2003 ; Pouliot et al. 2006 ; Korhonen and Pihlanto 2007a ). Fractionation and marketing of bioactive milk ingredients has emerged as a new lucrative sector for dairy industries and specialized bioindus- tries. At present many of these components are being exploited for both dairy and nondairy food formulations and even pharmaceuticals (Korhonen et al. 1998 ; Shah 2000 ; German et al. 2002 ; Playne
et al. 2003 ; Rowan et al. 2005 ; Krissansen 2007 ).
The dairy industry has achieved a leading role in the development of functional foods and has already commercialized products that boost, for example, the immune system; reduce elevated blood pressure;
combat gastrointestinal infections; help control body weight; and prevent osteoporosis (FitzGerald et al.
2004 ; Zemel 2004 ; Cashman 2006 ; Korhonen and Marnila 2006 ; Hartmann and Meisel 2007 ). There also is increasing evidence that many milk - derived components are effective in reducing the risk of metabolic syndrome, which may lead to various chronic diseases, such as cardiovascular disease and diabetes (Mensink 2006 ; Pfeuffer and Schrezenmeir 2006a ; Scholz - Ahrens and Schrezenmeir 2006 ).
Beyond essential nutrients milk seems thus capable of delivering many health benefi ts to humans of all ages by provision of specifi c bioactive components.
Figure 2.1 gives an overview of these components and their potential applications for promotion of human health.
This chapter reviews the current knowledge about technological and biological properties of milk - and colostrum - derived major bioactive components and their exploitation for human health. A particular emphasis has been given to bioactive proteins, pep- tides, and lipids, which have been the subjects of intensive research in recent years.
BIOACTIVE PROTEINS AND PEPTIDES
The high nutritional value of milk proteins is widely recognized, and in many countries dairy products
15
Figure 2.1. Bioactive milk components and their potential applications for health promotion.
Bioactive Milk Components Bioactive peptides
Calcium Heart health
Antimicrobial peptides Immunoglobulins Lactose derivatives
Lactoferrin
Digestive health
Bone health
Dental health Immune defense
Probiotic bacteria Bioactive peptides
Immunoglobulins Lactoperoxidase
GMP Calcium Bioactive peptides
Whey proteins Immunomodulatory peptides
Lactoferrin
Bioactive peptides
Probiotic bacteria Whey proteins
Calcium GMP CLA
Mood, memory, and stress Weight management
contribute signifi cantly to daily protein intake. The multiple functional properties of major milk proteins are now largely characterized (Mulvihill and Ennis 2003 ). Increasing scientifi c and commercial interest has been focused on the biological properties of milk proteins. Intact protein molecules of both major milk protein groups, caseins and whey proteins, exert dis- tinct physiological functions in vivo (Shah 2000 ; Steijns 2001 ; Walzem et al. 2002 ; Clare et al. 2003 ; Floris et al. 2003 ; Pihlanto and Korhonen 2003 ; Madureira et al. 2007 ; Zimecki and Kruzel 2007 ).
Table 2.1 lists the major bioactive proteins found in bovine colostrum and milk and their concentrations, molecular weights, and suggested biological func- tions. Many of the bioactive whey proteins, notably immunoglobulins, lactoferrin and growth factors, occur in colostrum in much greater concentrations than in milk, thus refl ecting their importance to the health of the newborn calf (Korhonen 1977 ; Pakkanen and Aalto 1997 ; Scammel 2001 ). Further- more, milk proteins possess additional physiological functions due to the numerous bioactive peptides that are encrypted within intact proteins. Their func- tions are discussed in the section “ Production and Functionality of Bioactive Peptides ” later in this chapter.
Fractionation and Isolation of Bioactive Milk Proteins
Increasing knowledge of the biological properties of individual milk - derived proteins and their fractions has prompted the need to develop technologies for obtaining these components in a purifi ed or enriched form. Normal bovine milk contains about 3.5% of protein, of which casein constitutes about 80% and whey proteins 20%. Bovine casein is further divided into α - , β - and κ - casein. Various chromatographic and membrane separation techniques have been developed for fractionation of β - casein in view of its potential use, e.g., in infant formulas (Korhonen and Pihlanto 2007a ). However, industrial manufac- ture of casein fractions for dietary purposes has not progressed to any signifi cant extent, so far. Whole casein is known to have a good nutritive value owing to valuable amino acids, calcium, phosphate and several trace elements. Individual casein fractions have been proven to be biologically active and also a good source of different bioactive peptides (see review by Silva and Malcata 2005 ).
The whey fraction of milk contains a great variety of proteins that differ from each other in their chemi- cal structure, functional properties and biological functions. These characteristics have been used to separate individual proteins, but the purity has proven a critical factor when evaluating the biological activ- ity of a purifi ed component. Membrane separation processes, such as ultrafi ltration (UF), reverse osmosis (RO) and diafi ltration (DF), are now indus- trially applied in the manufacture of ordinary whey powder and whey protein concentrates (WPCs) with a protein content of 30 – 80%. Gel fi ltration and ion - exchange chromatography techniques can be employed in the manufacture of whey protein iso- lates (WPI) with a protein content of 90 – 95% (Kelly and McDonagh 2000 ; Etzel 2004 ). The chemical composition and functionality of whey protein prepa- rations are largely affected by the method used in the process. Their biological properties are also affected and are diffi cult to standardize due to the complex nature of the bioactivities exerted by different pro- teins (Korhonen 2002 ; Foegeding et al. 2002 ). There- fore there is growing interest in developing specifi c techniques for isolation of pure whey protein com- ponents. Industrial or semi - industrial scale process- ing techniques are already available for fractionation and isolation of major native milk proteins, e.g., alpha - lactalbumin ( α - La), beta - lactoglobulin ( β - Lg), immunoglobulins (Ig), lactoferrin (LF) and gly- comacropeptide (GMP). Current and potential tech- nologies have been reviewed in recent articles (Korhonen 2002 ; Kulozik et al. 2003 ; Chatterton et al. 2006 ; Korhonen and Pihlanto 2007a ). Recently Konrad and Kleinschmidt (2008) described a novel method for isolation of native α - La from sweet whey using membrane fi ltration and treatment of the permeate with trypsin. After a second UF and dia- fi ltration of the hydrolysate, the calculated overall recoveries were up to 15% of α - La with a purity of 90 – 95%. In another recent method β - Lg was isolated from bovine whey using differential precipitation with ammonium sulphate followed by cation - exchange chromatography (Lozano et al. 2008 ).
The overall yield of purifi ed β - Lg was 14.3% and the purity was higher than 95%. The progress of proteomics has facilitated the use of proteomic techniques, for example, two - dimensional gel electrophoresis, in characterization of whey proteins (O ’ Donnell et al. 2004 ; Lindmark - M ồ sson et al.
2005). Using this technique, Fong et al. (2008)
molecular weight, and potential biological functions *
Protein
Concentration (g/L) Molecular Weight
Biological Activity Colostrum Milk Daltons
Caseins ( α s1 , α s2 , β , and κ )
26 28 14.000 – 22.000 Ion carrier (Ca, PO 4 , Fe, Zn, Cu), precursor for bioactive peptides immunomodulatory, anticarcinogenic
β - lactoglobulin 8.0 3.3 18.400 Vitamin carrier, potential antioxidant, precursor for bioactive peptides, fatty acid binding
α - lactalbumin 3.0 1.2 14.200 Effector of lactose synthesis in mammary gland, calcium carrier, immunomodulatory, precursor for bioactive peptides, potentially anticarcinogenic
Immunoglobulins 20 – 150 0.5 – 1.0 150.000 – 1000.000 Specifi c immune protection through antibodies and complement system, potential precursor for bioactive peptides
Glycomacro - peptide 2.5 1.2 8.000 Antimicrobial, antithrombotic, prebiotic, gastric hormone regulator
Lactoferrin 1.5 0.1 80.000 Antimicrobial, antioxidative, anticarcinogenic, anti - infl ammatory, iron transport, cell growth regulation, precursor for bioactive peptides, immunomodulatory, stimulation of osteoblast proliferation
Lactoperoxidase 0.02 0.03 78.000 Antimicrobial, synergistic effects with immunoglobulins, lactoferrin, and lysozyme Lysozyme 0.0004 0.0004 14.000 Antimicrobial, synergistic effects
with immunoglobulins, lactoferrin, and lactoperoxidase
Serum albumin 1.3 0.3 66.300 Precursor for bioactive peptides Milk basic protein N.A N.A 10.000 – 17.000 Stimulation of osteoblast
proliferation and suppression of bone resorption
Growth factors 50 μ g – 40 mg/L < 1 μ g – 2 mg/L 6.400 – 30.000 Stimulation of cell growth, intestinal cell protection and repair, regulation of immune system
* Compiled from Pihlanto and Korhonen (2003) and Korhonen and Pihlanto (2007b) ; N.A. = not announced.
18
identifi ed a large number of minor whey proteins after fi rst fractionating bovine whey by semicoupled anion and cation exchange chromatography. Such proteomic display appears useful in the future design of strategies for purifi cation of selected milk proteins or their fractions containing targeted bioactivities.
Biological Functions and
Applications of Major Milk Proteins Proteins contained in colostrum and milk are known to exert a wide range of nutritional, functional, and biological activities (Walzem et al. 2002 ; Pihlanto and Korhonen 2003 ; Marshall 2004 ; Tripathi and Vashishtha 2006 ; Yalcin 2006 ; Zimecki and Kruzel 2007 ). Apart from being a balanced source of valu- able amino acids, milk proteins contribute to the structure and sensory properties of various dairy products. A brief overview is given about the estab- lished and potential physiological functions of main milk proteins with special emphasis on whey pro- teins and bioactive peptides.
Whole casein and electrophoretic casein fractions have been shown to exhibit different biological activities, such as immunomodulation (Bennett et al.
2005 ; Gauthier et al. 2006b ) and provision of a variety of bioactive peptides, including antihyper- tensive (L ú opez - Fandi ủ o et al. 2006), antimicrobial (L ó pez - Exp ó sito and Recio 2006 ; Pan et al. 2006 ), antioxidative (Pihlanto 2006 ) and opioidlike (Meisel and FitzGerald 2000 ). These recent fi ndings suggest that casein hydrolysates provide a potential source of highly functional ingredients for different food applications. Examples of such products are the fer- mented milk products Calpis ® and Evolus ® , which are based on hypotensive tripeptides Val - Pro - Pro and Ile - Pro - Pro derived from both β - casein and κ - casein. Owing to emerging health properties and documented clinical implications, the bovine milk whey proteins have attained increasing commercial interest (Krissansen 2007 ; Madureira et al. 2007 ).
The total whey protein complex and several indi- vidual proteins have been implicated in a number of physiologically benefi cial effects, such as
1. Improvement of physical performance, recov- ery after exercise, and prevention of muscular atrophy (Ha and Zemel 2003 )
2. Satiety and weight management (Schaafsma 2006a,b); Luhovyy et al. 2007 )
3. Cardiovascular health (Yamamoto et al. 2003 ; FitzGerald et al. 2004 ; Murray and FitzGerald 2007)
4. Anticancer effects (Parodi 1998 ; Bounous 2000 ; Gill and Cross 2000 )
5. Wound care and repair (Smithers 2004 ) 6. Management of microbial infections and
mucosal infl ammation (Playford et al. 2000 ; Korhonen et al. 2000 ; Korhonen and Marnila 2006 )
7. Hypoallergenic infant nutrition (Crittenden and Bennett 2005 )
8. Healthy aging (Smilowitz et al. 2005 )
Although many of these effects remain still puta- tive, a few have been substantiated in independent studies. In the following discussion, the health - promoting potential of major whey proteins and examples of their commercial applications will be described briefl y.
Immunoglobulins
Immunoglobulins (Ig) carry the biological function of antibodies and are present in colostrum of all lactating species to provide passive immunity against invading pathogens. Igs are divided into different classes on the basis of their physicochemical struc- tures and biological activities. The major classes in bovine and human lacteal secretions are IgG, IgM, and IgA. The basic structure of all Igs is similar and is composed of two identical light chains and two identical heavy chains. These four chains are joined with disulfi de bonds. The complete basic Ig mole- cule displays a Y - shaped structure and has a molecu- lar weight of about 160 kilodaltons. Igs account for up to 70 – 80% of the total protein content in colos- trum, whereas in milk they account for only 1 – 2%
of total protein (Korhonen et al. 2000 ). The impor- tance of colostral Igs to the newborn calf in protec- tion against microbial infections is well documented (Butler 1994 ). Colostral Ig preparations designed for farm animals are commercially available, and colos- trum - based products have found a growing world- wide market as dietary supplements for humans (Scammel 2001 ; Tripathi and Vashishtha 2006 ; Struff and Sprotte 2007 ; Wheeler et al. 2007 ). Igs link various parts of the cellular and humoral immune system. They are able to prevent the adhesion of microbes, inhibit bacterial metabolism, agglutinate
bacteria, augment phagocytosis of bacteria, kill bac- teria through activation of complement - mediated bacteriolytic reactions, and neutralize toxins and viruses.
The concentration of specifi c antibodies against pathogenic microorganisms can be raised in colos- trum and milk by immunizing cows with vaccines made of pathogens or their antigens (Korhonen et al. 2000 ). Advances in bioseparation techniques have made it possible to fractionate and enrich these antibodies and formulate so - called immune milk preparations (Mehra et al. 2006 ). The concept of “ immune milk ” dates back to the 1950s when Petersen and Campbell fi rst suggested that orally administered bovine colostrum could provide passive immune protection for humans (Campbell and Petersen 1963 ). Since the 1980s, a great number of studies have demonstrated that such immune milk preparations can be effective in prevention of human and animal diseases caused by different pathogenic microbes, e.g., rotavirus, Escherichia coli, Candida albicans, Clostridium diffi cile, Shigella fl exneri, Streptococcus mutans, Cryptosporidium parvum, and Helicobacter pylori . The therapeutic effi cacy of these preparations has, however, proven to be quite limited (Weiner et al. 1999 ; Korhonen et al. 2000 ; Korhonen and Marnila 2006 ). A few commercial immune milk products are on the market in some countries, but the unclear regulatory status of these products in many countries has emerged as a con- straint for global commercialization (Hoerr and Bostwick 2002 ; Mehra et al. 2006 ). In view of the globally increasing problem of antibiotic - resistant strains causing endemic hospital infections, the development of appropriate immune milk products to combat these infections appears as a highly inter- esting challenge for future research.
α - Lactalbumin
α- La is the predominant whey protein in human milk and accounts for about 20% of the proteins in bovine whey. α - La is fully synthesized in the mammary gland where it acts as coenzyme for bio- synthesis of lactose. The health benefi ts of α - La have long been obscured, but recent research sug- gests that this protein can provide benefi cial effects through a) the intact whole molecule, b) peptides of the partly hydrolyzed protein, and c) amino acids of the fully digested protein (Chatterton et al. 2006 ).
α - La is a good source of the essential amino acids tryptophan and cystein, which are precursors of serotonin and glutathion, respectively. It has been speculated that the oral administration of α - La could improve the ability to cope with stress. A human clinical study with a group of stress - vulnerable sub- jects showed that an α - La – enriched diet affected favorably biomarkers related to stress relief and reduced depressive mood (Markus et al. 2000 ). In a later study the same researchers (Markus et al. 2002 ) observed that α - La improved cognitive functions in stress - vulnerable subjects by increased brain tryp- tophan and serotonin activity. In another clinical study (Scrutton et al. 2007 ) it was shown that daily administration of 40 g of α - La to healthy women increased plasma tryptophan levels and its ratio to neutral amino acids, but no changes in emotional processing was observed. Furthermore, there is sig- nifi cant evidence from animal model studies that α - La can provide protective effect against induced gastric mucosal injury. The protection was compa- rable to that of a typical antiulcer drug (Matsumoto et al. 2001 ; Ushida et al. 2003 ). Bovine α - La hydro- lysates and specifi c peptides derived from these hydrolysates have been associated with many biological activities, for example, antihypertensive, antimicrobial, anticarcinogenic, immunomodula- tory, opioid, and prebiotic (Pihlanto and Korhonen 2003 ; Chatterton et al. 2006 ).
Based on its high degree of amino acid homology to human α - La, bovine α - La and its hydrolysates are well suited as an ingredient for infant formulae.
A few α - La enriched formulae have already been launched on the market.
β - Lactoglobulin
β - Lg is the major whey protein in bovine milk and accounts for about 50% of the proteins in whey, but it is not found in human milk. β - Lg poses a variety of functional and nutritional characteristics that have made this protein a multifunctional ingredient mate- rial for many food and biochemical applications.
Furthermore, β- Lg has been proven an excellent source of peptides with a wide range of bioactivities, such as antihypertensive, antimicrobial, antioxida- tive, anticarcinogenic, immunomodulatory, opioid, hypocholesterolemic, and other metabolic effects (Pihlanto and Korhonen 2003 ; Chatterton et al.
2006 ). Of particular interest is the antihypertensive
peptide β - lactosin B [Ala - Leu - Pro - Met; f(142 – 145)], which has shown signifi cant antihypertensive activity when administered orally to SHR rats (Murakami et al. 2004 ) and a tryptic peptide [Ile - Ile - Ala - Glu - Lys; f(71 – 75)], which has exerted hypo- cholesterolemic activity in rat model studies (Nagaoka et al. 2001 ). Also an opioid peptide β - lactorphin [Tyr - Leu - Leu - Phe; f(102 – 105)] has been shown to improve arterial functions in SHR rats (Sipola et al. 2002 ). β - Lg – derived peptides carry a lot of biological potential, but further in vivo studies are essential to validate the physiological effects.
This view is supported by a recent study of Roufi k et al. (2006) , who showed that a β - Lg – derived ACE - inhibitory peptide [ALPMHIR; f(142 – 148)]
was rapidly degraded upon in vitro incubation with human serum, and after oral ingestion it could not be detected in sera of human subjects.
Lactoferrin
LF is an iron - binding glycoprotein found in colos- trum, milk, and other body secretions and cells of most mammalian species. LF is considered to be an important host defense molecule and is known to confer many biological activities, such as antimicro- bial, antioxidative, antiinfl ammatory, anticancer, and immune regulatory properties (L ử nnerdal 2003 ; Wakabayashi et al. 2006 ; Pan et al. 2007 ; Zimecki and Kruzel 2007 ). Furthermore, several antimicro- bial peptides, such as lactoferricin B f(18 – 36) and lactoferrampin f(268 – 284) can be cleaved from LF by the action of digestive enzyme pepsin. LF is considered to play an important role in the body ’ s innate defense system against microbial infections and degenerative processes induced, e.g., by free oxygen radicals. The biological properties of LF have been the subject of scientifi c research since its discovery in the early 1960s. Initially, the role was confi ned largely to antimicrobial activity alone, but now the multifunctionality of LF has been well recognized. The antimicrobial activity of LF and its derivatives has been attributed mainly to three mechanisms: a) iron - binding from the medium leading to inhibition of bacterial growth; b) direct binding of LF to the microbial membrane, especially to lipopolysaccharide in Gram - negative bacteria, causing fatal structural damages to outer membranes and inhibition of viral replication; and c) prevention of microbial attachment to epithelial cells or entero-
cytes. As reviewed by Pan et al. (2007) the bacteri- cidal effect of LF can be augmented by the action of lysozyme or antibodies. LF can also increase sus- ceptibility of bacteria to certain antibiotics, such as vancomycin, penicillin, and cephalosporins. The in vitro antimicrobial activity of LF and the derivatives has been demonstrated against a wide range of pathogenic microbes, including enteropathogenic E.
coli; Clostridium perfringens; Candida albicans;
Haemophilus infl uenzae; Helicobacter pylori; Liste- ria monocytogenes; Pseudomonas aeruginosa; Sal- monella typhimurium; S. enteriditis; Staphylococcus aureus; Streptoccccus mutans; Vibrio cholerae; and hepatitis C, G, and B virus; HIV - 1; cytomegalovi- rus; poliovirus; rotavirus; and herpes simplex virus (Farnaud and Evans 2003 ; Pan et al. 2007 ). The antitumor activity of LF has been studied intensively over the last decade and many mechanisms have been suggested, e.g., iron - chelation – related antioxi- dative property and immunoregulatory and antiin- fl ammatory functions. In in vitro experiments LF has been shown to regulate both cellular and humoral immune systems by 1) stimulation of proliferation of lymphocytes; 2) activation of macrophages, monocytes, natural killer cells, and neutrophils; 3) induction of cytokine and nitric oxide production;
and4) stimulation of intestinal and peripheral anti- body response (Pan et al. 2007 ).
During the past 2 decades it has become evident in many animal and human studies that oral admin- istration of LF can exert several benefi cial effects on the health of humans and animals. These studies have been compiled and reviewed in several excel- lent articles (Teraguchi et al. 2004 ; Wakabayashi et al. 2006 ; Pan et al. 2007 ; Zimecki and Kruzel 2007 ).
Animal studies with mice or rats have demonstrated that orally administered LF and related compounds suppressed the overgrowth and translocation of certain intestinal bacteria, such as E. coli, Strepto- coccus, and Clostridium strains but did not affect intestinal bifi dobacteria. Also, oral administration of LF and lactoferricin reduced the infection rate of H. pylori, Toxoplasma gondii, candidiasis, and tinea pedis as well as prevented clinical symptoms of infl uenza virus infection. Further animal studies have demonstrated that orally ingested LF and related compounds improved nutritional status by reducing iron - defi cient anemia and drug - induced intestinal infl ammation, colitis, arthritis, and decreased mortality caused by endotoxin shock.