Breast Milk Science: Toward Preemie Probiotics

“Breast is best,” so the mantra on infant feeding goes. The evolutionary case for that logic is easy—human milk is designed to feed human infants. But unraveling the chemistry that underpins the benefits of breast feeding versus using infant formula is still an adventure in complexity.

As researchers tease apart the roles of sugar, protein, and fat in milk, as well as the roles of beneficial gut microbes that help process the milk in babies’ tummies, they are finding many interrelated ways in which milk nourishes infants and protects their health.

One example is sugars. The most abundant sugar in human milk is the disaccharide lactose, from which babies derive energy. But breast milk also contains complex polymeric sugars called oligosaccharides that babies can’t break down. Oligosaccharides are metabolically costly to produce, and it makes no sense for mothers to make them if they don’t play an important role in infant survival, says David A. Mills, a food science professor at the University of California, Davis. Mills and his UC Davis colleagues have spent a decade developing a program to better understand breast milk, which has expanded from studying sugars to running clinical trials on infants and developing nutritional supplements.

The oligosaccharides are difficult to analyze, requiring new analytical methods using state-of-the-art lab instrumentation. UC Davis chemistry professor Carlito B. Lebrilla, for example, has developed a high-throughput liquid chromatography/mass spectrometry approach to separate, identify, and quantify about 200 oligosaccharides in milk. In collaboration with Mark A. Underwood, a professor of pediatric neonatology at UC Davis Children’s Hospital, Lebrilla and colleagues use feeding tubes that allow them to sample stomach contents to track which oligosaccharides get digested quickly in babies. They can also do what UC Davis food science professor J. Bruce German calls “diaper diagnostics,” taking a look at pee and poop to compare what goes into a baby with what comes out (Anal. Bioanal. Chem. 2013, DOI: 10.1007/s00216-013-6817-1).

In the long term, this combination of approaches could provide ways to diagnose digestive problems in babies, Lebrilla says.

It turns out that the major role of oligosaccharides in human milk is to serve as food for beneficial gut microbes. In particular, Bifidobacterium longum subsp. infantis appears to have coevolved with humans specifically to feed on the oligosaccharides. Genomic and biochemical studies in Mills’s lab have pinpointed the various genes and proteins that enable B. infantis to consume milk oligosaccharides (Microbiology 2013, DOI:10.1099/mic.0.064113-0).

Additionally, some of the cell wall proteins produced by B. infantis appear to have dual roles: They enable the bacteria to capture milk oligosaccharides for food and latch onto sugars residing on the cell walls of an infant’s intestines. Those capabilities enable B. infantis to outcompete and prevent colonization by other microbes, such as harmful Escherichia coli strains.

Capitalizing on what they’ve learned so far, the UC Davis breast milk researchers are developing B. infantis probiotics that could be used as nutritional supplements. And they are conducting clinical trials to see if the probiotics can improve the health of premature babies in neonatal intensive care units.

Studies elsewhere have already demonstrated that some probiotics support infant health. But those products have been prepared from bacterial strains that are easy to grow and generally thought to be health-promoting for adults, Underwood says. Instead, he and his colleagues are focusing on B. infantis, which is difficult to grow in a petri dish but clearly plays an important role in the intestines of healthy newborns.

For premature babies in particular, Underwood hopes the probiotics will help prevent necrotizing enterocolitis, a destructive intestinal disease that affects roughly 10% of premature babies. About 25% of the babies who contract the severe form of the disease die.

Doctors believe the disease is caused by pathogenic bacteria getting into the lining of the intestines. “When we compare the fecal composition of bacteria in a premature infant with a full-term infant, they are very different,” Underwood says. “The idea behind the probiotic is to try to give preemies more of the bacteria that we find in healthy term babies.”

To give B. infantis the best chance to thrive and keep pathogenic microbes at bay, the beneficial microbes must have the necessary oligosaccharides for food. For infants unable to get breast milk, the oligosaccharides have to come in the form of a prebiotic—a dose of oligosaccharides that could be added to formula.

The complexity of milk oligosaccharides defies large-scale chemical synthesis, but there may be ways to extract them from dairy cows. Work led by UC Davis animal genetics professor Juan F. Medrano and food science professor Daniela Barile demonstrated that cows make the needed oligosaccharides, although the amounts produced decrease after the first couple of weeks that a cow begins to produce milk (PLoS One 2011, DOI: 10.1371/journal.pone.0018895;Glycobiology 2013, DOI: 10.1093/glycob/cwt007). The researchers are working to find a way to capitalize on the massive dairy industry by isolating B. infantis prebiotics from waste streams. Another option might be to selectively breed cows to promote oligosaccharide production.

Oligosaccharides and B. infantis are not the end of the breast milk intrigue. After working out the sugar analysis, Lebrilla turned his attention to milk proteins. One protein in particular, lactoferrin, is glycosylated with its own set of sugars that turns it into a decoy to play a protective role. Pathogenic bacteria bind to lactoferrin’s sugars rather than sugars that reside on the walls of an infant’s intestinal cells (Mol. Cell. Proteomics 2012, DOI: 10.1074/mcp.m111.015248).

One quirk Lebrilla’s team discovered is that the types of sugars on lactoferrin change as bacterial populations change. This variability could be an evolutionary response to long-standing intestinal microbe patterns, or it could be the result of a feedback mechanism between an infant and its mother that responds to microbial changes in real time, Lebrilla says.

The researchers are also intrigued by hundreds of small peptides in milk. The peptides appear to be specifically cleaved from larger proteins in the mammary glands, rather than having been randomly produced. As a collection, they inhibit E. coli,Staphylococcus aureus, and possibly other microbes (J. Proteome Res. 2013, DOI: 10.1021/pr400212z).

These bacteria cause the breast infection mastitis, so the peptides likely help protect mothers from infection and perhaps protect infants as well, depending on how infants digest them. Newborns are flush with high amounts of peptides from their mothers for the first few days after birth, and then the concentrations drop dramatically. “It’s like the mother is dosing the infant to get rid of pathogenic bacteria while feeding the good ones and allowing them to get established,” Lebrilla says.

Beyond sugars and proteins, the science to study lipids in milk has lagged, German says. Lipids are clearly important in milk—the genes to secrete milk fat are similar in all mammals and have changed little over time. The lipids are also costly energy-wise for mothers to produce.

“You know you’ve got a little gold mine here,” German says about milk lipids. He believes they play a role in nutrient absorption and metabolism. But he doesn’t think that just looking at the individual lipids is the answer. He believes they have to be studied as particles, from small lactosomes that contain just phospholipids to larger globules in which the phospholipids encase triglycerides and cholesterol.

Although deciphering the composition of breast milk is critical for Underwood’s premature babies, that is only part of the breast milk equation. A better understanding of milk digestion is critical as well.

Preemies given straight breast milk don’t grow very well. Hospitals have to fortify breast milk with additional protein, phosphorus, and vitamin D. Research studies indicate, however, that premature guts may not be able to digest some material, regardless of how much a baby is fed, Underwood says. Rather than focusing on feeding them higher concentrations of certain nutrients, it may be better to figure out how to help preemies digest them.

Feeding tube and diaper studies of milk components will be crucial to this effort. In addition to Lebrilla’s oligosaccharide, protein, and peptide analyses, UC Davis food science professor Carolyn M. Slupsky uses nuclear magnetic resonance spectroscopy to look at how compounds in blood and urine respond to a baby’s diet, as well as to track the diversity of gut microbe species.

In a study comparing formula- and breast-fed infant rhesus monkeys, Slupsky found that the two groups not only had distinct gut microbiomes but they also had different insulin levels in the blood and appeared to be metabolizing amino acids differently (J. Proteome Res. 2013, DOI: 10.1021/pr4001702). She is now working to compare how compounds in breast milk differ between mothers and at different times during lactation, as well as how different formulas with varying nutrient content affect infant metabolism.

German emphasizes that the inspiration for the breast milk studies is to develop an overall understanding of what makes a baby healthy. On the preemie front in particular, medical science has saved so many babies from dying, German says. “But we still really don’t know how to best nourish them yet.”