Proteins and Non-protein Nitrogen in Human Milk

Stephanie Atkinson, Bo Lonnerdal

• Publisher: CRC Press
• Year: 2019
• ISBN:
  9781000722444
• Edition: 1
• Topic: Medicina
• Subtopic: Biochimica in medicina

For the first time, an entire publication has been dedicated to providing a critical review of the identification and analysis of the milk specific proteins such as lactalbumin, lactoferrin and casein; the non-milk specific proteins such as plasma and membrane proteins; and the minor nitrogen-containing components such as enzymes, hormones, and growth factors. Biological roles, whether nutritional, endocrinological or immunological, of the specific nitrogen compounds in mammary milk production and/or growth and development of the breast-fed infant are also presented. Identification of the molecular weight compounds that have led to questions about their function in milk and their inclusion in modern infant formulas is thoroughly discussed and of great value to scientists in sub-specialties of biochemistry, nutrition, physiology and immunology, as well as to pediatric practitioners with primary interests in the infant food industry, academia, or clinical nutrition. The thoroughness of each chapter, often providing an historical panorama of the specific aspect of milk composition, makes this book useful for both the uninitiated and expert audiences who are interested in advancing their knowledge of human milk biochemistry and its physiological significance to the recipient infant.

Section B: Proteins in Human Milk

Chapter 2

CASEIN MICELLES AND CASEIN SUBUNITS IN HUMAN MILK

Clemens Kunz and Bo Lönnerdal

TABLE OF CONTENTS

I.	Introduction
II.	Casein Micelles A. Bovine Submicelles and Micelles B. Human Micelles
III.	Casein Subunits A. β-Casein B. κ-Casein
IV.	Physiological Significance of Human Casein A. Casein Phosphopeptides B. Opioid-Like Activities and Immunostimulating Properties of Human Casein Peptides
V.	Quantitation of Casein
VI.	Conclusions

Acknowledgments

References

I. INTRODUCTION

Electron microscopy and physicochemical analysis show that proteins in human milk are present either in micelles, bound to the milk fat globule membrane (MFGM) or in soluble form in the whey fraction. The micelles, which give milk its white appearance, incorporate particular proteins, the caseins, and mineral ions, mainly calcium, phosphorus, and magnesium. Neither the composition nor function(s) of protein bound to the MFGM (about 1% is well understood (see Chapter 8). The remaining proteins, known as “soluble proteins” or whey proteins (see Chapter 3), are in true solution.

The traditional method for classifying milk proteins was developed by Rowland¹ in 1938 and was intended to be used primarily for bovine milk proteins. According to this approach, casein is precipitated at its isoelectric point (pH 4.6). This procedure has also been applied to human milk, but it was recognized early that human casein differs from cow’s casein in its physicochemical properties,² and that the precipitate formed by human casein at pH 4.6 is looser and softer than that formed by bovine casein.

As will be shown, human and bovine casein differ in their concentration in milk, individual components (α-, β-, κ-casein), degree of phosphorylation and glycosylation of components, and in micelle size and composition. Because of these differences the caseins of these two species may have different biological functions.

II. CASEIN MICELLES

Casein micelles are unusual among biological protein aggregates in that they display a distribution of sizes. This is in contrast to the functional aggregates of fixed size such as hemoglobin or fatty acid synthetase generally found in biological cells. The mechanism for the formation of casein micelles in cow’s milk, which includes suspension of the protein and sequestering of calcium and phosphate, has been the object of much research with a variety of models being proposed.^3,4,5,6 However, there are some reasons not to apply these models to the human casein system which has physical properties that are different from the bovine casein system: α-casein, a major part of bovine casein, is not present in human casein. Human casein micelles are smaller, sequester less calcium and phosphorus, and are hydrated to almost twice the extent of their bovine counterparts and also differ in the same respect from the micelles of milk from at least seven other species.⁷

Since there is no model proposed for the formation of human casein micelles, we will first briefly describe some characteristics of the bovine casein micelle, which may lead to a better understanding of the principal interactions of casein molecules, casein-submicelles, and casein micelles in human milk.

A. Bovine Submicelles and Micelles

Casein in uncooled cow’s milk is present in spherical particles with a diameter of about 20 to 600 nm, comprised of 20 to 150,000 casein molecules. The inorganic matter of casein micelles, mainly colloidal calcium phosphate, is about 8 g/100 g casein.⁸ When whole casein is in solution at a concentration, pH, and ionic strength as in milk, but at low Ca²⁺ activity, small aggregates are formed. The diameter of these aggregates varies from 10 to 20 nm and the molecular weights from 250,000 to 2,000,000. These aggregates have a dense hydrophobic core and a more hydrophilic outer layer, which contains most of the acidic and some of the basic groups. These small aggregates of whole casein are usually called submicelles, but they cannot be identical in composition to micelles, considering the ratio of the principal caseins (approx. α_s1:(β + γ):κ:α_s2 = 8:8:3:2). This ratio can change, particularly in the presence of a different κ-casein concentration of milk and differences in glycosylation and phosphorylation.

In addition, κ-casein probably exists in milk as an oligomer, containing several molecules and held together by covalent bonds. This means that there may be two types of submicelles, with and without κ-casein. Under the influence of calcium and phosphate in the same concentrations as in milk, the association increases considerably and the submicelles aggregate into micelles.⁸ κ-Casein has a protective function, preventing too high a calcium and/or phosphate stimulated casein micelle aggregation which would, without κ-casein, lead to the precipitation of the micelles. Further investigation of cow’s milk casein micelles is necessary to determine the function of two major kinds of submicelles (with and without κ-casein); the size of submicelles; the carbohydrate structure of κ-casein and its function; and the stability of the casein micelles.

A more detailed description of cow’s milk casein micelles can be found in the literature.^5,8,9

B. Human Micelles

The lack of information concerning human casein micelles is much greater than for bovine micelles. Until now only two casein subunits are known to be present in human milk: β- and κ-casein. There are several reports about the average micellar size of human casein. Due to difficulty in preparing casein micelles for studies of size, published values range from 30 to 75 nm, but are still lower than those reported for bovine micelles.^{10,11,12,13,14} Whether the subunit composition, and consequently the electrostatic forces of the human micelle, is causing this smaller size as compared to the cow’s milk micelle is not known. Another possibility would be a higher permeability of large micelles through the lacteal ducts in the bovine mammary gland as compared to the human breast.

There are some principal interactions between casein molecules, submicelles, and micelles which can occur (Table 1). Hydrogen bonds and van der Waal’s forces probably play only a minor role in the energetics of stabilization of protein aggregates, but are crucial for close packing and proper formation of specific aggregates. The major force behind favorable association is hydrophobic interaction. Among the caseins, ionic interactions such as calcium phosphate bridges and repulsive forces between sialic acid residues on κ-caseins play a greater role than they do in most other proteins.¹⁵

The micelle assembly is influenced by different phosphorylation and glycosylation of the casein subunits or calcium ion binding, although the exact mechanism is not well understood.

Studying the artificial casein micelle formation from human κ- and β-caseins and calcium ions, Azuma et al.¹⁶ re...

Table of contents

• Cover
• Title Page
• Copyright Page
• Dedication
• Preface
• The Editors
• Contributors
• Table of Contents
• Section A: Overview
• Section B: Proteins in Human Milk
• Section C: Non-Protein Nitrogen in Human Milk
• Section D: Physiological Significance of the Nitrogen Composition of Human Milk
• Index