Milk components possess a myriad of bioactive functions influencing an infant’s health, including  stimulation and modulation of the immune system.

J. Bruce German, Carlito Lebrilla and David A. Mills, Contributing Editors

The search for bioactive food ingredients is a major activity of scientists seeking to improve diet and human health. The search is challenging. Which food ingredients make healthy people healthier? Upon what targets should such ingredients work? What mechanisms act to protect people?

A group of researchers at the University of California, Davis (UCD), have taken a rather bold step and are asking the question: “What should we eat?” Milk emerged from evolution, as a remarkable biofluid to nourish mammalian infants. The scientists have taken milk apart, from the genes that make it to the molecules it contains to the biochemical effects it produces--and to the health benefits that result. What evolution came up with is proving to be astonishing and a practical blueprint for food research.

Genomics, Milk and Evolution of Nourishment
Scientists have now sequenced (at great expense) the entire genomes of a wide variety of species of organisms, including humans (Elsik et al., 2009). These genomes are being used to understand biological processes, from the pathogenicity of disease-causing viruses and bacteria to the molecules making up the immune system of animals that protect them (Collins et al., 2003). For each species, its genome is the roadmap of its history, the combined results of the Darwinian selective pressures that shaped its evolution.

Comprehending evolution means understanding these selective pressures and their consequences on genes, molecules, processes and organisms. For example, competition for food is a selection pressure that rewarded venom production in spiders, tongue speed in frogs and neck length in giraffes. So, too, can genomes be used to understand food itself and its value in nutrition and health. The emergence of lactation as a remarkable infant feeding strategy defines mammals (Lemay et al., 2007, 2009). Milk is responsible for much of mammalian success. Yet, through 200 million years, the constant selective pressure on milk was two-sided. As the sole source of nourishment for mammalian infants, evolution has certainly shaped the composition of breast milk for benefits to the infant. However, lactation is also a conflict between the infant’s survival needs and the cost of lactation to the mother. Each and every component in milk costs the mother. If it does not promote the success of the infant, its cost to the mother would result in its loss, through evolution. On the other hand, if anything in milk assists the infant in its survival against its peers in the complex, hostile environment, it will be retained, again through evolution. To the group at UCD, evolution has done the tough job of testing bioactives already. The scientists just have to determine what components are there, and how they work.

Milk is a model for understanding diet, not just a food. Human milk must be completely nourishing; traditionally, it is the only food the infant eats. Furthermore, although milk contains all essential nutrients, milk components do not solely nourish the infant with essential nutrients (Smilowitz et al., 2005). They provide myriad bioactive functions that influence an infant’s growth and development in all its systems, such as the growth, development, stimulation and modulation of the immune system (German et al., 2004). Milk also clearly evolved to be directly protective, shielding the infant from toxins and pathogenic diseases. Scientists at UCD are racing to discover how milk benefits infants and to use this knowledge to improve foods for everyone. One example makes this point very well: an obscure group of molecules termed oligosaccharides.

The Perplexing Abundance of Oligosaccharides
In many ways, human milk is similar to all mammalian milks, yet, in some respects, it is unusual. Its fat content is relatively high, at around 4%, and its lactose is conspicuously high, at about 7%. Its protein content is quite low, at around 1%. Yet, what genuinely surprised the UCD scientists was none of those. Recall that everything in milk costs the mother; if it is not valuable to the infant, it would be lost to evolutionary pressure. Imagine the surprise of the scientists, when they realized human milk is made up of 1% by weight, complex oligosaccharides that are completely indigestible by infants. This realization prompted Carlito Lebrilla, chair of chemistry, to develop the analytical toolset to detail the structures of these perplexing molecules (Ninonueva et al., 2005, 2006, 2007). The oligosaccharides turned out to be a complex array of sugar polymers, all based on a lactose backbone (see chart “Human Milk Oligosaccharides”).

To date, Lebrilla’s group has discovered over 200 different human milk oligosaccharide structures that differ in their size, charge and sequence (Ninonueva et al., 2008). The researchers working with professor Lebrilla have examined milks from other non-human primates and animals (Tao et al., 2009). While all mammals appear to produce oligosaccharides in their milk, human oligosaccharides are more complex and abundant than any other mammalian milk tested to date. The next question for the research group was obvious, though challenging: If the infant cannot digest oligosaccharides, what do they do? 

Milk Oligosaccharides and Bacteria
Since oligosaccharides are indigestible by infants, they pass through to the lower intestine. It has been proposed that bacteria in the intestine break down and consume the oligosaccharides there. If milk oligosaccharides act as “prebiotic” substrates that help shape the microbial content of the infant, Robert Ward, a graduate student, and David Mills, a professor and microbiologist, suggested commensal (normal intestinal) bacteria would possess the capacity to grow on these complex structures (Ward et al., 2006, 2007). Even Mills was not prepared for how selective the growth would prove to be. Among gut-related bacteria, including Lactobacillus, Clostridium, Eubacterium, E. coli, Veillonella and Enterococcus isolates, only a specific strain of Bifodobacterium, Bifidobacterium longum ssp infantis, and Bacteriodes species (normal constituents of the breast infant intestine) were able to consume human milk oligosaccharides (HMO) as a sole carbon source and achieve high cell densities (see chart “Selective Growth of Infant Gut Bacteria”). Professors Lebrilla and Mills teamed to develop methods that could simultaneously determine which bacteria could consume each oligosaccharide (Locascio et al., 2007). The highly abundant, lower molecular weight oligosaccharides were rapidly consumed and supported the growth of only those select bacteria. Detailed examination of the Bifidobacteria strain proved even more intriguing--they had evolved with their host.

The entire genome of Bifidobacterium longum ssp infanti was sequenced, and, in parallel, the enzymatic activities towards HMO metabolism were measured. The results of this research point to a host of genes, all together and co-regulated, that provide the means for  B. infantis to grow and flourish on milk oligosaccharides (see chart “Symbiotic Evolution”). B. infantis likely imports the oligosaccharides via an army of dedicated sugar-binding proteins and membrane transporters. Once inside the cell, these oligosaccharides are broken down by a complement of glycosidases that then enter the cell’s central metabolic pathways. Combining these various pieces of evidence, Mills and graduate student Dave Sela came to a dramatic realization: Humans and their oligosaccharides and Bifidobacterium longum ssp infantis co-evolved together, as a truly symbiotic partnership (Sela et al., 2009).

Lessons for Food
The strategies that emerged during mammalian evolution are truly remarkable. The example of the oligosaccharides illustrates many of the features that make milk such a compelling model for scientists to discover novel bioactivities for human health. Imagine, with this strategy, mothers are literally recruiting another life form, beneficial bacteria, to babysit their baby. What is this one example from a wide range of ongoing research teaching researchers about feeding humans in general? First, the bacteria that inhabit humans are important to their health. Second, evolution favored the considerable investment in producing indigestible oligosaccharides to ensure that only particular bacteria could thrive in the intestine of infants. Third, the bacteria that flourish in the competitive microbiological niche of the breast-fed infant’s intestines do so, because of an array of genes that provide the means to transport, digest and metabolize complex oligosaccharides. In other words, how the bacteria behave is important. Now, the race is on to find oligosaccharides with structures and functions similar to human milk. The beneficial bacterium, B. longum ssp infantis, is being examined to determine the features that prompted its co-evolution with humans. At the University of California, Davis Medical Center, Mark Underwood, Ph.D., is studying premature infants for the benefits that this combination of oligosaccharides and bacteria provide in immune regulation and development.

The research group at UCD is using its discoveries to guide ingredient development for people of all ages. It is now possible to consider innovative food products for hospitalized and antibiotic-treated patients, for the elderly and for various individuals with compromised intestinal functions. This one example is just a highlight of the multiple molecules, targets, mechanisms and benefits milk research is discovering. Milk is also a model for conjugated molecules, such as glycoproteins and glycolipids, that yield unique products during digestion (Seipert et al., 2008). Milk is a model for a new generation of biologically inspired food processing, such as how to bio-assemble complex structures, like casein micelles and milk fat globules, that control the digestive process and its dynamics (Ward et al., 2004; Argov et al., 2008a, 2008b). The future of foods that genuinely improve each person’s health is challenging. Nonetheless, the 200 million years that evolution has been working intensively on lactation provides humans with a silent partner with a lot of experience.  NS
Bruce German, Ph.D., is a professor in the Department of Food Science & Technology at the University of California, Davis. He serves as director of the Foods for Health Institute at UC Davis and chairs the Scientific Advisory Committee of the International Milk Genomics Consortium.
David Mills, Ph.D., is a professor in the Department of Viticulture and Enology at the College of Agricultural and Environmental Sciences, UC Davis, and the co-founder of the Lactic Acid Bacteria Genome Consortium.
Carlito Lebrilla, Ph.D., is a professor of chemistry in the Biochemistry and Molecular Medicine department in the School of Medicine at UC Davis.
The International Milk Genomics Consortium provides a collaborative and interactive platform for researchers and research end-users to accelerate the understanding of the biological processes underlying the mammalian milk genomics. Information about the consortium is available at The 7th International Symposium on Milk Genomics & Human Health will be held October 20-22, 2010, at the UC Davis Conference Center in Davis, Calif. Information is available at

Argov N, et al. Size-dependent lipid content in human milk fat globules. J Agric Food Chem. 2008a. 56(16):7446-50.
Argov N, et al. 2008b. Milk fat globule structure and function: nanoscience comes to milk production. Trends in Food Science & Technology. 
Collins FS, et al. 2003. US National Human Genome Research Institute. A vision for the future of genomics research. Nature. 24;422(6934):835-47.
Elsik CG, et al. 2009. Bovine Genome Sequencing and Analysis Consortium. The genome sequence of taurine cattle: a window to ruminant biology and evolution. Science. Apr 24;324(5926):522-8.
German JB, et al. 2004. International Milk Genomics consortium. Trends in Food Science & Technology. Volume 17, Issue 12, 656-661.
Lemay D, et al. 2006. Nutrition and food structure in food colloids. Self-Assembly and Material Science. pub Royal Society of Chemistry London UK 2007.
Lemay DG, et al. 2009. The bovine lactation genome: insights into the evolution of mammalian milk. Genome Biol. Apr 24;10(4):R43.
Lemay DG, et al. 2007. Gene regulatory networks in lactation: identification of global principles using bioinformatics BMC Systems. Biology. Nov 27;1:56.
Lemay DG, et al. 2007. Building the bridges to bioinformatics in nutrition research. Am J Clin Nutr. 86: 1261.
Locascio RG, et al. 2007. Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation. J. Ag. Food Chem. Oct 31;55(22):8914-9.
Ninonuevo M, et al. 2005. Nanoliquid chromatography mass spectrometry of oligosaccharides employing graphitized carbon chromatography on microchip with a high accuracy mass analyzer. Electrophoresis. 26(19): 3641–3649.
Ninonuevo MR, et al. 2007. Methods for the quantitation of human milk oligosaccharides in bacterial fermentation by mass spectrometry. Analytical Biochemistry. 361: 15–23.

Ninonuevo MR, et al. 2006. A strategy for annotating the human milk glycome. J Agric Food Chem. Oct 4;54(20):7471-80.
Ninonuevo, MR, et al. 2008. Daily variations in oligosaccharides of human milk determined by microfluidic chips and mass spectrometry. J Agric Food Chem. Jun 25;56(12):4854.
Seipert RR, et al. 2008. Factors that influence fragmentation behavior of N-linked glycopeptide ions. Anal Chem. May 15;80(10):3684-92.
Sela DA, et al. 2008. The complete genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proceedings of the National Academy USA Dec 2;105(48):18964-9.
Smilowitz JT, et al. 2005. Milk beyond essential nutrients: the metabolic food. The Australian Journal of Dairy Technology. 60(2): 77–83.
Tao N, et al. 2008. Bovine milk glycome. J Dairy Sci. Oct;91(10):3768-78.
Ward RE and German JB. 2004. Understanding milk’s bioactive components: a goal for the genomics toolbox. Journal of Nutrition. 134(4): 962S–967S.
Ward RE, et al. 2004. Bioguided processing: a paradigm change in food production. Food Technology. 58(5): 44–48.
Ward RE, et al. 2007. In vitro fermentability of human milk oligosaccharides by several strains of bifidobacteria Mol. Nutr. Food Res. 51, Nov;51(11):1398-405.< br>Ward, RE, et al. 2006. In vitro fermentation of breast milk oligosaccharides by Bifidobacterium infantis and Lactobacillus gasseri. Appl. Environ. Microbiol. 72: 4497-4499.

SIDEBAR: Referencing CLA and Whey Proteins for Health
While interest in milk oligosaccharides is growing, a multitude of other ingredients found in cows’ milk have captured attention for their health benefits. They include conjugated linoleic acids (CLA), a family of linoleic acid isomers and a variety of proteins.

CLAs are considered to have been discovered by Wisconsin researchers led by Michael Pariza, who began publishing papers on their health properties at least as early as the 1980s. (See also Pariza MW and Ha YL. 1990. Newly recognized anticarcinogenic fatty acids. Basic Life Sci. 52:167-70.) More recently, studies have supported the role of the cis-9, trans-11 form of CLA as an anti-inflammatory and its ability to reduce cardiovascular disease risk. A recent meta-analysis indicated that CLA also has a small impact on fat mass, potentially reducing body fat, but also, increasing lean muscle mass. (Whigham, L, et al. 2007. Efficacy of conjugated linoleic acid for reducing fat mass: a meta-analysis in humans. Am. J. Clin. Nutr. 85: 1203–11. [See]) In 2008, the FDA issued a no objection letter for use of CLA in certain foods and beverages (GRN 232). CLA content is also being promoted by at least one marketer of butter to consumers.

Much attention also has been paid to whey proteins, so called because they originally were the water-soluble proteins suspended in whey, after casein proteins coagulate into curds during cheese manufacturing. One recent paper provides an overview of the support for a number of health-promoting milk components. (Krissansen, GW. 2007. Emerging health properties of whey proteins and their clinical implications. J Am Coll Nutr 26: 713S–23S. See Some of the paper’s “key teaching points” include:
* beta-Lactoglobulin to inhibit allergy and carcinogenesis in animals.
* alpha-Lactalbumin protects against infection, directly kills cancer cells, improves morning alertness, and induces anxiolytic and rewarding effects.
* Glycomacropeptide inhibits colitis and enhances cognitive development.
* Lactoferrin enhances immunity to prevent cancer, suppresses immunity to block inflammatory disease, promotes bone growth and has anti-microbial activity. Also, certain milk antibodies bind to cholesterol, preventing its absorption.
* Whey proteins inhibit angiotensin-I-converting enzyme (ACE), helpful in managing high blood pressure. Whey proteins may augment cancer treatments, fight inflammatory disease, heal wounds, promote bone repair, lower blood pressure and cholesterol, and treat acne.
-- Claudia D. O’Donnell, Chief Editor