This article was downloaded by: [The University Of Melbourne Libraries] On: 30 April 2013, At: 02:50 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Critical Reviews in Food Science and Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/bfsn20 Milk Biologically Active Components as Nutraceuticals: Review Sindayikengera Séverin a & Xia Wenshui a a Southern Yangtze University, School of Food Science and Technology, Wuxi, China Published online: 18 Jan 2007. To cite this article: Sindayikengera Séverin & Xia Wenshui (2005): Milk Biologically Active Components as Nutraceuticals: Review, Critical Reviews in Food Science and Nutrition, 45:7-8, 645-656 To link to this article: http://dx.doi.org/10.1080/10408690490911756 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
Transcript
This article was downloaded by: [The University Of Melbourne Libraries]On: 30 April 2013, At: 02:50Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,37-41 Mortimer Street, London W1T 3JH, UK
Critical Reviews in Food Science and NutritionPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/bfsn20
Milk Biologically Active Components as Nutraceuticals:ReviewSindayikengera Séverin a & Xia Wenshui aa Southern Yangtze University, School of Food Science and Technology, Wuxi, ChinaPublished online: 18 Jan 2007.
To cite this article: Sindayikengera Séverin & Xia Wenshui (2005): Milk Biologically Active Components as Nutraceuticals:Review, Critical Reviews in Food Science and Nutrition, 45:7-8, 645-656
To link to this article: http://dx.doi.org/10.1080/10408690490911756
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
Milk Biologically Active Componentsas Nutraceuticals: Review
SINDAYIKENGERA SEVERIN and XIA WENSHUISouthern Yangtze University, School of Food Science and Technology, Wuxi, China
Milk contains components that provide critical nutritive elements, immunological protection, and biologically active sub-stances to both neonates and adults. Milk proteins are currently the main source of a range of biologically active peptides.Concentrates of these peptides are potential health-enhancing nutraceuticals for food and pharmaceutical applications.Several bioactive peptides may be used as nutraceuticals, for example, in the treatment of diarrhea, hypertension, throm-bosis, dental diseases, as well as mineral malabsorption, and immunodeficiency. Minor whey proteins, such as lactoferrin,lactoperoxidase, lysozyme, and immunoglobulins, are considered antimicrobial proteins. Milk also contains some naturalbioactive substances. These include oligosaccharides, fucosylated oligosaccharides, hormones, growth factors, mucin, gan-gliosides, and endogenous peptides, which are present in milk at secretion. Most of the claimed physiological properties ofmilk bioactive components have been carried out in vitro or in animal model systems, and these hypothesized propertiesremain to be proven in humans. Whether these milk bioactive components will replace drugs entirely in the immediate futureis still unclear, but the increasing appreciation of “drug foods” or nutraceuticals plays a complementary rather than asubstitutional role to the synthetic pharmacological drugs.
Milk is a polyphasic secretion of the mammalian gland, con-taining approximately 5% lactose, 3.2% protein, 4% lipid, and0.7% mineral salts. The nutritional value of milk and milk prod-ucts is due to these compounds. Milk contains components thatprovide critical nutritive elements, immunological protection,and biologically active substances to both neonates and adults(Warner et al., 2001). Milk remains one of the most elaboratelystudied of human food. Its composition within any mammalianspecies is indicative of the neonatal requirements of its offspring,presenting optimum composition of nutrients required duringthe newborn period of that species. Milk is a complete food fornewborn mammals, and it is the sole food during the early stageof rapid development.
The benefit of milk in preventing infection has been recog-nized for thousands of years. Much of this activity has beenattributed to antibodies, but the role of complex sugars in milkand of other minor proteins, such as lactoferrin and lactoperoxi-dase, as bioactive agents is only recently being recognized. Milkcontains various components with physiological functionality
Address correspondence to Xia Wenshui, School of Food Science and Tech-nology Southern Yangtze University, 170 Huihe Rd., Wuxi, 214036, China.E-mail: [email protected]
(Lindsay et al., 2002). It contains high levels of immunoglobu-lins and other physiologically active compounds for warding offinfection in the newborn. Similarly, colostrum is important fornewly born mammals, as it provides necessary immunity againstinfections.
Milk proteins are currently the main source of a range of bi-ologically active peptides, even though other animal and plantproteins contain potential bioactive sequences (Wu and Ding,2002). These peptides, which are encrypted within the sequenceof the parent proteins, can be released by enzymatic proteolysis,for example, during gastrointestinal digestion or during process-ing (Gobetti et al., 2002). Biologically active peptides derivedfrom milk proteins are inactive within the sequence of the precur-sor proteins. Once produced, bioactive peptides (BPs) may act inthe body as regulatory compounds with a hormone-like activity.Concentrates of these peptides are potential health-enhancingnutraceuticals for food and pharmaceutical applications.
Milk also contains some natural bioactive substances thatare extant in it by virtue of the physiological origin secretion.These include oligosaccharides, fucosylated oligosaccharides,hormones, growth factors, mucin and gangliosides, and endoge-nous peptides, which are present in milk at secretion (Cheisonand Wang, 2003). Nature factored these substances into milkwith a scheduled significance, they support two lines ofdefences (Lahov and Regelson, 1996). It would seem the first is
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Table 1 Concentration and biological functions of major milk proteins
Concentration (g/l)
Protein Cow Human Function
Total caseins 26.0 2.7 Ion carrier (Ca, PO4, Fe, Zn, Cu), precursors of bioactive peptidesα-Casein 13.0β-Casein 9.3κ-Casein 3.3Total whey protein 6.3 67.3β-Lactoglobulin 3.2 Retinol carrier, binding fatty acids, possible antioxidantα-Lactalbumin 1.2 1.9 Lactose-synthesis in mammary gland, Ca carrier, immunomodulation, anticarcinogenicImmunoglobulins (A, M, and G) 0.7 1.3 Immune protectionSerum albumin 0.4 0.4Lactoferrin 0.1 1.5 Antimicrobial, antioxidative, immunomodulation, iron absorption, anticarcinogenicLactoperoxidase 0.03 AntimicrobialLysozyme 0.0004 0.1 Antimicrobial, synergistic effect with immunoglobulins and lactoferrinMiscellaneous 0.8 1.1Proteose-peptone 1.2 Not characterizedGlycomacropeptide 1.2 Antiviral, bifidogenic
Adapted from Walstra and Jenness (1984), Yamauchi (1992), Korhonen et al. (1998).
an elaborate provision for milk’s initial self-defense against thedegradative army of microorganisms, while the second offersthe first line shield for the neonate. Among the former arelysozyme and lactoperoxidase (Haukland et al.,2001).
Oligosaccharides, glycolipids, and glycoproteins containingsialic acid residues may have a role as anti-infectives. Compo-nents, such as growth factors, may also be considered for futureproduct development, because of the economies of scale used inthe dairy industry.
With today’s sophisticated analytical, biochemical, and cell-biological research tools, the presence of many other compoundswith biological activity has been demonstrated. Achievements inseparation techniques in the dairy industry and enzyme technol-ogy offer opportunities to isolate, concentrate, or modify thesecompounds, so that their application in functional foods, dietarysupplements, nutraceuticals, and medical foods has become pos-sible.
This article provides a brief overview of the bioactive compo-nents in milk, their beneficial physiological effects, and potentialhealth implications.
MILK PROTEINS
The primary function of dietary proteins is to supply the bodyadequately with indispensable amino acids and organic nitro-gen. The amino acid sequences of bovine milk proteins, i.e.,caseins and whey proteins, were elucidated between 1972 and1977 by use of conventional methods. About 80% of bovinemilk proteins consists of caseins (2.7 g/100 g milk), a groupof phosphate-containing proteins named αs1-, αs2-, β-, and κ-casein (Fox and McSweeney, 2001; Modler, 2000). Caseins aresynthesized by the mammary secretory epithelium and are with-out any known biological activity. Human casein consists ofprimarily β- and κ-caseins. The bovine whey protein fraction
consists of α-lactalbumin, β-lactoglobulin, bovine serum albu-min, immunoglobulin, lactoferrin, transferrin, and the proteose-peptone fraction (0.08 g/100 g milk). Whey proteins have glob-ular three-dimensional structures and bear specific biologicalactivities. The concentration of casein, whey protein, and minorwhey proteins in milk is shown in Table 1.
Besides these main milk proteins, low amounts of minor pro-teins and peptides are natural constituents of milk with hormonalor other physiological activities, e.g., hormones releasing fac-tors and the endogenous antibacterial system (Table 2). Thesecompounds are secreted from the blood into the milk or aresynthesized in the mammary gland. A brief outline of bioactivepeptides derived from bovine milk, their precursors, and pos-sible bioactive role is shown in Table 3. These peptides havebeen obtained from casein as well as whey proteins by in vitrodigestion with proteolytic enzymes, or from in vivo digests afterfeeding the precursor protein.
Table 2 Bioactive proteins/peptides as naturalingredients of milk
Opioid peptides are those having pharmacological sim-ilarities to opium (morphine) and are derived from ca-sein called casomorphins (Nagendra, 2000). The ma-jor opioid peptides, as shown Tables 3 and 4, arefragments of β-Casein (β-CN). The major opioid pep-tides are fragments of β-CN, called β-casomorphins, dueto their exogenous origin and morphine-like properties(Brantl et al., 1979; Henschen et al., 1979; Yoshikawa et al.,1986); however, they have also been obtained from pepsin hy-drolysis of bovine αs1-CN fractions (Nagendra, 2000; Loukaset al., 1983; Paroli, 1988; Zioudrou et al., 1979). Similar pep-tides have been reported from human β-CN fractions (Greenberget al., 1984; Yoshikawa et al., 1984), and the Y-P-F sequence,which is common to bovine β-casomorphin, was also found tobe present in the primary structure of human β-CN. All κ-caseinfragments, known as casoxins, behave as opioid antagonists. Atetrapeptide amide, morphiceptin is the most active opioid ago-nist in the bovine β-casomorphin group. Two opioid antagonists,casoxin C and D, also belong to this group (Loukas et al., 1983).
Opioid peptides have been generated in vitro by enzymaticdigestion of β-caseins from cows, water buffalo, and sheep(Pihlanto-Leppala et al., 1994; Petrilli et al., 1983; Richardsonand Mercier, 1979). In general, the α- and β-CN fragments pro-
Table 4 Bioactive peptides derived from milk protein components: Opioid peptides
Peptides Origin Structure References
Agonistβ-Casomorphin 5 β-CN Tyr-Pro-Phe-Pro-Gly Brantl et al., 1981; Meisel, 1997β-Casomorphin 5 h β-CN Tyr-Pro-Phe-Val-Glu Yoshikawa et al., 1984Morphiceptin β-CN Tyr-Pro-Phe-Pro-NH2 Chang et al., 1985α-Casein exorphin αs1-CN Arg-Gly-Phe-Gln-Asn-Ala Loukas et al., 1983
TyrCasoxin B h κ-CN Tyr-Pro-Tyr-Tyr (O-CH3) Yamauchi, 1992Casoxin C κ-CN Tyr-Ile-Pro-Ile-Gln-Tyr-Val-Leu-Ser-Arg Chiba et al., 1989; Yamauchi, 1992Casoxin D h αs1-CN Tyr-Val-Pro-Phe-Pro-Pro-Phe Yamauchi, 1992Lactoferroxin A LF Tyr-Leu-Gly-Ser-Gly-Tyr (-OCH3) Tani et al., 1990
h = human, CN = casein, LF = lactoferrin.
duce agonist responses, while those derived from κ-CN elicitantagonist effects. It is noteworthy that bioactive peptides aregenerated from most of the major proteins in both bovine andhuman milk.
When opioid peptides are injected into the bloodstream, theyinduce an analgesic and sedative effect due to their action on thenervous system (Pihlanto-Leppala, 2001; Paroli, 1988). Stud-ies have suggested that opioid agonist and opioid antagonistsare formed in the gut as a result of in vivo hydrolysis of milkcasein. Opioid peptides released from casein during digestionshowed gastrointestinal motility (Daniel et al., 1990). Some ofthese peptides have been found to affect gastrointestinal transittime. Casomorphins have been found to prolong gastrointesti-nal transit time and to exert antidiarrhoeal action. The effect ofβ-casomorphins on the motility of rat intestinal tract was studiedusing the non-absorbable marker 141Ce. β-casomorphins havebeen detected in the plasma of pregnant or lactating women(Bicknell, 1985; Yen et al., 1985). Similarly, casomorphins havebeen detected in the duodenal chyme of minipigs and in the hu-man small intestine (Svedberg et al., 1985) as a result of in vivodigestion.
Casein Phosphopeptides (CPPs)
The micelles in bovine milk contain physiologically signif-icant amounts of calcium and phosphorous. This is because ofphosphorylated serine residues in αs1-, αs2-, and β-casein (Kellyet al., 2000). These so-called casein phosphopeptide (CPP) frag-ments help to create thermodynamically stable casein micellessupersaturated with calcium and phosphate, thus contributing tothe stability of milk during heat processing (Holt and Horne,1996).
By the cleavage of casein with enzymes, such as trypsinand alcalase, a number of CPP fragments can be obtained invitro (Adamson and Reynolds, 1995; Adamson and Reynolds,1996). The ability of CPP-containing hydrolysates to solubilizecalcium ions can be measured easily in vitro (Scholz-Ahrensand Schrezenmeir, 2000). CPP are mostly resistant to enzymatic
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hydrolysis in the gut and most often found in a complex with cal-cium phosphate (Renolds et al., 1994). Naito and Suzuki (1974)showed that this is indeed the case. Furthermore, it was shownthat the formation of CPP in the intestine could increase the con-centration of soluble calcium (Lee et al., 1980; Sato et al., 1986;Hirayama et al., 1992). Meisel and Frister (1989) found that CPPfragments were also released in the intestine of minipigs.
Cell culture and animal and human studies have subsequentlyaddressed the question of whether CPP in the diet improves theabsorption of calcium. The animal studies that tried to find pos-itive effects of dietary CPP on intestinal Ca absorption, Ca bal-ance, and bone formation showed controversial results (Tsuchitaet al., 2001). For example, no significant effects were foundby Brommage et al. (1991), Pointillart and Gueguen (1989), orScholz-Ahrens et al. (1990).
On the other hand, Japanese researchers reported a beneficialeffect of CPP using a postmenopausal bone loss rat ovariectomymodel (Tsuchida et al., 1996). In a randomized crossover design,Heany et al. (1994) studied the effect of a purified CPP prepa-ration on the Ca absorption in normal postmenopausal women(n = 35). No effect was seen when the population was analyzedas a whole; however, when the analysis was limited to womenwith low Ca absorption values (n = 17), the supplementationwith CPP resulted in a significant increase of 5.3% for themean absorption.
A rat pup model was applied by Hansen et al. (1996) to testthe effect of purified CPP on the passive absorption of both Caand Zn in an oat-based infant cereal and soy protein-based infantformula. These phytate-containing diets reduce the solubility ofZn and Ca. Compared to the nonsupplemented control diet, theaddition of CPP increased the absorption of Ca by 45% and 10%for the oat- and soy-based diets, respectively; the Zn absorptionincreased by 39% and 33%, respectively.
Subsequently, Hansen et al. (1997) investigated the absorp-tion of Ca and Zn in young men and women from rice-based(n = 11) or whole grain-based cereals (n = 11) with the labeledisotopes 47Ca and 65Zn. The actual quantity absorbed was calcu-lated from the whole-body retention, after excretion of the non-absorbed label via the faeces had taken place. CPP (1g) increasedthe actual quantity of absorbed Ca or Zn by approximately 30%when the low phytate rice-based meal was ingested; CPP of upto 2 g per serving could not increase the absorption of Ca orZn when the whole grain cereal, with approximately 10 timeshigher phytate content, was tested. A study by Yuan and Kitts(1991) in an animal system model could not demonstrate anyimprovement in calcium catabolism from diets containing CPP.CPPs-Ca complexes may enhance calcium absorption in thesmall intestine. However, further research is needed to confirmthis.
Several heat treatments of milk may cause dephosphoryla-tion of phosphoseryl residues and may affect the bioavailabilityof CPP. The addition of CPPs to toothpaste formulas has beensuggested to have anticariogenic effects and to prevent enameldemineralization (Grenby et al., 2001; Aimutis, 2004). CPPs arenot unpalatable and can be used as an anticariogenic additive
(Grenby et al., 2001; Scholz-Ahrens and Schrezenmeir, 2000).Commercial products with CPP include mineral drinks, nutri-tional supplements for children, confectionery, and products fordental care (Luo and Wong, 2000).
Angiotensin-I-converting enzyme (ACE) is a key enzyme in-volved in the regulation of blood pressure. Due to its activity,two amino acids are removed from angiotensin-I, yielding theoctapeptide angiotensin-II, which is a very potent vasoconstric-tor. Inhibition of the synthesis of angiotensin-II, thus lowersblood pressure. ACE also hydrolyzes bradykinin, which is avasodilator (Seppo et al., 2003). Thus, ACE inhibitors are an-tihypertensive peptides. Some specific inhibitors of ACE havebeen proven to be useful as antihypertensive drugs.
The antihypertensive effect of orally administered doses ofCalpis sour milk or peptides (Val-Pro-Pro or Ile-Pro-Pro) onspontaneous hypertensive rats was studied by Nakamura et al.(1995). The sour milk or peptides decreased systolic blood pres-sure 6–8 h postadministration. The antihypertensive effect ofsour milk containing two peptides, Val-Pro-Pro and Ile-Pro-Pro,was tested in hypertensive patients. Systolic blood pressure wasdecreased significantly at 4 and 8 wk after the beginning ofingestion, suggesting a mild pharmacological effect of antihy-pertensive peptides. These peptides can pass the intestinal tract,and after absorption, inhibit the production of angiotensin-II inblood. Sekiya et al. (1992) also studied the application of caseinhydrolysate against hypertension in human volunteers. ACE in-hibitors derived from casein or casokinins have been identifiedwithin the sequences of human β- and κ-CN (Kohmura et al.,1990; Kohmura et al., 1989). They are also generated by tryp-tic digestion of bovine αs1- and β-CN (Maruyama and Suzuki,1982). A synthetic seven amino acid peptide, equivalent to asegment found in the β-CN hydrolysate, exhibited potent anti-hypertensive activity in these rats over an 8-h interval, after oraladministration (Maeno et al., 1996).
Meisel et al. (1997) have measured ACE inhibition (ACEI)activities in various milk products, including pasteurized milk,yogurts, quarg, and fresh and ripened cheeses. Low activitywas found in products with a low degree of proteolysis, suchas yogurt, quarg, and fresh cheeses. Ripened cheese containedmore activity, but this was dependent on the degree of matura-tion; above a certain level in cheese maturation, ACEI activitydecreased. Therefore, dairy products may act as natural func-tional foods influencing blood pressure; this means that dairyproducts may be convenient vehicles for enrichment with natu-ral ACEI.
Antithrombotic Peptides
The clotting of blood and the clotting of milk are two phys-iologically important coagulation processes. A larger number
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MILK BIOLOGICALLY ACTIVE COMPONENTS AS NUTRACEUTICALS 649
of molecular similarities have been previously reported be-tween these two clotting phenomena (Jolles, 1975; Jolles andHenschen, 1982). Structural homologies between cow κ-caseinand human fibrinogen γ -chain were found by Jolles et al. (1978).The κ-casein fragment named casopiastrin, obtained from tryp-tic hydrolysates, shows antithrombotic activity by inhibiting fib-rinogen binding on platelets (Brody, 2000; Fiat et al., 1993). Dataon the pharmacological significance of peptides with antithrom-botic activity are not yet available.
Immunomodulating Peptides
Several immunomodulating peptides resulting from caseinhave been detected, including immunopeptides from αs-caseinand β-casein. In vitro activity of immunomodulating peptidesresulting from tryptic and chymotryptic hydrolysates of αs1- andβ-casein have been reported to stimulate the macrophage activityagainst red blood cells (Parker et al., 1984). Casein-derived im-munopeptides have been shown to stimulate the phagocytic ac-tivities of murine and human macrophages and to protect againstKlebsiella pneumoniae infection in mice (Smacchi and Gobetti,2000). The peptides may stimulate the proliferation and matu-ration of T cells and natural killer cells for the defense of thenewborn against a large number of bacteria, particularly entericbacteria (Clare and Swaisgood, 2000; Van, 2002). The injec-tion of casein or α-lactalbumin peptides has been found to havedirect immunomodulating activity against Klebsiella pneumo-niae in rats (Migliore-Samour et al., 1989). Lahov and Regelson(1996) have reported antibacterial activity of isracidin, the 1–23fragment of αs1-casein obtained from the action of chymosin,against Staphylococcus aureus and Candida albicans. The injec-tion of isracidin into the udder of sheep and cow gave protectionagainst mastitis.
In order to function physiologically in the human body,the active peptides must be absorbed from the intestine in anactive form. However, there is no evidence that these peptidescan be absorbed from the intestine in adults and the proposedproperties remain to be proven. Di- and tri-peptides can be eas-ily absorbed in the intestine; however, it is not clear that largerbioactive peptides containing excess of three amino acids areabsorbed from the intestine and reach the target organ.
Most of the claimed physiological properties of the casein-based bioactive peptides have been carried out in vitro or inanimal model systems and these hypothesized properties remainto be proven in humans.
Whey Protein-based Bioactive Substances
Whey proteins comprise approximately 20% of total milkproteins. The whey proteins are not coagulated by acid and areresistant to the action of chymosin. As a result, these proteinsremain present in acid and rennet wheys. α-Lactalbumin is oneof the main proteins in human milk. It contains readily digestibleamino acids. β-Lactoglobulin represents about half the total pro-
tein in whey of cow’s milk. However, it is absent from humanmilk.
Many whey proteins are claimed to possess physiologicalproperties. Bovine whey contains metal binding proteins, im-munoglobulins, growth factors, and hormones (Walzem, 2002).Bioactive peptides obtained from whey proteins, and their phys-iological effects have been less extensively studied than thosefrom caseins. Most of the functions of whey proteins are relatedto the immune or digestive systems.
α-Lactorphin has been shown to exert a weak opioid activ-ity to smooth muscles. β-Lactorphin has been shown to havea smooth muscle-contracting effect (Antila et al., 1991). Wongand Watson (1995) have shown immunostimulatory functions ofwhey proteins. McIntosh et al. (1995) have reported anticarcino-genic effects of whey proteins in mice and rats. However, furtherresearch is warranted to substantiate these findings. Antimicro-bial properties of these whey protein fractions are well estab-lished. Lactoperoxidase, lactoferrin, and immunoglobulins havealready been commercialized. Some of the bioactive peptidesobtained from whey proteins include α-lactorphin, β-lactorphin,albutensin A, and β-lactotensin (Table 5).
Serorphins obtained from bovine blood serum albumin haveshown opioid activity. In studies by McIntosh et al. (1993) andRegester et al. (1996), whey-protein fed rats showed the lowestincidence of colon cancer (30%), compared to 55% for meat dietand 60% for soy diet. α-Lactalbumin and β-Lactoglobulin havephysiological properties of whey proteins, including immuno-enhancing effects. The possible role of α-lactalbumin as anantitumor agent is being investigated (Nagendra, 2000). Thetwo peptides, α-lactorphin and β-lactorphin, have been shownto cause contraction of smooth muscles similar to morphine.Albutensin and β-lactotensin cause contraction of guinea-pigileum longitudinal muscle. Whey proteins including α- and β-lactorphin and albutensin appear to have ACE inhibitory activity.β-Lactoglobulin can bind retinol and can transport this into thesmall intestine (McLeod et al., 1996).
Minor Bioactive Proteins
Minor whey proteins, such as lactoferrin, lactoperoxidase,lysozyme, and immunoglobulins, are considered antimicrobialproteins. The specific roles of various immunoglobulins, lacto-ferrin, and lactoperoxidase in protecting the newborn calf arewell established.
Immunoglobulins. Immunoglobulins are not transferredacross the placenta to the mammalian fetus; hence, babiesand calves are born with very low concentrations of serumimmunoglobulins. Immunoglobulins occur in high concentra-tion in bovine or human colostrum. The absorption of immuno-globulins occurs from colostrum to provide passive immunityafter birth. The antibodies protect the newborn against infec-tions. Immunoglobulins are particularly rich in colostrum, butdecline in concentration during normal lactation. In the cow, themajor class of immunoglobulin is IgG1, with concentrations ofabout 48 g/l in colostrum and 0.6 g/l in normal milk (Farrell et al.,
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Table 5 Bioactive peptides derived from whey proteins
Precursorprotein Fragment Peptide sequence Name Function References
α-Lactalbumin 50–53 Tyr-Gly-Leu-Phe α-Lactorphin Opioid agonist ACE inhibition Mullally et al., 1996β-Lactoglobulin 102–105 Tyr-Leu-Leu-Phe β-Lactorphin Non-opioid stimulatory effect on ileum Mullally et al., 1996
142–148 Ala-Leu-Pro-Met- — ACE inhibition Mullally et al., 1997His-Ile-Arg
146–149 His-Ile-Arg-Leu β-Lactotensin Ileum contraction Korhonen et al., 1998Bovine serum 399–404 Tyr-Gly-Phe-Gln- Serorphin Opioid Tani et al., 1994
albumin Asp-Ala208–216 Ala-Leu-Lys-Ala- Albutensin A Ileum contraction Korhonen et al., 1998
Trp-Ser-Val-Ala-Arg ACE inhibition
Lactoferrin 17–42 Lys-Cys-Arg-Arg- Lactoferricin Antimicrobial Shin et al., 1998; Tomita et al., 1991Trp-Glu-Trp-Arg-Met-Lys-Lys-Leu-Gly-Ala-Pro-Ser-Ile-Pro-Ser-Ile-Thr-Cys-Val-Arg-Arg-Ala-Phe
2004). In human milk, the predominant class of immunoglobu-lins is IgA, with levels of about 17 g/l in colostrum and about 1 g/lin normal breast milk (Kulkarni and Pimpale, 1989). Cow’s milkimmunoglobulins have been put forward as a possible effectivemeans of preventing or combating diarrhea (Reddy et al., 1988).Many studies, both animal and human, have been performed withimmunoglobulins either from nonimmunized cows or from cowshyperimmunized against specific pathogens. The conclusion tobe drawn from these studies is that immunoglobulin preparationsfrom vaccinated cows are more effective, because the titres ofthe pathogen-inactivating immunoglobulins are higher.
Several excellent articles summarize the various clinicalstudies conducted using bovine immunoglobulin concentrates(BIC) against oral pathogens (Streptococcus mutans, Candidaalbicans), the human-specific gastric pathogen Helicobacterpylori, rotavirus infections in children, Cryptosporidiumparvum-induced diarrhea in immunocompromised individuals,and enteropathogenic Eschericia coli (EPEC) infections in chil-dren (Bostwick et al., 2000; Bogsted et al., 1996; Weiner et al.,1999). BIC for human application is only in its infancy, becausethere are still uncertainties with respect to dosage and timingof administration. Furthermore, immunoglobulins are heatsen-sitive molecules, subject to human digestive action and maypresent difficulties with respect to palatability or shelf life offood products due to contaminating enzymes of a proteolyticnature. However, several digestion studies show that 20% ormore of the ingested antibodies will survive and retain biologi-cal activity (Roos et al., 1995; Kelley et al., 1997). To increasesurvival rate, it is important to protect against gastric digestion.Encapsulating the BIC with acid-resistant coatings may be asimple way to achieve this.
Natural antimicrobials, like BIC, may have a number of ad-vantages over synthetic antibiotics: lower cost; their polyclonalnature (in principle) enhances their resistance against bacterialinactivation; their activity is most likely to be limited to the in-
testinal lumen; they place less of a burden of the microbial gutecology (Jan, 2001). From a commercial point of view, theirentry into the market is faster because of less stringent regula-tory issues and their use in dietary supplements or special foodproducts.
Lactoferrin. Lactoferrin is a dominant whey protein inhuman milk and plays an important role in iron uptake in theintestine (Hutchens et al., 1994; Viljoen, 1995). Bovine lacto-ferrin is homologous to human lactoferrin. The concentration oflactoferrin in bovine colostrum and milk is about 1.5–5 mg/mland 0.1 mg/l, respectively (Tsuji et al., 1990). In human milk andcolostrum, the reported levels are 2–4 g/l and 6–8 g/l, respec-tively. This indicates that lactoferrin is even more important forhumans than for bovine species. The presence of high concentra-tions of lactoferrin in colostrum and the passing on to the new-born suggests the involvement of lactoferrin in the primary de-fense system against pathogenic bacteria (Kussendrager, 1993).
Lactoferrin has also been identified in the milks of othermammalian species, such as the cow, pig, horse, buffalo, goat,and mouse. On a commerical basis, lactoferrin is isolated fromcow’s milk. Lactoferrin can also be found in tears, synovial flu-ids, saliva, and seminal fluid, with concentrations ranging fromabout 10 µg/ml to about 2 mg/ml (Steijns and Van Hooijdonk,2000). Blood plasma lactoferrin is derived from the neutrophilsthat degranulate and synthesize lactoferrin during inflammation(Britigan et al., 1994).
Lactoferrins are single-chain polypeptides of about80000 Da, containing 1–4 glycans, depending on the species(Spik et al., 1994). Bovine and human lactoferrins consist of689 and 691 amino acids, respectively; the sequence iden-tity is 69% (Pierce et al., 1991). The three-dimensional con-formations of both human and bovine lactoferrin are nowknown in great detail (Moore et al., 1997). The structureelucidation of the N-bound glycans attached to lactoferrinsfrom various species has been the result of the extensive
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studies of Montreuil and Spik and colleagues (Spik et al.,1994; Coddeville et al., 1992). The sugars found in bovinelactoferrin are N-acetyllactosamine, N-acetylglucosamine,galactose, fucose, mannose, and neuraminic acid.
The three-dimensional structures of bovine and human lacto-ferrin are very similar, but not entirely superimposable. Eachlactoferrin comprises of two homologous lobes, called N- andC- lobe, referring to the N- terminal and C- terminal part of themolecule, respectively. Each lobe also consists of two sublobesor domains, which form a cleft where the ferric ion (Fe3+) isbound tightly in synergistic cooperation with a (bi)carbonateanion (Jan, 2001). In the natural state, bovine lactoferrin is onlypartly saturated with iron (15–20%) and has a salmon pink color,the intensity of which depends on the degree of iron saturation.Iron-depleted lactoferrin with less than 5% iron saturation iscalled apolactoferrin, whereas iron-saturated lactoferrin is re-ferred to as holo-lactoferrin. The lactoferrin found in breast milkis essentially apo-lactoferrin. Lactoferrin’s affinity for iron isvery high (about 260 times that of blood serum transferrin), withan affinity constant of about 1020 (Baker et al., 1994). The iron-binding capacity of lactoferrin is dependent upon the presenceof (small amounts of) (bi)carbonate. The binding site appearsto be optimized for the binding of ferric iron and (bi)carbonate,with respect to size, charge, and stereochemistry (Jan, 2001).
The cationic N-terminus of bovine lactoferrin is of specialinterest because of the reported antibacterial activity (Bellamyet al., 1992). Lactoferrin exhibits both bacteriostatic and bac-teriocidal activity against a range of microorganisms. Lacto-ferrin also causes the release of lipopolysaccharide moleculesfrom outer membrane of the Gramnegative bacteria and actsdirectly as an antibiotic (Diarra et al., 2001). Lactoferrin hasbeen found to inhibit the growth of Escherichia coli, Salmonellatyphimurium, Shigella dysenteriae, Listeria monocytogenes,Bacillus stearothermophilus, and Bacillus subtilis (Batish et al.,1988; Payne et al., 1990; Saito et al., 1991). The antimicrobialeffect is mainly on the organisms that require iron as lactofer-rin chelates iron, thereby, depriving the organisms of a sourceof this nutrient. Lactoferrin interacts with the bacterial cellmembrane, leading to permeability changes and causes releaseof lipopolysaccharide from the outer membrane of the Gram-negative bacteria (Nibbering et al., 2001).
Lactoferricin, a peptide derived from bovine lactoferrin dueto action of pepsin, has been found to have antimicrobial activ-ity against various bacteria and Candida albicans (Jones et al.,1994). Lactoferricin is a single peptide consisting of 25 aminoacid residues. A similar active peptide consisting of 47 aminoacid residues has been obtained from human lactoferrin. Themolecule is folded into two globular units, each capable of bind-ing one ferric (Fe3+) ion.
In cats, bovine lactoferrin is used for the treatment of in-tractable stomatitis: the phagocytic activity of neutrophils is ap-parently stimulated (Sato et al., 1996). In rainbow trout, bovinelactoferrin also stimulates the phagocytic cells (Sakai et al.,1995). In mice, bovine lactoferrin induces both the mucosaland systemic immune response (Debbabi et al., 1998). One
may conclude that lactoferrin makes an important contribu-tion to the host defense system. It eliminates pathogens, suchas bacteria, viruses, and fungi, stimulates and protects cells in-volved in the host defense mechanisms, and controls the cytokineresponse.
The occurrence of lactoferrin in biological fluids like milks,tears, saliva, and seminal fluids suggests that it could have a rolein the nonspecific defense against invading pathogens. Its broadantimicrobial spectrum (including Gram-positive and Gram-negative bacteria, yeasts and fungi), and the recently discoveredantiviral activity (including cytomegalovirus, herpes, influenza,HIV, rotavirus, hepatitis C) support this envisaged role (Supertiet al., 1997; Harmsen et al., 1995; Marchetti et al., 1996).
Current commercial applications of bovine lactoferrin in-clude infant formulas, nutritional iron supplements and drinks,fermented milks, chewing gums, immune-enhancing nutraceu-ticals, cosmetic formulas, and feed and pet care supplements(Jan, 2001).
Lactoperoxidase
Lactoperoxidase is a major antibacterial agent in colostrum.It is a very effective bactericidal agent in the presence of SCN−
and H2O2. The enzyme, in the presence of H2O2, catalyses theoxidation of thiocyanate (SCN−) and produces an intermediateproduct with antimicrobial properties (Visalsok et al., 2004).The concentration of lactoperoxidase in colostrum and milkis about 11–45 mg/l and 13–30 mg/l, respectively (Korhonen,1977).
Indigenous lactoperoxidase in milk may be exploited for thecold-sterilization of milk, while the isolated enzyme may beadded as a bactericidal agent to milk replacers for young calvesor piglets. Lactoperoxidase may be a useful additive for infantformulae, perhaps because human milk contains very little or nolactoperoxidase (Fox, 2001).
Lysozyme
Lysozyme is an antimicrobial enzyme found in colostrumand human milk (Vannini et al., 2004). The enzyme hydrolysesβ- 1 → 4 linkages between N-acetylmuramic acid and 2-acetyl-amino-2-deoxy-D-glucose residues in bacterial cell walls, re-sulting in cell lysis. The concentration of lysozyme in colostrumand normal milk is about 0.14–0.7 and 0.07–0.6 mg/l, respec-tively (Korhonen, 1977). Lysozyme is a 15 kDa single-chainprotein. The amino acid content of bovine milk lysozyme isdifferent from that of human milk and egg white lysozyme.
Milk lysozyme is active against a number of Gram-positiveand some Gram-negative bacteria. There seems to be a synergis-tic action of lysozyme and lactoferrin against E. coli, as the latterdamages the outer membrane of Gram-negative bacteria and theorganism becomes susceptible to lysozyme. A direct interac-tion between lysozyme and lactoferrin was observed with Mi-crococcus luteus (Yamauchi, 1992). Combinations of lysozymeand lactoferrin are more bacteriostatic than either of the proteins
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alone (Suzuki et al., 1989). The enzyme is toxic to Gram-positiveand Gram-negative bacteria, such as Pseudomonas aeruginosa,Salmonella typhimurium, and Listeria monocytogenes. Gram-negative organisms may be inhibited or killed. Gram-positivebacteria are more resistant and are generally only inhibited intheir growth.
MILK SUGARS AND OTHER MILK-BASED BIOACTIVECOMPONENTS
Human milk contains a number of protective factors, in-cluding oligosaccharides, mucin, gangliosides, and other N-acetylneuraminic acid-containing components (Table 6). Theseoligosaccharides are complex sugar structures attached to lac-tose. The concentration of oligosaccharides in human milk canrange from 10 to 20 g/l. Human milk contains more types andhigh amounts of oligosaccharides than cow milk. Oligosaccha-ride structures can also comprise the carbohydrate portion ofglycoconjugates, such as glycolipids and glycoproteins. Glyco-protein in human milk inhibits the binding of enterohaemor-rhagic E. coli (Newburg and Newbauer, 1995).
Lactose can be used to produce lactulose and lacto-oligosaccharides and has been reported to enhance calcium ab-sorption. Lactulose is used as a promoter of probiotic bacteria,and its use is now widespread in infant formulas. Lacto-oligosaccharides are also used as probiotic growth promoters.The oligosaccharides in human milk inhibit the binding of hostcells by enteropathogenic E. coli, Campylobacter jejuni, andStreptococcus pneumoniae to the target cells.
Mucin is a long macromolecule in human milk and linkswith oligosaccharides. Human milk mucin complex binds to
rotavirus, and inhibition of rotavirus has been reported. Mucin-associated glycoprotein (lactadherin) is responsible for bindingwith rotavirus (Yolken et al., 1992).
Calcium is thought to play a role in the regulation of bloodpressure. There is some epidemiological evidence that higher in-takes of calcium, especially from dairy products, are associatedwith maintenance of blood pressure. However, more researchis needed to substantiate this. The possible protective role ofcalcium in the prevention of colon cancer has been investigatedby researchers at the Dutch Dairy Research Institute (NIZO) inEde. It has been hypothesized that the main factor involved inthe promotion of colon cancer is the presence of bile salts. Milkappears to play some role in providing calcium phosphate thatbinds bile salts in order to prevent their toxic effect (Van derMeer and Lapre, 1991). Lipid-based bioactive compounds inmilk include fatty acids. The role of conjugated linoleic acid forinhibition of cancer and atherosclerosis has been studied (Pariza,1997; Schmelz and Merrill, 1997).
Breast milk contains hundreds of complex oligosaccharidesthat are involved in growth-promotion of Bifidogenic bacteria,act as receptor analogues for epithelial cells to prevent the ad-hesion of pathogens, or can inactivate toxins. Human milk gly-copeptides and glycoproteins are also thought to be stimulatingthe growth of bifidobacteria. These factors are absent from cowmilk. Protective effects of fucosylated oligosaccharides and gly-coproteins and glycolipids against enterotoxigenic E. coli havebeen reported (Newburg et al., 1990). This inhibition appearsto be associated with acidic glycolipids that contain sialic acidgangliosides.
CONCLUSION
Over the past few years, a number of new food ingredients la-beled as being nutraceuticals have been launched on the food andpharmaceutical market. These include components that have aproven beneficial effect on human health, such as bioactive sub-stances from milk (Silva and Verdalet, 2003). They gain accep-tance as functional foods and should provide a new convergencefor food science, pharmaceutical industry, and nutrition. The riseof customer awareness about the deleterious effects of chemi-cal preservatives and the increasing preference for natural, or“green,” components should give these milk bioactive compo-nents an ever-increasing role in the field of food preservationand nutraceuticals.
Most of the claimed physiological properties of milk bioac-tive components have been carried out in vitro or in animal modelsystems; these hypothesized properties remain to be proven inhumans. It is important to gain sufficient data based on humanstudies as well as human cell culture models to demonstrate thehealth enhancing effect of a bioactive substance.
It is hoped that milk will continue to find increasing use for itsvariety of available products, and that the functional propertiesof its bioactive components in the consumer’s gut will exert adisease suppressing effect, making it an invaluable and cheap,functional food.
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ACKNOWLEDGEMENT
The authors would like to thank the National Innovation ofTechnology Program (1997-17) for financial support.
REFERENCES
Adamson, N.J. and Reynolds, E.C. 1995. Characterization of tryptic casein phos-phopeptides prepared under industrially relevant conditions. Biotechnologyand Bioengineering, 45:196–204.
Adamson, N.J. and Reynolds, E.C. 1996. Characterization of casein phospho-peptides prepared using alcalase: Determination of enzyme specificity. En-zyme and Microbial Technology, 19:202–207.
Aimutis, W.R. 2004. Bioactive properties of milk proteins with particular focuson anticariogenesis. Journal of Nutrition, 134:989S–995S.
Antila, P., Paakkari, I., Jarvinen A., Mattila, M.J., Laukkanen, M., Pihlanto-Leppala, A., Mantsala, P., and Hellman, J. 1991. Opioid peptides derived fromin vitro proteolysis of bovine whey proteins. International Dairy Journal,1:215–229.
Baker, E.N., Anderson, B.F., Baker, H.M., Day, C.L., Haridas, M., Norris, G.E.,Rumball, S.V., Smith, C.A., and Thomas, D.H. 1994. Three-dimensionalstructure of lactoferrin in various functional states. In: Advances in Experi-mental Medicine and Biology. pp. 1–12. Hutchens, T.W., Rumball, S.V., andLonnerdal, B., Eds., Plenum Press, New York.
Batish, V.K., Chander, H., Zumdegeni, K.C., Bhatia, K.L., and Singh, R.S.1988. Antibacterial activity of lactoferrin against some common food-bornepathogenic organisms. Australian Journal of Dairy Technology, 5:16–18.
Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K., andTomita, M. 1992. Identification of the bactericidal domain of lactoferrin.Biochemica Biophysica. Acta., 1121:130–136.
Bogsted, A.K., Johansen, K., Hatta, R., Kim, M., Casswall, M., Svensson, L., andHammerstrom, L. 1996. Passive immunity against diarrhea. Acta. Pediatrica.,85:125–128.
Bostwick, E.F., Steijns, J., and Braun, S. 2000. Lactoglobulins. In: Natural foodantimicrobial systems. pp. 133–158. Naidu, A. S., Ed., Boca Raton: CRCPress.
Brantl, V., Teschemacher, H., Blasıg, J., Henschen, A., and Lottspeich, F. 1981.Opioid activities of β-casomorphins. Life Sciences, 28:1903–1909.
Brantl, V., Teschemacher, H., Henschen, A., and Lottspeich, F. 1979. Novel opi-oid peptides derived from casein (β-casomorphins). Hoppe Seylers Z. Physiol.Chem., 360:1211–1216.
Britigan, B.E., Serody, J.S., and Cohen, M.S. 1994. The role of lactoferrin asan anti-inflammatory molecule. In: Advances in Experimental Medicine andBiology. pp. 143–156. Hutchens, T.W., Rumball, S.V., and Lonnerdal, B.,Eds., Plenum Press, New York.
Brody, E.P. 2000. Biological activities of bovine glycomacropeptide. BritishJournal of Nutrition, 84:S39–S46.
Brommage, R., Juillerat, M.A., and Jost, R. 1991. Influence of casein phospho-peptides and lactulose on intestinal calcium absorption in adult female rats.Lait, 71:173–180.
Chang, K.-J., Su, Y.F., and Brent, D.A. 1985. Isolation of a specific µ-Opiatereceptor peptide, Morphiceptin, from an enzymatic digest of milk proteins.The Journal of Biological Chemistry, 260:9706–9712.
Cheison, S.C. and Wang, Z. 2003. Bioactive milk peptides: Redefining the food-drug interface—Paper 1: Antimicrobial and immunomodulating peptides.Africa Journal of Food, Agriculture, Nutrition and Development, 3:29–38.
Chiba, H., Tani, F., and Yoshikawa, M. 1989. Opioid antagonist peptides derivedfrom κ-casein. Journal of Dairy Research, 56:363–366.
Clare, D.A. and Swaisgood, H.E. 2000. Bioactive milk peptides: A prospectus.Journal of Dairy Science, 83:1187–1195.
Coddeville, B., Strecker, G., Wieruszeski, J.M., Vliegenthart, J.F.G., Van Hal-beek, H., Peter-Katalinic, J., Egge, H., and Spik, G. 1992. Heterogeneity of
bovine lactotransferrin glycans: Characterization of α-D-Gal p-(1->3)-β-D-Gal- and α-NeuAc-(2->6)-β-D-GalpNAc-(1->4)-β-D-GlcNAc-substitutedN-linked glycans. Carbohydrate Research, 236:145–164.
Daniel, H., Vohwinkel, M., and Rehner, G. 1990. Effect of casein andβ-casomorphin on gastrointestinal motility in rats. Journal of Nutrition,120:252–257.
Debbabi, H., Dubarry, M., Rautureau, M., and Tome, D. 1998. Bovine lactoferrininduces both mucosal and systemic immune response in mice. Journal ofDairy Research, 65:283–293.
Diarra, M.S., Lacasse, P., and Petitclerc, D. 2001. Method and composition fortreatment and/or prevention of antibiotic-resistant microorganism infections.International Patent Application No. PCT/CA00/01517.
Farrell, H.M.Jr., Jimenez-Flores, R., Bleck, G.T., Brown, E.M., Butler, J.E.,Creamer, L.K., Hicks, C. L., Hollar, C.M., Ng-Kwai-Hang, K.F., andSwaisgood, H.E. 2004. Nomenclature of the proteins of cows’ milk—SixthRevision. Journal of Dairy Science, 87:1641–1674.
Fiat, A.M., Migliore-Samour, D., Jolles, P., Drouet, I., Bal Dit Soitier, C., andCaen, J. 1993. Biologically active peptides from milk proteins with emphasison two examples concerning antithrombotic and immunomodulating activi-ties. Journal of Dairy Science, 76:301–310.
Fox, P.F. 2001. Milk proteins as food ingredients. International Journal of DiaryTechnology, 54:41–55.
Fox, P.F., and McSweeney, P.L.H. 2001. Advanced Dairy Chemistry: 1. Proteins.2nd ed., Gaithersburg, MD: Aspen Publishers, Inc.
Gobetti, M., Stepaniak, L., De Angelis, M., Corsetti, A., and Di Cagno, R. 2002.Latent bioactive peptides in milk proteins: Proteolytic activation and signif-icance in diary processing. Critical Reviews in Food Science and Nutrition,42:223–239.
Greenberg, R., Groves, M.L., and Dower, H.J. 1984. Human beta-casein. Aminoacid sequence and identification of phosphorylation sites. J. Biol. Chem.,259:5132–5138.
Grenby, T.H., Andrews, A.T., Mistry, M., and Williams, R.J.H. 2001. Dentalcaries-protective agents in milk products: Investigations in vitro. Journal ofDentistry, 29:83–92.
Hansen, M., Sandstrom, B., and Lonnerdal, B. 1996. The effect of casein phos-phopeptides on zinc and calcium absorption from high phytate infant dietsassessed in rat pups and caco-2 cells. Pediatric Research, 40:547–552.
Hansen, M., Sandstrom, B., Jensen, M., and Sorensen, S.S. 1997. Casein phos-phopeptides improve zinc and calcium absorption from rice-based but notfrom whole-grain infant cereal. Journal of Pediatric Gastroenterology andNutrition, 24:56–62.
Harmsen, M.C., Swart, P.J., DeBethune, M.P., Pauwels, R., De Clercq, E., Hauw,T., and Meijer, D.K.F. 1995. Antiviral effects of plasma and milk proteins:Lactoferrin shows potent activity against both human immunodeficiency virusand human cytomegalovirus replication in vitro. Journal of Infectious Dis-eases, 172:380–388.
Haukland, H.H., Ulvatne, H., Sandvik, K., and Vorland, L.H. 2001. The an-tibacterial peptides lactoferricin B and Magainin 2 cross over the bacterialcytoplasmic membrane and reside in the cytoplasm. FEBS Lett., 508:389–393.
Heany, R.P., Saito, Y., and Orimo, H. 1994. Effect of casein phosphopeptideon absorbability of co-ingested calcium in normal postmenopausal women.Journal of Bone Mineral Metabolism, 12:77–81.
Henschen, A., Lottspeich, F., Brantl, V., and Teschemacher, H. 1979. Novelopioid peptides derived from casein, β-casomorphins. II. Structure of activecomponents from bovine casein peptone. Hoppe-Seyler’s Physiol. Chem.,360:1217–1224.
Hirayama, M., Toyota, K., Hidaka, H., and Naito, H. 1992. Phosphopeptidesin rat intestinal digests after ingesting casein phosphopeptides. BioscienceBiotechnology Biochemistry, 56:112–1129.
Holt, C. and Horne D.S. 1996. The hairy casein micelle: Evolution of the conceptand its implications for dairy technology. Netherlands Milk & Dairy Journal,50:85–112.
Hutchens, T.W., Rumball, S.V., and Lonnerdal, B. 1994. Lactoferrin: Struc-ture and function. Advances in Experimental Medical and Biological, 357:1–298.
Dow
nloa
ded
by [
The
Uni
vers
ity O
f M
elbo
urne
Lib
rari
es]
at 0
2:50
30
Apr
il 20
13
654 S. SEVERIN AND X. WENSHUI
Jan, M.S. 2001. Milk ingredients as nutraceuticals. International Journal ofDairy Technology, 54:81–88.
Jolles, P. 1975. Structural aspects of the milk clotting process. Comparativefeatures with the blood clotting process. Mol. Cell. Biochem., 7:73.
Jolles, P., and Henschen, A. 1982. Comparison between the clotting of bloodand milk. Trends Biochem. Sci., 7:325.
Jolles, P., Loucheux-Lefebvre, M. H., and Hanschen, A. 1978. Structural relat-edness of κ-casein and fibrinogen γ -chain. J. Mol. Evol., 11:271.
Jones, E.M., Smart, A., Bloomberg, G., Burgess, L., and Millar, M.R. 1994.Lactoferricin, a new antimicrobial peptide. Journal of Applied Bacteriology,77:208–214.
Kelley, C.P., Chetman, S., Keates, S., Bostwick, E.F., Roush, A.M., Castagliuolo,I., Lamont, J.T., and Pothoulakis, C. 1997. Survival of anti-Clostridium diffi-cile bovine immunoglobulin concentrate in the human gastrointestinal tract.Antimicrobial Agents and Chemotherapy, 41:236–241.
Kelly, P.M., Kelly, J., Mchra, R., Oldfield, D.J., Raggett, E., and O’Kennedy,B.T. 2000. Implementation of integrated membrane processes for pi-lot scale development of fractionated milk components. Lait, 80:139–153.
Kohmura, M., Nio, N., and Ariyoshi, Y. 1990. Inhibition of angiotensin I-converting enzyme by synthetic peptides of human κ-casein. Agric. Biol.Chem., 54:835–836.
Kohmura, M., Nio, N., Kubo, K., Minoshima, Y., Munekata, E., and Ariyoshi,Y. 1989. Inhibition of angiotensin I-converting enzyme by synthetic peptidesof human β-casein. Agric. Biol. Chem., 53:2107–2114.
Korhonen, H. 1977. Antimicrobial factors in bovine colostrum. Journal of theScientific Agricultural Society of Finland, 49:434–447.
Korhonen, H., Pihlanto-Leppala, A., Rantamaki, P., and Tupasela, T. 1998. Thefunctional and biological properties of whey proteins: prospects for the de-velopment of functional foods. Agriculture and Food Science in Finland,7:283–296.
Kulkarni, P.R., and Pimpale, N.V. 1989. Colostrum—A review. Indian Journalof Dairy Science, 42:216–224.
Kussendrager, K. 1993. Lactoferrin and lactoperoxidase: Bioactive milk pro-teins. International Food Ingredients, 6:17–21.
Lahov, E., and Regelson, W. 1996. Antibacterial and immunostimulating ca-seinderived substances from milk: Caseicidin, isracidin peptides. Food andChemical Toxicology, 34:131–145.
Lee, Y.S., Noguchi, G., and Naito, H. 1980. Phosphopeptides and soluble cal-cium in the small intestine of rats given a casein diet. British Journal ofNutrition, 43:457–467.
Lindsay, S., Paul, N.B., Michael, J.B., Lawrence, K.C., Helen, D., Patrick, J.B.E.,Carl, H., Geoffrey, B.J., George, K., Gillian, E.N., Stanislava, U., and Su-Ying,W. 2002. Milk protein structure—What can it tell the dairy industry? Inter-national Dairy Journal, 12:299–310.
Loukas, L., Varoucha, D., Zioudron, C., Straty, R.A., and Klee, W.A. 1983. Opi-oid activities and structures of alpha-casein-derived exorphins. Biochemistry,22:4567–4573.
Luo, S.J. and Wong, L.L. 2000. Oral care chewing gums and confections. Inter-national Patent Application No. PCT/US00/02142.
Maeno, M., Yamamoto, N., and Takano, T. 1996. Identification of anantihypertensive peptide from casein hydrolysate produced by a pro-teinase from Lactobacillus heleveticus CP790. J. Dairy Science, 79:1316–1321.
Marchetti, M., Longhi, C., Conte, M.P., Pisani, S., Valenti, P., and Seganti, L.1977. Lactoferrin inhibits herpes simplex virus type I adsorption to Vero cells.Antiviral Research, 29:221–231.
Maruyama, S., and Suzuki, H. 1982. A peptide inhibitor of angiotensin I-converting enzyme in the tryptic hydrolysate of casein. Agric. Biol. Chem.,46:1393–1394.
McIntosh, G.H., Jorgensen, L., and Royle, P.J. 1993. The potential of an insolubledietary fibre-rich source from barley to protect from DMH-induced intestinaltumors in rats. Nutrition and Cancer, 19:213.
McIntosh, G.H., Regester, G.O., Le Leu, R.K., Royle, P.J., and Smithers, G.W.1995. Dairy proteins protect against dimethylhydrazine-induced intestinaltumors in rats. Journal of Nutrition, 125:809–816.
McLeod, A., Fedio, W., Chu, L., and Ozimek, L. 1996. Binding of retionoic acidto β-lactoglobulin variants A and B: Effect of hepatic and tryptic digestionon the protein-ligand complex. Milchwissenschaft, 51:3–7.
Meisel, H. 1997. Biochemical properties of regulatory peptides derived frommilk proteins. Biopolymers, 43:119–128.
Meisel, H. and Frister, H. 1989. Chemical characterization of bio-active peptidesfrom in vivo digests of casein. Journal of Dairy Research, 56:343–349.
Meisel, H., Goepfert, A., and Gunther, S. 1997. ACE-inhibitory activities inmilk products. Milchwissenschaft, 52:307–311.
Migliore-Samour, D., Floch, F., and Jolles, P. 1989. Biologically active ca-sein peptides implicated in immunomodulation. Journal of Dairy Research,56:357.
Modler, H.W. 2000. Milk processing. In: Food Proteins: Processing Applica-tions. pp. 1–88. Nakai, S., Modler, H. W., Eds., Willey, New York.
Moore, S.A., Anderson, B.F., Groom, C.R., Haridas, M., and Baker, E.N. 1997.Three-dimensional structure of diferric bovine lactoferrin at 2.8 A resolution.Journal of Molecular Biology, 274:222–236.
Mullally, M.M., Meisel, H., and Fitzgerald, R.J. 1996. Synthetic peptidescorresponding to alpha-lactalbumin and beta-lactoglobulin sequences withangiotensin-I-converting enzyme inhibitory activity. Biological ChemistryHoppeseyler, 377:259–260.
Mullally, M.M., Meisel, H., and Fitzgerald, R.J. 1997. Identification of a novelangiotensin-I-converting enzyme inhibitory peptide corresponding to trypticfragment of bovine beta-lactoglobulin. FEBS Letters, 402:99–101.
Nagendra, P.S. 2000. Effects of milk-derived bioactives: An overview. BritishJournal of Nutrition, 84:S3–S10.
Naito, H. and Suzuki, H. 1974. Further evidence for the formation in vivo ofphosphopeptide in the intestinal lumen from dietary β-casein. Agriculturaland Biological Chemistry, 38:1543–1545.
Nakamura, Y., Yamamoto, N., Kumi, S., and Takano, T. 1995. Antihyperten-sive effect of sour milk and peptides isolated from it that are inhibitorsto angiotensin-I converting enzyme. Journal of Dairy Science, 78:1253–1257.
Newburg, D.S., and Newbauer, S.H. 1995. Carbohydrates in milk. In: Handbookof Milk Composition. pp. 273–349. Jensen, R.G., Ed., Academic Press, SanDiego.
Newburg, D.S., Pickering, L.K., McCluer, R.H., and Cleary, T.G. 1990. Fuco-sylated oligosaccharides of human milk protect sucling mice from heat stableenterotoxin of Escherichia coli. Journal of Infectious Diseases, 162:1075–1080.
Nibbering, P.H., Ravensbergen, E., Welling, M.M., Van Berkel, L.A., VanBerkel, P.H.C., Pauwels, E.K., and Nuijens, J.H. 2001. Human lactoferrin andpeptides derived from its N-Terminus are highly effective against infectionswith antibiotic resistant bacteria. Infection and Immunity, 69:1469–1476.
Pariza, M.W. 1997. Conjugated linoleic acid: A newly recognized nutrient. IFTAnnual Meeting, Orlando, FL, Abstract, pp. 15–3.
Parker, F., Migliore-Samour, D., Floch, F., Zerial, A., Werner, G.H., Jolles, J.,Casaretto, M., Zahn, H., and Jolles, P. 1984. Immunostimulating hexapeptidefrom human casein: Amino acid sequence, synthesis, and biological proper-ties. European Journal of Biochemistry, 145:677.
Paroli, E. 1988. Opioid peptides from food (the exorphins). World Rev. Nutr.Diet, 55:58–97.
Payne, K.D., Davidson, P.M., and Oliver, S.P. 1990. Influence of bovine lacto-ferrin on the growth of Listeria monocytogenes. Journal of Food Protection,53:468–472.
Petrilli, P., Addeo, F., and Chianese L. 1983. Primary structure of water buffalobeta-casein tryptic and CNBr peptides. Ital. J. Biochem., 32:336–344.
Pierce, A., Colavizza, D., Benaissa, M., Maes, P., Tartra, A., Montreuil, J., andSpik, G. 1991. Molecular cloning and sequence analysis of bovine lactoferrin.European Journal of Biochemistry, 196:177–184.
Pihlanto-Leppala, A. 2001. Bioactive peptides derived from bovine whey pro-teins: Opioid and ACE-Inhibitory peptides. Trends in Food Science & Tech-nology, 11:347–356.
Pihlanto-Leppala, A., Antila, P., Mantsala, P., and Hellamn, J. 1994. Opioidpeptides produced by in vivo proteolysis of bovine caseins. Int. Dairy J.,4:291–301.
Dow
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by [
The
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vers
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Lib
rari
es]
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2:50
30
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MILK BIOLOGICALLY ACTIVE COMPONENTS AS NUTRACEUTICALS 655
Pointillart, A. and Gueguen, L. 1989. Effect of casein phosphopeptides on cal-cium and phosphorous utilization in growing pigs. Reproduction NutritionDevelopment, 29:477–486.
Reddy, N.R., Roth, S.M., Eigel, W.N., and Pierson, M.D. 1988. Foods andfood ingredients for prevention of diarrheal disease in children in developingcountries. Journal of Food Protection, 51:66–75.
Regester, G.O., McIntosh, G.H., Lee, V.W.K., and Smithers, G.W. 1996.Whey proteins as nutritional and functional food ingredients. Food Australia,48:123–126.
Reynolds, E.C., Riley, P.F., and Adamson, N.J. 1994. A selective purificationprecipitation procedure for multiple phosphoseryl-containing peptides andmethods for their identification. Anal. Biochem., 217:277–284.
Richardson, B.C. and Mercier, J.C. 1979. The primary structure of the ovinebeta-caseins. Eur. J. Biochem., 99:285–297.
Roos, N., Mahe, S., Benamouzig, R., Sick, H., Rautureau, J., and Tome, D. 1995.15N-labelled immunoglobulins from bovine colostrum are partially resistantto digestion in human intestine. Journal of Nutrition, 125:1238–1244.
Saito, H., Miyakawa, H., Tamura, Y., Shimamura, S., and Tomita, M. 1991.Potent bacteriocidal activity of bovine lactoferrin hydrolysate reproduced byheat treatment at acidic pH. Journal of Dairy Science, 74:3724–3730.
Sakai, M., Kobayashi, M., and Yoshida, T. 1995. Activation of rain-bow trout, Oncorhynchus mykiss, phagocytic cells by administration ofbovine lactoferrin. Comparative Biochemistry and Physiology, 110B:755–759.
Sato, R., Inanami, O., Tanak, Y., Takase, M., and Naito, Y. 1996. Oral admin-istration of bovine lactoferrin for treatment of intractable stomatitis in fe-line immunodeficiency virus (FIV)-positive and FIV-negative cats. AmericanJournal of Veterinary Research, 57:1443–1446.
Sato, R., Noguchi, T., and Naito, H. 1986. Casein phosphopeptide (CPP) en-hances calcium absorption from the ligated segment of rat small intestine.Journal of Nutrition Science and Vitaminology, 32:67–76.
Schlimme, E. and Meisel, H. 1995. Bioactive peptides derived from milk pro-teins: Structural, physiological, and analytical aspects. Die Nahrung, 39:1–20.
Schmelz, E.M. and Merrill, A.H. 1997. Milk sphingolipids: A new category offunctional food. IFT Annual Meeting, Orlando, FL, Abstract. pp. 15–4.
Scholz-Ahrens, K.E. and Schrezenmeir, J. 2000. Effects of bioactive substancesin milk on mineral and trace element metabolism with special reference tocasein phosphopeptides. British Journal of Nutrition, 84:S147–S153.
Scholz-Ahrens, K.E., Kopra, N., and Barth, C.A. 1990. Effect of casein phos-phopeptides on utilization of calcium in minipigs and vitamin-D deficientrats. Zeitschrift fur Ernahrungswissenschaft, 29:295–298.
Sekiya, S., Kobayashi, Y., Kita, E., Imamura, Y., and Toyama, S. 1996. Anti-hypertensive effects of tryptic hydrolysates of casein on normotensive andhypertensive volunteers. Journal of Japanese Society of Nutrition and FoodScience, 45:513.
Seppo, L., Jauhiainen, T., Poussa, T., and Korpela, R. 2003. A fermented milkhigh in bioactive peptides has a blood pressure-lowering effect in hypertensivesubjects. Am. J. Clin. Nutr., 77:326–330.
Shin, K., Yamauchi, K., Teraguchi, S., Hayasawa, H., Tomita, M., Otsuka, Y.,and Yamazaki, S. 1998. Antibacterial activity of bovine lactoferrin and itspeptides against enterohaemorrhagic Escherichia coli 0157:H7. Letters onApplied Microbiology, 6:407–411.
Silva, H.E.R. and Verdalet, G.I. 2003. Functional foods and ingredients derivedfrom milk. Arch. Latinoam. Nutr., 53:333–347.
Smacchi, E. and Gobbetti, M. 2000. Bioactive peptides in dairy products: Synthe-sis and interaction with proteolytic enzyme. Food Microbiology, 17:129–141.
Spik, G., Coddeville, B., Mazurier, J., Bourne, Y., Cambillaut, C., and Montreuil,J. 1994. Primary and three-dimensional structure of lactotransferrin (lactofer-rin) glycans. In: Advances in Experimental Medicine and Biology. pp. 21–32. Hutchens, T.W., Rumball, S.V., and Lonnerdal, B., Eds., Plenum Press,New York.
Steijns, J.M. and Van Hooijdonk, A.C.M. 2000. Occurrence, structure, biochem-ical properties and technological characteristics of lactoferrin. British Journalof Nutrition, 84:S1–S8.
Superti, F., Ammendolia, M.G., Valenti, P., and Seganti, I. 1997. Antirotavi-ral activity of milk proteins: Lactoferrin prevents rotavirus infection in the
enterocyte-like cell line HT-29. Medical Microbiology and Immunology,186:83–91.
Suzuki, T., Yamauchi, K., Kawase, K., Tomita, M., Kiyasawa, I., and Okongi, S.1989. Collaborative bacteriostatic activity of bovine lactoferrin with lysozymeagainst E. coli 0111. Agriculture and Biological Chemistry, 53:1705–1706.
Svedberg, J., De Haas, J., Leimenstoll, G., Paul, F., and Teschemacher, H. 1985.Demonstration of β-casomorphin immunoreactive materials in in vivo digestsof bovine milk and in small intestine contents after bovine milk ingestion inadults humans. Peptides, 6:625.
Tani, F., Iio, K., Chiba, H., and Yoshikawa, M. 1990. Isolation and characteriza-tion of opioid antagonist peptides derived from human lactoferrin. Agr. Biol.Chem., 54:1803–1810.
Tani, F., Shiota, H., Chiba, H., and Yoshikawa, M. 1994. Serophin, an opioidpeptide derived from serum albumin. In: β-Casomorphins and related pep-tides: Recent developments. pp. 49–53. Brantl, V. and Teschemacher, H., Eds.,VCH-Weinheim, Germany.
Tomita, M., Bellamy, W.R., Takase, M., Yamauchi, K., Wakabayashi, H., andKawase, K. 1991. Potent antibacterial peptides generated by pepsin digestionof bovine lactoferrin. Journal of dairy Science, 74:4137–4142.
Tsuchita, H., Goto, T., Shimizu, T., Yonehara, Y., and Kuwata, T. 1996. Di-etary casein phosphopeptides prevent bone loss in aged ovariectomized rats.Journal of Nutrition, 126:86–93.
Tsuchita, H., Suzuki, T., and Kuwata, T. 2001. The effect of casein phospho-peptides on calcium absorption from calcium-fortified milk in growing rats.British Journal of Nutrition, 85:5–10.
Tsuji, S., Hirata, Y., Mukai, F., and Ohtagaki, S. 1990. Comparison of lactoferrincontent in colostrum between different cattle breeds. Journal of Dairy Science,73:125–128.
Van der Meer, R. and Lapre, J.A. 1991. Calcium and colon cancer. Bulletin ofthe International Dairy Federation, 255:55–59.
Van der Ven, C. 2002. Biochemical and functional characterization of casein andwhey protein hydrolysates. A study on the correlations between biochemi-cal and functional properties using multivariate data analysis. Ph.D thesis,Wageningen University, The Netherlands.
Vannini, L., Lanciotti, R., Baldi, D., and Guerzoni, M.E. 2004. Interactionsbetween high pressure homogenization and antimicrobial activity of lysozymeand lactoperoxidase. International Journal of Food Microbiology, 94:123–135.
Viljoen, M. 1995. Lactoferrin: A general review. Haematologica, 80:252–267.
Visalsok, T., Shigeru, H., Satoshi, Y., and Souichi, K. 2004. Effects of alactoperoxidase-thiocyanate-hydrogen peroxide system on Salmonella enter-itidis in animal or vegetable foods. International Journal of Food Microbiol-ogy, 93:175–183.
Walstra, P. and Jenness, R. 1984. Dairy chemistry and physics. John Willey &Sons, Inc., New York.
Walzem, R.L., Dillard, C.J., and German, J.B. 2002. Whey components: Mil-lennia of evolution create functionalities for mammalian nutrition: What weknow and what we may be overlooking. Critical Reviews in Food Science andNutrition, 42:353–375.
Warner, E. A., Kanekanian, A.D., and Andrews, A.T. 2001. Bioactivity of milkproteins: 1. Anticariogenicity of whey proteins. International Journal of DairyTechnology, 54:151–153.
Weiner, C., Pan, Q., Hurtif, M., Boren, T., Bostwick, E., and Hammerstrom, L.1999. Passive immunity against human pathogens using bovine antibodies.Clinical Experimental Immunology, 116:193–205.
Wong, C.W. and Watson, D.L. 1995. Immunomodulatory effects of dietary wheyproteins in mice. Journal of Dairy Research, 62:359–368.
Wu, J.P. and Ding, X.L. 2002. Characterization of inhibitory and stability of soyprotein-derived Angiotensin-I-Converting Enzyme inhibitory peptides. FoodResearch International, 35:367–375.
Yamauchi, K. 1992. Biologically functional proteins of milk and peptides derivedfrom milk proteins. Bulletin of the International Dairy Federation, 272:51.
Yen, S.S.C., Quingley, M.E., Reid R.L., Ropert, J.F., and Cetei, N.S. 1985.Neuroendocrinology of opioid peptides and their role in the control of
Dow
nloa
ded
by [
The
Uni
vers
ity O
f M
elbo
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Lib
rari
es]
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2:50
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il 20
13
656 S. SEVERIN AND X. WENSHUI
gonadotrophin and prolactin secretion. American Journal of Obstetrics andGynaecology, 152:485.
Yolken, R.H., Peterson, J.A., Vonderfech, S.L., Fouts, E.T., Midthun, K., andNewburg, D.S. 1991. Human milk mucin inhibits rotavirus replication and pre-vents experimental gastroenteritis. Journal of Clinical Investigation, 90:1984–1991.
Yoshikawa, M., Tani, F., Yoshimura, T., and Chiba, H. 1986. Opioid peptidesfrom milk protein. Agric. Biol. Chem., 50:2419–2421.
Yoshikawa, M., Yoshimura, T., and Chiba, H. 1984. Opioid peptides from humanβ-casein. Agric. Biol. Chem., 48:3185–3187.
Yuan, Y.V. and Kitts, D.D. 1991. Confirmation of calcium absorption and femoralutilization in spontaneously hypertensive rats fed casein phosphopeptide diets.Nutrition Research, 11:1257–1272.
Zioudrou, C., Streaty, R.A., and Klee, W.A. 1979. Opioid pep-tides derived from food. The exorphins. J. Biol. Chem., 254:2446–2449.
