Bioactive proteins in breast milk


Correspondence: Dr. Bo Lönnerdal, Professor of Nutrition & Internal Medicine, Department of Nutrition, University of California, One Shields Avenue, Davis, CA 95616, USA. Fax: +1-530-752-3564; email:


Human milk contains many proteins that have been shown to be bioactive, but it is still not known whether these activities are exerted in breast-fed infants. These bioactivities include enzyme activities, enhancement of nutrient absorption, growth stimulation, modulation of the immune system and defence against pathogens. The antimicrobial activities are very diverse, ranging from stimulation of beneficial microorganisms (i.e. prebiotic effects), killing or inhibition of growth of pathogens, to mechanisms preventing attachment or invasion of harmful microorganisms. Among the bioactive proteins are lactoferrin, lysozyme, secretory immunoglobulin A, haptocorrin, lactoperoxidase, α-lactalbumin, bile salt stimulated lipase, β- and κ-casein, and tumour growth factor β. Human milk proteins may be largely resistant against digestion in the gastrointestinal tract, be partially digested into bioactive peptides, or be more or less completely digested and utilised as a source of amino acids. These events can be studied using an in vitro digestion model, which is useful for predicting results in human infants. Some bovine milk proteins, for example, lactoferrin and tumour growth factor β, may also resist proteolysis and be capable of exerting bioactivities similar to those of human milk proteins.

Key Points

  1. Bioactive proteins in breast milk are likely to contribute to the advantages of breast-feeding through enzyme activities, enhancement of nutrient absorption, growth stimulation, modulation of the immune system and defence against pathogens.
  2. Some bioactive proteins in human breast milk remain stable and are digested only in part or not at all in the infant gut.
  3. Recombinant human milk proteins may be used in future applications, provided that safety and efficacy can be documented.

Milk, both cow's milk and breast milk, is an excellent source of nutrients, such as calcium, phosphorus, magnesium, protein, water- and fat-soluble vitamins and lipids, all necessary for growth, particularly in infants. However, it is also a source of bioactive proteins. Further, breast milk has been shown to be the superior source of such proteins for infants.[1]

Even though the composition of infant formulas has been adjusted over the years, breast-fed as opposed to formula-fed infants reveal significantly different growth patterns, nutritional status and health. Although the difference in health is more pronounced in developing countries, it is still the case in developed countries such as the United States.[2] Breast-fed infants experience fewer and shorter periods of infection, such as upper respiratory infections and otitis media.[2] They also have different gut microflora profiles,[3] which is an area where current research is very active. However, there is much to be learnt if we are going to ‘normalize’ formula-fed infants so they have similar growth, development and nutritional status as breast-fed infants.

While breast-feeding will always be the preferred option for the development of healthy infants, those mothers, who for whatever reason choose not to breast-feed their infants, should have available to them the best possible infant formula. Therefore, whatever advances are made in infant nutrition research on breast milk, they should, whenever possible, be adapted to infant formulas.

What are bioactive proteins?

They are proteins with roles beyond nutrition and include enzyme activities, enhancement of nutrient absorption, growth stimulation, modulation of the immune system and defence against pathogens.

Some of the physiological activities provided by milk bioactive proteins in the gastrointestinal tract include:

  • Enhancement of nutrient absorption by specific binding proteins that facilitate the uptake of nutrients
  • Inhibitors of enzymes such as trypsin inhibitors which may limit digestion
  • Active enzymes, some of which assist in nutrient digestion and absorption
  • Growth stimulation
  • Modulation of the immune system
  • Defence against pathogens.

A very complex set of protective mechanisms against pathogenic bacteria and viruses provided by bioactive proteins include:

  • The prebiotic effect, that is, stimulation of beneficial microorganisms in the gut (e.g. Lactobacilli, Bifidobacteria etc.)
  • Outright killing of pathogens
  • Inhibition of pathogens, and thus the limitation of their effects
  • Neutralising mechanisms preventing attachment or invasion by the pathogen into the intestinal mucosa

Bioactive proteins provide defense against infections through: immunological factors (antibodies such as secretory immunoglobulin A (sIgA), live cells, cytokines or signalling molecules); active proteins and enzymes (lactoferrin, lysozyme); oligosaccharides/glycoproteins; gut microflora (through prebiotic effect); and nutrients to optimise the infant's immune system.

Milk proteins are classified into whey proteins, caseins and mucins. Mucins specifically surround milk fat globules, forming a chemical barrier: milk fat globule membrane proteins. They are only about 1–2% of all breast milk proteins, but are nonetheless an intricate part of the milk and contain fascinating components. Unfortunately, no infant formula contains these milk fat globule membrane proteins. They are usually lost or discarded in dairy processing.

When whole milk is fractionated through centrifugation, the sedimented products are separated into a clear solution of whey (proteins and non-protein nitrogen), a pellet containing active cells and caseins, and fat, containing the milk fat globule membrane, as previously mentioned.

Concentrations of nitrogen and total protein in human milk

Breast milk is an active fluid; it changes in composition during the course of lactation. Breast milk produced immediately after delivery, the early milk or colostrum, is different from milk produced later in lactation. Total proteins in colostrum are high, but decline sharply after about the first month, then more slowly in subsequent months. Total nitrogen also declines after the first month, whereas non-protein nitrogen (mostly urea) remains at a constant low level throughout.

Whey protein content in early milk is also very high, containing many proteins, such as immunoglobulins, lactoferrin and α-lactalbumin, and declines as lactation progresses, at first sharply after the colostrum period, then levelling out through the remainder of lactation. In comparison, caseins can often be totally absent in early colostrum,[4] although after 4–5 days will rise sharply, then more slowly decline. The whey-to-casein ratio at the start of breast-feeding can be as much as 80:20 and then decreases to something like 60:40 and reaches virtual parity in later lactation.[5]

Thus, if we seek to emulate breast milk in infant formula, in particular for the newborn infant, casein should be kept at low levels. In fact, the changing nature of breast milk through the lactation period suggests that infant formulas should be staged in narrower intervals much more than they are at present in order to meet the infant's needs.

Bioactive proteins in human milk

The bioactive proteins (whey proteins, enzymes and caseins) in breast milk include:

  • lactoferrin
  • lysozyme
  • secretory IgA (sIgA)
  • haptocorrin (vitamin B12-binding protein)
  • α-lactalbumin
  • bile salt stimulated lipase
  • κ-casein, β-casein

But what are their actions?


Some oligosaccharides act as soluble receptor-analogues; that is, they act as decoys, as some sit on proteins or are in free-form in the intestinal lumen, and trick bacteria into attaching to them rather than on those oligosaccharides which are attached to the brush-border membrane of the small intestinal mucosa. This has been demonstrated in several studies from varying countries.[6-8] Some of the literature particularly emphasises the role of proteins as well as oligosaccharides in resistance to infection (glycolipids,[6] high molecular weight protein,[6] sIgA[7] and oligosaccharide-enriched fraction along with sIgA[8]).


Lactoferrin is a multi-functional protein. It is bacteriostatic; that is, it can inhibit bacterial growth, particularly by binding to iron and making it inaccessible to pathogens which require iron to grow. It is also bactericidal, and the protein fragment lactoferricin has been shown to be particularly potent in this way.[9]

Lactoferrin has antiviral effects and also facilitates iron absorption into cells. Further, it has immunomodulatory effects and is a growth factor.

Arnold et al.[10] showed that the iron free-form of lactoferrin, the most common form of lactoferrin in breast milk, can kill Streptococcus mutans, Streptococcus pneumoniae, Escherichia coli, Vibrio cholerae, Pseudomonas aeroginosa and Candida albicans.

Lactoferrin is a tough protein with a structure that makes it difficult to digest. In a study by Davidson and Lönnerdal,[11] significant quantities of intact lactoferrin was found in infant stools, even up to 4 months of age, suggesting that lactoferrin survives and is active in the small intestine. Whole lactoferrin declined in infant stools from 155 mg/24 h at 1 week of age, to 20 mg/24 h at 14 weeks, while lactoferrin intake from breast milk was 2.5 g/24 h at 1 week to 1.4 g/24 h at 14 weeks.[11] Of course, digestive capacity and efficiency increase as an infant grows. But early in life, limited capacity allows tough proteins such as lactoferrin, and sIgA, to resist digestion to some extent, although some parts of these proteins may still be absorbed into the small intestine through the mucosa. This has led to the hypothesis that there are structures attached to the mucosa that lactoferrin can bind to, and these have been named lactoferrin receptors, and such a receptor was later isolated by biochemical methods.[12]

The gene for the lactoferrin receptor was cloned and gene expression in various tissues was explored (Fig. 1), and significant expression was found in fetal and young neonatal small intestinal tissue.[13] This correlates with the fact that lactoferrin is the dominant protein in breast milk (20–25%).

Figure 1.

Lactoferrin receptor expression – human tissues (Reproduced with permission from S. Karger AG).

With the use of molecular and cell biology, the gene for the lactoferrin receptor was inserted into Caco-2 cells, and the receptor expression was increased via transfection. These cells took up iron from lactoferrin much more than non-transfected cells, indicating that lactoferrin is involved in cellular iron acquisition, although cell homeostasis subsequently dictates how much iron is absorbed into the body.[13]

Molecular evidence indicates that lactoferrin inside the cell acts as a transcription factor on DNA in the cell nucleus of small intestine cells.[14] Thus, lactoferrin, as a dietary protein, is going into the cell and affects synthesis of other valuable proteins, such as immune proteins and cell-signalling proteins.


Lysozyme is an active enzyme which is present in high concentration in breast milk, as much as 3000-fold higher in human than in bovine milk.[15] It acts as an antibacterial enzyme and it cleaves to β,1–4 glycoside linkages in the cell wall of bacteria.

Lysozyme has a special relationship with lactoferrin. Lactoferrin first binds tightly to components of the outer cell membrane, that is, lipopolysaccharides, of Gram-negative bacteria, and creates holes in the membrane, through which lysozyme then enters the glycomatrix of the bacteria, degrading it and effectively killing the pathogen.[16]


sIgA accounts for 90% of total immunoglobulins in milk (total IgA, IgG, IgM).

Whatever specific bacterial and viral pathogens that the mother was exposed to, antibodies she has developed against them will be transferred to the infant (via the enteromammary immune pathway).

sIgA, as opposed to other types of IgA, is stable against proteolytic enzymes in infant gut, and there binds to bacterial and viral antigens, promoting inhibition of attachment to the mucosal lining.

Other immunoglobulins such as IgA, IgM and IgG are present, but in lower concentrations, and are easily digested and will not survive in the small intestine in the manner of sIgA.


Haptocorrin (vitamin B12-binding protein) is largely unsaturated in human milk; that is, there is much more haptocorrin than there is vitamin B12. Intrinsic factor, which is required for absorption of vitamin B12, is absent in the newborn infant, and so haptocorrin may facilitate vitamin B12 absorption.[17]

Haptocorrin has anti-microbial properties. Even at very low concentrations, it has been shown to kill E. coli.[18] Haptocorrin is also stable against proteolytic digestive enzymes.

Bile salt-stimulated lipase (BSSL)

BSSL comprises only 1–2% of total milk proteins, but this is substantial for an active enzyme. It is present in milk of some species, notably humans, but not in cow's or goat's milk.

In the gut lumen it hydrolyzes milk fat (triglycerides, diglycerides, monoglycerides, and hydrolysis of these is very important for early and preterm infants), vitamin A esters, cholesterol esters and lyso-phospholipids. Thus, it is a workhorse in the role of lipid digestion.

Andersson et al.[19] conducted an open randomised cross-over clinical study on healthy preterm infants with a birthweight of 900–1500 g, after 1 week's feeding with fortified mother's milk or fortified pasteurized mother's milk, in order to examine fat absorption and anthropometry. They found that significantly less fat was absorbed from pasteurized milk. Thus, BSSL was being destroyed by the pasteurizing process, and without this enzyme being active, preterm infants could not absorb lipids to the maximum extent. The anthropometric summary also revealed that infants receiving non-pasteurized breast milk had greater weight and length gain and heel-to-knee measurement was increased.

Phase III clinical trials are currently being undertaken in Europe and the United States to examine the effects of addition of BSSL in pasteurized milk.


Caseins make up 20–40% of proteins in breast milk, which is less than in cow's milk, but still play an important role in infant development.

β-casein is unique among the caseins in that it has multiple phosphorylated amino acids along its backbone. When digested, smaller casein phosphopeptides (CPP) are formed and facilitate calcium absorption. Also formed are small caseinomorphins, that is, opioid peptides that have affinity for opiate receptors, which may be involved in sleep–wake patterns.

In examining human intestinal cells, calcium has been seen to be taken up, but with the addition of very small amounts of CPP purified from digested breast milk casein, calcium absorption increased substantially (S Rudloff, B Lönnerdal, unpubl. obs. 1990). This may be one of the reasons for significantly better uptake of calcium from breast milk as opposed to formula.

κ-casein in breast milk is heavily glycosylated (∼40%) and is an inhibitor of bacterial adhesion (e.g. Helicobacter pylori);[20] H. pylori has been shown to be much less frequent in breast-fed than formula-fed infants.

Glycans of κ-casein have a structure similar to that of surface-exposed carbohydrates of cells in the mucosa of the gastrointestinal tract and there can act as soluble ‘decoys’ for pathogens.

Studies have found that κ-casein, as well as sIgA, can inhibit H. pylori adhesion to human gastric mucosa[20] and that lactoferrin can inhibit H. pylori growth.[21] This indicates that proteins in the milk work together in a synergistic manner to protect against pathogens.

Milk fat globule membrane proteins

The active components in milk fat globule membrane proteins include lactadherin, butyrophylin, xanthine oxidase and mucins.

Human milk mucins can bind to rotaviruses and inhibit viral replication[22] and membrane mucins on milk fat globules inhibit binding of S-fimbriated E. coli to buccal epithelia.[23]

A study of the effect of a complementary food enriched with a bovine milk fat globule membrane fraction on incidence and duration of diarrhoea in Peruvian infants was undertaken.[24] This was a double-blind randomised clinical trial in Lima, Peru, with 6 to 12-month-old infants (n = 550) given complementary food, twice daily, with the milk fat globule membrane fraction (n = 277) or placebo (skim milk protein) (n = 273) for 6 months. Micronutrients were also added to the diets and anthropometry, morbidity, bacteriology/virology and nutritional status (Fe, Zn, vitamins A, B12, folate) were all evaluated. In each group, 250 participants completed the study. Results were: significantly reduced incidences of diarrhoea and severe diarrhoea, its prevalence and duration, and numbers of children with persistent diarrhoea. This indicates that the addition of milk fat globule membrane fraction to the diet will reduce the incidence of gastrointestinal diseases.

Proteolytic fate of human milk proteins

Most of the proteins in breast milk will be totally broken down and utilised by the infant as a supply of amino acids. However, some proteins are subject to partial proteolysis, leaving biologically active fragments (e.g. CPPs), and some undergo no (or limited) proteolysis (e.g. lactoferrin and sIgA) and can survive digestion and can be found totally intact in the stool.

Biological functions of α-lactalbumin

α-Lactalbumin provides an example of this variation in proteolytic processes. In the upper tract of the small intestine, partial digestion is occurring, forming various types of peptides (Fig. 2). These may exert bioactivities for some time in the small intestine, but eventually α-lactalbumin is digested, forming amino acids. This is a scenario in which digestion is slow, with transitory peptides that perform certain functions for a certain length of time.

Figure 2.

Biological functions of α-lactalbumin (Reproduced with permission from the American Chemical Society Publications).

Quantitation of human milk proteins

To explore the stability of proteins from human milk, fecal samples from exclusively breast-fed infants were collected and soluble proteins were extracted in phosphate-buffered saline. Insoluble matter was removed by centrifugation, producing a supernatant. From this, total protein and total nitrogen were quantified. Specific milk proteins were also quantified, including lactoferrin, sIgA and lysozyme.[11]

Subsequently, an in vitro proteolytic stability assay was developed and used on haptocorrin without (Apo-Hc) and with vitamin B12 (Holo-Hc).[17] In each solution, the human infant stomach was replicated by adjusting pH to 3.5, adding pepsin, the major stomach enzyme involved in protein digestion, and incubating at 37°C for 30 min. Then, to replicate the small intestinal environment, pH was raised to 7.0 and pancreatic enzymes were added, and the mixture was then shaken at 37°C for 60 min. Analysis was then performed by sodium dodecyl sulfate – polyacrylamide gel elctrophoresis (SDS-PAGE) and Western blotting (Fig. 3).

Figure 3.

Stability of haptocorrin without vitamin B12 (Apo-Hc) and with vitamin B12 (Holo-Hc) in human milk by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.

Following the procedure in Figure 3, it was found that both apo-Hc and holo-Hc remained largely undigested by both SDS-PAGE and Western blotting analyses, as opposed to serum albumin, caseins and α-lactalbumin, of which only small segments remained. In fact, under Western blotting, using antibodies to haptocorrin, haptocorrin remained after digestion, with no degradation whatsoever. This indicates that some proteins do have the capacity to survive the digestive system and are available to perform other functions than providing nutrients.

Other proteins from breast milk are being analysed in the same manner.

The test systems being used to perform analyses of bioactive proteins in breast milk include:

  • in vitro activity (e.g. enzyme activity, Fe-binding) as discussed above
  • cells (human intestinal cells in culture)
  • experimental animals
    • rat pups
    • infant non-human primates (rhesus monkeys)
  • humans (infants)

To conduct these experiments, and improve the degree of evidence required, ever-increasing quantities of breast milk proteins would be required, and such a supply is not possible.

Thus, production of recombinant, or genetically engineered, human milk proteins has been explored. Expression systems for human milk proteins have included milk from animals, such as in cows, sheep and goats, but this is complicated and does not provide recombinant human milk proteins in sufficient quantities. Other systems could include microorganisms, but the risks of contamination are high and ultra-purity is complex and expensive to obtain. Likewise, the use of non-edible plants, such as tobacco, exposes the product to toxic compounds which have to be eliminated at great expense and time.

Edible plants, such as rice and barley, as expression systems for recombinant human milk, have greater potential. At the University of California at Davis, experiments have been undertaken to insert human milk protein genes into rice.[25, 26] The advantages of using rice is that there is a very high level of expression, it can be expressed as a germination product or storage protein, it has very low risk of toxicity/allergenicity (very important for clinical trials on infants), and the rice industry has high production and processing capacity.

From this recombinant system, both lactoferrin and lysozyme have been successfully and rapidly produced in quantity.

Using these products, a randomised controlled trial was performed. Children hospitalised with acute watery diarrhoea (n = 140) at the Oral Rehydration Unit, Children's Hospital and Institute of Nutritional Investigation, Lima, and the Belen Hospital, Trujillo, Peru, were separated into three treatment groups receiving one of the following:

  • standard World Health Organization (WHO) oral rehydration solution (ORS: glucose and salt)
  • WHO rice-based ORS or
  • rice-based ORS with recombinant human lactoferrin (rhLF) and recombinant human lysozyme (rhLZ)

After 48 h treatment in the hospital and up to 12 days follow-up, in the rice-based ORS with rhLF and rhLZ group, there was significant reduction in volumes of diarrhoea (measured by diaper weight), reduction in duration of diarrhoea (measured by days until appearance of solid stools) and reduction in relapse rate (measured by recurrence of diarrhoea).[27] Thus, the addition of rhLF and rhLZ to ORS helps in the treatment of diarrhoea in infants.


Bioactive proteins in breast milk are likely to contribute to the advantages of breast-feeding. In some cases, purified cow's milk proteins, such as a-lactalbumin and milk fat globule membrane proteins, may also provide bioactivities through α-lactalbumin and milk fat globule membrane. Recombinant human milk proteins may be used in future applications, provided safety and efficacy can be documented. This may be the only way in which sufficient quantities of such proteins may be available for research and trial purposes.

Multiple Choice Questions

  • 1.Vitamin B12 in breast milk is bound to:
    1. Lactoferrin
    2. α-lactalbumin
    3. Lysozyme
    4. β-casein
    5. Haptocorrin

Answer: E. β-casein is involved in calcium absorption and the other options are involved in antibacterial, antiviral and bioactivities rather than nutritional provision.

  • 2.Bile salt-simulated lipase can hydrolyze all of these except one. Which one?
    1. Cholesterol esters
    2. Oligosaccharides
    3. Triglycerides
    4. Diglycerides
    5. Vitamin A esters

Answer: B. Oligosaccharides have antibacterial rather than nutritional roles, such as in esters and the glycerides.

  • 3.Maternal immunity is transferred to the breast-fed infants by:
    1. Transferrin
    2. Mucins
    3. Immunoglobulin G (IgG)
    4. Secretory IgA (sIgA)
    5. IgM

Answer: D. Unlike other immunoglobulins, sIgA is found in large quantities in milk, and is not readily absorbed by the infant gut, but rather binds to antigens and prevents their attachment to the infant mucosal lining, providing direct immunity from mother to infant. Mucins are involved in the creation of milk fat globule membrane protein rather than direct immunity, and transferrins are iron-binding blood plasma glycoproteins that control the level of free iron in the body.


The author was invited by Pfizer Australia Pty Ltd to speak at a Pfizer-sponsored educational meeting in November 2012. This supplement is a review article based on the presentation provided by the author at this meeting. The content for this article was presented by the author, and a medical writer, Rodrick Faulkner, undertook the writing of the first draft. The author contributed to reviewing and revising the manuscript with the medical writer coordinating the author amendments. While Pfizer had the opportunity to review the manuscript prior to submission, the author takes full responsibility for the content and expression of the submitted manuscript. The Editor-in-Chief of the Journal of Paediatrics & Child Health has reviewed the article and feels there is no significant Conflict of Interest.