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.
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. Breast-fed infants experience fewer and shorter periods of infection, such as upper respiratory infections and otitis media. They also have different gut microflora profiles, 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, 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.
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.
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 and membrane mucins on milk fat globules inhibit binding of S-fimbriated E. coli to buccal epithelia.
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. 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.
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.
Subsequently, an in vitro proteolytic stability assay was developed and used on haptocorrin without (Apo-Hc) and with vitamin B12 (Holo-Hc). 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.
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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). Thus, the addition of rhLF and rhLZ to ORS helps in the treatment of diarrhoea in infants.