HUMAN MILK AND INFANT FORMULA

Chapter 9 HUMAN MILK AND INFANT FORMULA Finding the perfect alternative to Mother’s milk has proven to be a very complicated task that continues today with an ever-growing assortment of modified and specialized formulas. If you were born in the 1930’s and 40’s and not breastfed as an infant, there is a good chance that you were fed a formula created by mixing 13 oz of unsweetened evaporated milk with 19 oz of water and two tablespoons of either corn syrup or table sugar. Every day, parents prepared a day’s worth of this formula, transferred it to bottles that they had sterilized in a pan of boiling water, and stored it in a refrigerator until used. In addition to this formula, infants received supplemental vitamins and iron. Supplemental infant nutrition has a fascinating history that began long before pediatricians recommended evaporated milk formula as alternatives to breastfeeding. Prior to the use of evaporated milk as a substitute for breast milk, wet nursing was the method of choice; breastfeeding was the preferred method of feeding infants, just as it is today. But if a mother’s milk supply was inadequate or she chose not to nurse, the family often employed a “wet nurse” to nourish infants. This practice was common in Europe during the 18th century and in America during the colonial period. Families would hire a wet nurse to reside in the home or sometimes send the infant to live in the wet nurse’s home and retrieve the baby after he or she had been weaned, perhaps three to six months later. Wet nurses were selected with the utmost care, because it was believed the quality of milk the baby received determined his or her future “disposition”. Brunette wet nurses were preferred to redheads or blondes because their breast milk was thought to be more nutritious and their disposition more “balanced”. This is perhaps the genesis of all those bad blonde jokes we still hear today.

300

FUNCTIONAL FOODS

In 18th century Europe, the demand for wet nurses was so great that bureaus were established where wet nurses could register and reside until their services were needed. The British governments regulated the bureaus very strictly; passing laws mandating that wet nurses undergo routine health examinations and prohibiting them from nursing more than one infant at a time. Eventually, wet nursing fell out of favor and attention turned to finding an adequate substitute for mother’s milk. The practice of feeding human babies milk from animals; “dry nursing”, began to flourish in the 19th century and milk from a variety of animals was tried: goats, cows, mares, and donkeys. Cow’s milk quickly became the most widely used because of its ready availability, although donkey’s milk was thought to be healthier because its appearance more closely resembled that of human milk. Physicians at the time argued about the best way to prepare the milk. Some promoted giving fresh raw milk to the infant. Others recommended that it be warmed or boiled first, and still others suggested diluting it with water and adding sugar or honey. When baby bottles were adopted during the Industrial Revolution, many popular designs evolved. Prior to that, the milk was spoon fed to infants or given via an improvised feeding device such as a hollowed out cow’s horn fitted with a leather nipple. Rubber nipples became widely available and very popular after their invention by Elijah Pratt in 1845. After weaning the infant from breast milk or a substitute animal’s milk, he or she was given an infant food called pap which consisted of boiled milk or water, thickened with baked wheat flour and sometimes egg yolk. A more elaborate infant food, called panada, was made from bread, flour, and cereals cooked in a milk or water-based broth. Detailed recipes for various kinds of infant paps and panadas have been published in cookbooks throughout history. In 1930, three Canadian doctors; Frederick Tisdall, Theodore Drake, and Alan Brown developed Pablum at the Hospital for Sick Children in Toronto. During the 1920s and 1930s, considerable time and effort were spent studying the science of artificial feeding. Society seemed to welcome the scientific approach to infant feeding and food and bought products that advertised increased nutritional value for their children. In 1931, Pablum, an infant cereal fortified with minerals and vitamins, became commercially available in Canada and the United States. The food was heralded as an excellent cereal addition to the infant’s diet, and it was a major commercial success. Modern infant formula is an industrially produced milk substitute designed for infant consumption. Usually based on either cow or soy

HUMAN MILK

301

milk, infant formula strives to duplicate the nutrient content of natural human breast milk. Since the exact chemical and biological properties of breast milk are still not fully understood, ‘formula’ is necessarily an imperfect approximation. Besides breast milk, infant formula is the only other infant milk which the medical community considers nutritionally acceptable for infants under the age of one year. However, its use particularly in the third world is somewhat contentious. Cow’s milk is not recommended because of its high protein and electrolyte (e.g., sodium) contents which may put a strain on an infant’s immature kidneys. Evaporated milk, although perhaps easier to digest due to denaturing of the proteins, is still nutritionally inadequate. Further, the biological benefits of human milk is a potent defense mechanism in breast-fed infants, particularly in third world countries where it helps to control severe diarrhea which is often lethal (Hendricks and Guo, 2006). Human Milk Chemistry Human milk is the “gold” reference standard for infant nutrition and is recognized as the preferred food for infants due to the nutrient balance, immunological protection, and other growth-promoting substances. Infant formula formulation is a mimic of human milk chemistry. Gross Composition The gross composition of human milk, cow’s milk and infant formula is shown in Figure 9.1. The protein content of human milk is approximately 1.0%, with approximately 70% of the protein being provided by whey proteins. Human milk has the highest level of lactose (7.0%) among mammals, providing 40% of human milk’s total energy. Fat provides approximately 50% of the gross energy of human milk, with an average content of 3.8%. Ash content in human milk is only 0.2% compared with 0.7% in bovine milk. Water content in human milk is similar to bovine milk at about 87%. Infants need approximately 125 ml of water /kg body weight per day supplied from either breast milk or a commercial formula (water loss is estimated at 20 ml/kg body weight per day). As lactation progresses, the chemical composition of human milk changes as a result of physiological and external factors. Some external factors may contribute negatively to the quality of human milk, for example, some environmental pollutants, such as heavy metals, can be detected in human milk, as well as many drugs. It is difficult to fully measure the impact of maternal diet on milk composition. Maternal

302

FUNCTIONAL FOODS

malnutrition plays a role in many developing countries where the food supply is limited, infections are common due to poor hygiene, and economic situations do not allow for the choice of properly used infant formulas. Dehydration can affect water fluxes in the body and thus, reduce the volume of milk produced (Hendricks, 2001). FIGURE 9.1 — Composition Of Human Milk, Bovine Milk And Infant Formula (%) Human Milk Protein CN:WP Fat Lactose Total Solids Ash

1.00 30:70 3.80 7.00 12.40 0.20

Bovine Milk 3.40 80:20 3.50 5.00 12.50 0.70

Formula 1.50 40:60 3.80 7.20 13.0 0.30

Proteins In Human Milk The level of total protein in milk is approximately 0.9 - 1.2%, of which approximately 70% is whey protein and 30% is casein along with small amount of proteins associated with the milk fat globules. There is no blactoglobulin in human milk. The primary whey proteins are
-lactalbumin, lactoferrin, and secretory IgA (SIgA). The whey proteins human milk lacks are
-caseins, b-casein and -casein (Figure 9.2).
-Lactalbumin is one of the major whey proteins and is required for the biosynthesis of lactose. Human
-lactalbumin can bind both Ca and Zn. However, only a small part of the total calcium found in human milk is bound to
-lactalbumin. It is possible that
-lactalbumin may generate peptides that facilitate the absorption of divalent cations, thus exerting a positive effect on mineral absorption. Supplementation of infant formula with bovine
-lactalbumin may increase the absorption of iron and zinc. Lactoferrin tightly binds iron, presumably limiting the availability of iron to potentially pathogenic microflora. SIgA can bind specific antigens in the infant gastrointestinal tract, preventing infection. Lysozyme is another human milk protein that plays a specific role in host protection, by lysing the cell walls of potential pathogens, preventing infection. Caseins contribute to the amino acid pattern of human milk, and they are also highly digestible. Functionally, their most important property is the ability to form stable aggregates that include calcium and phosphorus, which allows for greater concentrations of these minerals in human milk than is possible by

303

HUMAN MILK

their solubility alone. Non-protein nitrogen components consist of urea, peptides, nucleotides, nucleosides, and free amino acids, and remain after milk protein has been precipitated with 12% trichloroacetic acid. Casein exists as micelles in colloidal dispersion. Micelles of human milk range from 20 to 55 nm in size compared with those from 100 to 150 nm of bovine milk. FIGURE 9.2 — Protein Components Of Human And Bovine Milk (%)

Total Caseins
S1-Casein
S2-Casein β-Casein -Casein Micelle Size (nm) Whey Proteins
-Lactalbumin β-Lactoglobulin Lactoferrin Serum albumin Lysozyme Immunoglobulins Others

Human Milk

Bovine Milk

0.3 g/100 g — — 85 15 50 0.7g/100g 26 26 10 10 16 (IgA) 12

2.6 g/100 g 40 8 38 12 150 0.8g/100g 17 43 trace 5 trace 10 (IgG) 24

Proteins in human milk provide an important source of amino acids to the growing infant, and also play a very important role in facilitating the digestion and uptake of many other components in human milk. Lactoferrin, β-casein, and haptocorrin may enhance the absorption of iron, calcium, and vitamin B12, respectively. Other activities of human milk proteins include immune function enhancement, defense against pathogenic bacteria, viruses and yeasts, and gut development and function (Lonnerdal, 2003). The contents of casein and whey proteins change profoundly in the early stages of lactation; whey protein concentration is very high and casein is virtually undetectable during the initiation of lactation. As lactation progresses, casein synthesis in the mammary gland and milk casein concentration increases, while the concentration of whey proteins decreases, in part due to a larger volume of milk produced. Therefore, the ratio of whey: casein is not constant, but fluctuates between 70:30 or 80:20 in early lactation, to 50:50 in late lactation (Lonnerdal, 2003). The amino acid profile of caseins and whey proteins are different, thus, the amino acid profile of human milk varies during lactation.

304

FUNCTIONAL FOODS

Historically, the protein content of human milk was overestimated due to the large proportion of non-protein nitrogen (NPN) in human milk relative to the milk of other species. In the milk of most species, NPN makes up a small fraction (~5%) of the total nitrogen (TN); therefore, it is fairly accurate to estimate the protein content by total nitrogen analysis. In cases such as this, the true milk protein content is estimated by multiplying the nitrogen content of the milk by a conversion factor of 6.38, which takes into account the fraction of NPN in dairy products. In human milk, NPN accounts for approximately 20 - 25% of TN, thus, the use of the 6.38 conversion factor with the total nitrogen in milk yields an overestimate of total protein (Lonnerdal, 2003). To obtain a more accurate estimate, it is best to determine the TN content, subtract the NPN, then multiply the remaining nitrogen by the conventional Kjeldahl factor of 6.25. The protein content in human milk ranges from 1.4 – 1.6 g/100 ml during early lactation, 0.8-1.0g/100ml by 3 - 4 months of lactation, and 0.7-0.8 g/100 ml after 6 months. The levels of protein and corresponding intakes may not accurately represent the amount of utilizable amount of amino acids supplied to infants, as intact breast-milk proteins have been found in the stool of the breastfed infant, indicating that they are incompletely digested, and that available amino acids do not represent utilized amino acids. Undigested, biologically active proteins may have physiological benefits for the breastfed infant, therefore, the nutritional loss of the amino acids in these proteins may be insignificant, depending on the quantity lost. It is commonly understood that nutrients in human milk are exceptionally well utilized by the breastfed infant. Human milk proteins play many roles in the absorption of these nutrients. Proteins bind essential nutrients, aid in maintaining their solubility, and facilitate their uptake by intestinal mucosa. Protease inhibitors may assist in this process by limiting proteolytic enzyme activity, which can preserve the physiological function of some relatively stable binding proteins. In addition, some enzymes in human milk affect the digestion and utilization of some micronutrients. Human milk proteins involved in digestive function include bile saltstimulated lipase, amylase, and
1-antitrypsin (Lonnerdal, 2003). Bile salt-stimulated lipase may aid in the digestion of lipids in newborns, especially in premature infants, who experience reduced lipase activity and poor lipid utilization. Bile salt-stimulated lipase hydrolyzes di- and triacylglycerols, cholesterol esters, diacylphosphatidylglycerols, and micellar and water-soluble substrates. Human milk has a significant

HUMAN MILK

305

amount of
-amylase, although there is not a substrate for amylase in human milk. It has been suggested that the amylase in human milk may compensate for low salivary and pancreatic amylase activity in newborns, and may aid in the digestion of complex carbohydrates when complementary foods are fed close to the breastfeeding session. The protease inhibitors
1-antitrypsin and antichymotrypsin are both present in human milk. It is thought that they may collaborate to limit the activity of pancreatic enzymes in breast fed infants. Proteins such as
1-antitrypsin may escape complete digestion, and can be found in the stool of breastfed infants. In in vitro experiments, the addition of
1-antitrypsin results in a larger amount of lactoferrin resisting proteolytic degradation, although data on total nitrogen balance of breastfed infants are not substantially affected. This suggests that the protease inhibitor effect of
1-antitrypsin and antichymotrypsin may simply delay breakdown of these proteins, rather than preventing it completely (Lonnerdal, 2003). β-Casein is the major constituent of caseins in human milk, and it is a highly phosphorylated protein. Phosphopeptides formed during digestion have been shown to keep Ca soluble, thus enhancing calcium absorption. Clusters of phosphorylated threonine and serine residues are located close to the N-terminal end of β-casein, and can complex Ca ions. Thus, phosphopeptides formed from β-casein contribute to the high bioavailability of calcium in breast milk (Lonnerdal, 2003). Casein phosphopeptides may also affect the absorption of zinc and other divalent cations. Lactoferrin, a major iron-binding protein capable of binding two ferric irons, binds a major portion of the iron in human milk. It facilitates human intestinal cell iron uptake in cultured cells, which is most likely mediated by a specific enterocyte lactoferrin receptor (Suzuki, et al, 2002). Studies investigating the addition of bovine lactoferrin to infant formula have not revealed an enhancing effect on either iron uptake or iron status. Therefore, it appears that bovine lactoferrin does not bind to a human lactoferrin receptor, or that lactoferrin only exerts a benefit in human milk, and that when added to infant formula, other constituents interfere with iron utilization from lactoferrin. Heat treatment processing in formula after lactoferrin is added may contribute to the lack of effect observed when lactoferrin is added to human milk (Lonnerdal, 2003). Haptocorrin, previously known as vitamin B12 binding protein, binds nearly all the vitamin B12 found in human milk. Haptocorrin exists at a much higher level than vitamin B12 in human milk, which results in this protein being found most commonly in the unsaturated form. This

306

FUNCTIONAL FOODS

may have important antimicrobial benefits, as research indicates that haptocorrin may inhibit bacterial growth. Holohaptocorrin, the complex of vitamin B12 and haptocorrin, appears to bind in a saturable manner to human intestinal brush border membranes, and human intestinal cells in culture take up haptocorrin-associated vitamin B12 (Adkins & Lonnerdal, 2002). This collectively suggests a role for haptocorrin in vitamin B12 absorption early in life. Intrinsic factor is a substance secreted by the gastric mucosa that facilitates vitamin B12 absorption. Although intrinsic factor is present in the stool of breastfed infants at a young age, it may not be present in amounts adequate to facilitate the uptake of vitamin B 12 via the intrinsic factor receptor, therefore, haptocorrin is the main route for vitamin B12 absorption (Adkins & Lonnerdal, 2002). Folate-binding protein (FBP) has been found in human milk, both in particulate and soluble forms. When found as the soluble form, FBP is ~22% glycosylated which may aid this protein in resisting proteolytic digestion. In newborn goats, FBP has been found to resist proteolysis and tolerate low gastric pH, and it is possible that it behaves in a similar manner in human infants (Salter & Mowlem, 1983). Experiments performed with rat intestinal cells observed an increased uptake of folate when it was complexed with FBP than when it was provided in the free form (Colman, Hettiarachchy, & Herbert, 1981). It has been theorized that FBP may slow the release of folate in the small intestine, allowing for a slow absorption of folate, which could increase tissue use. Insulin-like growth factors (IGFs) I and II are also present in human milk, most commonly associated with IGF-binding proteins. These IGFbinding proteins may protect against IGF being digested, prolong their half-life, and control their interaction with intestinal receptors. Lipids In Human Milk Lipids play a diverse role in human nutrition and development (e.g., energy source, energy storage, vehicles for the absorption and transport of fat-soluble compounds). Fat is the most variable component of human milk and although the fat content in human breast milk is markedly influenced by lactational stage, fatty acid composition remains relatively stable. Normal growth and weight gain of infants is dependent on an adequate supply of fats in the diet. Especially the essential fatty acids, a group of naturally occurring unsaturated fatty acids with chain lengths of 18, 20, and 22 carbon atoms and containing between two and six methylene interrupting double bonds (Hendricks and Guo, 2006). Of

HUMAN MILK

307

these, Oleic (18:1), Palmitic (16:0), Linoleic (18:2 -6), and
-linolenic acid (18:3 -3) are most abundant in mature breast milk. With the latter two generally recognized as dietary essential fatty acids because of the inability of tissues to introduce the necessary double bonds in the carbon chains before carbon 9. Human milk contains about 3 to 5% total lipid, existing as emulsified globules, 1-4 µm in diameter, covered with a phospholipid-protein membrane derived from the mammary cells that line ducts of the glands and are released with the milk during lactation. The main function of the phospholipids in milk is as emulsifying agents and stabilizers of the milk fat globule membrane. They readily bind cations like calcium, sodium and magnesium, and possibly interact with digestive enzymes. Bovine milk contains substantial quantities of C4:0 to C10:0 short chain, saturated fatty acids, about 2% (w/w of fat) C18:2 (linoleic), and almost no other long-chain polyunsaturated fatty acids (Figure 9.3). The fatty acid composition is not altered by ordinary changes in diet. In contrast, human milk contains very little short chain fatty acids (C4:0 to C10:0), 10 to 14% (w/w of fat) linoleic (18:2 -6), and small quantities of other polyunsaturated fatty acids. The triacylglycerol structure differs as well, with much of the sn-2 position in human milk lipids occupied by C16:0 (palmitic). Human milk also contains the long chain polyunsaturated fatty acids docosahexanoic (DHA) (22:6 -3) and eicosapentaenoic (EPA) (20:5 -3) which have been shown to be important in the development of retinal and brain tissue. The major sterol in both human and bovine milk is cholesterol. Trace amounts of other sterols are present also, e.g., lanosterol in bovine milk and desmosterol and some phytosterols in human milk. The amount of cholesterol present in human milk is 10 to 15 µg/100ml. Since the role of dietary cholesterol is still not fully defined, an intake similar to that obtained through breast feeding is generally recommended. EPA and DHA are the predominant long chain polyunsaturated fatty acids in human milk, and are known to be essential to normal development of infants. These fatty acids may also be formed from precursors, even in preterm infants. The studies indicated that DHA supplementation increased DHA levels and there are correlations between DHA levels in maternal plasma and human milk, and between milk and infant plasma phospholipids. Until recently, infant formulas did not contain any significant levels of DHA, even though it is present in human milk. DHA can be synthesized from linoleic acid, but high intakes of linoleic acid can also inhibit this process. Thus, a preformed source of DHA in

308

FUNCTIONAL FOODS

the infant diet may be more efficient in assuring the supply of an adequate amount (Lonnerdal, 1986). FIGURE 9.3 — Fatty Acid Profiles Of Human And Bovine Milks (%, w/w) Human

Bovine

1.4 6.2 7.8 22.1 6.7 44.2

3.5 1.9 1.3 2.5 2.8 10.7 27.8 12.6 63.1

Monounsaturated Palmitoleic (16:1) Oleic (18:1) Gadoleic (20:1) Cetoleic (22:1) Total

3.1 35.5 0.96 Trace 39.8

2.5 26.5 Trace Trace 30.3

Polyunsaturated Lineoleic (18:2) Linolenic (18:3) Parinaric (18:4) Arachidonic (20:4) Eicosapentenoic (20:5) Total

8.9 1.2 0.72 Trace 10.82

2.9 1.6 Trace Trace Trace 4.5

Saturated Butyric (4:0) Caproic (6:0) Caprylic (8:0) Capric (10:0) Lauric (12:0) Myristic (14:0) Palmitic (16:0) Stearic (18:0) Total

Carbohydrates In Human Milk Carbohydrate in human milk is comprised of monosaccharides, such as glucose and galactose; disaccharides, such as lactose and lactulose; oligosaccharides; and some more complex carbohydrates, such as glycoproteins. Lactose is the primary carbohydrate in human milk and most likely to contribute to malabsorption and intolerance syndromes resulting from metabolic disturbances, such as lactose intolerance, lactose malabsorption, and galactosemia. Monosaccharides in milk are primarily made up of glucose and galactose, and are found at levels of approximately 100 mg/100ml in human milk. Lactose is the nutrient least likely to be affected by maternal nutrition, including malnutrition or energy supplementation. The concentration of lactose in human milk is relatively stable at about 7%. Total oligosaccharide levels comprise of up to 10% of total carbohydrates.

HUMAN MILK

309

Lactose together with mineral constituents, is crucial to maintaining a constant osmotic pressure in milk. An exclusively breastfed baby receives approximately 10 - 14 g lactose per day/ kg body weight. Lactose in human milk has been reported to exert a beneficial effect on the absorption of minerals, most notably calcium, which is most likely due to its conversion to lactic acid by intestinal flora, which lowers the pH, causing increased solubility of calcium salts. This is possible because human milk has a low buffering capacity and a low content of protein and phosphorus. Neither lactose nor lactulose are hydrolyzed in the upper GI tract, and only to a very small extent in the proximal intestinal tract, but are hydrolyzed in the distal intestines. Lactulose, a disaccharide of galactose and fructose, is a growth promoting factor and energy source to Lactobacillus bifidus and Lactobacillus acidophilus. In addition, the production of lactic acid has a slight laxative effect. Oligosaccharides in human milk, ranged from tri- to octasaccharides at levels of 0.8-1.4%. At least 21 different types of oligosaccharides have been identified in human milk, composed of many different molecules, including simple sugars and sugar derivatives such as uronic acid. These can be acidic, neutral, linear, or branched. Oligosaccharides in human milk have been divided into nitrogen-free oligosaccharides, or oligosaccharides containing either N-acetylglucosamine or Nacetylneuraminic acid (sialic acid). Small oligosaccharides are common in human milk, as well as a high content of complex and fucosylated and sialylated oligosaccharides. More than 130 components have been characterized in human milk. Some components are thought to be involved with the immune system, while others may be involved with the development of a specific intestinal microflora. The oligosaccharide component of human milk is thought to be the main energy source for the intestinal flora of the breast-fed infant, which is rich in bifidobacteria and lactobacilli. Lactobacilli ferment lactose to lactic acid which, along with a low pH, promotes the growth of Lactobacillus bifidus, as well as the bifidus factors lactulose, oligosaccharides, glycoproteins, and glycopeptides. The bifidus factor is most likely found in the nitrogencontaining oligosaccharides. Oligosaccharides added to cow’s milk based infant formula include galacto-oligosaccharides and inulin, and have been shown to stimulate the growth of bifidi and lactobacilli. Vitamins In Human Milk All water-soluble and fat-soluble vitamins are found in human milk. Human milk contains more vitamin A, E, C, nicotinic acid, and inositol than bovine milk, however, it has a lower content of vitamins B1, B2,

310

FUNCTIONAL FOODS

B6, B12, K, biotin, pantothenic acid, and choline (Figure 9.4). Human milk appears to contain adequate amounts of most vitamins to support normal infant growth, with the exception of vitamin D and possibly, vitamin K. Exclusively breastfed infants of mothers on a total vegetarian diet may require vitamin B12 supplementation to prevent a deficiency, which results in severe and permanent neurologic damage. FIGURE 9.4 — Vitamin Content In Human And Bovine Milk (mg/L) Human Milk Vitamin A Carotene Cholecalciferol (D) Tocopherol (E) Vitamin K Thiamin (B1) Riboflavin (B2) Pyridoxine (B6) Cobalamin (B12) Niacin Folic acid Ascorbic acid (C) Biotin Pantothenic acid Inositol

0.53 0.24 0.001 5.4 0.015 0.15 0.37 0.10 0.0003 1.7 0.043 47 0.007 2.1 300

Bovine Milk 0.37 0.21 0.0008 1.1 0.03 0.42 1.72 0.48 0.0045 0.92 0.053 18 0.036 3.6 160

Fat-Soluble Vitamins Vitamin A is comprised of a family of compounds in which the basic constituent is all-trans-retinol. Vitamin A is required for a large number of life processes and a deficiency has been associated with clinical disorders unique to infants. Compounds with vitamin A activity present in human milk include retinyl esters, retinol, and β-carotene. When maternal nutritional status is good, human milk supplies adequate amounts of vitamin A. Although vitamin A content of milk decreases as lactation progresses, milk ingestion volumes increase; therefore, the infant receives adequate amounts of vitamin A. Poor maternal nutritional status results in milk with a low vitamin A content, which can place the infant at risk for deficiency. Mechanisms regulating storage, mobilization, and secretion of retinoids from mammary cells have yet to be determined; although, there is indication that the concentration of retinal binding protein in serum determines the amount of retinol delivered to milk. Research indicates that vitamin A supplementation just preceding or following parturition can

HUMAN MILK

311

significantly increase vitamin A levels in human milk, especially in situations of low intake levels. Vitamin D plays an essential role in bone metabolism and may also be implicated in immune system regulation. The serum concentration of 25-OH-D (25-OH cholecalciferol), the active metabolite of vitamin D, is generally used to measure vitamin D status. Dietary ergocalciferol (D2) and cholecalciferol (D3) are converted to the active metabolite, 25OH-D, in the body. Infants can synthesize vitamin D endogenously in the epidermis upon exposure to sunlight, or they can receive it through dietary intake. Seasonal variations in vitamin D synthesis in infants have been observed, and light-skinned infants are more likely to benefit from sunlight exposure than dark-skinned infants. The level of 25-OHD in human milk is low, corresponding with both maternal serum 25OH-D levels and maternal dietary vitamin D intake, and can also be affected by race, season, and latitude. Infants who are exclusively breastfed receive below the minium recommended intake of vitamin D, and much lower than the recommended dietary intake, and as such, are at risk for deficiency, rickets, and improper bone mineralization, especially if sunlight exposure is poor. Normal vitamin D stores present at birth are depleted within 8 weeks, and formula-fed infants have higher serum concentrations of vitamin D metabolites than breast fed infants. Maternal supplementation with 400 - 2000 IU of vitamin D daily increases the vitamin D content of human milk, however, only the 2000 IU dose achieves satisfactory levels of 25-OH-D in the infant. Adequate sunlight exposure levels have not been clearly established, and due to the low level of vitamin D in human milk, vitamin D supplementation is recommended for breast-fed infants in Europe and the northern United States. Vitamin E is comprised of a group of compounds with different degrees of biological activity, with the most active being
-tocopherol. Vitamin E is an antioxidant, acting as a free radical scavenger and protecting against PUFA peroxidation in cell membranes. The transport of vitamin E across the placenta is limited, thus, neonatal tissues have low levels of vitamin E. Hemolytic anemia can result in neonates with a vitamin E deficiency. The vitamin E content of human milk is adequate for a term infant, but may not be sufficient for preterm infants, that have even lower levels of vitamin E at birth than the term infants. Decreased vitamin E levels in preterm infants may be related to PUFA, iron, and selenium concentrations, and hemolytic anemia is observed more frequently in preterm infants than term infants, presumably due

312

FUNCTIONAL FOODS

to lower vitamin E levels in infants fed formula supplemented with PUFAs and iron. Therefore, preterm infants who are not breastfed should receive formula enriched with vitamin E as well as LC-PUFA, although these fatty acids may provoke early postnatal decreases in both serum vitamin E and total lipids ratio. The vitamin E content of human milk is dependent on many factors, including individual variation, stage of lactation, and large amounts of dietary vitamin E. However, maternal supplementation with vitamin E has not been shown to affect the vitamin E content of human milk in mothers with moderate vitamin E intake. In addition, in populations with low vitamin E status, adequate vitamin E content in human milk has been observed, suggesting that maternal stores of vitamin E can be mobilized during lactation to ensure adequate supply in human milk. Vitamin K activity is provided by several different naturally occurring compounds, including vitamin K1, which is provided in the diet as phylloquinone, and vitamin K 2, menaquinones, which are synthesized by bacteria in the gastrointestinal tract. Vitamin K is essential to the proteins involved in blood coagulation, and some plasma proteins and organs have been shown to be dependent on vitamin K, including proteins that are involved in the maintenance of bone structure. The transfer of vitamin K across the placenta is very limited, thus, newborn infants generally exhibit extremely low concentrations of vitamin K. However, vitamin K levels remain constant in human milk over 6 months of lactation. Vitamin K is localized in the lipid core of the milk fat globule, and not the membrane. Even in situations where maternal vitamin K consumption exceeds the recommendations, exclusively breastfed infants do not receive the recommended dietary intake, and their plasma concentrations are low compared with formula-fed infants. In addition, breast-fed infants more frequently report the development of hemorrhagic disease. Due to the low content of vitamin K in human milk, and the low concentration of vitamin K in neonates, vitamin K supplementation is recommended after birth. Studies investigating the relationship between maternal vitamin K intake and content in human milk have mixed results, as some indicate no correlation, while another observed that maternal supplementation with vitamin K appeared to increase maternal plasma and breast milk concentrations, unless the supplemental dose of vitamin K was low. Preterm infants may require vitamin K supplementation as they tend to develop deficiencies more easily than term infants.

HUMAN MILK

313

Water-Soluble Vitamins Water-soluble vitamins are not effectively stored; therefore, it might be expected that maternal dietary intake would affect the contents of water-soluble vitamins in human milk more readily than fat-soluble vitamins. Thiamin content in human milk is average 0.15 mg/L. Six weeks of supplementation with thiamin from 1.3 to 3.4 mg/day did not increase milk thiamin levels in women from the United States who were adequately nourished. Urinary excretion of thiamin was higher in supplemented women, compared to unsupplemented women, suggesting that a limit exists in the transfer of this vitamin into human milk. Early studies indicated that a maternal thiamin deficiency could lead to low levels of thiamin in milk. Low maternal intake of riboflavin can produce low concentrations of riboflavin in breast milk. Supplementation with a modest amount of riboflavin (2 mg/day) increased milk riboflavin levels. A maternal intake of 2.5 mg/day was considered sufficient to maintain riboflavin status during lactation. The concentration of biotin in human milk from women in the U.S. was reported to be between 5-12 mg per liter. Supplementation with high levels of other B vitamins does not appear to affect biotin levels in milk. Supplementation with biotin increases biotin level in milk when its level is low, and has no effect when biotin levels are in the normal range. Vitamin B6 concentration in milk of mothers with vitamin B6 intakes around the RDA (2.5 mg/day) appears to be approximately 210 µg/L. Vitamin B6 level in the milk of U.S. women with low socioeconomic status and low vitamin B6 intake was 120 µg/L. Supplementation of vitamin B6 at levels above the RDA (5.3 mg/day) did not alter the vitamin B6 level in milk. However, it is important to note that supplementation of vitamin B6 at high levels to lactating women should be avoided as this can suppress lactation. Folate concentration in human milk increases with lactation time, ranging from 15 – 20 g/L in early lactation, to 40 - 70 g/L in mature milk. Supplementation of 0.8 g/L per day of folate to well-nourished U.S. women did not change milk folate concentration. However, when women of lower socioeconomic status and concomitant low folate intake (60% of the RDA) were supplemented with folate, the folate level in their milk was increased. Low intakes of vitamin B12, cyanocobalamin, are most likely reflected in lower milk concentration of this vitamin. Mean vitamin B12 levels in

314

FUNCTIONAL FOODS

well nourished U.S. women range between 0.97 - 1.10 µg/mL, and women of low socioeconomic status averaged 0.55 µg/mL. Maternal supplementation for 40 days with additional vitamin B12 raised the milk levels to only 0.79 µg/mL in the women of lower socioeconomic status, suggesting that long-term impaired maternal vitamin B12 status may not be completely alleviated in this time period. Supplementation of well-nourished women with additional vitamin B12 does not appear to augment milk concentration. Vitamin B12 levels can be low in the milk of U.S. women, especially when the mother follows a vegetarian diet, and can cause a vitamin B12 deficiency in the infant. Vitamin B12 in human milk is found as a protein-bound vitamin. Vitamin C level in the milk of women from the United States is about 50 mg/L. In well-nourished U.S. women, neither short-term nor longterm (6 months) supplementation of high levels (800 mg/day) of vitamin C affected milk concentration of this vitamin. Therefore, there appears to be an upper limit on the transfer of vitamin C into human milk, past which additional supplementation will not further augment levels in human milk. However, women with a niacin level of 1.96 mg/L with an intake between 15 - 23 mg niacin/day, showed a substantial increase in their milk to 3.9 mg/L with 120 mg niacin/day for 6-14 days. Pantothenic acid level in human milk appears to be influenced by maternal daily dietary intake. Milk pantothenic acid level correlated strongly with the maternal intake of pantothenic acid over the previous 24 hours. Minerals In Human Milk Minerals exist in the body in several chemical forms, including inorganic ions and salts, or as constituents of other organic molecules, including proteins, fats, and nucleic acids. They contribute to a variety of physiological functions, including structural components of body tissues to essential parts of many enzymes and biologically important molecules. Sodium, potassium, chloride, calcium, magnesium, phosphorus, and sulfate make up the macrominerals found in human milk. Citrate is not a mineral, but it is found as a water-soluble portion of human milk that can bind some minerals. The primary determinant of macromineral concentration in human milk is duration of lactation, during which sodium and chloride decrease, and potassium, calcium, magnesium, and free phosphate increase over time. However, the mineral content of human milk is also influenced by nutritional status of the mother and environmental and other factors. The concentrations

315

HUMAN MILK

of macro- and microelements in human milk and bovine milk are compared in Figure 9.5. FIGURE 9.5 — Mineral Composition Of Mature Human And Bovine Milk (Per Liter)

Sodium (mg) Potassium (mg) Chloride (mg) Calcium (mg) Magnesium (mg) Phosphorus (mg) Iron (mg) Zinc (mg) Copper (mg) Manganese (g) Iodine (g) Fluoride (g) g Selenium (g) Cobalt (g) Chromium (g) Molybdenum (g)

Human Milk

Bovine Milk

150 600 430 300 30 130 0.3 1.5 0.3 12 70 16 16 0.1 0.3 3

500 1500 950 1200 120 950 0.5 3.5 0.1 30 260 20 12 0.5 3 73

(Adapted from Guo, 1990; Flynn, 1992)

Microelements Sodium is the main cation of extracellular fluid, and it is also the main controller of extracellular volume. It is involved in the regulation of osmolarity, acid-base balance, active transport across cells, and the membrane potential across cells. Potassium is the primary intracellular cation, as its concentration is 30 times greater concentration inside the cell than in the extracellular fluid. Potassium in the extracellular fluid is involved in the transmission of nerve impulses, maintenance of blood pressure, and control of skeletal muscle contraction. Chloride is also essential in the maintenance of fluid and electrolyte balance, as it is the principal extracellular anion (Flynn, 1992). Under normal circumstances, a dietary deficiency of sodium, potassium, or chloride does not occur. However, depletion of sodium and chloride can occur during extreme conditions, such as chronic diarrhea, heavy perspiration, or renal disease, and depletion of potassium can occur in situations where there are large alimentary or renal losses. The concentrations of sodium, potassium, and chloride in breast milk decrease with duration of lactation, from a reported 480, 740, and 850 mg/L in colostrum,

316

FUNCTIONAL FOODS

respectively, to 160, 530, and 400 mg/L respectively. No relationship has been identified between maternal intake and concentrations of these electrolytes in milk. Sodium, potassium, and chloride in human milk are present almost entirely as free ions. Calcium comprises about 1.5 - 2% of body weight in an adult, which has accrued approximately 1200 g of calcium. About 99% of this calcium is found in teeth and bones, providing structure and strength as calcium phosphate. The remaining calcium is found in extracellular fluids, intracellular structures, and cell membranes, and is involved in several regulatory functions, such as maintenance of a normal heart beat, hormone secretion, blood coagulation, nerve conduction, muscle contraction, activation of enzymes, and integrity of membranes. Human milk supplies approximately 200 mg of calcium in an average daily milk secretion of 750 ml, which appears to be sufficient for the term infant, but may not be adequate for a preterm infant. Supplementation of 1000 mg calcium/day in lactating women does not affect milk calcium or lactation-associated bone mineral changes. The calcium content of human milk increases in early lactation, from 250 at day 1, to 320 mg/ L by day 5, and remains constant at approximately 300 mg/L up to day 36 of lactation. Studies on calcium concentration during lactation reveal an approximate 30% decrease between the first and ninth months of lactation. There is no correlation between maternal dietary consumption of calcium and its concentration in human milk. Calcium binds with phosphate and casein in human milk to produce calcium phosphate linkages in casein micelle subunits, and it can also bind to citrate, or be found in the ionized form. The calcium in human milk is more available for absorption. Magnesium plays an essential role in a variety of physiological processes, including neuromuscular transmission, muscle contraction, protein and nucleic acid metabolism, and as a cofactor for many enzymes. Magnesium, along with calcium and phosphate, supports skeletal growth. A deficiency of magnesium is not common except in conditions of severe malnutrition and certain disease states. Mature human milk contains magnesium at a concentration of approximately 30-35 mg/L. With a normal range of dietary magnesium intake, there is no relationship between maternal magnesium consumption and concentration of magnesium in human milk. It has been reported to be about 30% higher in colostrum than in mature milk. Some magnesium associates with phosphate and caseins in human milk. Phosphorus is a nutrient essential to humans, as it serves a number of important biological functions. It exists as organic and inorganic

HUMAN MILK

317

phosphates in all tissues and fluids, and is essential to many body components, including lipids, proteins, carbohydrates, and nucleic acids, and also plays an important role in metabolism. It is an important part of calcium phosphate, a major structural component of teeth and bones. Dietary deficiencies of phosphorus do not usually occur as phosphorus is contained in a wide variety of foods of plant and animal origin. In human milk, phosphorus content increases from 100 mg/L on day 1 to 170 mg/L by day 8, and decreases to 130 mg/L by day 36 of lactation. Trace Elements Trace elements, also known as microminerals, are substances that make up less than 0.01% of the body mass. In human milk these include iron, zinc, copper, manganese, selenium, iodine, fluorine, molybdenum, cobalt, chromium, and nickel. Iron is an essential component of heme in hemoglobin, myoglobin, cytochromes, and other proteins; therefore, it plays a role in the transport, storage, and utilization of oxygen. Iron deficiency anemia affects about 30% of the world’s population, including Western and underdeveloped countries. The mean iron concentration in human milk is 0.3 mg/L. The iron content of human milk decreases over the duration of lactation; colostrum iron level is about 1 mg/L, and decreases to 0.3 - 0.6 mg/L in mature milk. Dietary intake of iron has no relationship with iron concentration in human milk, and supplementation with iron at levels up to 30 mg/day does not affect milk iron concentration. Human milk iron is bound to three main components: lactoferrin, a low molecular weight compound, and a component of the milk fat globule membrane. Lactoferrin is the primary iron-binding protein in human milk, possessing a high affinity for the ferric ions, which bind two sites together with bicarbonate or carbonate ions. Lactoferrin concentration in human milk is much higher than iron concentration, so although one-third of iron is bound to lactoferrin, only 3-5% of lactoferrin is saturated with iron. However, iron released from other components during digestion may become bound to lactoferrin, especially when bicarbonate from pancreatic fluid is present. Citrate in the low molecular weight fraction and xanthine oxidase in the fat globule membrane may be among these other iron-binding components. Very little iron in human milk is bound to casein (Lonnerdal, 1989). Zinc is essential to proper growth and development, sexual maturation, wound healing, and it may play a role in immune system function and other physiological processes. Zinc assists several hormones involved in reproduction, is required for DNA, RNA, and

318

FUNCTIONAL FOODS

protein synthesis, and is a cofactor for many enzymes involved in most major metabolic processes (Flynn, 1992). Human zinc deficiency was first reported in the 1960s in the Middle East, resulting in dwarfism, impaired sexual development, and anemia. It is difficult to detect mild deficiencies of zinc, although they have been shown to occur in Western countries, especially in infants and children, and give rise to suboptimal growth, poor appetite, impaired taste acuity, and low hair zinc levels. Mean zinc concentration in mature human milk during the first six months of lactation is about 2 mg/L, although large variations in zinc have been reported at 0.65 - 5.3 mg/L. Dietary zinc intake has no correlation to zinc content of human milk, and zinc supplementation of a zinc adequate diet does not significantly affect human milk zinc concentration. Zinc in human milk is found in three major components: serum albumin and citrate in the whey, and in alkaline phosphatase in the fat globule membrane. Copper is required for iron utilization and is a cofactor for enzymes involved in glucose metabolism, as well as the synthesis of hemoglobin, phospholipids, and connective tissue. Copper deficiency is rare except in conditions of severe malnutrition. Mature human milk contains copper at a concentration of 0.3 mg/L. Copper concentration decreases with advancing lactation, from 0.6 mg/L in weeks one and two of lactation, to 0.36 mg/L by 6 - 8 weeks, and 0.21 - 0.25 mg/L by 20 weeks of lactation. No significant correlation exists between milk copper concentrations and dietary copper intake. Copper in human milk is bound to serum albumin and citrate. Copper has also been found in the fat globule membrane, however, the ligand has not yet been identified. Manganese is a cofactor for glycosyl transferases, which play a role in mucopolysaccharide synthesis, and is a nonspecific cofactor for many other enzymes. Two manganese metalloenzymes have been identified: mitochondrial superoxide dismutase and pyruvate carboxylase (Hurley & Keen, 1987). As manganese is widely distributed in foods, a dietary deficiency is not known to occur in humans (Flynn, 1992). In mature human milk, the mean concentration of manganese is approximately 10 µg/L and manganese is known to decrease with duration of lactation. No cases of manganese deficiency in human infants have been reported, thus, fully breastfed infants appear to receive adequate manganese (Lonnerdal et al., 1983). Manganese in human milk is mainly bound to lactoferrin, however, it exists at such a low concentration that approximately 2000 times more iron is bound to lactoferrin than manganese. Therefore, very little of the metal-binding capacity of lactoferrin is occupied by manganese (Lonnerdal, 1989).

HUMAN MILK

319

Selenium is an important component of the enzyme glutathione peroxidase. Glutathione peroxidase is present in many tissues, where it works with vitamin E, catalase, and superoxide dismutase as an antioxidant, protecting cells against oxidative damage. Selenium concentration in the milk of U.S. women is approximately 16 µg/L. Selenium concentration is higher in colostrum, at 41 µg/L, than in mature milk, 16 µg/L. A correlation was observed between human milk selenium content and both maternal plasma selenium concentration and plasma glutathione peroxidase activity, suggesting that milk selenium content is influenced by maternal selenium status (Levander et al., 1987). The average selenium content of milk of North American women is considered more than sufficient for breastfed infants. Iodine is essential to the thyroid hormones, thyroxine and triiodothyronine, which play an important role in the regulation of basal energy metabolism and reproduction. Iodine deficiency causes the thyroid gland to enlarge and form a goiter, while excess iodine in the diet reduces uptake of iodine by the thyroid gland, which yields signs of thyroid deficiency. In the United States, mean iodine concentrations in human milk have been reported as 142 µg/L (range: 21 – 281 µg/L). A correlation between milk iodine concentration and dietary iodine intake has been observed, therefore, the use of iodized salt can augment milk iodine content (AAP, 1981). North American women have an elevated iodine intake, and thus, the amounts of iodine in their milk are adequate. Molybdenum is a crucial component of several enzymes, including aldehyde oxidase, xanthine oxidase, and sulfite oxidase, where it exists in the prosthetic group molybdopterin. It has yet to be determined whether the human requirement is specifically for molybdenum, or whether it is for molybdopterin or a precursor. Dietary deficiency has not been observed in humans, except for a patient on long-term total parenteral nutrition. Molybdenum content of human milk is strongly correlated with the stage of lactation, decreasing from 15 µg/L on day 1, to 4.5 µg/L by day 14, and finally to a concentration of approximately 2 µg/L by one month and thereafter. Chromium is considered essential to human health, and the earliest sign of a deficiency is impaired glucose tolerance. Chromium deficiency has been observed exclusively in patients receiving long-term total parenteral nutrition. These patients respond to intravenous trivalent chromium with amelioration of glucose intolerance. The mean chromium content of mature human milk is 0.27 µg/L.

320

FUNCTIONAL FOODS

The only function of cobalt identified in humans is its presence as an essential part of vitamin B12. Vitamin B12 is synthesized from bacteria. Therefore, inorganic cobalt is essential for all animals that rely completely on their bacterial flora for vitamin B12 supply. Mature human milk contains cobalt at a concentration of approximately 0.1 µg/L. Dietary supplementation of cobalt increases the vitamin B12 level of human milk only when the maternal diet is cobalt deficient. Fluoride is considered a beneficial element, rather than an essential element, to human health, as it protects against dental caries and accumulates in bones and teeth. However, excessive fluoride intake leads to fluorosis, which causes mottling of the teeth, and also affects bone health and kidney function. In mature human milk, the mean fluoride content is about 16 µg/L. Infants who are breast fed or consuming concentrated or powdered formula prepared with nonfluorinated water have a low fluoride intake, and should receive fluoride supplements (NRC, 1989). Substantial evidence exists to establish the necessity of nickel, silicon, arsenic, and boron in animals, and it is most likely that these trace elements are also essential to humans. However, the nutritional functions of these elements are yet to be determined (NRC, 1989). Nickel is found in mature human milk at a level of 1.2 µg/L, silicon is found at 700 µg/L, arsenic is found at 0.2 - 0.6 µg/L (Renner, 1983). Biological Functions Of Human Milk A main function of some important human milk proteins is to provide antimicrobial activity against pathogenic bacteria, viruses, and fungi. The major immunoglobulin (>90%) in human milk is secretory immunoglobulin A (SlgA), which is a dimer linked to a secretory component and a joining chain. This molecular arrangement allows the molecule to resist intestinal proteolysis, which is confirmed by the detection of modest amounts of SlgA in the stool of breastfed infants. SlgA binds to bacteria and viruses in the intestine and prevents attachment to mucosal epithelial cells, limiting infection and colonization. Concentrations of SlgA in human milk range from 1-2 g/L in early lactation, and remain steady at 0.5 - 1 g/L up to the late stage of lactation (Goldman, 1993). Maternal immunity against many general pathogens can be transferred to the infant in the form of SlgA in the milk, mediated via the enteromammary pathway. Antibodies against bacterial pathogens including Escherichia coli, Vibrio cholera, Streptococcus pneumonia, Clostridium difficile, Haemophilus influenzae, and Salmonella; against rotavirus, cytomegalovirus, HIV,

HUMAN MILK

321

respiratory syncytial virus, and influenza virus; and against yeasts such as Candida albicans, have been found in human milk, demonstrating the extent of this defense (Goldman, 1993). Lactoferrin may be responsible for many antimicrobial activities of human milk. It was originally thought that lactoferrin, which is largely unsaturated with iron yet has a high affinity for it, could withhold iron from iron-requiring pathogens, thereby exerting a bactericidal activity against pathogens (Lonnerdal, 2003). This may be possible, however, studies have observed a strong bactericidal activity of lactoferrin without dependence on iron saturation. This activity may be related to the production of lactoferricin, a potent bactericidal peptide formed during lactoferrin digestion. Recent research revealed that lactoferricin inhibits enteropathogenic Escherichia coli from attaching to intestinal cells. There appear to be several defense contributions by lactoferrin against bacterial infection (Lonnerdal, 2003). In vitro, lactoferrin has been shown to have activity against viruses, including HIV, and fungi, such as Candida albicans, however, the mechanism of these activities is not known. In vitro digestion of human milk produced two bifidogenic peptides that originated from lactoferrin. These peptides were stable against further digestion with pepsin, trypsin, and chymotrypsin, were active at low concentrations, and possessed a bifidogenic effect approximately 100 times stronger than N-acetyl-glucosamine (Liepke et al., 2002). Advantages of the bifidogenic effect include potentially decreasing the allergenicity of non-digestible proteins, decreasing the incidence of rotavirus-induced diarrhea, antibacterial activity, and increased production of short chain fatty acids in the colon. Lysozyme is a major enzymatic component of human milk that can degrade the outer cell wall of gram-positive bacteria. In synergistic action with lactoferrin, lysozyme has also been shown to kill gramnegative bacteria in vitro. This is accomplished when lactoferrin binds to the lipopolysaccharide and removes it from the outer cell membrane of bacteria, allowing lysozyme to access and degrade the inner proteoglycan matrix of the membrane, which destroys the bacteria. Lactoperoxidase in human milk may contribute to the defense against infection in the mouth and the upper GI tract (Lonnerdal, 2003). In the presence of hydrogen peroxide, lactoperoxidase catalyzes the oxidation of thiocyanate to hypothiocyanate, which can render inactive both grampositive and gram-negative bacteria. Hydrogen peroxide is produced in small quantities by cells, and thiocyanate is provided by saliva. Lactoperoxidase in cow’s milk has been used in developing countries for many years to maintain microbial quality, and although human milk

322

FUNCTIONAL FOODS

does contain lactoperoxidase, the physiological significance is not yet determined. Exposure of
-lactalbumin to intestinal tract proteases yields peptide fractions, three of which have been implicated in antimicrobial activity against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermis, Klebsiella pneumonia, Streptococci, and Candida albicans. These findings may explain the inhibitory effect of
-lactalbumin-supplemented infant formula on diarrhea, caused by enteropathogenic Escherichia coli, in infant rhesus monkeys (Kelleher et al., 2003). Breastfeeding appears to provide protection against Helicobacter pylori infection in young children (Stromquist, et al., 1995). The heavily glycosylated -casein molecule in human milk, has been shown to inhibit the adhesion of Helicobacter pylori to human gastric mucosa (Stromquist et al., 1995). The mechanism by which -casein prevents attachment is that it acts as a receptor analogue, thus halting bacterial attachment to the mucosal lining. Human milk proteins are also involved in the immune system function of breastfed infants. Human milk contains many cytokines, including tumor necrosis factor a, transforming growth factor b, and interleukins (IL) 1 b, IL-6, IL-8, and IL-10. All of these cytokines are immunomodulatory, and most of them are anti-inflammatory, which may mitigate the effect of infections. The cytokines are found in free form, and also may be released from cells in breast milk (Lonnerdal, 2003). Human milk also contains lactoferrin, which has been shown to increase the production and release of cytokines such as IL-1, IL-8, tumor necrosis factor a, nitric oxide, and granulocyte-macrophage colony stimulating factor, which may also affect the immune system (Kelleher & Lonnerdal, 2001). When lactoferrin binds to its receptor in the small intestine, this may either cause signaling events that affect cytokine production downstream, or it is possible that the internalized lactoferrin can bind to the nucleus, which could affect nuclear transcription factor B, and subsequently, cytokine expression. Lactoferrin was recently shown to activate the transcription of IL-1b in mammalian cells, which indicates that lactoferrin may interact directly with the nucleus. Several proteins are also implicated in the development of the infant gut and its functionality, including growth factors, lactoferrin, and casein-derived peptides. Research has shown that IGF-I and IGF-II stimulate DNA synthesis and promote the growth of many types of cells in culture; therefore, they may play a role in the development of the infant gastrointestinal tract.

HUMAN MILK

323

Several peptides that possess physiological activity have been generated from human casein, and especially from β-casein (Lonnerdal, 2003). Although these proteins have been generated in vitro, they have also been detected from intestinal contents, suggesting that they are formed in vivo as well (Lonnerdal, 2003). Weight gain has been higher in infants who are fed formula supplemented with bovine lactoferrin than unsupplemented formula (Hernell & Lonnerdal, 2002). In support of this theory, administration of lactoferrin has been shown to enhance cell proliferation in the small intestine of experimental animals, and also to affect crypt cell development. The rapid development of intestinal mucosa in suckling newborns has been hypothesized to be in part due to the mitogenic effect of lactoferrin. Breastfed premature infants excrete intact lactoferrin in their urine, demonstrating that intact lactoferrin is absorbed by the infant gut (Goldman, 1989). Infant Formula Human milk is the best reference standard by which all infant formula is compared, and it has always been considered a speciesspecific food. In addition to nutritional components, human milk also contains immunoglobulin SlgA, peptide and non-peptide hormones, growth factors, proteins, peptides, lipids, and milk membrane fractions. Each discovery regarding infant formula, including formulation and processing, allows for the improvement of a product that continues to be increasingly similar to human milk. Although much is still unknown about human milk, and how to produce the optimum infant formula, new information is constantly being discovered. Some of the recent progress made in infant formula formulation and processing includes fortification with arachidonic and docosahexaenioc acids, nucleotides, and ingredients that promote healthy colonic microflora; effect of removal of phytate on soy formulas; trace mineral solubility and availability; component distribution and interactions; addition of whey peptides fractions. Ingredient Selection For Infant Formula Infant formula is designed to substitute for breast milk when mothers are not able to breast-feed their infants. It is most commonly prepared with cow’s milk, whose composition is modified to be more similar to human milk. Milk-based infant formulas include ingredients such as milk and whey protein, and soy formulas are based on soy protein isolate. Protein

324

FUNCTIONAL FOODS

hydrolysate formulas include protein that has been hydrolyzed to peptides and amino acids. Milk-free formulas are used in exceptional cases of intolerance to cow’s milk, and they exclude cow’s milk components. Medical formulations of infant formula exist for infants with special needs, including those caused by congestive heart failure, inborn errors of metabolism, and steatorrhea. Soy-based formulas contain soy protein isolate, in which methionine is a limiting amino acid, therefore, supplemental methionine must be added to achieve a more appropriate amino acid profile. Milk-based formulas are based on 0.6% casein and 0.9% whey proteins, yielding a 40:60 ratio of casein to whey proteins. Protein levels in milk-based infant formula are approximately 15 g/ L, providing 10% of total energy. Protein in milk-based formula is provided by non-fat milk and whey protein products. Protein in soybased formula provides approximately 11-13% of total energy, at 18 21 g/L. Soy-based formulas also contain additional L-methionine, Lcarnitine, and taurine. In special use formulas, protein is provided by casein hydrolysates, whey, and skim milk. It is contained at levels of 15 - 30 g/L accounting for 9 -12 % of the total energy. The carbohydrate content of milk-based infant formulas ranges from 70 - 72 g/L providing 40% of total kilocalories. In soy-based infant formula, carbohydrate levels average 67 - 69 g/L, providing about 40% of energy. Special use formulas contain carbohydrates at levels of 70 109 g/L, accounting for 40 - 45% of the total energy. Lactose is the major carbohydrate source in milk-based infant formulas, whereas carbohydrate in soy-based and special use formulas is provided by sucrose, corn syrup solids, dextrins, hydrolyzed corn starch, glucose, and glucose polymers. Fat levels in milk-based formulas range from 36 - 38 g/L providing about 50% of total energy. In milk-based formulas, fat is provided by oleo, coconut, soy, palm, sunflower, and safflower oils. In soy-based formulas, fat content ranges from 36 - 38 g/L, providing 47 - 49 % of the total energy. Soy-based formulas contain fat from oleo, soybean, safflower, coconut, palmolein, and high oleic safflower oil. Special use formulas contain fats from corn, safflower, soy, palm, coconut, sunflower, and high oleic safflower oils, and medium chain triglycerides. These fats are contained at levels of 34 - 49 g/L, accounting for 44 - 50 % of the total energy (Feldhausen et al., 1996). Formulation Aspects Of Infant Formula Infant formula is a complex and balanced food for infants. In the

HUMAN MILK

325

quest to produce an infant formula that closely mimics human milk, there are many steps to consider. The formulation of infant formula is a complicated task, with many details regarding composition, physicochemical properties, and shelf stability. Infant formula does vary in composition, but within narrow and precise limits. Formula should provide protein of an appropriate biological quality at levels of 10 -15% of calories, fat at 45 - 50% of calories, linoleic acid at 2 - 3% of total calories, and carbohydrate should make up the remaining calories. Milk, or milk and whey-based formulas must take into account the need to alter the natural composition of bovine milk. The changes that must be employed include lowering protein content, while maintaining biological quality, raising carbohydrate content, changing fat composition, and lowering the mineral content. In general, the guidelines for infant formula formulation are listed as follows: • • • • • • • • • •

All ingredients proven by FDA regulations Protein: fat: CHO ≈ 1: 2: 4 C18:2 accoutanting for 2-3% of total energy CN: WP = 40: 60 Ca: P = 1.5: 1 Minerals and Vitamins fortified Functional nutrients: -3, carnitine, nucleotides, prebiotics pH ≈ 7.0-7.2 Osmolarity ≈ 270 mOsm/L Processing damages to nutrients

Osmolarity is the measure of osmotically active substances, including sugars, amino acids, and mineral contents per liter of a solution. The osmolarity of human milk ranges between 270 – 290 mOsm/L, while bovine milk and infant formulas range between 200 – 400 mOsm/L, although osmolarities above 350 mOsm/L, should be avoided, as they can stress the newborn kidney and increase water loss. The osmolarity of commercial infant formula is approximately 230 - 270 mOsm/L, or 300 mOsm/kg water. Osmolarity, along with renal solute load, plays an important role on the efficacy of the food. The renal solute load is defined as the sum of solutes that must be excreted by the kidney. The renal solute load is most commonly expressed in mOsm/day, and the concentration in urine is expressed as osmolality (mOsm/kg water). If the renal solute load is too high, hypernatremic dehydration can occur, and if the renal solute load is too low, hyponatremia can occur (Fomon, 1993). Osmolarity has a relationship with renal solute load in that it relates to the amount of osmotically active substance that is contributed

326

FUNCTIONAL FOODS

by the infant formula. In a typical formula containing 67 kcal and 7 grams of lactose per 100 mL, lactose contributes approximately 200 mOsm/kg water, which is approximately 70% of solutes. By replacing lactose with glucose, the osmotic solute load would be doubled (Fomon, 1993). This type of substitution is important when formulating infant formula, as the osmolarity range is narrow. Therefore, when formulating infant formula it is crucial to keep the osmolarity within an appropriate range. Components in milk, including proteins, carbohydrates, fats, and mineral salts, which provide pH buffering properties. Buffering groups in milk include protein bound residues and salts. Protein bound residues include aspartic acid, glutamic acid, histidine, tyrosine, lysine, esterphosphate, N-acetyl neuraminic acid, and terminal groups. The salts native to milk that possess buffering capabilities include phosphate, phosphate esters, citrate, carbonate, various carboxylic acids, various amines, and lactic acid. Human milk has an average pH of 7.00 -7.25, which is higher than bovine milk. Therefore, infant formula is modeled on human milk, and the optimal pH for infant formula would be similar to that of human milk. When bovine milk products are used as a base for infant formula, careful consideration of ingredients and mineral salts must be employed to produce a product that follows all the necessary guidelines and achieves the proper pH. Bovine milk-based infant formulas are prepared using skim milk powder, demineralized whey, lactose, vegetable oils, essential fatty acids, lecithin, nucleotides, vitamins, and minerals. The American Academy of Pediatrics Committee on Nutrition 1982 Task Force and 1987 FDA Recommendations established recommended nutrition levels of infant formulas per 100 kcal (Figure 9.6). The recommended global standard for the composition of infant formula is shown in Figure 9.7. These guidelines provide the acceptable and safe ranges for the composition of infant formula, including energy-contributing and non-energy containing nutritive ingredients (Committee on Nutrition, American Academy of Pediatrics, 1998). Some nutrients have wide ranges that are considered acceptable. For example, sodium, potassium, and chloride, where the maximum limit can be approximately three times as high as the minimum limit. Other nutrients, such as selenium, have a very narrow safe range. Nitrate salts are not usually added to infant formula, due to the potential to cause methemoglobinemia. In regards to trace elements, such as iron, copper, and zinc, sulfate forms have been traditionally used in commercial production. The process of infant formula formulation begins with determination of the target nutrient

327

HUMAN MILK

levels that will make up the composition of the formula. The ingredients are then selected, and calculations are performed to determine the amount of each ingredient that will contribute to the gross energy composition of the formula, including protein, fat, and carbohydrate. Once these main ingredients of the formula are calculated, the composition of each ingredient is examined to determine the contribution of minerals and vitamins provided by the main ingredients, and vitamins and mineral salts can be added as necessary to achieve the target nutrient profile. FIGURE 9.6 — Nutrient Specifications For Infant Formulas

Protein (g) Fat (g) Linoleic acid (g) Vitamin A (IU) Vitamin D (IU) Vitamin E (IU) Vitamin K (g) Thiamin (g) Riboflavin (g) Vitamin B6 (g) Vitamin B12 (g) Niacin (g) Folic acid (g) Pantothenic acid (g) Biotin (g) Vitamin C (mg) Choline (mg) Inositol (mg) Calcium (mg) Phosphorus (mg) Magnesium (mg) Iron (mg) Zinc (mg) Manganese (g) Copper (g) Iodine (g) Sodium (mg) Potassium (mg) Chloried (mg) (Fomon, 1993)

Minimum

Maximum

1.8 3.3 0.3 250 40 0.7 4 40 60 35 0.15 250 4 300 1.5 8 7 4 60 30 6 0.15 0.5 5 60 5 20 80 55

4.5 6.0 750 100 3.0 75 60 200 150

328

FUNCTIONAL FOODS

FIGURE 9.7 — Proposed Compositional Requirements Of Infant Formula (Koletzko, et al, 2005)

Energy Proteins Cows’ milk protein Soy protein isolates Hydrolyzed cow’s milk protein Lipids Total fat Linoleic acid
-linolenic acid Ratio linoleic acid/
-linolenic acid Lauric + myristic acids Trans fatty acids Erucic acid Carbohydrates Total carbohydrates1 Vitamins Vitamin A Vitamin D3 Vitamin E Vitamin K Thiamin Riboflavin Niacin2 Vitamin B6 Vitamin B12 Pantothenic acid Folic acid Vitamin C Biotin Minerals and trace elements Iron - (formula based on cows’ milk protein and protein hydrolysate) Iron (formula based on soy protein isolate) Calcium Phosphorus (formula based on cows’ milk protein and protein hydrolysate) Phosphorus (formula based on soy protein isolate) Ratio calcium/phosphorous

Unit

Minimum

Maximum

kcal/100mL

60

70

g/100 kcal g/100 kcal g/100 kcal

1.8* 2.25 1.8†

3 3 3

g/100 kcal g/100 kcal mg/100 kcal % of fat % of fat % of fat

4.4 0.3 50 5:1 NS NS NS

6.0 1.2 NS 15:1 20 3 1

g/100 kcal

9.0

14.0

g RE/100 kcal‡ g/100 kcal mg
-TE/100 kcal2 g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal g/100 kcal mg/100 kcal g/100 kcal

60 1 0.5¶ 4 60 80 300 35 0.1 400 10 10 1.5

180 2.5 5 25 300 400 1500 175 0.5 2000 50 30 7.5

mg/100 kcal

0.3**

1.3

mg/100 kcal mg/100 kcal

0.45 50

2.0 140

mg/100 kcal

25

90

mg/100 kcal mg/mg

30 1:1

100 2:1

329

HUMAN MILK

FIGURE 9.7 — Proposed Compositional Requirements Of Infant Formula (Koletzko, et al, 2005) - Continued Unit

Minimum

Maximum

Minerals and trace elements - continued Magnesium mg/100 kcal Sodium mg/100 kcal Chloride mg/100 kcal Potassium mg/100 kcal Manganese g/100 kcal Fluoride g/100 kcal Iodine g/100 kcal Selenium g/100 kcal Copper g/100 kcal Zinc mg/100 kcal

5 20 50 60 1 NS 10 1 35 0.5

15 60 160 160 50 60 50 9 80 1.5

Other substances Choline Myo-inositol L-carnitine

7 4 1.2

50 40 NS

mg/100 kcal mg/100 kcal mg/100 kcal

*

The determination of the protein content of formulae based on non-hydrolyzed cows’ milk protein with a protein content between 1.8 and 2.0 g/100 kal should be based on measurement of true protein ([total N minus NPN] ×6.25). † Formula based on hydrolyzed milk protein with a protein content less than 2.25 g/ 100kcal should be clinically tested. 1

Sucrose (saccharose) and fructose should not be added to infant formula.

‡

1g RE (retinol equivalent) = 1g all-trans retinol = 3.33 IU vitamin A. Retinol contents shall be provided by performed retinol, while any contents of carotenoids should not be included in the calculation and declaration of vitamin A activity. 2

1mg
-TE (
-tocopherol equivalent) = 1 mg d-
-tocopherol.

¶Vitamin E content shall be at least 0.5 mg
-TE per g PUFA, using the following factors of equivalence to adapt the minimal vitamin E content to the number of fatty acid double bonds in the formula: 0.5 mg
-TE/g linoleic acid (18:2n-6); 0.75 mg
-TE/g
-linoleic acid (18:3n-3); 1.0 mg
-TE/g arachidonic acid (20:4n-6); 1.25 mg
-TE/g eicosapentaenoic acid (20:5n-3); 1.5 mg
-TE/g docosahexaenoic acid (22:6n-3). 2

Niacin refers to performed niacin.

**

In populations where infants are at risk of iron deficiency, iron contents higher than the minimum level of 0.3 mg/100 kcal may be appropriate and recommended at a national level. NS = not specified.

In order to achieve the proper pH, different mineral salts should be used. Some mineral salts contribute to a pH that is more acidic, while other mineral salts contribute to a more neutral or basic pH. For example, when the pH of infant formula is lower than optimal (i.e., 6.8 - 7.0), mineral salts that include bicarbonates (i.e., sodium bicarbonate) and oxides (i.e., magnesium oxide) can be substituted to increase pH to an optimal level, and chlorides (i.e., magnesium chloride) and citrates

330

FUNCTIONAL FOODS

(i.e., potassium citrate) can be used to reduce a higher pH, or in a formula where the pH is already acceptable (Smith, 2004). As skim milk powder and whey protein products are important functional ingredients of milk-based infant formulas, their physical and chemical composition also contributes to the properties that are observed in infant formulas. Bovine milk proteins play an important role in infant formula. Casein micelles will irreversibly aggregate at temperatures above the boiling point, and heating also results in precipitation of proteins onto the fat globule surface. Acid causes casein micelles to destabilize and/or aggregate due to decreased electric charge around that of the isoelectric point. Acid also increases the solubility of minerals so that organic calcium and phosphorus within the micelle slowly become more soluble. Heat causes whey proteins to adsorb onto the surface of the casein micelle. The buffering capacity of milk salts change with heating, releasing carbon dioxide, producing organic acids, and precipitating tricalcium phosphate and casein phosphate with the subsequent release of hydrogen ions. Clearly, pH and heat treatment are two important areas to consider when formulating and processing infant formula, as they can both exert considerable impacts on the properties of milk proteins. In infant formulas and milk-based nutritional supplements, dairy ingredients are used extensively to build a nutritional base, and are used in conjunction with additional proteins, lipids, carbohydrates, vitamins, minerals, and other ingredients, when merited, to achieve the desired nutrient profile. Clearly, dairy ingredients possess many functional characteristics in addition to nutrition, which is important during both processing and reconstitution. Lactose, a reducing sugar, can also contribute to Maillard reactions in sterilized products. In addition to carbohydrate, protein is a very important part of infant formula and other milk-based nutritional supplements. Some infant formulas are based solely on bovine milk proteins, with an approximate whey protein: casein ratio of 60:40, which is more similar to human milk (80:20), although the whey protein systems in human and bovine milks are markedly different. The addition of whey protein to infant formula creates additional stability issues, such as the heat stability of the final product. The main objective in manufacturing heat-stable infant formulas with added whey proteins is to control the formulation and processing variables that can prevent whey protein self-aggregation. It is most effective to begin with a whey protein source that contains low levels of heat-denatured protein, and to employ factors such as processing (heat treatment) and formulation

HUMAN MILK

331

(salt balance) in order to produce the best heat stable final product. To form a successful protein-stabilized emulsion with optimum heat stability, solubilization of the protein is essential. Heat treatment alters the solubility of the calcium phosphate in milk, and calcium salts play a role in protein aggregation at pH > 6.5, which is the pH range for infant formula. Casein enhances the protein-stabilized emulsion in milkbased products, while viscosity can be controlled to prevent creaming during storage, which results from milk protein reactivity. Another factor to consider is that divalent cations may contribute to the instability of milk protein systems during heating. This effect can be controlled by proper selection of the minerals used in fortification, and by balancing the divalent cations with other mineral salts, such as citrates or phosphates. Clearly, many factors come into play when formulating a milk-based formula or nutritional supplement. Once the proper considerations and preparations have been made, the infant formula should yield the desirable properties. Infant Formula Processing Infant formula is processed as ready-to-feed and concentrated liquids, and as dried powder. Liquid products must be sterilized in order to prevent spoilage during long-term storage. For commercial liquid infant formula, this would be classified as a retort product, which is sterilized inside the container. Sterilization is accomplished by heating in a commercial pressure cooker (retort) at temperatures of 115 - 123°C for 12 - 20 minutes. The sterile liquid formula may then be stored for 6-12 months without spoiling or exhibiting textural changes. However, it is difficult to control the textural changes in sterile liquid infant formula, as sediment and gelation may occur. Even though microbial spoilage may not occur, textural and chemical changes may render the formula less usable or less nutritious. In order to prevent these changes, appropriate formulation, homogenization, and heat treatment must be employed. In processing both liquid and dry ingredients, processes include intense preheating conditions, nonfat dry milk or condensed milks should be the “high-heat” type, indicating that heat applied during the manufacture of these ingredients was sufficient to denature most whey proteins. If ingredients used under these conditions are not “high-heat”, then they may contribute to sediment formation during storage of the finished product. When fresh milk is used to produce evaporated products, vitamin and mineral addition should be delayed until after

332

FUNCTIONAL FOODS

evaporation, and before sterilization, in order to mitigate loss of vitamins. Two procedures exist for the processing portion of infant formula manufacturing, the “dry procedure”, which consists of ingredients being blended in the dry form, and the “wet procedure”, in which liquid ingredients are mixed and then dried. The dry procedure consists of blending all ingredients in dry form, producing a homogeneous blend, which is its most favorable feature. This is accomplished by completing the entire mixing in a batch plant, with precise dosing and filling in a continuous plant. The wet procedure follows a specific chain of events in which liquid ingredients are mixed prior to drying. In the wet method, the procedure consists of selection and reception of raw materials (skim milk), clarification, deaeration, separation, pasteurization, evaporation, blending with oil and other components (including fat-soluble vitamins, emulsifiers, and stabilizers), mixing, homogenization, addition of water soluble vitamins and minerals, and drying. There is also a combined method of these two processes that has the advantages of both and is more commonly applied. The combined method involves adding watersoluble components to milk prior to drying, and adding less soluble components in a dry form to the blend after drying. The wet procedure allows for optimal mixing, while the dry procedure is less costly for operation and investment. From a nutritional standpoint, ultra-high-temperature (UHT) is preferred for heat treatment of liquid infant formula as nutrient and vitamin loss is minimized, as well as the browning that takes place between reducing sugars and amino groups of protein, which can lower protein quality. Pretreatment of the mix at an intense preheating temperature prior to UHT processing may mitigate the likelihood of gelation, which can be caused by enzymes associated with bacteria found in the original milk ingredients. Powdered infant formula is produced either by blending dry ingredients or by drying a mixture of liquid ingredients. Microbiological quality is easiest to control when the infant formula powder is produced by drying the liquid mixture. Fresh milk can be used, and it is filtered, clarified, deaerated, separated into skim milk and cream, and pasteurized (74 - 77°C, 15 - 20 seconds). Then these ingredients, or dried milk products, can be combined with warmed vegetable oils, followed by emulsifiers, stabilizers, and possible fat-soluble vitamins. Vitamin and mineral fortification can be delayed until the product is dried and cooled, although this is not ideal as thorough mixing is difficult (Packard, 1982). When vitamins and minerals are added prior to drying,

HUMAN MILK

333

some overdosages of heat-labile vitamins are required to account for processing losses. Ideally, fat-soluble vitamins are added prior to evaporation, while water-soluble vitamins and minerals are added after evaporation and before drying. Concentrated liquid formula is prepared by blending the ingredients to the desired solids level, or by evaporating excess liquid. Most infant formulas contain high carbohydrate content, which has the potential to stick to the dryer walls at high temperatures and moisture levels. Thus, low inlet air temperature, low solids in-feed, heating the concentrated mix prior to in-feed, and using an insulated or cooledwall drying system is recommended. In general, a batch mix of 45% or less solids and an in-feed temperature of approximately 70°C is ideal. Two-stage drying can also be performed, producing a moist powder that is then dried on a surface dryer to yield the desired moisture content. Following the drying process, the product is cooled and bagged in bulk or into consumer size units. RECENT DEVELOPMENTS IN INFANT FORMULA FORMULATION Essential Fatty Acids Human milk contains small amounts of arachidonic acid (AA) and docosahexaenoic acid (DHA). DHA is a long-chain polyunsaturated fatty acid that plays a major structural role in the grey matter of the brain and in the retina of the eyes. AA, another long-chain polyunsaturated fatty acid, is the principal omega-6 fatty acid of the brain. It is important in brain development and growth in infants, and is a precursor to eicosanoids, which are involved in the regulation of immunity, blood clotting, and other functions in the body and the precursor to the prostaglandin hormones. Infants were fed either infant formula, infant formula fortified with these fatty acids, or human milk exclusively for 17 weeks. Visual acuity was measured at 6, 17, 26, and 52 weeks, and electroretinography was used to measure retinal maturity at 17 and 52 weeks. Blood levels of DHA and AA were measured and correlated with the results from the visual and developmental tests. The results from this study indicate that infants fed infant formula fortified with the fatty acids had more mature retinal function and improved visual function at 6 and 17 weeks, respectively. When followed at one year, the supplemented groups maintained higher levels of visual function than unsupplemented groups (Hoffman et al., 2000).

334

FUNCTIONAL FOODS

In another study by Birch, Garfield, Hoffman, & Uauy (2000), supplementation of term infant formula with 0.36% DHA and 0.72% AA (weight percent of fat) during the first four months of life was associated with a mean increase of 7 points on the Mental Development Index of the Bayley scores at 18 months of age compared with control formula infants. Based on studies such as this, companies in the US have produced commercial infant formulas that include added DHA and AA. The levels of DHA are approximately 0.32% (weight percent of fat), and the levels of AA are approximately 0.64% (weight percent of fat). These natural DHA and AA are extracted from the algae Crypthecodinium cohnii and the fungal source Mortierella alpina, respectively. Nucleotides Nucleotides are one component of human milk identified as having an effect on immune function. The effect of human milk followed by infant formula, and infant formula fortified with nucleotides were compared with respect to their effect on response to immunizations as an indicator of immune development. The level of nucleotides (72 mg/ L) and ratio of individual nucleotides were patterned after those found in human milk. Results showed that infant formula fortified with nucleotides enhanced H influenza type b and diphtheria humoral antibody responses post vaccination. The consumption of human milk also enhanced antibody response to oral polio virus (Pickering et al., 1998). These results indicated that infant formula supplemented with nucleotides enhanced immune function in infants compared to the control infant formula. Prebiotic Compounds Infants who consume breast milk have gastrointestinal flora that are richer in bifidobacteria and lactobacilli than infants who consume bovine milk-based formula, and both of these species are considered to be potentially beneficial to the health of the host. The absence of oligosaccharides from infant formula, another major component in human milk, may be responsible for the differences in colonic flora. The addition of two oligosaccharides, galacto-oligosaccharides and inulin, to bovine milk-based infant formula has been shown to stimulate the growth of bifidi and lactobacilli, and to have a bifidogenic effect (Vandenplas, 2002). Therefore, the addition of oligosaccharides to infant formula could improve the colonic balance of microflora, and possibly,

HUMAN MILK

335

the health of the infant host (Vandenplas, 2002). The addition of oligosaccharides to bovine milk-based infant formula is one more improvement that brings infant formula one step closer to the gold standard of human milk. However, prebiotic oligosaccharides are presently not recommended to be supplemented in infant formulas according to ESPGHAN Committee on Nutrition and FDA due to inadequate information. It is clear that progress is still being made in the way of infant formula formulation and improvement. However, there are still many areas that merit further research in the quest to formulate and produce an infant formula that really mimics human milk. Component interactions that occur during processing should be considered since such interactions could lead to the loss of nutritional value in the final product. Summary Human milk is the best reference standard by which all infant formula is compared, and it has always been considered a speciesspecific food. Modern infant formulas are designed for infants based on our knowledge of human milk. There are numerous differences in chemical and biological properties between human milk and infant formula since we still do not fully understand chemical and biological properties of human milk. In addition to nutritional components, human milk also contains immunoglobulin SlgA, lactoferrin, peptide and nonpeptide hormones, growth factors, peptides, lipids, and other fractions. It is in fact a living tissue much like blood or plasma. Each advance in infant formula, including formulation and processing, allows for the improvement of a product that continues to be increasingly similar to human milk. Although much is still unknown about human milk, and how to produce the optimum infant formula, new information is constantly being discovered. Some of the recent progress made in infant formula formulation and processing includes fortification with -6 fatty acids such as arachidonic and -3 fatty acids including docosahexaenioc acid and eicosapentaenoic acid, nucleotides, and ingredients that promote healthy colonic microflora; effect of removal of phytate on soy formulas; trace mineral solubility and availability; component distribution and interactions; modification of whey protein profile and addition of bioactive peptide fractions.

336

FUNCTIONAL FOODS

References AAP (American Academy of Pediatrics) 1981. Nutrition and lactation. Pediatrics, 68, 435-443. Adkins, Y. & Lonnerdal, B. 2002. Mechanisms of vitamin B12 absorption in breast fed infants. J. of Pediatric Gastroenterology and Nutrition, 35, 192-198. Agostoni, C., Axelsson, I., Goulet, O., Koletzko, B., Michaelsen, K. F., Puntis, J. W. L., Rigo, J., Shamir, R., Szajewska, H., and Turck, D. 2005. Prebiotic oligosaccharides in dietetic products for infants: A commentary by the ESPGHAN Committee on Nutrition. J. of Pediatric Gastroenterology and Nutri. 39, 465-473. Colman, N., Hettiarachchy, N., and Herbert, V. 1981. Detection of a milk factor that facilitates folate uptake by intestinal cells. Science, 211, 1427-1428. Committee on Nutrition, American Academy of Pediatrics 1998. Iron fortified infant formulas. Pediatrics, 84, 1114-1115. Feldhausen, J., Thomson, C., Dunca, B. and Taren, D. 1996. Pediatric Nutrition Handbook. Chapman & Hall. NY. USA. Flynn, A. 1992. Minerals and trace elements in human milk. Advances in Food & Nutrition Research, 36, 209-252. Fomon, S.J. 1993. Nutrition of Normal Infants. Boston: Mosby-Yearbook, Inc. St. Louis, USA. Goldman, A.S., and Goldblum, R.M. 1989. Immunoglobulins in human milk. In: Protein and Non-Protein Nitrogen in Human Milk. S.A. Atkinson and B. Lonnerdal (Eds), pp 44-51. CRC Press Inc, Boca Raton, Forida. Goldman, A. 1993. The immune system of human milk: antimicrobial, anti-inflammatory, and immunomodulating properties. Pediatric Infectious Disease J. 12, 664-672. Guo, M.R. 1990 Heat-Induced Modification of Milk Protein. Ph.D. Thesis. The National University of Ireland, Ireland. Hendricks, G. M. 2001. Solubility and Relative Absorption of Copper, Iron, and Zinc in Infant Formulae. Ph.D. Thesis. University of Vermont, USA. Hendricks, G.M. and Guo, M. 2006. The significance of milk fat in infant formula. Advanced Dairy Chemistry, Volume 2: Lipids, 3rd edition. pp 467-479.Spring, New York. Hernell, O. & Lonnerdal, B. 2002. Iron status of infants fed low iron formula: no effect of added bovine lactoferrin or nucleotides. Am. J. of Clini. Nutri., 76, 858-864. Hoffman, D.R., Birch, E.E., Brich, D.G., Uauy, R, Castaneda, Y.S., Lapus, M.G. & Wheaton, D.H. 2000. Impact of early dietary intake and blood lipid composition of long-chain polyunsaturated fatty acids on later visual development. J. of Pediatric Gastroenterology and Nutri, 31 (5), 540-553. Hurley, L.S. & Keen, C.L. 1987. Manganese. In W. Mertz, Trace elements in human and animal nutrition, 5th Ed, Vol. 1 pp. 185-223. Academic Press. San Diego, USA. Kelleher, S.L. & Lonnerdal, B. 2001. Immunological activities associated with milk. In: B. Woodward & H.H. Draper, Advances in nutritional research. Immunological properties of milk, Vol. 10, pp. 39-65. Plenum Press, New York, USA. Kelleher, S.L.,Chatterton, D, Neilsen, K., and Lonnerdal, B. 2003. Glycomacropeptide and
-lactalbumin supplementation of infant formula affects growth and nutritional status in infant rhesus monkeys. Am. J. of Clini. Nutri, 77, 126-128. Koletzko, B., Baker, S., Cleghorn, G., Neto, U. F., Gopalan S., Hernell O., Hock Q. S., Jirapinyo P., Lonnerdal, B., Pencharz, P., Pzyrembel, H., Ramirez-Mayans, J., Shamir, R., Turck, D., Yamashiro, Y., and Z. Ding. 2005. Golbal standard for the composition of infant formula: Recommendations of an ESPGHAN coordinated international expert group. J. of Pediatric Gastroenterology and Nutri 41:584-599.

HUMAN MILK

337

Levander, O.A.; Moser, P.B.; & Morris, V.C. 1987. Dietary selenium intake and selenium concentrations of plasma, erythrocytes and breast milk in pregnant and postpartum lactating and nonlactating women. Am. J. of Clini. Nutri., 46, 694-698. Liepke, C.; Adermann, K.; Raida, M.; Magert, H-J.; Forssmann, W-G.; & Zucht, H-D. 2002. Human milk provides peptides highly stimulating the growth of bifidobacteria. Europ. J. of Biochem., 269, 712-718. Lonnerdal, B.; Keen, C.L.; Ohtake, M.; & Tamura, T. 1983. Iron, zinc, copper, and manganese in infant formulas. Am. J. of Dis. of Children, 137, 433-437. Lonnerdal, B. 1986. Effect of maternal dietary intake on human milk consumption. J. of Nutri., 116, 499-513. Lonnerdal, B. 1989. Trace element nutrition in infants. Ann. Rev. Nutri. 9, 109-125. Lonnerdal, B. 2003. Nutritional and physiological significance of human milk proteins. Am. J. of Clini. Nutri., 77 (Suppl), 1537S-43S. NRC National Research Council. 1989. Recommended daily allowances. 10th ed. National Academy of Science, National Research Council. Washington, DC. Packard, V.S. 1982. Human Milk and Infant Formula. Academic Press, New York, USA. Pickering, L.K.; Granoff, D.M.; Erickson, J.R.; Masor, M.L.; Cordle, C.T.; Schaller, J.P.; Winship, T.R.; Paule, C.L. & Hilty, M.D. 1998. Modulation of the immune system by human milk and infant formula containing nucleotides. Pediatrics, 101 (2), 242249. Renner, E. 1983. Milk and Dairy Products in Human Nutrition. Volkswirtschaftlicher Verlag, Munich, Germany. Smith, C. R. 2004. Solubility and Relative Bioavailability of Iron and Zinc in Whey Protein Dominated Infant Formulas. Ph. D. Thesis. University of Vermont. Stromquist, M.; Falk, P.; Bergstrom, S. et al. 1995. Human milk -casein and inhibition of Helicobacter pylori adhesion to human gastric mucosa. J. of Pediatric Gastroenterology and Nutri., 21, 288-296. Suzuki, Y.A.; Shin, K.; & Lonnerdal, B. 2002. Molecular cloning and functional expression of a human intestinal lactoferrin receptor. Biochem., 40, 15771-15779. Vandenlas, Y. 2002. Oligosaccharides in infant formula. Brit. J. of Nutri, 87 (Suppl. 2): S293-S296. (Guo, M. R. Hendricks, G.M.)