heat stability of alpha-lactalbumin.     

Investigation of the lactosylation of whey proteins by liquid chromatography-mass spectrometry

Heat treatment of milk induces a reaction between the milk proteins and lactose, resulting in lactosylated protein species. The lactosylation of the two major whey proteins alpha-lactalbumin and beta-lactoglobulin was investigated by reversed phase liquid chromatography-mass spectrometry (LC-MS). Three sample series, consisting of aqueous model solutions of each whey protein separately and in mixture and whole milk, were heated for different time periods, and the progression of the lactosylation reaction was monitored. The observed degrees of lactosylation and the reaction kinetics showed that the lactosylation of beta-lactoglobulin was not influenced by the presence of other components, whereas the lactosylation of alpha-lactalbumin was enhanced in whole milk compared to the aqueous model systems. An in-depth evaluation of the LC-MS data yielded information regarding changes of physicochemical properties of the whey proteins upon lactosylation. Whereas retention time shifts indicated changes in hydrophobicity for both alpha-lactalbumin and beta-lactoglobulin, changes in the charge state distribution denoting conformational alterations were observed only for beta-lactoglobulin. The analysis of different liquid and solid milk products showed that the lactosylation patterns of the whey proteins can be used as indicators for the extent of heat treatment.     

Changes in structures of milk proteins upon photo-oxidation

Changes in protein structures as a result of riboflavin-induced photo-oxidation were studied for six milk proteins: alpha-casein, beta-casein, kappa-casein, lactoferrin, alpha-lactalbumin, and beta-lactoglobulin. The milk proteins showed significant variability in sensitivity to photo-oxidation. After photo-oxidation, an increase in carbonyl content because of oxidation of tryptophan, histidine, and methionine, as well as formation of dityrosine, was observed for all proteins studied, although at very different levels. Generally, the increment was highest for alpha- and beta-casein and was lowest for lactoferrin. Loss of tryptophan because of photo-oxidation was well-correlated with the formation of the tryptophan oxidation products, N-formylkynurenine and kynurenine. Changes at the tertiary protein structure level were observed after photo-oxidation of the globular proteins, where tryptophan fluorescence emission indicated unfolding of alpha-lactalbumin and beta-lactoglobulin, whereas lactoferrin achieved a more compact tertiary structure. Changes in secondary structure were observed for alpha-lactalbumin and beta-lactoglobulin, whereas the secondary structure of lactoferrin did not change. Polymerization of alpha- and beta-casein and of lactoferrin was observed, whereas kappa-casein, alpha-lactalbumin, and beta-lactoglobulin showed little tendency to polymerize after photo-oxidation. Lability toward photo-oxidation is discussed according to the structural stabilities of the globular proteins.     

Monitoring of five sulfonamide antibacterial residues in milk by in-tube solid-phase microextraction coupled to high-performance liquid chromatography

A simple, rapid, and sensitive method for the quantitative monitoring of five sulfonamide antibacterial residues in milk was developed by coupling in-tube solid-phase microextraction (SPME) to high-performance liquid chromatography with an ultraviolet detector. A poly(methacrylic acid-ethylene glycol dimethacrylate) monolithic capillary column was selected as the extraction medium for this on-line technique. To obtain optimum extraction efficiency, several parameters relating to in-tube SPME were investigated. By simple extraction with ethanol, dilution with phosphate buffer solution, and centrifugation, the sample solution then could be directly injected into the device for extraction. The calculated detection limits for sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium were 2.0, 2.8, 1.7, 2.5, and 22 ng/mL, respectively. The method was linear over the range of 20-5000 ng/mL (100-5000 ng/mL for sulfacetamide sodium) with a correlation coefficient R (2) value >0.9980. Excellent method reproducibility was found by intra- and interbatch precisions, yielding the relative standard deviations of <10.0 and <9.94%, respectively. The proposed method was proved to be robust in monitoring sulfadiazine, sulfamethazine, sulfamethoxazole, sulfamonomethoxine sodium, and sulfacetamide sodium residues in milk.     

Effect of pH on the thermal denaturation of whey proteins in milk

The effect of pH on thermal denaturation of four main whey protein fractions in skim milk was examined by gel permeation FPLC. On heating skim milk at 80 degrees C for 0.5-20.0 min over the pH range 5.2-8.8, the extent of denaturation, based on loss of solubility at pH 4.6, increased with heating time and was usually in the order immunoglobulins > serum albumin/lactoferrin > beta-lactoglobulin > alpha-lactalbumin. Rates of denaturation of the immunoglobulins and the serum albumin/lactoferrin fraction were highest at the lower end of this pH range, whereas those of beta-lactoglobulin and alpha-lactalbumin increased over most of the pH range. The effects of pH, addition of Ca, and reduction of disulfide bonds on the rates of the unfolding and aggregation stages of denaturation are discussed.     

Reduction of stale flavor development in low-heat skim milk powder via epicatechin addition

The influence of epicatechin (EC) on off-flavor development in low-heat skim milk powder samples during processing and storage was investigated. Milk powder samples were prepared from a concentrated skim milk (control) plus a concentrated skim milk with EC (treatment). Volatile extracts of the powders were analyzed by aroma extract dilution analysis (AEDA) at 0 days and after 17 months of storage in conjunction with sensory analysis of the flavor attributes. The treatment milk powders with EC added prior to drying reported a reduction in the formation of three main compounds, 4-hydroxy-2,5-dimethyl-3-(2H)-furanone, o-aminoacetophenone, and furfural, by 8-, 4-, and 4-fold for the 0 day old samples, while for the 17 month aged samples o-aminoacetophenone was the major compound reduced in formation by 8-fold, respectively, based on the flavor dilution factors reported. The sensory evaluations indicated that the treatment milk powders for 0 day old and 17 month aged samples were statistically lower (alpha = 0.05) in stale flavor intensity in comparison to the respective control samples, while no differences were noted in bitterness intensity.     

Rapid fingerprinting of milk thermal processing history by intact protein mass spectrometry with nondenaturing chromatography

Thermal processing of foods results in proteins undergoing conformational changes, aggregation, and chemical modification notably with sugars via the Maillard reaction. This can impact their functional, nutritional, and allergenic properties. Native size-exclusion chromatography with online electrospray mass spectrometry (SEC-ESI-MS) was used to characterize processing-induced changes in milk proteins in a range of milk products. Milk products could be readily grouped into either pasteurized liquid milks, heavily processed milks, or milk powders by SEC behavior, particularly by aggregation of whey proteins by thermal processing. Maillard modification of all major milk proteins by lactose was observed by MS and was primarily present in milk powders. The method developed is a rapid tool for fingerprinting the processing history of milk and has potential as a quality control method for food ingredient manufacture. The method described here can profile milk protein oligomeric state, aggregation, and Maillard modification in a single shot, rapid analysis.     

Liquid chromatographic determination of sulfamethazine in milk

A simple, relatively rapid liquid chromatographic method has been developed for the determination of sulfamethazine (SMZ) in milk at levels in the low ppb range. The method is based on extracting SMZ from milk with chloroform, evaporating the chloroform, dissolving the residues in hexane, extracting into buffers, and chromatographing the buffer solution. The method has been shown to determine levels as low as 5 ppb reliably. Levels greater than or equal to 7 ppb have been confirmed by gas chromatography/mass spectrometry after derivatization of extracts from fortified, incurred, and shelf milk. Intralaboratory recoveries and percent coefficients of variation are satisfactory. Sulfadimethoxine and sulfaquinoxaline can also be determined by the method. Application of the method to other dairy products is being investigated.     

Preparation of antibodies and development of an enzyme-linked immunosorbent assay (ELISA) for the determination of doxycycline antibiotic in milk samples

This paper reports the development of an immunoassay for the specific analysis of doxycycline (DC), a congener of the tetracycline antibiotic family (TCs), in milk samples. This is the first time that DC antibody production is reported, based on a rationally designed and well-characterized immunizing hapten. The chemical structure of the immunizing hapten (13-[(2-carboxyethyl)thiol]-5-hydroxy-6-α-deoxytetracycline, TC1) was designed to maximize recognition of the tetracycline characteristic moiety defined as lower periphery of the TCs plus the region of the upper periphery composed by the hydroxyl group at position C(5) (B ring) and the dimethylamino group in ring A. Polyclonal antibodies raised against TC1 coupled to horseshoe crab hemocianyn (HCH) were used to develop a homologous indirect competitive enzyme-linked immunosorbent assay (ELISA). The microplate ELISA can detect DC in buffer down to 0.1 μg L(-1). The ELISA has been proven to tolerate a wide range of ionic strengths and pH values. The assay is very selective for DC with a minor recognition of methacycline (32% of cross-reactivity). Experiments performed with whole milk samples demonstrate that samples can be directly analyzed after a simple treatment method, reaching detectability values below 5 μg L(-1).     

Direct biosensor immunoassays for the detection of nonmilk proteins in milk powder.

The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.     

Front-face fluorescence spectroscopy allows the characterization of mild heat treatments applied to milk. Relations with the denaturation of milk proteins

Emission and excitation spectra of intrinsic fluorophores present in milk were used to evaluate changes in milk following thermal treatments in the 57-72 degrees C temperature range from 0.5 min up to 30 min. Alternatively, the concentrations of native alkaline phosphatase, lactoferrin, immunoglobulin G, bovine serum albumin, beta-lactoglobulin, and alpha-lactalbumin were determined in the same samples by enzymatic and immunochemical techniques. As principal component analysis applied to the normalized fluorescence spectra successfully discriminated different milk samples according to the temperature and time of thermal treatment, principal component regression was applied to predict the amounts of the native proteins investigated using fluorescence data. The results showed strong correlations between measured and predicted data for alkaline phosphatase and beta-lactoglobulin. This study has demonstrated that front-face fluorescence spectroscopy has a promising potential to become a rapid and nondestructive analytical technique for the evaluation of physicochemical changes in milk induced by low thermal treatment.     

Simultaneous determination of oxytetracycline, tetracycline, and chlortetracycline in milk by liquid chromatography

A simple, rapid procedure is described for the simultaneous quantification of 3 tetracycline drugs in bovine milk. Samples are prepared by dilution with an EDTA/phosphate buffer solution and filtration through a molecular weight cutoff filter. Analytes are concentrated on-column using a reverse-phase gradient elution system of oxalic acid, acetonitrile, and methanol. The limits of quantitation are approximately 15-50 ng/mL and the limits of detection are 10-20 ng/mL, depending on the compound. For oxytetracycline, over the range 50-1200 ng/mL, the average recovery and intralaboratory coefficient of variation were 97% and 4.1%, respectively. Over the same range, these parameters were, respectively, 97% and 5.0% for tetracycline, and 89% and 6.4% for chlortetracycline. The applicability of this procedure is demonstrated by separation and detection of incurred tetracycline residues in milk from treated 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.     

Luminex-based triplex immunoassay for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powder

An automated fluorescent microsphere-based flow cytometric triplex immunoassay, using the Luminex 100 flow analyzer with MultiAnalyte Profiling (xMAP) technology, was developed for the simultaneous detection of proteins from three vegetable sources as potential fraudulent adulterants in milk powder. In the final triplex inhibition immunoassay, soluble wheat proteins (SWP) and proteins from soy and pea were coupled to three different microsphere sets. A mixture of these microsphere sets was transferred to a microtiter plate well together with the sample and a mixture of three affinity-purified polyclonal antibodies raised against the proteins and labeled with a fluorophore (Alexa 532). After incubation for 1.5 h at room temperature in the dark, the fluorescence intensities on the microspheres were directly measured (no wash procedure) in the Luminex during 10 s per well (100 microspheres per set). The sensitivities of the three assays for plant protein extracts were determined as 0.5-0.6 microg/mL at 50% inhibition. For the detection of the vegetable proteins in milk powder, the samples were dissolved in buffer (0.1 g in 10 mL) and further diluted (20 times) to create a 50% inhibition at approximately 0.5% of the vegetable proteins in the total protein content of milk powder. With the help of calibration standards, prepared under conditions comparable to those for sample materials, the triplex immunoassay proved to be quantitative above 0.1%, although concentrations in high-heated milk powders were underestimated. Due to the xMAP technology, in which 100 different microsphere sets can be distinguished, this triplex immunoassay can easily be extended to detect other possible adulterants.     

Effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible thermal denaturation of beta-lactoglobulin and alpha-lactalbumin in skim milk

The effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible denaturation of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) in reconstituted skim milk samples was studied over a wide temperature range (75-100 degrees C). The irreversible thermal denaturation of beta-LG had a reaction order of 1.5 and that of alpha-LA had a reaction order of 1.0 in all systems and under all conditions. The rates of irreversible denaturation of beta-LG and alpha-LA were markedly dependent upon the composition of the milk. At all temperatures, the irreversible denaturations of beta-LG and alpha-LA were enhanced at a higher protein concentration and were retarded when the nonprotein-soluble components and lactose concentrations were increased. The effects of increasing the concentrations of lactose and nonprotein-soluble components were interpreted using the preferential hydration theory and allowed for the interpretation of the changes in the denaturations of beta-LG and alpha-LA when the milk total solids concentration was increased.     

Improved liquid chromatographic determination of riboflavin in milk and dairy products

Reported here is a simple liquid chromatographic (LC) method for the determination of riboflavin in milk (liquid, evaporated, and dry), yogurt, and cheese. The method involves passing liquid samples or filtrates of semisolid and solid samples through a C18 cartridge. Retained riboflavin is then eluted with an aliquot of 50% methanol in 0.02M acetate buffer of pH 4. A volume of the eluate is injected into the LC system consisting of a C18 column, a solvent of water-methanol-acetic acid (65 + 35 + 0.1, v/v) with a flow rate of 1 mL/min, and a UV detector set at 270 nm. The method is precise and accurate and compares favorably with the present AOAC method. Moreover, it involves fewer sample preparation steps and has a total analysis time of less than 1 h.