Isolation and physical characterization of an exocellular polysaccharide.

The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40 were investigated. Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes. Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water. Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide. Fractions were analyzed by multiangle static light scattering in aqueous 0.10 M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 +/- 0.06).10(3) and (1.62 +/- 0.07).10(3) kg/mol, respectively, were calculated so that Mw/Mn approximately 1.13. The number-averaged radius of gyration was found to be 86 +/- 2 nm. From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained. Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 +/- 4 nm was calculated. Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 +/- 3 nm, as found with GPC.

Determination of denatured serum proteins in the casein fraction of heat-treated milk by capillary zone electrophoresis.

The amount of heat-denatured serum proteins in heat-treated milk could be estimated by analyzing the casein fraction, obtained by isoelectric precipitation at pH 4.6, by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at pH.3. Optimization of the sample and running buffer minimized adsorption of serum proteins, especially that of bovine serum albumin (BSA). This afforded a detection limit down to ca. 5-65 micrograms/mL of the three main serum proteins in milk. The detector response (UV at 214 nm) was linear in the range of 0.05-0.35 and 0.05-0.85 mg/mL for alpha-lactalbumin and beta-lactoglobulin, respectively. BSA showed a slightly less linear behavior, due to residual adsorption to the capillary wall. The recovery of serum proteins was in the range of 89-107%. The method was evaluated by analyzing Dutch commercial milks and cheese milk, which had received increasing heat loads. The addition of milk powder to pasteurized milk could be detected by this method as well as the serum protein to casein ratio in various products.

Selective detection in RP-HPLC of Tyr-, Trp-, and sulfur-containing peptides by pulsed amperometry at platinum.

A technique for electrochemical detection of Trp-, Tyr-, and sulfur-containing peptides, using a two-step potential waveform at a platinum wall-jet electrode, has been developed. The detection is fully compatible with reversed-phase HPLC employing gradients of acetonitrile in water/trifluoroacetic acid. At approximately +1.2 V (first potential) versus Ag/AgCl, Trp-, Tyr-, and Cys-containing peptides are predominantly detected, while at +1.4 - 1.6 V, Met- and (Cys)2-containing peptides are additionally detected. The electrode surface is cleaned by the second potential (+2.0 V). the linearity is at least 2 orders of magnitude. The sensitivity is in the picomole range. By using postcolumn electrochemical conversion, the selectivity toward Met and Cys-containing peptides can be enhanced. Applications are shown for the determination of caseinomacropeptide (6.6 kDa) and a tryptic map of beta-casein.

Determination of caseinomacropeptide with capillary zone electrophoresis and its application to the detection and estimation of rennet whey solids in milk and buttermilk powder.

The presence of rennet whey solids in milk powder and buttermilk powder could be detected by analyzing the caseinomacropeptide (6.7 kDa) content by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at low pH. Under these conditions, genetic A and B variants migrated as a single peak. This afforded a detection limit of 0.4% of rennet whey solids in combination with large volume injections and on-column isotachophoretic concentration of the sample. The detector response (UV at 214 nm) was linear in the range of 1-20% rennet whey solids and the recovery of caseinomacropeptide was 98%. Pseudo-caseinomacropeptide, lacking the N-terminal Met-residue, could be separated from caseinomacropeptide, thus preventing false positive results for some types of acid buttermilk powder.

Determination of milk proteins by capillary electrophoresis.

The determination of milk proteins by capillary zone electrophoresis (CZE) is hampered by the adsorption of the solute on the capillary wall. The effects of pH, ionic strength of the buffer and polymeric additives were studied in combination with a hydrophilically coated capillary. Optimum separations were obtained at low pH (2.5-3) in aqueous solutions containing 6 M urea and methylhydroxyethylcellulose, resulting in a complete separation of the serum proteins and caseins, including some genetic variants. The results were compared with those achieved with reversed-phase HPLC. With CZE, theoretical plate numbers in the range 300,000-700,000 were obtained. The relative standard deviations for migration times were below 0.08% and for peak areas were 2-4%. The separation of cow, goat and sheep milk proteins and of heat-damaged casein is reported.

Assessment of the capabilities of capillary zone electrophoresis for the determination of hippuric and orotic acid in whey.

A rapid method was developed for the simultaneous determination of hippuric and orotic acid in rennet whey by capillary zone electrophoresis using an uncoated capillary utilizing a 0.04 M amino-2-methyl-1,3-propanediol (AMPD)-N,N-bis(2-hydroxyethyl) glycine (BICINE) buffer (pH 8.8) with UV detection at 254 and 280 nm. Whey proteins were removed by ultrafiltration. The method was evaluated for external, internal and standard addition procedures for both peak areas and peak heights. The use of an internal standard (sorbic acid) eliminated injection errors and gave, when applied to peak areas, the same levels for hippuric and orotic acid in those obtained with high-performance liquid chromatography. Relative standard deviations were 1-2%. Peak heights gave erratic results owing to sample matrix effects on peak widths.

Determination of biogenic amines in cheese and some other food products by high-performance liquid chromatography in combination with thermo-sensitized reaction detection.

A simple high-performance liquid chromatographic (HPLC) analysis is described for biogenic amines in cheese and other food products. The sample clean-up consists of a precipitation-extraction step with trichloroacetic acid, which gives recoveries of amines in cheese in the range of 85-105%. The HPLC analysis is performed by reversed-phase ion-pair chromatography, using a ninhydrin-containing eluent, which eliminates the need for an extra reagent pump for the post-column derivatization. Band broadening is minimized by using a poly(tetrafluoroethylene) knitted tube reactor at 145 degrees C. The detection limit for amines is 2 mg/kg cheese and the method is linear for 0.1-4 micrograms of amine injected. Examples are given of the analysis of amines in cheese, wine and sauerkraut.

Study of the high-performance liquid chromatographic separation of reducing sugars, applied to the determination of lactose in milk.

The loss of reducing sugars by formation of Schiff bases on amino-bonded silica or on dynamically coated amino-silica columns was investigated at two column temperatures. Depending on the type, age and temperature of the column, these losses were in the range of 0-100%. A dimethylamino-bonded silica column did not cause loss of reducing sugars, but the retention of sugars was too weak to allow separation. The analysis of reducing sugars can be carried out on diol-modified silica with diisopropylethylamine in the eluent to enhance mutarotation. It is also possible to use a cation-exchange resin (Ca2+) column, equipped with a pre-column packed with a mixed-bed ion-exchange to remove interfering salts and acids. In combination with an acetate-acetonitrile sample clean-up, this method results in coefficients of variation of less than 1% for the determination of lactose in skim-milk.