Biological Functions and Applications of Whey Proteins

Health - Promoting Proteins and Peptides in Colostrum and Whey

4. Biological Functions and Applications of Whey Proteins

It is well documented that intact bovine whey proteins exert distinct physiological functions in vivo (Walzem et al. 2002 ; Clare et al. 2003 ; Floris et al.

2003 ; Pihlanto and Korhonen 2003 ; Madureira et al.

2007 ; Zimecki and Kruzel 2007 ; M ö ller et al. 2008 ).

Increasing evidence from animal model and human clinical studies suggests that milk whey proteins provide many health benefi ts and attract, therefore, increasing commercial interest (Smithers 2008 ). In fact, several whey proteins or their combinations are already commercially available (Playne et al. 2003 ; Rowan et al. 2005 ; Mehra et al. 2006 ; Krissansen 2007 ). These formulated products are targeted to boost, for example, the immune system, reduce elevated blood pressure, combat gastrointestinal infections, and help control body weight (FitzGerald et al. 2004 ; Korhonen and Marnila 2006 ; Hartmann and Meisel 2007 ; Luhovyy et al. 2007 ). Also, there is increasing evidence that many milk - derived components are effective in reducing the risk of meta- bolic syndrome, which may lead to various chronic diseases, such as cardiovascular disease and diabe- tes (Mensink 2006 ; Pfeuffer and Schrezenmeir 2006 ; Scholz - Ahrens and Schrezenmeir 2006 ).

The potential benefi cial health effects attributed to the total whey protein complex include the following:

1. Improvement of physical performance, recovery after exercise, and prevention of muscular atrophy (Ha and Zemel 2003 ; Krissansen 2007 ) 2. Satiety and weight management (Schaafsma

2006a, 2006b ; Luhovyy et al. 2007 )

3. Cardiovascular health (Murray and FitzGerald 2007 ; M ö ller et al. 2008 )

4. Anticancer effects (Parodi 1998 ; Bounous 2000 ; Gill and Cross 2000 )

5. Wound care and repair (Smithers 2004, 2008 ) 6. Management of microbial infections and mucosal

infl ammation (Korhonen et al. 2000b ; Korhonen and Marnila 2006 )

Roufi k et al. 2006 ), antibacterial (L ó pez - Exp ó sito and Recio 2006 ; L ó pez - Exp ó sito et al. 2007 ), antioxidative (Pihlanto 2006 ), immunomodulatory (Gauthier et al. 2006a ; Saint - Sauveur et al. 2008 ), and opioid - like (Meisel 2005 ).

Many industrially employed lactic acid bacteria (LAB) – based starter cultures are highly proteolytic and can release different bioactive peptides from milk proteins through microbial proteolysis (Matar et al. 2003 ; Fitzgerald and Murray 2006 ; Gobbetti et al. 2007 ). In particular, Lactobacillus helveticus strains have been shown capable of releasing anti- hypertensive peptides, the best known of which are casein - derived ACE - inhibitory tripeptides Val - Pro - Pro and Ile - Pro - Pro. The hypotensive capacity of these peptides has been demonstrated in many in vitro, rat model and human studies (Nakamura et al.

1995 ; Hata et al. 1996 ; Sipola et al. 2002 ; Seppo et al. 2003 ; Jauhiainen et al. 2005 ). Also yogurt bacteria, cheese starter bacteria, and commercial probiotic bacteria have been demonstrated to produce different bioactive peptides in milk during fermentation (Fuglsang et al. 2003 ; Gobbetti et al.

2004 ; Virtanen et al. 2006 ; Donkor et al. 2007 ).

Recently, Chen et al. (2007) showed that fermentation of milk with a commercial starter culture mixture of fi ve LAB strains followed by hydrolysis with a microbial protease increased ACE inhibitory activity of the hydrolysate, and two strong ACE - inhibitory tripeptides (Gly - Thr - Trp and Gly - Val - Trp) were identifi ed. The hypotensive effect of the hydrolysate containing these peptides was demonstrated in an animal model study using spontane- ously hypertensive rats (SHR). It can be speculated that events, as described in the above study, may happen also under in vivo conditions in the gastrointestinal tract, resulting in the release of peptides with different bioactivities.

Over the last decade, large - scale technologies, based on membrane separation techniques, have been developed for the purpose of enrichment and isolation of peptides with a specifi c molecular weight range (Kitts and Weiler 2003 ; Pouliot et al.

2006 ; Korhonen and Pihlanto 2007a ). In particular, nanofi ltration and ultrafi ltration techniques are often employed to fractionate and enrich specifi c

and Petersen 1963 ). Since the late 1980s, a great number of clinical studies have demonstrated that such immune milk preparations can be effective in prevention of human and animal diseases caused by different pathogenic microbes, for example, 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 appears, however, to be quite limited (for reviews see Weiner et al. 1999 ; Korhonen et al. 2000b ; Korhonen and Marnila 2006 ; Hammarstr ö m and Kr ü ger - Weiner 2008 ). 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 constraint for global commercialization (Hoerr and Bostwick 2002 ; Mehra et al. 2006 ). During the last decade, antibiotic - resistant strains causing endemic hospital infections have emerged as global problems. The development of appropriate immune milk products to combat these infections appears to be a highly interesting challenge for future research. To this end, encouraging results have been obtained from preliminary intervention trials with immune milk preparations against Clostridium diffi cile enterotox- ins (Numan et al. 2007 ; Young et al. 2007 ).

4.2. α - lactalbumin

α - la is the major 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 biosynthesis of lactose. The biological functions of α - la have long been obscured, but recent research suggests that this protein can provide benefi cial health effects through (1) the intact whole molecule, (2) peptides of the partly hydrolyzed protein, and (3) 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- 7. Hypoallergenic infant nutrition (Crittenden and

Bennett 2005 )

8. Healthy aging (Smilowitz et al. 2005 ).

In the following sections, the health - promoting properties of major bovine whey proteins and exam- ples of their commercial applications will be described briefl y.

4.1. Immunoglobulins

The biological function of colostral Igs is to give the offspring an immunological protection against microbial pathogens and toxins and to protect the mammary gland against infections. To this end, the major mechanisms provided by colostral and milk Igs are augmenting phagocytosis and cell - mediated cytotoxicity reactions by leukocytes, agglutination of bacteria, neutralization of microbes and toxins, and activation of the complement system in milk (Korhonen and Marnila 2000a ). The importance of colostral Igs to the newborn calf in protection against microbial infections is well documented (Butler 1998 ; Hurley 2003 ). Colostral Ig preparations designed for farm animals are commercially available, and colostrum - based products have found a growing worldwide market as dietary supplements for humans (Scammel 2001 ; Tripathi and Vashishtha 2006 ; Struff and Sprotte 2007 ; Wheeler et al. 2007 ).

Bovine colostrum contains natural antibodies against a wide variety of nonpathogenic and poten- tially pathogenic microorganisms found in the cow ’ s environment and feeds. Specifi c antibodies can be raised in colostrum and milk by immunizing cows with vaccines made of different microorganisms or their antigenic components (Korhonen et al. 2000b ).

Recent advances in bioseparation techniques have made it possible to fractionate and enrich these antibodies and formulate so - called immune milk preparations to treat humans against microbial infections (Korhonen 2004 ; 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

4.4. Lactoferrin

Lactoferrin is an iron - binding glycoprotein found in different biological fl uids of mammals and in neutrophils. Lf is considered to be an important host defense molecule and exhibits a diverse range of physiological functions, such as antimicrobial, anti - infl ammatory, antioxidant, and immunomodulatory activity. Experimental and clinical research carried out over the last 30 years has accumulated increasing evidence about the potential benefi cial health effects of Lf and its derivatives. These benefi ts may include, for example, anti - infective, anticancer, and anti - infl ammatory effects, as reviewed in many articles (L ö nnerdal 2003 ; Wakabayashi et al. 2006 ; Pan et al. 2007 ; Weinberg 2007 ; Zimecki and Kruzel 2007 ). Furthermore, several antimicrobial 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 pro- cesses induced, for example, by free oxygen radi- cals. 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 major known or speculated in vivo activities of Lf in lacteal secretions are

1. Defense against infections of the mammary gland and the gastrointestinal tract (antimicrobial activity, regulation of the immune system) 2. Nutritional effects (bioavailability of iron, source

of amino acids)

3. Mitogenic and trophic activities on the intestinal mucosa and gastrointestinal tract associated lym- phoid tissue and on bone tissue

4. Antineoplastic activity in the gastrointestinal tract.

The antimicrobial activity of Lf and its derivatives has been attributed mainly to three mechanisms: (1) iron binding from the medium leading to inhibition of bacterial growth, (2) direct binding of Lf to the microbial membrane, especially to lipo- polysaccharide in Gram - negative bacteria, causing jects showed that an α - la enriched diet favorably

affected 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 tryptophan and serotonin activity. In another recent clinical study, Scrutton et al. (2007) demonstrated 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 were observed. Moreover, many animal model studies suggest that α - la can provide a protective effect against induced gastric mucosal injury (Matsumoto et al. 2001 ; Ushida et al. 2003 ).

Bovine α - la exhibits a high degree of amino acid homology to human α - la. Therefore, α - la enriched or purifi ed from cow ’ s milk and hydrolysates of this protein are well suited as an ingredient for infant formulas. A few bovine α - la enriched formulas are already commercially available.

4.3. β - lactoglobulin

β - lg is the predominant whey protein in bovine milk, accounting for about 50% of the proteins in whey, but it is not found in human milk. β - lg exerts a variety of functional and nutritional characteristics that have made this protein a multifunctional ingredient material for many food and biochemical applications (Smithers 2008 ). β - lg has excellent heat - set gelation properties that make it suitable for a wide range of applications in products where water binding and texturization are required. The putative biological activities of β - lg have been reviewed recently by Chatterton et al. (2006) and include, for example, the following: antiviral, prevention of pathogen adhesion, anticarcinogenic and hypocho- lesterolemic, and ability to bind hydrophobic components, including retinol and long - chain fatty acids.

It has been speculated that this protein may also play a role in the absorption and subsequent metabolism of fatty acids. Furthermore, β - lg has proven an excellent source of peptides with a wide range of bioactivities (see chapter 3 ).

Over the last 4 decades, a great number of animal and human studies have shown that oral administration of Lf can exert many benefi cial health effects.

These studies have been compiled and reviewed in several excellent articles (Teraguchi et al. 2004 ; Wakabayashi et al. 2006 ; Pan et al. 2007 ; Weinberg 2007 ; Zimecki and Kruzel 2007 ). Animal studies with mice or rats have demonstrated that orally administered Lf and related compounds can sup- press the overgrowth and translocation of certain intestinal bacteria, such as E. coli, Streptococcus, and Clostridium strains, but does not affect the growth of intestinal bifi dobacteria. Also, oral administration of Lf and lactoferricin reduces the infection rate of H. pylori, Toxoplasma gondii, candidiasis, and tinea pedis, as well as prevents clinical symptoms of infl uenza virus infection. Further animal studies have demonstrated that orally ingested Lf and related compounds can reduce iron - defi cient anemia and drug - induced intestinal infl ammation, colitis, and arthritis and decrease mortality caused by endotoxin shock. Also, Lf stimulates weight gain in preweaning calves. A recent animal study by Cornish et al. (2004) has shown that oral Lf administration to mice regulates the bone cell activity and increases bone formation. In addition, animal studies have shown benefi cial effects of Lf ingestion on inhibition of carcinogen - induced tumors in colon, esophagus, lung, tongue, bladder, and liver. Clinical studies in infants have demonstrated that oral administration of bovine Lf preparations increases the number of bifi dobacteria in fecal fl ora and the serum ferritin level, while the ratios of Enterobacteriaceae, Streptococcus, and Clostridium tend to decrease. A recent clinical study (King et al. 2007 ) has shown that Lf supplementation to healthy infants for 12 months was associated with fewer lower respiratory tract illnesses and higher hematocrits as compared to the control group, which received regular infant formula. Other human studies have shown that orally administered Lf increases eradication rate of H. pylori gastritis when given in connection with triple therapy. Also, in further human studies, Lf ingestion has been demonstrated to decrease the incidence of bacteremia and severity of infection in neutropenic patients, alleviate symptoms of fatal structural damage to outer membranes and

inhibition of viral replication, and (3) prevention of microbial attachment to epithelial cells or entrocytes. As reviewed by Pan et al. (2007) , the bactericidal effect of Lf can be augmented by the action of lysozyme or antibodies. Lf can also increase susceptibility of bacteria to certain antibiotics, such as vancomycin, penicillin, and cephalospo- rins. 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, Helico- bacter pylori, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, S. enteriditis, Staphylococcus aureus, Streptoccccus mutans, Vibrio cholerae, and hepatitis C, G, and B virus, HIV - 1, cytomegalovirus, poliovirus, rotavirus, and herpes simplex virus (Farnaud and Evans 2003 ; Pan et al. 2007 ). One possible in vivo effect of Lf is blocking biofi lm development of bacteria by chelat- ing iron from the environment. Iron deprivation stimulates bacterial surface motility, causing the bacteria to wander across the surface instead of forming cell clusters and biofi lms. The bacterial transition from a free - living form into biofi lms can be devastating for the host, because biofi lms protect bacteria effectively from the host defense mechanisms and antibiotics. Lf has been shown to block biofi lm development of Pseudomonas aeruginosa at concentrations below those that kill or prevent growth.

The antitumor activity of Lf has been studied intensively over the last decade and many mechanisms have been suggested, for example, iron chelation related antioxidative property, immu- noregulatory, and anti - infl ammatory functions (Wakabayashi et al. 2006 ). 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, as well as (4) stimulation of intestinal and peripheral antibody response (Pan et al.

2007 ).

bial system. In this system, which was originally suggested in the 1960s by Reiter and Oram (1967) , LP catalyzes peroxidation of thiocyanate anion or some halides in the presence of a hydrogen peroxide source to generate short - lived oxidation products of SCN - , primarily hypothiocyanate (OSCN - ), which kill or inhibit the growth of many species of microorganisms, including bacteria, viruses, fungi, molds, and protozoa (Seifu et al. 2005 ). The hypothiocyanate anion causes oxidation of sulphydryl (SH) groups of microbial enzymes and other membrane proteins, leading to intermediary inhibition of growth or killing of susceptible microorganisms.

Nowadays, the LP/SCN/H2O2 antimicrobial system is considered to be an important part of the natural host defense system of mammals (Boots and Floris 2006 ). One possible in vivo function of the LP system is related to the presence of LP in human airway epithelia, where it is likely to actively combat respiratory infections caused by microbial invaders (Gerson et al. 2000 ).

The LP system is known to be bactericidal against Gram - negative pathogenic and spoilage bacteria, such as E. coli, Salmonella spp., Pseudomonas spp., and Campylobacter spp. On the other hand, the system is bacteriostatic against many Gram - positive bacteria, such as Listeria spp., Staphylococcus spp., and Streptococcus spp. Also, this mechanism is inhibitory to Candida spp. and the protozoan Plasmodium falciparium and it has been shown to inactivate in vitro the HIV type 1 and polio virus (Seifu et al. 2005 ). The LP system is considered to provide a natural method for preserving raw milk, as milk contains naturally all necessary components to make the system functional. The natural concentrations of thiocyanate and H2O2 can, however, be critical in milk, and the system usually requires activation by addition of a source of these components.

The effectiveness of the activated LP system in raw, uncooled milk has been demonstrated in many pilot studies and fi eld trials worldwide (Anonymous 2005 ). Since 1991, the LP system has been approved by the Codex Alimentarius Committee for preserva- tion of raw milk under conditions where facilities for milk cooling are insuffi cient. The method is now being utilized in practice in a number of developing hepatitis C virus infection, and reduce small intes-

tine permeability in drug - induced intestinal injury.

Lf ingestion has also been shown to promote the cure of tinea pedis (Tomita et al. 2008 ). Recent human studies reviewed by Zimecki and Kruzel (2007) suggest that oral bovine Lf administration could be benefi cial in stress - related neurodegenera- tive disorders and treatment of certain cancer types.

Lf has attracted increasing commercial interest and many products containing added Lf have already been launched on the market in Asian countries, in particular. Lf is produced industrially by many com- panies worldwide, and it is expected that its use as an ingredient in functional foods and pharmaceuti- cal preparations will increase drastically in the near future (Tamura 2004 ; Tomita et al. 2008 ). Current commercial applications of Lf include, for example, yogurt products marketed in Japan and Taiwan, and baby foods and infant formulas marketed in South Korea, Japan, and China. In addition, Lf has been applied in different dietary supplements that combine, for example, Lf and bovine colostrum and/

or probiotic bacteria. Due to potential synergistic actions, Lf has been incorporated together with LZM and LP into human oral health care products, such as toothpastes, mouth rinses, moisturizing gels, and chewing gums (Wakabayashi et al. 2006 ). The U.S. Food and Drug Administration (FDA) has approved the use of bLf (at not more than 2% by weight) as a spray to reduce microbial contamina- tion on the surface of raw beef carcasses. FDA has granted a “ Generally Recognized as Safe ” (GRAS, GRN 67) status to bLf, and this determination accounts for uses at defi ned levels in beef carcasses, subprimals, and fi nished cuts (Taylor et al. 2004 ).

4.5. Lactoperoxidase

LP is a glycoprotein that occurs naturally in colostrum, milk, and many other human and animal secretions. LP is the most abundant enzyme in milk and can be recovered in substantial quantities from whey using chromatographic techniques (Kussendrager and van Hooijdonk 2000 ). The biological function of LP has been associated mainly with its ability to catalyze an unspecifi c antimicro-

Dalam dokumen Peptides in Food and Health (Halaman 155-160)