Casein content of human milk

Three methods for estimating the casein content of human milk were tested; isoelectric precipitation with washing and correction for co-precipitating proteins, sedimentation by ultracentrifugation, and indirect analysis (ie analyzing for the content of the major whey proteins and subtracting these from the total protein content). Gel electrophoresis and amino acid analysis were used to confirm some of the results. The casein content (mg/ml) of mature human milk (n = 9) was 2.33 +/- 1.69 by isoelectric precipitation, 1.80 +/- 0.48 by sedimentation and 2.96 +/- 1.08 by the indirect approach. A probable partition of nitrogen in breast milk would be casein N: whey protein N: non-protein N of 20:50:30; ie the correct ratio of casein nitrogen: whey nitrogen is approximately 20:80. Analysis of trace elements and minerals demonstrates that of total Ca 10%, Mg 5%, Zn 28%, Cu 17%, and Fe 27% is bound to casein when prepared by ultracentrifugation while isoelectric precipitation causes a redistribution of some of these elements. Since the protein ratio of human milk is considered a guideline when manufacturing infant formulas, these findings should be considered with regard to infant nutrition.     

Determination of total proteins in cow milk powder samples: a comparative study between the Kjeldahl method and spectrophotometric methods

In the present paper, a comparative study between the Kjeldahl method and several spectrophotometric methods was carried out for the determination of total proteins in a range of milk powder samples (skim milk powder, whole milk powder, whey protein powder, buttermilk powder). The most important finding of this paper was that the Bradford method could be used for the determination of total proteins in skim milk powder and whole milk powder samples (without extraction of lipids) instead of the Kjeldahl method. The Bradford method showed the highest sensitivity of the spectrophotometric methods. Using casein and BSA as standard proteins, the Lowry method showed the lowest variation of specific absorbance indicating either casein or BSA could be used as a standard. The UV-220 nm method with previous extraction of lipids showed the best results for the determination of total proteins in all the samples; all the results were not statistically different (P>0.05) from those obtained by the total protein nitrogen (TPN) without extraction of the lipids. The Bradford method (without extraction of lipids) showed the best results for the determination of total proteins in all the samples whose results were not statistically different (P>0.05) from those obtained for total nitrogen by the Kjeldahl method. However, when these results were compared to TPN they were statistically different from each other (P<0.05) for the buttermilk powder and whey protein powder samples. This means that the high sensitivity of the Bradford method was enough to overcome the turbidity of solutions due to the fat material in the samples. The determination of total proteins using the Bradford method for the whole milk powder and whey protein powder samples with and without the extraction of lipids was not statistically different (P<0.05) from each other.     

Quantitation of the major whey proteins in human milk,and development of a technique to isolate minor whey proteins

Several whey proteins of human milk have important nutritional and physiological roles for the breast fed infant. Only 75 to 85% of the proteins in whey have been identified and quantitated, leaving a remaining protein fraction containing many proteins of probable nutritional and physiological significance. In this study, colostrum and mature milk samples were collected, and the concentrations of five major whey proteins were measured using immunoelectrophoresis or immunodiffusion. Based on Kjeldahl nitrogen analysis, the total concentration of whey proteins in colostrum was 17.05 mg/ml and in mature milk 5.95 mg/ml. The concentration of α-lactalbumin was 3.7 mg/ml and 1.7 mg/ml in colostrum and mature milk, respectively; lactoferrin concentration was 7.3 and 1.5 mg/ml; and secretory IgA concentration was 4.7 and 1.2 mg/ml. In both colostrum and mature milk, concentrations of serum albumin and lysozyme were 0.37 and 0.07 mg/ml, respectively. The sum of these major whey proteins expressed as a percentage of the total whey protein accounted for 81% of mature whey protein, and 94% of colostral whey protein, leaving 19% and 6% in the remaining protein fraction. A method was developed to isolate this remaining protein fraction from the major whey proteins using gel filtration and immunoaffinity chromatography. The quantitatively minor whey protein fraction obtained was further characterized using polyacrylamide gel electrophoresis and ion exchange chromatography.     

Human-milk proteins: analysis of casein and casein subunits by anion-exchange chromatography, gel electrophoresis, and specific staining methods.

Casein in human milk is believed to serve several biological functions in newborns. However, the content and subunit composition of human casein has so far received little attention. We recently developed a method to separate human-milk whey and casein by adjustment of whole human milk to pH 4.3 and addition of calcium followed by ultracentrifugation. In this study we analyzed and evaluated human casein prepared by different methods. We used fast protein liquid chromatography (FPLC) with an anion-exchange column (Mono-Q) and polyacrylamide gradient gel electrophoresis techniques to analyze the casein subunit composition. Total casein in human milk, as determined by the Kjeldahl method, varies during lactation; the casein content is approximately 20% of the total protein content in early lactation and 45% in late lactation. We found differences in both glycosylation and phosphorylation patterns of kappa-caseins and beta-caseins from premature and term milk samples.     

Nutrient composition of banked human milk in Brazil and influence of processing on zinc distribution in milk fractions

OBJECTIVE AND METHODS: We measured the contents of fat, protein, lactose, calcium, phosphorus, zinc, iron, copper, and vitamin A in processed mature milk samples (individual, n = 60, and pooled, n = 10) from a reference human milk bank in Brazil and assessed the effect of pasteurization followed by freezing on the nutrient composition and the pattern of zinc distribution in fractions (fat, whey, and casein) of milk samples (n = 15). RESULTS: Mean nutrient concentrations were within expected ranges in mature milk from healthy women, except fat, which was lower. Interindividual variability of nutrient concentrations was high (coefficient of variation, 21-62%) but reduced overall in pooled samples. Processing of milk samples did not affect the nutrient contents but did cause a significant shift (P < 0.04) in the relative distribution of zinc, with a decrease in the whey fraction and an increase in the fat fraction. CONCLUSIONS: Redistribution and possible alterations in the zinc-binding pattern during processing in human milk banks may reduce zinc bioavailability to the infant.     

Manganese binding proteins in human and cow's milk

Manganese nutrition in the neonatal period is poorly understood, due in part to a lack of information on the amount of manganese in infant foods and its bioavailability. Since the molecular localization of an element in foods is one determinant of its subsequent bioavailability, we have studied the binding of manganese in human and cow's milk. An extrinsic label of 54Mn was shown to equilibrate isotopically with native manganese in milks and formulas. Milk samples were separated into fat, casein and whey by ultracentrifugation. In human milk, the major part (71%) of manganese was found in whey, 11% in casein and 18% in the lipid fraction. In contrast, in cow's milk, 32% of total manganese was in whey, 67% in casein and 1% in lipid. Within the human whey fraction, most of the manganese was bound to lactoferrin, while in cow's whey, manganese was mostly complexed to ligands with molecular weights less than 200. The distribution of manganese in formulas was closer to that of human milk than of cow's milk. The bioavailability of manganese associated with lactoferrin, casein and low molecular weight complexes needs to be assessed.     

A low-molecular-weight factor in human milk whey promotes iron uptake by Caco-2 cells

The iron bioavailability of human milk (HM) is substantially greater than that of cow's milk (CM), but the factor responsible for this high bioavailability is unknown. This study evaluated the effects of various HM and CM fractions on iron bioavailability. Milk was separated into fat, casein and whey fractions by ultracentrifugation. Whey was further fractionated by ultrafiltration with a 10-kDa membrane to produce a 10-kDa retentate (10kR) and a 10-kDa filtrate (10kF). Samples were prepared by mixing various combinations of the fractions, bringing the samples to prefractionation weight with minimum essential medium (MEM), and adding iron (10 micro mol/L) as ferrous sulfate. Samples were divided into two aliquots: one was subjected to in vitro digestion, the other was not. Bioavailability was assessed by applying the samples to Caco-2 cell monolayers and incubating for 24 h. Ferritin formation in the cells was used as an index of iron uptake. Removing the fat from undigested HM samples doubled the ferritin formation, but removing the whey or casein had no effect. Results with digested HM samples were similar, except that removing the whey decreased ferritin formation by 48%. Removing the fat from digested CM samples had no effect, but removing the casein doubled the ferritin formation. Removing the 10kF from HM reduced ferritin formation by 60%, but removing the 10kR had no effect. These data suggest that a low-molecular-weight factor (<10 kDa) in human milk enhances iron absorption.     

Binding form of vitamin B2 in bovine milk: its concentration, distribution and binding linkage

The contents of total, free, and bound vitamin B2 (B2) in bovine milk and their distribution in four separate milk fractions, including milk during the early lactation stage, were estimated. The total B2 content in whole mature milk was 179 +/- 25 micrograms/100 g (n = 16), and its distribution in the cream, whey, skim milk membrane, and casein fractions was 6, 67, 9, and 18%, respectively. The amount of flavins bound to protein in the total B2 was 13.6% in whole milk and rich in membrane fraction. The total B2 content (micrograms/100 g of milk) was higher in colostrum at 1-3 days (287 +/- 120) than in colostrum at 4-7 days (173 +/- 27), in transitional milk (182 +/- 33), and in mature milk (179 +/- 44). The bound flavin content decreased slightly as lactation progressed (20-30 micrograms/100 g), but the ratio of bound/total B2 did not vary (12-15%). Milk fat globule membrane (MFGM) contained 414 +/- 65 micrograms of B2/g of protein, most of it being bound to protein (92%). Market milks contained as much total B2 as raw whole milk, but the amount of bound form was only 2%. Guanidine HC1, urea, sodium dodecyl sulfate, pH at 3.0-3.5, delipidation, and boiling released most of the B2 bound to protein, suggesting that bound flavins bind to milk proteins by a hydrophobic linkage.     

Human milk proteins: separation of whey proteins and their analysis by polyacrylamide gel electrophoresis, fast protein liquid chromatography (FPLC) gel filtration, and anion-exchange chromatography

Human milk proteins are of nutritional and physiological significance to the newborn infant. To further study these proteins, a rapid procedure to separate and analyze human milk whey proteins was developed using fast protein liquid chromatography (FPLC). First, to separate whey proteins from casein, different variables such as low- or high-speed centrifugation at different temperatures with or without adjustment of pH to 4.6 or 4.3 and with or without addition of calcium to whole milk or skim milk were tested. Each variable was evaluated by gel filtration, anion-exchange chromatography, sodium dodecyl sulphate (SDS)-polyacrylamide gradient gel electrophoresis, immunoelectrophoresis, and immunodiffusion. The optimum method for a discrete separation of whey and casein is the adjustment of whole milk to pH 4.3 with addition of 60 mmol calcium/L, followed by ultracentrifugation. Rapid and sensitive separation and analysis of whey proteins was achieved by FPLC gel filtration and anion-exchange chromatography.     

Comparison of solubility of pea protein hydrolysate by three analytical methods

Pea protein hydrolysate was obtained by enzymatic hydrolysis with trypsin. The degree of hydrolysis (DH) was controlled by using the pH-stat method. Solubility of the trypsin-treated hydrolysate was tested at nine different pH values starting from 2 up to 10. Protein determinations were carried out using Kjeldahl, Lowry and modified Lowry methods. The results revealed that samples analysed with either the Kjeldahl or Lowry method gave similar values. However, systematic consistent differences existed for those results obtained by the Kjeldahl and the modified Lowry as well as between those results obtained by the Lowry and the modified Lowry.     

Comparison of solubility of pea protein hydrolysate by three analytical methods

Pea protein hydrolysate was obtained by enzymatic hydrolysis with trypsin. The degree of hydrolysis (DH) was controlled by using the pH-stat method. Solubility of the trypsin-treated hydrolysate was tested at nine different pH values starting from 2 up to 10. Protein determinations were carried out using Kjeldahl, Lowry and modified Lowry methods. The results revealed that samples analysed with either the Kjeldahl or Lowry method gave similar values. However, systematic consistent differences existed for those results obtained by the Kjeldahl and the modified Lowry as well as between those results obtained by the Lowry and the modified Lowry.     

Postprandial changes in the content and composition of nonprotein nitrogen in human milk

The effect of a meal on human milk (HM) total nitrogen (TN) and nonprotein nitrogen (NPN) content and composition was examined. Two studies were performed in which milk and blood samples were collected 2-3 h after subjects consumed either a test breakfast or lunch. To monitor the rate of transfer of plasma urea into milk, two women were given [15N]2-urea with the meal. Milk TN concentrations were not significantly different from premeal values. However, concentrations of milk NPN, urea nitrogen, and alanine were increased by greater than or equal to 20% over premeal values. [15N]2-Urea appeared in plasma and milk within 15 min and reached maximum enrichments of 10% and 5.5% in plasma and milk, respectively. Several HM NPN components increase in concentration postprandially; however, these concentrations were not always correlated with changes in plasma concentrations, suggesting that milk NPN may also reflect metabolic activities within the mammary gland.     

Proteomic Analysis of Human Milk Reveals Nutritional and Immune Benefits in the Colostrum from Mothers with COVID-19

Despite the well-known benefits of breastfeeding and the World Health Organization’s breastfeeding recommendations for COVID-19 infected mothers, whether these mothers should be encouraged to breastfeed is under debate due to concern about the risk of virus transmission and lack of evidence of breastmilk’s protective effects against the virus. Here, we provide a molecular basis for the breastfeeding recommendation through mass spectrometry (MS)-based proteomics and glycosylation analysis of immune-related proteins in both colostrum and mature breastmilk collected from COVID-19 patients and healthy donors. The total protein amounts in the COVID-19 colostrum group were significantly higher than in the control group. While casein proteins in COVID-19 colostrum exhibited significantly lower abundances, immune-related proteins, especially whey proteins with antiviral properties against SARS-CoV-2, were upregulated. These proteins were detected with unique site-specific glycan structures and improved glycosylation diversity that are beneficial for recognizing epitopes and blocking viral entry. Such adaptive differences in milk from COVID-19 mothers tended to fade in mature milk from the same mothers one month postpartum. These results suggest that feeding infants colostrum from COVID-19 mothers confers both nutritional and immune benefits, and provide molecular-level insights that aid breastmilk feeding decisions in cases of active infection.     

Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS

Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants.

The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.     

Vitamin K in colostrum and mature human milk over the lactation period--a cross-sectional study

Vitamin K was quantitated in the milk of four groups of 15 mothers from 1 d to 6 mo postpartum in a cross-sectional study. Concentrations were 7.52 +/- 5.90 and 6.36 +/- 5.32 nmol/L (3.39 +/- 2.66 and 2.87 +/- 2.40 micrograms/L) in colostrum and mature milk, respectively. Differences between colostrum and mature milk or among samples of mature milk collected at 1, 3, and 6 mo were not statistically significant. Because of significantly increased volumes of milk over the lactation period, approximately twice as much vitamin K was delivered in mature milk as in colostrum. Within normal ranges, concentrations of vitamin K in milk were not predicted by dietary intake of vegetables or fat. Vitamin K was correlated with fat in colostrum and was localized in the lipid core of the milk fat globule but was not associated with membranes. Vitamin K in human milk is insufficient to meet recommended intakes for infants aged less than 6 mo. Population and clinical studies are needed to assess the vitamin K status of exclusively breast-fed infants and to evaluate current recommendations.     

Comparative Study of Cadmium Transfer in Ewe and Cow Milks During Rennet and Lactic Curds Preparation

Cadmium transfer from whole milk to cream, rennet, or lactic curds was studied before and following a repeated oral cadmium administration to three lactating ewes and one cow. Before cadmium administration, the cadmium levels in milk were around 0.4 μg/L in ewes and less than 0.2 μg/L in cow. Throughout cadmium administration the mean cadmium levels in milk were 3.3 ± 1.4 μg/L in ewes and 2.5 ± 1 μg/L in cow. During cadmium administration, 86% of cadmium in ewe milk was dispersed in the skimmed milk and 17% in the cream, whereas only 72% was dispersed in the cow skimmed milk and 27% in the cow cream. Most of milk cadmium was associated with casein fractions. About 70% of milk cadmium was transferred to the rennet or lactic curds of ewe. The remaining cadmium present in whole milk, about 9%, was transferred to the rennet or lactic curd whey. In cow, the proportion of cadmium associated with rennet or lactic curds, rennet curd whey, and lactic curd whey was, respectively, 60%, 56%, 14% and 12% of total milk cadmium. The fraction of total cadmium transferred from milk to its milk products, whatever the species, ranged from 94% to 103%. The factor of concentration of cadmium from whole milk to milk products ranged from three to six. We suggest that the excretion of cadmium into milk is mainly achieved via the milk casein secretion. This is, to our knowledge, the first in vivo study where the cadmium transfer from milk to its milk products after repeated cadmium oral administration to ewe and cow has been studied. Received: 27 October 1998/Accepted: 1 April 1999     

High-calcium diet with whey protein attenuates body-weight gain in high-fat-fed C57Bl/6J mice

An inverse relationship between Ca intake and BMI has been found in several studies. It has been suggested that Ca affects adipocyte metabolism via suppressing 1,25-dihydroxycholecalciferol (1,25(OH)2-D3) and decreases fat absorption. We studied the effect of Ca and milk proteins (whey and casein) on body weight in C57Bl/6J mice. Male mice, age 9 weeks, were divided into three groups (ten mice per group) receiving modified high-fat (60% of energy) diets. Two groups received a high-Ca diet (1.8% calcium carbonate (CaCO3)), with casein or whey protein (18% of energy), and one group received a low-Ca diet (0.4% CaCO3) with casein for 21 weeks. Food intake was measured daily and body weight twice per week. Body fat content (by dual-energy X-ray absorptiometry) of all mice and faecal Ca and fat excretion of seven mice/group were measured twice during the study. Final body weight (44.1 (SEM 1.1) g) and body fat content (41.6 (SEM 0.6) %) were significantly lower (P < 0.05) in the high-Ca whey group than in the low-Ca casein group (48.1 (SEM 0.8) g and 44.9 (SEM 0.8) %). Body weight and body fat content of the high-Ca casein group did not differ significantly from the low-Ca casein group even though serum 1,25(OH)2-D3 levels were significantly lower (P < 0.001) in both high-Ca groups than in the low-Ca casein group. Thus changes in serum 1,25(OH)2-D3 do not seem to affect body weight in this animal model. There was a significant difference in fat excretion between the high-Ca whey and low-Ca casein groups (3.9 (SEM 0.9) % in the high-Ca whey v. 1.4 (SEM 0.2) % in the low-Ca casein group; P < 0.05), which may partly explain the effect on body weight.     

THE NUTRITIONAL VALUE OF BREAST MILK FROM NON-PREGNANT MOTHERS

The whey proteins and secretory immunoglobulin A pattern of breast milk from non-biological mothers in the first days of relactation bear more resemblance to transitional and mature human milk than to colostrum.     

Infant formulas with increased concentrations of alpha-lactalbumin

Human and bovine milk differ substantially in the ratio of whey to casein protein (approximately 60:40 in human milk and approximately 20:80 in bovine milk) and in the proportions of specific proteins. Although current infant formulas closely mimic the ratio of total whey to casein inhuman milk, the concentration of a-lactalbumin (the dominant protein in human milk) is relatively low in formula, whereas beta-lactoglobulin, a protein not found in human milk, is the most dominant whey protein in formula. Because of the differences in the protein profiles of human milk and infant formula, amino acid profiles also differ. To meet all essential amino acid requirements of infants, formula concentrations of protein must be higher than those in human milk. Recently, whey sources with elevated concentrations of alpha-lactalbumin have become available, which permitted the development of formulas with increased concentrations of this protein and decreased concentrations of beta-lactoglobulin. alpha-Lactalbumin is rich in tryptophan, which is typically the limiting amino acid in formula, and as a result, formulas have been developed with lower protein but higher tryptophan concentrations. This type of formula may offer a number of advantages to the neonate, which include producing plasma tryptophan concentrations equal to those found in breastfed infants and obviating the need for the body to dispose of excess nitrogen loads.     

Isolation of the nonprotein nitrogen fraction from human milk by gel-filtration chromatography and its separation by fast protein liquid chromatography

Human milk (HM) is unique compared with the milk of other species in that nonprotein nitrogen (NPN) constitutes 20-25% of the total N. The NPN fraction consists of a diverse group of compounds with molecular masses less than 10,000 Da (in the picogram to microgram per milliliter range), which have only been partially characterized. We developed a methodology to separate and concentrate the NPN fraction for further analysis. NPN was initially separated from other milk components by Sephadex G-25 gel filtration. Further isolation and separation was carried out by fast protein liquid chromatography gel filtration and ion-exchange chromatography. Molecular masses of unknown peaks were determined by using known molecular mass markers and standards. The methodologies developed lead to the discrete separation of NPN from other milk compounds and can be particularly valuable for isolating peptides in HM.