Direct determination of citric acid in milk with an improved pyridine-acetic anhydride method.

Summary The determination of citric acid with pyridine and acetic anhydride has been investigated at reaction temperatures from 17 to 60° C. The optimum proportions of pyridine, acetic anhydride, water, and acetic acid for maximum color intensity and stability are given for each temperature. The procedure has been modified to eliminate the violent nature of the reaction, even when the analysis is done at a reaction temperature of 60° C. Details of a method for the determination of 25–200 μ g. of citric acid, at a reaction temperature of 32° C., are presented. In comparison with previously published methods based on the reaction, the recommended technique results in improved sensitivity, stability, and reproducibility without requiring careful timing. The method has been successfully applied to the routine analysis of milk and milk products. Milk and serum can be analyzed directly, after suitable dilution. Corrections for the interference caused by fat in homogenized milk, and by trichloroacetic acid in T.C.A. serum, can be made easily. Results of direct analysis of milk were from 5 to 15% higher than those for the corresponding sera and are believed to represent the true values for the citric acid content of milk

Solubility of Tricalcium Citrate in Solutions of Variable Ionic Strength and in Milk Ultrafiltrates

Summary Solubility of tricalcium citrate has been determined at 21 and 95° C. by dissolution in water and by precipitation from supersaturated solutions containing various proportions of calcium to citrate and at pH 4.4 to 8.8. Solubility product in solutions at equilibrium varied with ionic strength, according to the relation pks = 17.63 − 10.84 μ , but was unaffected by variations in pH and temperature and by the presence of magnesium or phosphate ions. Milk ultrafiltrates were shown to be saturated with tricalcium citrate, i.e., the calculated pk s values agreed with the solubility product in all but two of the 15 samples tested, and composition of two ultrafiltrates was unaffected by agitation with crystals of tricalcium citrate

Direct Analysis of Lactose in Milk and Serum

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Analysis for Citric Acid in Presence of Casein

Summary Factors affecting citric acid determination in the presence of casein were studied with the pyridine-acetic anhydride method. Although the opalescence in skimmilk tests affected results by only 2%, the turbidity introduced by solutions of isolated casein could cause gross overestimation. A pH above 12.3 in the test aliquot assured a clear reaction mixture with both skimmilk and casein solutions. It is, therefore, recommended that test aliquots containing casein should comprise sufficient alkali to insure a clear reaction. Acid casein required seven resuspensions in fresh washing solution at pH 4.5 for complete removal of occluded citrate, lactose, and calcium. Adsorption of citric acid by casein precipitated at the iso-electric point, or with trichloroacetic acid, was demonstrated and appears to account for the lower citrate concentration in acid sera, as compared to skimmilk. These results extend the conclusions of a previous paper

Incomplete Release of Citrate During Acid Precipitation of Casein

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Sialic Acid as an Index of the κ-Casein Content of Bovine Skimmilk

Summary The sialic acid content of skimmilk proteins has been examined, using a modification of Warren's thiobarbituric acid method. Lactose interferes with this method and was removed by thorough washing of the proteins. κ -casein and proteose-peptone appear to be the only two skimmilk proteins containing significant amounts of sialic acid. Treatments with DEAE cellulose almost completely removed the low concentrations of sialic acid present in α s - and β -caseins, and examination of these caseins by starch gel electrophoresis indicates that such removal involved traces of κ -casein. Since the stability of α s - κ -casein mixtures in the presence of calcium appeared to be directly related to the sialic acid content of the mixture at all levels below the 0.37% (uncorrected for moisture) needed to give complete stability, it is concluded that sialic acid measurements can be used as an index of the intact κ -casein content of such mixtures. Proteose-peptone is precipitated by 12% TCA, but remains in solution at pH 4.5, so analysis of sialic acid can also be used as an index of the κ -casein concentration of whole acid casein. Values of 0.26–0.59% sialic acid were obtained in washed whole acid caseins prepared from individual lots of milk, indicating that the proportion of κ -casein is variable. The sialic acid content of κ -casein appears to be close to 2.3%; on this basis, the proportion of κ -casein in whole acid casein varies between 11–26%

Effect of lactation stage and concurrent pregnancy on milk composition in the bottlenose dolphin

Although many toothed whales (Cetacea: Odontoceti) lactate for 2–3 years or more, it is not known whether milk composition is affected by lactation stage in any odontocete species. We collected 64 pooled milk samples spanning 1–30 months postpartum from three captive bottlenose dolphins Tursiops truncatus. Milks were assayed for water, fat, crude protein (TN × 6.38) and sugar; gross energy was calculated. Ovulation and pregnancy were determined via monitoring of milk progesterone. Based on analysis of changes in milk composition for each individual dolphin, there were significant increases (P<0.05) in fat (in all three dolphins) and crude protein (in two of three), and a decrease (P<0.05) in water (in two of three) over the course of lactation, but the sugar content did not change. In all three animals, the energy content was positively correlated with month of lactation, but the percentage of energy provided by crude protein declined slightly but significantly (P<0.05). At mid-lactation (7–12 months postpartum, n=17), milk averaged 73.0±1.0% water, 12.8±1.0% fat, 8.9±0.5% crude protein, 1.0±0.1% sugar, 1.76±0.09 kcal g−1 (=7.25 kJ g−1) and 30.3±1.3% protein:energy per cent. This protein:energy per cent was surprisingly high compared with other cetaceans and in relation to the growth rates of calves. Milk progesterone indicated that dolphins ovulated and conceived between 413 and 673 days postpartum, following an increase in milk energy density. The significance of these observed compositional changes to calf nutrition will depend on the amounts of milk produced at different stages of lactation, and how milk composition and yield are influenced by sampling procedure, maternal diet and maternal condition, none of which are known

Turbidimetric Micro-Determination of Magnesium in Milk

Summary From 5 to 40 μ g. of magnesium can be determined by measuring the turbidity formed when the potassium salt of erucic acid is added in the presence of potassium oxalate. Preliminary removal of calcium as oxalate is achieved rapidly at room temperature; other milk components do not interfere. Analysis of various samples of milk and serum by the present technique showed that, compared with ashed samples, direct analysis gave results which agreed within±3.3%. The method is simple, convenient, and suitable for routine analysis

Preparation of a Standard for Citric Acid Analysis

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Fractionation of κ-Casein on a Diethylaminoethyl Cellulose Column

Summary A complex chromatogram showing seven overlapping peaks was obtained from κ -casein by gradient sodium chloride elution (0 → 0.4 m), followed by 0.25 N sodium hydroxide, from a diethylaminoethyl cellulose column. The material recovered from the two peaks eluted most quickly contained no κ -casein, but contained several components with low mobility in starch gel electrophoresis (SGE), and low sialic acid and phosphate contents. A later fraction contained a high proportion of a casein resembling α s -casein, with κ -casein and some components that moved more rapidly than α s -casein in SGE. Recoveries from the four other peaks were mainly κ -caseins that differed in their elution characteristics but not in SGE patterns, sialic acid content, or phosphate content. One was not eluted with sodium chloride but was eluted in advance of the sodium hydroxide front; another was eluted with the sodium hydroxide front. Both latter peaks may represent an aggregated form of κ -casein. Chromatography achieved only limited purification and was accompanied by a deleterious aging of the κ -casein. This aging released a sialic acid-containing compound soluble in 12% trichloracetic acid, and lowered the ability of the κ -casein to stabilize α s -casein in the presence of calcium