Review Article Milk Proteins, Peptides, and...

Review ArticleMilk Proteins Peptides and Oligosaccharides Effects againstthe 21st Century Disorders

Chia-Chien Hsieh1 Blanca Hernaacutendez-Ledesma2 Samuel Fernaacutendez-Tomeacute2

Valerie Weinborn34 Daniela Barile34 and Juliana Mariacutea Leite Nobrega de Moura Bell34

1Department of Human Development and Family Studies (Nutritional Science amp Education) National Taiwan Normal UniversityNo162 Section 1 Heping East Road Taipei 106 Taiwan2Instituto de Investigacion en Ciencias de la Alimentacion (CIAL CSIC-UAM CEI UAM+CSIC) Nicolas Cabrera 928049 Madrid Spain3Food Science Department University of California Davis One Shields Avenue Davis CA 95616 USA4Foods for Health Institute University of California Davis One Shields Avenue Davis CA 95616 USA

Correspondence should be addressed to Blanca Hernandez-Ledesma bhernandezcsices

Received 21 October 2014 Accepted 21 December 2014

Academic Editor Jinsong Ren

Copyright copy 2015 Chia-Chien Hsieh et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Milk is the most complete food for mammals as it supplies all the energy and nutrients needed for the proper growth anddevelopment of the neonate Milk is a source of many bioactive components which not only help meeting the nutritional require-ments of the consumers but also play a relevant role in preventing various disorders Milk-derived proteins and peptides have thepotential to act as coadjuvants in conventional therapies addressing cardiovascular diseases metabolic disorders intestinal healthand chemopreventive properties In addition to being a source of proteins and peptides milk contains complex oligosaccharidesthat possess important functions related to the newbornrsquos development and health Some of the health benefits attributed to milkoligosaccharides include prebiotic probifidogenic effects antiadherence of pathogenic bacteria and immunomodulation Thisreview focuses on recent findings demonstrating the biological activities of milk peptides proteins and oligosaccharides towardsthe prevention of diseases of the 21st century Processing challenges hindering large-scale production and commercialization ofthose bioactive compounds have been also addressed

1 Introduction Role of Milk in Human Health

Milk as the first food for mammals supplies all the energyand nutrients needed for the proper growth and developmentof the neonate For all mammalians the consumption ofmilk ends at the weaning period with the exception ofhumans that continue consuming milk throughout theirlife Milk and derived dairy products are considered animportant constituent of a balanced diet Moreover it is asource of many bioactive components such as high-qualityproteins lipids carbohydrates lactose vitamins mineralsenzymes hormones immunoglobulins and growth factorsamong others These components not only help meetinghuman nutritional requirements but also play a relevantrole in preventing various disorders such as hypertension

and cardiovascular diseases [1] obesity [2] osteoporosis [3]dental caries [4] poor gastrointestinal health [5] colorectalcancer [6] ageing [7] and others [8]

Milk proteins supply nitrogen and amino acids to youngmammals and possess multiple physiological properties inthe intact form Moreover studies carried out in the pastdecades have demonstrated the role of these proteins asa source of biologically active peptides Bioactive peptidesare inactive within the sequence of the parent protein butonce released by in vitro processing conditions or by in vivogastrointestinal digestion are capable of acting as regulatorycompounds exerting a positive impact on body functions andultimately promoting health benefits to the consumer [9]

Human milk is undoubtedly the most complete source ofnourishment for the newborn Breastfed infants have been

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 146840 16 pageshttpdxdoiorg1011552015146840

2 BioMed Research International

Table 1 Commercial milk products containing peptides with proven antihypertensive activity

Commercial name Obtention process Protein source Active sequence(s) Publicationnumber [reference]

Peptide C12 Hydrolysis with trypsin 120572s1-Casein FFVAPFPEVFGK JP62270533 [31]Biozate Hydrolysis with trypsin Whey proteins Whey peptides US6998259 [157]

Lowpept Hydrolysis with pepsin 120572s1-Casein RYLGY AYFYPEL WO2005012355[158]

Calpis Fermentation 120573-Casein VPP IPP US5449661A[37 38]

Evolus Fermentation 120573-Casein VPP IPP US6972282 [159]

shown to be less susceptible to diseases (ie diarrhea andrespiratory diseases) than those that were not breastfed Thisprotective effect which was previously attributed to humanmilk antibodies is today strongly correlatedwith the presenceof complex oligosaccharides (OS) the third most abundantcomponent of human milk [10] Human milk is composedof OS in concentrations varying according to different stagesof lactation 20ndash23 gL in colostrum and 12ndash14 gL in maturemilk [11] being even more abundant than proteins (12 gL)[12] Human milk oligosaccharides (HMO) are complexsugars having 3 to 20 monosaccharide units [13] that are notdigestible by human enzymes [14] These compounds haveimportant functions related to the newbornrsquos developmentand health at local and systemic levels including prebioticprobifidogenic effects and antiadherence of pathogenic bac-teria [15] brain development [16] and immunomodulatoryproperties [17] among others

In the last fifty years chronic disorders have become theleading cause of morbidity and mortality in industrializedcountries with increasing incidence also observed in devel-oping countries Chronic disorders include cardiovascularand neurological diseases stroke cancers immune disor-der and chronic respiratory disease obesity diabetes andmetabolic syndrome [18] In Europe 87 of all deaths occurdue to chronic diseases and the number of people affectedis expected to rise considerably over the next few decadesThe majority of chronic diseases are caused by risk factorswhich are mostly preventable Diet and lifestyle are twoenvironmental factors that strongly affect these diseases thusmodifications of these habits are becoming a new strategy fordisease preventiontreatment

The aim of this paper is to review the recent literature onthe physiological effects of proteins peptides and oligosac-charides with special emphasis on animal and human trialsOther aspects such as the limited availability of in vivo studiesdemonstrating the biological activities of OS frombovine andcaprine milk and the current challenges associated with therecovery and commercial production of these compoundshave also been addressed

2 Impact of Milk Proteins and Peptides onthe 21st Century Diseases

21 Milk-Derived Peptides against Cardiovascular DiseasesCardiovascular diseases (CVD) have become the leading

cause of morbidity and mortality worldwide representingan important medical and public health issue [19] Althoughearlier studies associated the consumption of wholemilk withhigher incidence of CVD it has been demonstrated that milkcontains a plethora of bioactive substances which may con-tribute to the prevention of most of the risk factors of CVD[20] Recently bioactive milk peptides have gained interestbecause of their notable antihypertensive antioxidant anti-inflammatory and hypocholesterolaemic effects In this sec-tion the most current scientific information regarding invitro and in vivo studies on the role of milk proteins-derivedpeptides on CVD is summarized and discussed

211 Milk Peptides with Antihypertensive Activity Epidemi-ological studies suggest that the dietary intake of milk anddairy foods is related to decreased risk of hypertension [21]In addition to their high mineral content (eg calciumpotassium and magnesium) that can lower blood pressure[22] other milk components such as proteins and theirhydrolyzed products have been also linked to the antihy-pertensive effect of milk and dairy products Angiotensin-converting enzyme (ACE) is a multifunctional enzyme thatacts as one of the main regulators of blood pressureThus ACE inhibition is currently considered as one of thebest strategies for hypertension treatment Most biologicallyactive peptides generated from milk proteins have demon-strated ACE inhibitory activity In the last two decadesantihypertensive effects of some of these peptides have beenevaluated in spontaneously hypertensive rats (SHR) andhypertensive humans and the peptide sequences doses andmaximum decreases of systolic blood pressure (SBP) havebeen summarized in several reviews [23ndash25]The hydrolyzateobtained by the action of pepsin on casein containing the120572s1-casein-derived peptides RYLGY and AYFYPEL has beenpatented and commercialized under the name of Lowpeptby its antihypertensive properties demonstrated in bothSHR [26] and hypertensive humans [27] (Table 1) Pepsinhas been also used to hydrolyze whey protein lactoferrinwith the release of peptides containing ACE activity andACE-dependent vasoconstriction inhibitory properties [28]Antihypertensive effects in SHR after short-term and long-term treatments have been also observed for those peptides[29 30] Trypsin is another gastrointestinal enzyme used torelease the antihypertensive peptide 120572s1-casein peptide f(23ndash34) from casein during the manufacture of the commercial

BioMed Research International 3

ingredient peptide C12 [31 32] (Table 1) In addition to the useof gastric and pancreatic enzymes alone or in combinationto produce antihypertensive peptides the use of food-gradeenzymes derived frommicroorganisms has become commonfor the release of peptides with demonstrated SBP loweringeffects in SHR [33ndash36]

Milk fermentation is another strategy to produce anti-hypertensive peptides by the proteolytic action of lactic acidbacteria on milk proteins The most representative peptidesare those derived from 120573-casein and identified in sourmilk fermented by Lactobacillus helveticus and Saccharomycescerevisiae (Calpis Table 1) These tripeptides with sequencesVPP and IPP have demonstrated an ability to exert potentdecreasing effects on the SBP of SHR [37 38] A numberof clinical trials have been conducted to confirm their anti-hypertensive properties in humans although controversialresults have been found Three meta-analyses performedwith the published data of 17 [39] 12 [40] and 28 [41]clinical trials have reported an average decrease in SBP of51 48mm and 17mm of Hg respectively However noeffects were found in Dutch and Danish subjects consumingfermented milk containing peptides VPP and IPP [42 43] Arecent meta-analysis including 18 trials has reported higherantihypertensive effects for these two tripeptides in Asianthan in Caucasian people [44] Those findings suggest thatgenetics andor dietary patterns might exert an importantinfluence on the antihypertensive effects of peptides IPPand VPP Similarly the age has been described as anothermajor influencing factor [45] With the evidence presentedto date the European Food Safety Authority (EFSA) Panelon Dietetic Products Nutrition and Allergies (NDA) [46]concluded that there are no sufficient data to establish acauseeffect relationship between the consumption of pep-tides VPP and IPP and the control of hypertension andfurther studies are thus required Other peptides derivedfrom 120573-casein during milk fermentation with Enterococcusfaecalis in which sequences are LHLPLP and HLPLP havealso shown antihypertensive effects in SHR [47] In recentstudies fermentedmilkwith Lactococcus lactisNRRLB-50571and NRRLB-50572 has presented important SBP diastolicblood pressure (DBP) and heart rate-lowering effects in SHR[48 49] although the peptides responsible for the activityhave not been identified

Accumulating evidence built in animal and clinical stud-ies is currently available on the antihypertensive activity ofmilk-derived peptides However much work is still neededIdentification of the active form reaching the target organsand elucidation of its bioavailability after oral ingestion andits complete mechanism of action are two of the main aspectsrequired to be deeply investigated in the future to supporthealth claims

212 Antioxidant and Anti-Inflammatory Milk-Derived Pep-tides Oxidative stress is one of the main responsible factorsfor the initiation or evolution of CVD The search of naturalantioxidants providing additional benefits to the endogenousantioxidant defense system is gaining interest [50] Amongfood-derived peptides with antioxidant properties without

harm side effects those derived from milk proteins are mostfrequently studied The majority of the studies carried outto characterize antioxidant peptides derived from casein andwhey proteins have only used in vitro chemical assays [51 52]However their limited similarity to physiological conditionsmakes the in vitro assays very restrictive and reported effectsneed to be confirmed by animal models andor humantrials Nevertheless to date just few in vivo trials have beencarried out to demonstrate the antioxidant effects of milk-derived peptides related to benefits on cardiovascular healthZommara et al [53] reported the antiperoxidative action offermented milk on rats fed a vitamin-E deficient diet Theconsumption of fermented milk by healthy subjects has beenalso demonstrated to lower the levels of oxidized low-densitylipoprotein isoprostanes and the glutathione redox ratioImprovements of total plasma antioxidant activity and ofthe resistance of the lipoprotein fraction to oxidation haveresulted in enhanced antiatherogenicity [54]The compoundsresponsible for the observed effects have not been identifiedyet although milk peptides liberated during fermentationprocess might have a crucial role Thus further studiesfocused on evaluating the potential of milk-derived peptidesas antioxidant at cardiovascular level should be of greatrelevance

Chronic inflammation is another responsible factor forthe development of CVD The downregulation of cytokinesinvolved in the inflammation-associated endothelial dys-function by food components including peptides may delayor alleviate inflammation thus exerting favorable effectsagainst CVD [55] A recent study using lipopolysaccharide-(LPS-) stimulated mouse macrophages has reported theability of a yak casein hydrolyzate to reduce the secretionof proinflammatory cytokines and the production of nitricoxide and to scavenge free radicals suggesting a potential roleas preventive agent against inflammation related disorders[56] To date only one human trial has been conductedto demonstrate the anti-inflammatory properties of milkpeptides This study reported an improvement in the vas-cular function through modulation of the glucose levelsand inflammation and oxidative stress biomarkers after theconsumption of the commercial whey derived peptide NOP-47 by healthy individuals [57] This finding opens a newdoor towards searching of new milk-derived peptides withantioxidant and anti-inflammatory activity

213 Hypocholesterolaemic Milk Peptides Blood lipids arerepresented in various forms including total cholesteroltriglycerides lipoproteins (high-density lipoproteins orHDLlow-density lipoproteins or LDL and very-low-densitylipoproteins or VLDL) and free fatty acids An inappropriateratio of these lipids is one of the most important risk factorsfor developing CVD Therefore CVD therapypreventionstrategies focus on reaching an optimal lipid balance inorder to achieve a positive cardiovascular health Thosetherapies aim at increasing the physiological levels of desir-able lipids (eg HDL cholesterol) while reducing the othersassociated with atherogenic functions (eg LDL choles-terol triglycerides) Milk proteins mainly whey proteins


and derived hydrolyzates or peptides have been reportedto exert hypocholesterolaemic effects in different animalmodels The ingestion of whey protein was correlated witha significant reduction of total cholesterol levels in rats fedwith cholesterol-free and cholesterol-enriched diets [58 59]Nagaoka et al [60] have reported similar effects for a 120573-lactoglobulin tryptic hydrolyzate administered to rats fedwith a diet rich in cholesterol The hydrolyzate reduced totalcholesterol and increased HDL cholesterol and fecal steroidexcretionThe fragment f(71ndash75) of this whey protein knownas lactostatin with sequence IIAEK has been reported asthe main factor responsible for the observed effects [60]120573-Lactotensin another 120573-lactoglobulin peptide released bychymotrypsin hydrolysis decreased total cholesterol LDLand VLDL cholesterol content in mice fed with a cholesterol-enriched diet [61] Although themechanismof action of thosepeptides has not been completely elucidated preliminaryresults suggest a key role played by the amino acid compo-sition [50] Further studies are clearly needed to corroboratethose results The exact mode of this hypocholesterolaemicaction needs to be determined in clinical trials

22 Milk-Derived Hydrolyzates and Peptides on IntestinalHealth The gastrointestinal tract (GIT) serves as a special-ized interface between the body and the external environ-ment The GIT is strategically covered by a monolayer ofspecially designed epithelial cells continually exposed to ahigh concentration of food components and substances alongthe gut luminal surface Hence the modulator effect of thediet on GIT functions has been accepted as essential formaintaining and improving the general health of the host[62] Interestingly more than 70 of the current ldquofood forspecified health uses productsrdquo (FOSHU) are related to GITfunctions [63]

Dairy proteins hydrolyzates and peptides have beendemonstrated to transform the dynamics of mucus mainlyvia influencing the mucin secretion and expression and thenumber of goblet cells In ex vivo preparations of rat jejunumcasein hydrolyzates increased mucin secretion [64 65] The120573-casein derived peptide 120573-casomorphin 7 produced thesame effects which have been suggested to be mediated byinteraction with opioid receptors Also this peptide has beenreported to stimulate the expression of mucin Muc2 andMuc3 genes in rat intestinal DHE cells and MUC5AC genein human intestinal HT29-MTX cells [66] Another 120573-caseinfragment f(94ndash123) identified in commercial yoghurt alsohad the ability to increase the mucin output and the mRNAlevels of MUC2 and MUC4 genes in HT29-MTX cells [67]Casein and whey proteins hydrolyzates have been reportedto be a source of peptides with capacity to induce mucinsecretion andMUC5AC gene expression in HT29-MTX cells[68] Among these peptides the 120572s1-casein fragments f(143ndash149) and f(144ndash149) and the 120573-lactoglobulin fragment f(102ndash105) known as 120573-lactorphin were suggested as the majorpeptides responsible for the observed effects

A few in vivo studies have also pointed out the regulationof the protective mucus layer by dairy proteins and productsthereof Rats fed with a diet based on casein hydrolyzates as

the exclusive source of nitrogen were found to enhance theirendogenous nitrogen flow and expression of mucin genesMuc3 andMuc4 in the small intestine and colon respectively[69] Plaisancie et al [67] reported the capacity of the 120573-casein fragment f(94ndash123) once orally ingested by rats toupregulate the Muc2 Muc4 rat defensing 5 and lysozymemRNA transcripts expression the goblet cells recounts andthe number of crypts containing Paneth cells in the rat smallintestine In the dextran sulphate sodium- (DSS-) inducedmodel of rat colitis the studies of Sprong et al [70] and Faureet al [71] demonstrated the gut-protective effects exerted bya cheese whey protein diet and a diet supplemented withThr Ser Cys and Pro residues respectively Moreover thisprotection has been reported for a whey protein isolate and120572-lactalbumin hydrolyzate against chemical-induced ulcerativegastric lesions [72 73]

Enhancement of the mucosal immune response is alsoa dietary modulating strategy of the defense systems pro-tecting the GIT Animal models have proved the improve-ment of the mucosal immunity by promotion of gut-relatedimmunoglobulin (Ig) levels after ingestion of lactoferrin orits derived peptides lactoferricin and lactoferrampin [74 75]Likewise immunomodulatory effects have been reported fora trypsin casein hydrolyzate in newborn calves [76] andcasein phosphopeptides (CPPs) and peptides released fromLactobacillus helveticusR389-fermentedmilk inmice [77 78]Furthermore Kitamura and Otani [79] demonstrated thatingestion by healthy humans of CPPs-enriched cakes inducedan increase in the faecal IgA content suggesting a positiveeffect on mucosal immunity

Oxidative and inflammatory imbalances are bothinvolved in the etiology of several human chronic gut-relateddisorders such as ulcerative colitis and Crohnrsquos disease Thesearch of natural preventive treatments against these imbal-ances is being prompted [80 81] Whey protein has beensuggested to exert beneficial effects through enhancementof antioxidant enzymes and downregulation of both oxi-dative markers and proinflammatory cytokines [82] Theseprotective findings were found in animal [83 84] and humanstrials [85 86] The whey-derived peptide caseinomacro-peptide has been proven to have protective properties in the246-trinitrobenzene sulphonic acid (TNBS) and DSS-induced model of rat ileitis and colitis through immunomo-dulation of the regulatory T helper cells activation and inter-leukin secretions [87 88] Turbay et al [89] demonstrated inthe TNBS-induced murine colitis model the anti-inflam-matory effects exerted by 120573-casein hydrolyzates generatedby the cell envelope-associated proteinase of Lactobacillusdelbrueckii ssp lactis CRL 581 However peptides releasedand responsible for the observed bioactivity have not beenidentified yet

23 Milk Proteins and Peptides against Metabolic DisordersDiabetes mellitus is considered one of the most commonmetabolic disorders and one of the major health problemsworldwide It affects almost 6 of the worldrsquos populationwith type 2 diabetes representing approximately 90ndash95 ofthe diagnosed cases [90] Diet and lifestyle interventions


are the preferred treatment strategies for this metabolicdisorder with pharmacotherapy being prescribed only ifsupervised lifestyle intervention fails [91] Epidemiologicalevidence supports that consumption of milk and dairy foodsis associated with a lower incidence of type 2 diabetes Thesebeneficial effects on metabolic and inflammation factorslinked to diabetes and insulin resistance have been alsodemonstrated by cell and animal models being multiplemilk components such as calcium medium-chain fattyacids linoleic conjugated acid lactose citrate proteins andpeptides characterized as the main responsible factors forthe observed effects acting through different mechanisms ofaction [92]

During the ingestion of a meal the presence of nutri-ents at gastrointestinal level stimulates the secretion of twoincretins hormones the glucagon-like peptide-1 (GLP-1)and the glucose-dependent insulinotropic polypeptide (GIP)Both hormones are implicated in the stimulation of theinsulin secretion from the pancreatic 120573-cells secretion ofgastric and pancreatic enzymes and modulation of gutmotility and nutrient absorption allowing the clearance ofthe absorbed glucose [93] Type 2 diabetes is characterized bydifferent disorders including progressive dysfunction of pan-creatic cells insulin resistance and augmented production ofhepatic glucose [94] Continuous intravenous administrationof GLP-1 has been demonstrated to normalize blood glucoselevels in diabetic subjects [95] However the rapid degrada-tion of this hormone by the enzyme dipeptidyl peptidase-IV(DPP-IV) and its consequent inactivation makes this type 2diabetes treatment strategy impracticable Currently specificDPP-IV inhibitors are thus incorporated to GLP-1 analoguesin new oral therapies against this metabolic disease [96]

Diet supplementation with whey protein is currentlyunder preclinical and clinical trials as a promising alternativein the prevention andor treatment of type 2 diabetes andrelated diseases [97 98] Several mechanisms of action havebeen suggested for whey protein including the stimulation ofinsulin release improvement of glucose tolerance in diabeticpatients reduction of body weight and modulation of guthormones such as cholecystokinin leptin and GLP-1 [99] Inthe last years the role of peptides released during the transitof whey proteins through the GIT on the observed effectshas been hypothesized [100] Cell culture and animal modelshave been used to confirm this hypothesis A dose-dependentinsulinotropic activity of whey protein hydrolyzates has beenobserved in a cell-based coculture using pancreatic BRIN-BD11 cells and Caco-2 cells monolayers [101] These authorsalso observed that the oral administration of the hydrolyzatesto obese mice evoked an improvement of blood glucoseclearance reduction of hyperinsulinemia and restorationof the pancreatic capacity to secrete insulin in response toglucose The main mechanism of action suggested for thesehydrolyzates is the DPP-IV inhibitory activity exerted bythe peptides contained in them [102] Among the bioac-tive peptides described to date sequences derived from 120573-lactoglobulin IPA and IPAVF are the most potent as DPP-IV inhibitors [103 104] Another 120573-lactoglobulin fragmentwith sequenceVAGTWYhas been also demonstrated to exerthypoglycemic effects in the oral glucose tolerance test inmice

[105] Likewise both in vitro DPP-IV inhibitory and in vivohypoglycemic effects have been reported for peptides releasedfrom caseins [106] Recent in silico studies have shown thatboth caseins and whey proteins might serve as precursorsof DPP-IV inhibitory peptides because of the high numberof fragments contained within them that match DPP-IVinhibitory sequences [107 108]Thus this research area holdsa great potential and currently a number of investigations arefocused on the identification of new milk proteins-derivedpeptide with capacity to prevent diabetes and associatedmetabolic syndromes

24 Chemopreventive Role of Milk Proteins and PeptidesCancer is the second leading cause of mortality worldwideand its incidence will continue rising in the next few years inspite of the important advances achieved in the developmentof cancer therapies It has been estimated that by 2020approximately 15 million new cancer cases will be diagnosedand 12 million cancer patients will die [109] It is well knownthat 35 of cancer deaths are attributed to diet and itsfood components [110] However cell culture and animaland human trials results have shown that an importantnumber of food constituents can lower cancer risk and evensensitize tumor cells against anticancer therapies [111] Inthe last few years food proteins and derived peptides havebecome one of the food components with themost promisingpreventive properties against cancer initiation promotionand progression stages [112]

Among the milk proteins lactoferrin and its derivedpeptide lactoferricin are the most studied For both com-pounds their antioxidant immunomodulatory and anti-inflammatory activities are closely linked to their protectiveeffects against cancer (Table 2) Lactoferrin acts by inducingapoptosis inhibiting angiogenesis and modulating carcino-gen metabolizing enzymes in addition to its antioxidant andimmunomodulatory properties [113] Moreover lactoferricinhas shown potent anticancer properties in different cell linesincluding breast colon fibrosarcoma leukemia and oral andovarian cancer cells without harming normal lymphocytesfibroblasts or endothelial or epithelial cells [114] Also animalmodels have confirmed the beneficial properties of thismilk-derived peptide The possible mechanism of bovinelactoferricin in anticarcinogenesis has been shown to berelated to its ability to induce apoptosis It is its stronglycationic nature that allows this peptide to target negativelycharged cancer cells with the outer membrane [115] Thesuppressed ability in angiogenesis of bovine lactoferricinwas in vitro and in vivo demonstrated to contribute to itschemopreventive properties [116] A significant inhibition oftumor growth and of liver and lung metastasis was reportedafter subcutaneous administration of bovine lactoferricin inboth spontaneous and experimental metastasis mice mod-els [117] Similar results were observed after subcutaneoustreatment and repeated injections of this peptide on Meth Afibrosarcoma xenograftsmice and established neuroblastomaxenografts respectively [118 119]120572-Lactalbumin is a whey protein with anticancer proper-

ties which has been reported when it forms a complex with


Table2Ch

emop

reventivep

ropertieso

flactoferrin

andits

deriv

edpeptidelactoferricin

againstcancerd

emon

strated

bycellcultu

reexperim

entsandanim

alsm

odels

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Breastcancer

Hum

anlactoferrin

MDA-

MB-231cells

Inhibitio

nof

cellgrow

thCellcyclearrest

[160]

Bovine

lactoferrin

4T1x

enograftBa

lbcmice

Improvem

ento

ftam

oxifenchem

opreventivee

ffects

Dow

nregulationof

proinfl

ammatorycytokines

[161]

Bovine

lactoferrin

-oleicacid

complex

MCF

-7cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[162]

Bovine

lactoferric

inMCF

-7T

-47D

and

MDA-

MB-435

cells

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]

Colon

cancer

Camellactoferrin

HCT

-116

cells

Inhibitio

nof

cellproliferatio

nAntioxidant

activ

ityInhibitio

nof

DNAdamage

[164

]

Bovine

lactoferrin

Caco-2

xeno

graft

mou

semod

elInhibitio

nof

tumor

grow

th[165]

Bovine

lactoferrin

-oleicacid

complex

HT-29

cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[166]

C26cells

Cytotoxica

ctivity

[118]

Caco-2

cells

Inhibitio

nof

cellproliferatio

n[166]

Cell

cycle

arrestby

downregulationof

cyclinE1

Bovine

lactoferric

inUltraviolet-irradiatedCa

co-2

cells

Redu

ctionof

DNAdamage

Cell

cycle

arrestby

downregulationof

cyclinE1

[167]

Colo-35

andHT-29

cells

Cytotoxica

ctivity

indu

ctionof

apop

tosis

[163]

Cervicalcancer

Bovine

lactoferrin

HeLac

ells

Inhibitio

nof

cellgrow

thIndu

ctionof

nucle

araccumulationof

Smad-2

[168]

Fibrosarcoma

Bovine

lactoferric

inMethAcells

Cytotoxica

ctivity

Tumor

cellmem

braned

isrup

tion

[118]

Headandneck

cancer

Hum

anlactoferrin

Squamou

scarcino

maO

12tumor

bearingmice

Redu

ctionof

tumor

Immun

omod

ulatoryeffects

[169]

Hepatocarcino

ma

Bovine

lactoferrin

-oleicacid

complex

HepG2cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[162]


Table2Con

tinued

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Leuk

emia

Bovine

lactoferric

in

THP-1h

uman

mon

ocyticleuk

emic

cells

Indu

ctionof

apop

tosis

Activ

ationof

ROSgeneratio

nandCa

2+M

g2+ -depend

ent

endo

nucle

ases

[170]

JurkatTleuk

emiacells

Indu

ctionof

apop

tosis

bytriggerin

gmito

chon

drial

swellin

gandreleaseo

fcytochrom

ecIndu

ctionof

cellmem

branep

ermeabilization

[163171]

Activ

ationof

ROSgeneratio

nandcaspase-3andcaspase-9

activ

ityRe

ductionof

DNAmethyltransfe

rasese

xpression

[172]

Lymph

oma

RajiandRa

mos

BurkittrsquosB-lymph

oma

cells

Indu

ctionof

apop

tosis

Stim

ulationof

DNAfragmentatio

nchromatin

cond

ensatio

nandnu

clear

disin

tegration

[114]

Ramos

B-lymph

omac

ellsxeno

graft

sin

SCID

beige

mice

Extensionof

survivalof

mice

[114]

Bovine

lactoferric

inA20

celllymph

omas

insyngeneic

Balbcmice

Tumor

necrosisandregressio

nof

thetum

ors

Indu

ctionof

long

-term

specificc

ellularimmun

ity[173]

B16-BL

6melanom

aand

L5178Y

-ML2

5lymph

omac

ellsmetastasis

mod

elsin

syngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Lung

cancer

Bovine

lactoferrin

A549cells

Transgenicmiceo

verexpressing

hVEG

F-A165

Dow

nregulationof

proinfl

ammatorycytokines

Supp

ressionof

tumor

developm

ent

[174]

Melanom

aBo

vine

lactoferric

inB16F

10cells

Cytotoxica

ctivity

[118]

Spon

taneou

sB16-BL6

metastasis

mod

elsinsyngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Nasop

haryngeal

carcinom

aHum

anlactoferrin

5ndash8FC

NE2

and

HONE1

cells

Xeno

graft

Balbcmice

Supp

ressionof

tumorigenesisthroug

hinhibitio

nof

the

AKT

pathway

[175]

Neuroblastoma

Bovine

lactoferric

in

Hum

anMYC

N-amplified

and

non-MYC

N-amplified

neurob

lasto

ma

cells

Cytotoxica

ctivity

Destabilizationof

thec

ytop

lasm

icmem

brane

Activ

ationof

caspase-6caspase-7andcaspase-9

[119]

SH-SY-5Y

neurob

lasto

max

enografts

innu

derats

Redu

ctionof

thetum

orgrow

th[119]

Oralcancer

Bovine

lactoferric

inOralsqu

amou

scarcino

maS

AScell

Indu

ctionof

apop

tosis

Cleavage

ofcaspase-3andpo

ly-ADPrib

osep

olym

erase

Phosph

orylationof

extracellularsignal-regulated

kinase

andc-JunN-te

rminalkinasestre

ssactiv

ated

protein

kinase

[176]

Ovaria

ncancer

Bovine

lactoferric

inSkov3andCa

ov3

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]


oleic acid known as ldquohuman alpha-lactalbumin made lethalto tumor cells HAMLETrdquo or ldquobovine alpha-lactalbuminmade lethal to tumor cells BAMLETrdquo It has been recognizedthat both protein and fatty acid are required to show cytotoxicactivity against cancer cells [115] Treatment of cancer cellswith HAMLET provokes morphological changes typical ofapoptotic cells through caspase activation and causes mito-chondrial permeability transition resulting in mitochondrialswelling loss of mitochondrial membrane potential andcytochrome c release [120]These authors also found that thiscomplex induced autophagic cell death and changes in theproteasome structure and function Similar effects resultingfrom chromatin condensation and cell shrinkage have beenobserved after treatment of cancer cells with the complexBAMLET The efficacy of both complexes has been shown tobe influenced by the type of cancer cell line [120] In the lastyears the therapeutic effects against bladder cancer have beenstudied in animal models as preliminary step for BAMLETuse in human trials It has been demonstrated that intravesicaladministration of HAMLET delays tumor progression in amurine bladder cancer model although no preventive effectson tumor formation were observed [121]

Intact caseins have not been characterized as chemopre-ventive proteins but they have been suggested as an importantsource of peptides with anticancer properties CPPs areable to bind calcium to inhibit cell proliferation and toinduce apoptosis of intestinal tumor HT-29 and AZ-97 cellsthrough activation of voltage-activated calcium channelswhichmediate the calcium flood according to the depolariza-tion state of the cell [122] However in differentiated epithelialintestinal cells a protective effect from programmed celldeath is observed after treatment with these peptides [123]120573-Casomorphin 7 and 120573-casomorphin 5 two casein-derivedsequences with opioid properties have shown antiprolifer-ative and cell cycle arresting activities on breast and coloncancer cells [115 124 125] It has been suggested that theseeffects are mediated by interaction with specific opioid andsomatostatin receptors although further studies confirmingthis mode of action are needed

3 Impact of Milk Oligosaccharides onHuman Health

Despite the important role of HMO in infant health thelimited supply of human milk has hindered its use in com-mercial infant formula [126] and in large-scale clinical trialsPresumably the health benefits provided by HMO to infantscould be extended to humans of all ages if alternative sourcesof these complex OS are identified [127] In that view theneed of finding other sources of human-likeOS has promptedthe identification characterization and quantification ofunknown OS present in many other types of milk and theirrespective industrial streams [128 129]

31 Alternative Sources of Oligosaccharides Major Sourcesof Nonhuman Milk Oligosaccharides and Their IndustrialEffluents Increasing interest on plant- and lactose-derivedOS has been observed in the past decade as an alternative

source for complex HMO Some of these OS include galacto-OS (GOS) fructo-OS (FOS) and lactulose among others[130] These indigestible OS are considered prebiotics dueto their ability to confer health benefits to the host throughthe selective growth and activity of commensal bacteria[131] One such example is inulin an oligofructan with D-fructofuranosyl 120573(1-2) links that cannot be broken down byhuman digestive enzymes thus exerting several intestinalphysiological effects that contribute to the host health GOScommonly produced by transgalactosylation of lactose by120573-galactosidases are another example of a current availablesource of OS for use by the infant formula industry

Despite the fact that some health promoting effects suchas improved bifidogenic activity have been attributed to someof those OS [131] little similarity has been observed betweencommercially available GOS and HMO except that they areboth built on a lactose core [127] GOS and FOS are composedof a simple linear core being devoid of structures havinghigh biological activity such as fucose sialic acid and N-acetyl glucosamine Because GOS and FOS do not possessthe intrinsic structural complexity observed in HMO it isexpected that domestic farm animals and their processingstreams such as whey permeate from cheese manufacturingcan be a source of OS more similar to the ones present inhuman milk [132]

World milk production is almost entirely derived fromcattle (83) buffaloes (13) goats (2) sheep (1) andcamels (03) (httpwwwfaoorgagriculturedairy-gate-waymilk-productiondairy-animalsenVA95gvldXXs)Considering that cow milk accounts for 83 of the worldmilk production the enormous interest of the scientificcommunity to identify quantify and characterize the OSpresent in cattle milk and their industrial byproducts isnot surprising A comprehensive review by Urashima etal [132] shows that approximately 25 bovine milk OS(BMO) structures had been characterized before 2011The development of advanced analytic techniques suchas several mass spectrometric methods and hydrophilicinteraction liquid chromatography-high performance liquidchromatography has enabled significant improvement in theidentification of new BMO as many as 40 BMO have beencharacterized [133 134]

The low concentration of BMO makes it challenging toidentify and characterize these compounds when comparedwith HMO The OS concentration can reach values as highas 07ndash10 gL in bovine colostrum or can be detected asjust trace amounts in bovine milk [135] being much lowerthan the OS concentration in human milk Caprine milk isanother type of milk which contains complex OS similarto HMO The discovery of the presence of fucosylated andsialylated OS that are considered as prebiotics and whichhave the ability to reduce pathogen adherence to the intestinewall has opened up translational opportunities to humanhealth [136] Approximately 37 caprine milk OS (COS) havebeen identified of which nearly half of them have had theirstructural complexity elucidated Similar to bovinemilk COSare present in very small concentrations when comparedwithHMO However they have been found in concentrations of025ndash03 gL which is 4-5 times higher than BMO [137]


From those two alternative sources of HMO-like OS(BMO and COS) industrial streams arising from cheesemanufacturing and production of whey protein concen-trates (WPC) and isolates (WPI) have been considered asa more realistic source of OS for future commercialization[129 138] Considering the enormous worldwide productionof whey (180ndash190 times 106 tonnesyear httpwwwadpiorgPortals0PDF09ConferenceTAGEAFFERTSHOLTpdf)and the fact that the major industrial application of wheyto produce WPC and WPI generates a new byproductcontaining the target OS the development of economicallyfeasible processes to recover these compounds represents akey step in enabling the large-scale production of OS

32 Biological Activities of Oligosaccharides While a widerange of biological functions has been attributed to HMOless information is available regarding the biological activitiesof BMO and COS The limited availability of large quantitiesof OS with high degree of purity can be inferred by thelimited number of in vivo studieswith those compounds withthe majority of milk OS biological activities being describedby in vitro studies Recent reports of some of the biologicalactivities of HMO BMO and COS are reported in Table 3

321 Prebiotic Activity One of the main features of HMOis that they can only be consumed by very specific bacteriastrains that possess the appropriate set of enzymes to cleavetheir complex structure This prebiotic effect is associatedwith improved health outcomes A prebiotic is ldquoa selectivelyfermented ingredient that allows specific changes both inthe composition andor in the activity in the gastrointestinalmicroflora conferring benefits upon host well-being andhealthrdquo [139] Because HMO are only partially digested inthe small intestine they can reach the colon intact wherethey selectively stimulate the development of bifidogenicflora A recent study has demonstrated the bifidogenic effectof major fucosylated and sialylated HMO when fed as asole source of carbon to 25 major isolates of the humanintestinal microbiota [140] Most of the Bifidobacteria sppand Bacteroides spp were able to consume those OS and toproduce short chain fatty acids while common pathogenicbacteria were not able to grow on those OS In vitro biologicalactivities of HMO have been supported by in vivo studiesOne of the newest publications in this topic demonstrated theability of 2-fucosyllactose and 3-fucosyllactose to selectivelyincrease some intestinal bacteria populations like Barnesiellathe major bacterial genus in mice [141] being this effectcorrelated with reduced level of colitis

Prebiotic activities of COS recovered from caprine wheyhave been evaluated by in vitro studies [142] The purifiedCOS fraction favored the development of Bifidobacteriumspp and produced short chain fatty acids such as lactic andpropionic acids but presented no inhibition of Staphylococcusaureus and Escherichia coli grown in human faeces

322 Antipathogenic Activity A second feature of OS is theability to reduce pathogen biding to the intestinal mucosaThe intestinal mucosa is heavily glycosylated and covered

with complex glycans including glycoproteins glycolipidsandmucins among others [143 144] Bacteria and viruses areable to recognize certain types of fucosylated and sialylatedOS and adhere to them [130] therefore acting as anti-infectiveagentsMilk OS are also fucosylated and sialylated so bacteriaand viruses in presence of OS will attach less to intestinalcells The ability of pathogens to bind to specific OS seemsto be intrinsically correlated with their structure NeutralOS containing HexNAc block adhesion of pathogens thatcause diarrhea (Vibrio cholerae) and pneumonia (Strepto-coccus pneumoniae) [15 145] while neutral fucosylated OShave been shown to inhibit adhesion of other pathogens(ie Campylobacter jejuni and diarrheagenic E coli) thatcause gastrointestinal disorders [146] Acidic OS containingsialic acid are able to block adhesion of Helicobacter pyloriwhich causes peptic ulcers and other gastric diseases [147]Staphylococcus aureus and Clostridium botulinum [148]

Recent in vitro studies have demonstrated that BMO alsopossess antibacterial properties as observed for HMO BMOfrom colostrum permeate proved to be effective in protect-ing HEp-2 cells from enteropathogenic E coli Cronobactersakazakii and Salmonella enterica serovar typhimurium [149]It has also been demonstrated that BMO can inhibit thepili-mediated adhesion of Neisseria meningitidis in vitro[150] Several studies have demonstrated the inhibition ofthe attachment of enteric pathogens such as E coli andCampylobacter jejuni and noroviruses with HMO [151] Thiseffect has also been demonstrated by in vivo studies in whichisolated HMO were fed to suckling mice before and afterinfection with enteropathogenic E coli Mice that receivedHMO significantly reduced colonization of this species com-pared with untreated controls [152]

323 Anti-Inflammatory Activity OS have been also con-sidered as anti-inflammatory agents due to their prebioticactivities and their ability to act as receptors of microorgan-isms In vivo studies have demonstrated that COS possessanti-inflammatory properties towards the development ofexperimental colitis in rats Pretreatment of the rats withisolated COS reduced the typical signs of induced colitisincluding less anorexia better body weight gain and lessmacroscopic intestinal lesions among others [153] Similarresults were observed by Lara-Villoslada et al [154] whereCOS were shown to play an important role in intestinalprotection and repair after a damage caused by DSS in rats

4 Future Prospects

Milk has long been recognized as a source of macro- andmicronutrients Recent identification of many important bio-logically active substances on milk and its derivatives hasattracted much attention from the scientific community Notonly are many of these bioactive compounds associated withgrowth but they also confer many health benefits that mightsupport disease prevention Milk proteins and peptides areusually well tolerated and demonstrate oral bioavailability Inthis view they have the potential to act as health promotingingredients and to participate in auxiliary therapies to boost


Table 3 Biological activities of human bovine and goat oligosaccharides

Microorganismsanimals Molecule used Dose Durationdetails Outcome measured ReferenceBifidobacterium sppBacteroides sppClostridium sppLactobacillus sppEnterococcus sppStreptococcus sppStaphylococcus sppEnterobacter spp andEscherichia coli

HMO (21015840-FL 31015840-FLLDFT 31015840-SL and

61015840-SL)05ndash2 gL 48 hrs OS incubation

SCFA quantificationbacterial growth andOS consumption

[140]

Mice HMO (21015840-FL and31015840-FL)

500mM starting with5mL increasing by25mL every 3 dreaching a daily

amount of 25mL onday 20

From day 1 to day 20after birth

Bacterial amountcolitis signs [141]

Bacteria from human feces Pooled GOS During incubation Bacterial amount [142]

Mice Pooled HMO 15mgdayOne day before andafter infection with

EPEC

Intestinalcolonization of EPEC [152]

HEp-2 cells Pooled BMO fromcolostrum

20mgL of totalcarbohydrate in

cultureDuring incubation Adherence inhibition [149]

Bovine thyroglobulin andhuman salivary agglutininglycoproteins

Pooled HMO andBMO 40 gL During incubation Neisseria meningitidis

Pili attachment [150]

Rats Pooled GOS 500mg(kglowastd)2 days before and 6days after induced

colitisColonic damage [153]

HMO human milk oligosaccharides FL fucosyllactose LDFT lacto-difucosyl-tetraose SL sialyllactose GOS galacto-oligosaccharides BMO bovine milkoligosaccharides

overall success in chronic diseases However this researcharea is only at its beginning and more peptides with physi-ological effects are to be discovered in the future Confirmingthe health benefits of these bioactive compounds requiresthe design of clinical trials based on metabolomic genomicsproteomics transcriptomics and epigenetic data in orderto explore new biomarkers related to the observed healthbenefits

While larger data for in vivo biological activities of milkand peptides is observed the same is not observed for OS Todate few studies have demonstrated the safety and efficacyof OS supplementation [155 156] The reduced number ofbiological activities evaluated for BMO and COS reveals thechallenges associated with the production of OS in adequatequantities and purity needed for clinical trials The develop-ment of new synthetic pathways to produce highly purifiedOS and of large-scale processes to recover those OS fromtheir respective industrial streams will likely improve theelucidation of their biological activities and determine theirsafety and efficacy in clinical trials with humans Moreoverthe development of more environmentally friendly processesthat are also economically feasible not only will enable the

production of a new generation of prebiotics but will addressenvironmental issues associated with the disposal of OS-containing waste streams and poor economic viability of ourfood industry

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors acknowledge funding from theNational Instituteof Health the ldquoBill and Melinda Gates Foundationrdquo and thePeter J Shields Chair in Dairy Food Science at the Universityof California at Davis Chia-Chien Hsieh acknowledgesthe Ministry of Science and Technology for National Sci-ence Council of Taiwan (MOST 103-2320-B-003-003-MY3)Samuel Fernandez-Tome and Blanca Hernandez-Ledesmaacknowledge the Ministry of Economy and Competitiveness(MINECO) for their FPI fellowship and ldquoRamon y Cajalrdquopostdoctoral contract respectively


References

[1] B Lamarche ldquoReview of the effect of dairy products on non-lipid risk factors for cardiovascular diseaserdquo Journal of theAmerican College of Nutrition vol 27 no 6 pp 741Sndash746S2008

[2] C Jaffiol ldquoMilk and dairy products in the prevention andtherapy of obesity type 2 diabetes and metabolic syndromerdquoBulletin de lrsquoAcademie Nationale de Medecine vol 192 no 4 pp749ndash758 2008

[3] K Uenishi ldquoPrevention of osteoporosis by foods and dietarysupplements Prevention of osteoporosis by milk and dairyproductsrdquo Clinical Calcium vol 16 no 10 pp 1606ndash1614 2006

[4] Y Shimazaki T Shirota K Uchida et al ldquoIntake of dairy pro-ducts and periodontal disease the Hisayama studyrdquo Journal ofPeriodontology vol 79 no 1 pp 131ndash137 2008

[5] C M Weaver ldquoShould dairy be recommended as part of ahealthy vegetarian diet PointrdquoTheAmerican Journal of ClinicalNutrition vol 89 no 5 pp 1634Sndash1637S 2009

[6] M Pufulete ldquoIntake of dairy products and risk of colorectalneoplasiardquo Nutrition Research Reviews vol 21 no 1 pp 56ndash672008

[7] E Ginter ldquoVegetarian diets chronic diseases and longevityrdquoBratislavske Lekarske Listy vol 109 no 10 pp 463ndash466 2008

[8] R Nagpal P V Behare M Kumar et al ldquoMilk milk productsand disease free health an updated overviewrdquo Critical Reviewsin Food Science and Nutrition vol 52 no 4 pp 321ndash333 2012

[9] R Nagpal P Behare R Rana et al ldquoBioactive peptides derivedfrom milk proteins and their health beneficial potentials anupdaterdquo Food and Function vol 2 no 1 pp 18ndash27 2011

[10] D S Newburg ldquoNeonatal protection by an innate immunesystem of human milk consisting of oligosaccharides andglycansrdquo Journal of Animal Science vol 87 no 13 pp 26ndash342009

[11] G V Coppa P Pierani L Zampini I Carloni A Carlucci andO Gabrielli ldquoOligosaccharides in human milk during differentphases of lactationrdquoActa Paediatrica vol 88 no 430 pp 89ndash941999

[12] O Ballard and A L Morrow ldquoHumanmilk composition nutri-ents and bioactive factorsrdquo Pediatric Clinics of North Americavol 60 no 1 pp 49ndash74 2013

[13] D S Newburg and S H Neubauer ldquoCarbohydrates in milksanalysis quantities and significancerdquo in Handbook of MilkComposition R G Jensen Ed chapter 4 pp 273ndash349 Aca-demic Press San Diego Calif USA 1995

[14] M B Engfer B Stahl B Finke G Sawatzki and H DanielldquoHuman milk oligosaccharides are resistant to enzymatichydrolysis in the upper gastrointestinal tractrdquoAmerican Journalof Clinical Nutrition vol 71 no 6 pp 1589ndash1596 2000

[15] G V Coppa L Zampini T Galeazzi et al ldquoHuman milk oligo-saccharides inhibit the adhesion to Caco-2 cells of diarrhealpathogens Escherichia coli Vibrio cholerae and Salmonellafyrisrdquo Pediatric Research vol 59 no 3 pp 377ndash382 2006

[16] S E Carlson and S G House ldquoOral and intraperitoneal admin-istration of N-acetylneuraminic acid effect on rat cerebral andcerebellar N-acetylneuraminic acidrdquo Journal of Nutrition vol116 no 5 pp 881ndash886 1986

[17] N Klein A Schwertmann M Peters C Kunz and S StrobeldquoImmunomodulatory effects of breastmilk oligosaccharidesrdquo inShort and Long Term Effects of Breast Feeding on Child HealthB Koletzko K F Michaelsen and O Hernell Eds pp 251ndash259Springer New York NY USA 2002

[18] D Divajeva T Marsh S Logstrup et al ldquoEconomics of chronicdiseases protocol cost-effectiveness modelling and the futureburden of non-communicable disease in Europerdquo BMC PublicHealth vol 14 no 1 article 456 2014

[19] W Y Huang S T Davidge and J Wu ldquoBioactive natural con-stituents from food sources-potential use in hypertension pre-vention and treatmentrdquo Critical Reviews in Food Science andNutrition vol 53 no 6 pp 615ndash630 2013

[20] S G Chrysant andG S Chrysant ldquoAn update on the cardiovas-cular pleiotropic effects of milk and milk productsrdquoThe Journalof Clinical Hypertension vol 15 no 7 pp 503ndash510 2013

[21] M F Engberink M A H Hendriksen E G Schouten et alldquoInverse association between dairy intake and hypertension therotterdam studyrdquo The American Journal of Clinical Nutritionvol 89 no 6 pp 1877ndash1883 2009

[22] L A J van Mierlo L R Arends M T Streppel et al ldquoBloodpressure response to calcium supplementation a meta-analysisof randomized controlled trialsrdquo Journal of Human Hyperten-sion vol 20 no 8 pp 571ndash580 2006

[23] R J Fitzgerald BAMurray andD JWalsh ldquoHypotensive pep-tides from milk proteinsrdquo Journal of Nutrition vol 134 no 42004

[24] D Martınez-Maqueda B Miralles I Recio and B Hernandez-Ledesma ldquoAntihypertensive peptides from food proteins areviewrdquo Food amp Function vol 3 no 4 pp 350ndash361 2012

[25] B Hernandez-Ledesma M J Garcıa-Nebot S Fernandez-Tome L Amigo and I Recio ldquoDairy protein hydrolysatespeptides for health benefitsrdquo InternationalDairy Journal vol 38no 2 pp 82ndash100 2014

[26] M D M Contreras R Carron M J Montero M Ramos andI Recio ldquoNovel casein-derived peptides with antihypertensiveactivityrdquo International Dairy Journal vol 19 no 10 pp 566ndash5732009

[27] I Recio M M Contreras B Gomez-Sala C Vazquez HFernandez-Escribano and R del Campo ldquoEffect of a caseinhydrolysate containing novel peptides in hypertensive subjectsrdquoAnnals of Nutrition and Metabolism vol 58 no 3 pp 16ndash172011

[28] P Ruiz-Gimenez M C Burguete M Castello-Ruiz et alldquoBovine lactoferrin pepsin hydrolysate exerts inhibitory effecton angiotensin I-converting enzyme-dependent vasoconstric-tionrdquo International Dairy Journal vol 17 no 10 pp 1212ndash12152007

[29] P Ruiz-Gimenez J B Salom J F Marcos et al ldquoAntihy-pertensive effect of a bovine lactoferrin pepsin hydrolysateidentification of novel active peptidesrdquo Food Chemistry vol 131no 1 pp 266ndash273 2012

[30] R Fernandez-Musoles P Manzanares M C Burguete EAlborch and J B Salom ldquoIn vivo angiotensin I-convertingenzyme inhibition by long-term intake of antihypertensivelactoferrin hydrolysate in spontaneously hypertensive ratsrdquoFood Research International vol 54 no 1 pp 627ndash632 2013

[31] R Sugai U Murakami and Y Yamori ldquoAngiotensin convertingenzyme inhibitorsrdquo Japanese Patent 62270533 1995

[32] J A Cadee C-Y Chang C-W Chen C-N Huang S-L Chenand C-K Wang ldquoBovine casein hydrolysate (c12 Peptide)reduces blood pressure in prehypertensive subjectsrdquo AmericanJournal of Hypertension vol 20 no 1 pp 1ndash5 2007

[33] A Abubakar T Saito H Kitazawa Y Kawai andT Itoh ldquoStruc-tural analysis of new antihypertensive peptides derived fromcheese whey protein by proteinase K digestionrdquo Journal of DairyScience vol 81 no 12 pp 3131ndash3138 1998


[34] G-W Chen J-S Tsai and B Sun Pan ldquoPurification of angi-otensin I-converting enzyme inhibitory peptides and antihy-pertensive effect of milk produced by protease-facilitated lacticfermentationrdquo InternationalDairy Journal vol 17 no 6 pp 641ndash647 2007

[35] B Hernandez-Ledesma M Miguel L Amigo M A Aleixan-dre and I Recio ldquoEffect of simulated gastrointestinal digestionon the antihypertensive properties of synthetic 120573-lactoglobulinpeptide sequencesrdquo Journal of Dairy Research vol 74 no 3 pp336ndash339 2007

[36] J-S Tsai T-J Chen B S Pan S-D Gong and M-Y ChungldquoAntihypertensive effect of bioactive peptides produced byprotease-facilitated lactic acid fermentation of milkrdquo FoodChemistry vol 106 no 2 pp 552ndash558 2008

[37] Y Nakamura N Yamamoto K Sakai A Okubo S YamazakiandT Takano ldquoPurification and characterization of angiotensinI-converting enzyme inhibitors from sour milkrdquo Journal ofDairy Science vol 78 no 4 pp 777ndash783 1995

[38] Y Nakamura N Yamamoto K Sakai and T Takano ldquoAntihy-pertensive effect of sour milk and peptides isolated from it thatare inhibitors to angiotensin I-converting enzymerdquo Journal ofDairy Science vol 78 no 6 pp 1253ndash1257 1995

[39] A H Pripp ldquoEffect of peptides derived from food proteinson blood pressure a meta-analysis of randomized controlledtrialsrdquo Food and Nutrition Research vol 52 pp 1ndash9 2008

[40] J-Y Xu L-Q Qin P-Y Wang W Li and C Chang ldquoEffectof milk tripeptides on blood pressure a meta-analysis ofrandomized controlled trialsrdquoNutrition vol 24 no 10 pp 933ndash940 2008

[41] L-Q Qin J-Y Xu J-Y Dong Y Zhao P van Bladeren andWZhang ldquoLactotripeptides intake and blood pressuremanage-ment a meta-analysis of randomised controlled clinical trialsrdquoNutrition Metabolism and Cardiovascular Diseases vol 23 no5 pp 395ndash402 2013

[42] M F Engberink E G Schouten F J Kok L A J van MierloI A Brouwer and J M Geleijnse ldquoLactotripeptides show noeffect on human blood pressure results from a double-blindrandomized controlled trialrdquo Hypertension vol 51 no 2 pp399ndash405 2008

[43] L Usinger L T Jensen B Flambard A Linneberg and HIbsen ldquoThe antihypertensive effect of fermented milk in indi-viduals with prehypertension or borderline hypertensionrdquo Jour-nal of Human Hypertension vol 24 no 10 pp 678ndash683 2010

[44] A F G Cicero B Gerocarni L Laghi and C Borghi ldquoBloodpressure lowering effect of lactotripeptides assumed as func-tional foods a meta-analysis of current available clinical trialsrdquoJournal of HumanHypertension vol 25 no 7 pp 425ndash436 2011

[45] A F G Cicero F Aubin V Azais-Braesco and C Borghi ldquoDothe lactotripeptides isoleucine-proline-proline and valine-pro-line-proline reduce systolic blood pressure in european sub-jects A meta-analysis of randomized controlled trialsrdquo Ameri-can Journal of Hypertension vol 26 no 3 pp 442ndash449 2013

[46] EFSA Panel on Dietetic Products Nutrition and Allergies(NDA) ldquoScientific opinion on the substantion of health claimsrelated to isoleucineproline-proline (IPP) and valine-proline-proline (VPP) and maintenance of normal blood pressure(ID 615 661 1831 1832 2891) and maintenance of the elasticproperties of the arteries (ID 1832) pursuant to Article 13(1) ofregulation (EC) No 19242006 on request from the EuropeanCommissionrdquo EFSA Journal vol 7 pp 1259ndash1277 2009

[47] M Miguel I Recio M Ramos M A Delgado and M AAleixandre ldquoAntihypertensive effect of peptides obtained from

Enterococcus faecalis-fermented milk in ratsrdquo Journal of DairyScience vol 89 no 9 pp 3352ndash3359 2006

[48] J C Rodrıguez-Figueroa A F Gonzalez-CordovaM J Torres-Llanez H S Garcia and B Vallejo-Cordoba ldquoNovel angiot-ensin I-converting enzyme inhibitory peptides produced infermented milk by specific wild Lactococcus lactis strainsrdquoJournal of Dairy Science vol 95 no 10 pp 5536ndash5543 2012

[49] J C Rodrıguez-Figueroa A F Gonzalez-Cordova H Astiaz-aran-Garcıa and B Vallejo-Cordoba ldquoHypotensive and heartrate-lowering effects in rats receivingmilk fermented by specificLactococcus lactis strainsrdquo British Journal of Nutrition vol 109no 5 pp 827ndash833 2013

[50] K Erdmann B W Y Cheung and H Schroder ldquoThe possibleroles of food-derived bioactive peptides in reducing the risk ofcardiovascular diseaserdquoThe Journal of Nutritional Biochemistryvol 19 no 10 pp 643ndash654 2008

[51] A Pihlanto ldquoAntioxidative peptides derived from milk pro-teinsrdquo International Dairy Journal vol 16 no 11 pp 1306ndash13142006

[52] O Power P Jakeman and R J Fitzgerald ldquoAntioxidativepeptides enzymatic production in vitro and in vivo antioxidantactivity and potential applications ofmilk-derived antioxidativepeptidesrdquo Amino Acids vol 44 no 3 pp 797ndash820 2013

[53] M Zommara H Toubo M Sakono and K Imaizumi ldquoPre-vention of peroxidative stress in rats fed on a low vitamine-containing diet by supplementing with a fermented bovinemilk whey preparation effect of lactic acid and 120573-lactoglobulinon the antiperoxidative actionrdquo Bioscience Biotechnology andBiochemistry vol 62 no 4 pp 710ndash717 1998

[54] T Kullisaar E Songisepp M Mikelsaar K Zilmer T Viha-lemm and M Zilmer ldquoAntioxidative probiotic fermentedgoatsrsquo milk decreases oxidative stress-mediated atherogenicityin human subjectsrdquo British Journal of Nutrition vol 90 no 2pp 449ndash456 2003

[55] G Tompa A Laine A Pihlanto H Korhonen I Rogelj andP Marnilab ldquoChemiluminescence of non-differentiated THP-1promonocytes developing an assay for screening anti-inflam-matory milk proteins and peptidesrdquo The Journal of Biologicaland Chemical Luminiscence vol 26 no 4 pp 251ndash258 2010

[56] X-YMao X Cheng XWang and S-JWu ldquoFree-radical-scav-enging and anti-inflammatory effect of yak milk casein beforeand after enzymatic hydrolysisrdquo Food Chemistry vol 126 no 2pp 484ndash490 2011

[57] K D Ballard R S Bruno R L Seip et al ldquoAcute ingestion ofa novel whey-derived peptide improves vascular endothelialresponses in healthy individuals a randomized placebo con-trolled trialrdquo Nutrition Journal vol 8 article 34 2009

[58] S Nagaoka Y Kanamaru and Y Kuzuya ldquoEffects of wheyprotein and casein on the plasma and liver lipids in ratsrdquoAgricultural and Biological Chemistry vol 55 no 3 pp 813ndash8181991

[59] S Nagaoka Y Kanamaru Y Kuzuya T Kojima and T KuwataldquoComparative studies on the serum cholesterol lowering actionof the whey protein and soybean protein in ratsrdquo BioscienceBiotechnology and Biochemistry vol 56 no 9 pp 1484ndash14851992

[60] S Nagaoka Y Futamura K Miwa et al ldquoIdentification of novelhypocholesterolemic peptides derived from bovine milk 120573-lactoglobulinrdquo Biochemical and Biophysical Research Commu-nications vol 281 no 1 pp 11ndash17 2001


[61] R Yamauchi K Ohinata and M Yoshikawa ldquo120573-Lactotensinand neurotensin rapidly reduce serum cholesterol via NT2receptorrdquo Peptides vol 24 no 12 pp 1955ndash1961 2003

[62] M Shimizu ldquoInteraction between food substances and theintestinal epitheliumrdquo Bioscience Biotechnology and Biochem-istry vol 74 no 2 pp 232ndash241 2010

[63] M Shimizu ldquoFunctional food in Japan current status andfuture of gut-modulating foodrdquo Journal of Food and DrugAnalysis vol 20 no 1 pp 213ndash216 2012

[64] J Claustre F Toumi A Trompette et al ldquoEffects of peptidesderived from dietary proteins on mucus secretion in rat jeju-numrdquo American Journal of PhysiologymdashGastrointestinal andLiver Physiology vol 283 no 3 pp G521ndashG528 2002

[65] A Trompette J Claustre F CaillonG Jourdan J AChayvialleand P Plaisancie ldquoMilk bioactive peptides and 120573-casomorphinsinduce mucus release in rat jejunumrdquo Journal of Nutrition vol133 no 11 pp 3499ndash3503 2003

[66] S Zoghbi A Trompette J Claustre et al ldquo120573-Casomorphin-7regulates the secretion and expression of gastrointestinalmucins through a 120583-opioid pathwayrdquo American Journal ofPhysiologymdashGastrointestinal and Liver Physiology vol 290 no6 pp G1105ndashG1113 2006

[67] P Plaisancie J Claustre M Estienne et al ldquoA novel bioactivepeptide from yoghurts modulates expression of the gel-formingMUC2 mucin as well as population of goblet cells and Panethcells along the small intestinerdquo Journal of Nutritional Biochem-istry vol 24 no 1 pp 213ndash221 2013

[68] D Martınez-Maqueda B Miralles M Ramos and I RecioldquoEffect of 120573-lactoglobulin hydrolysate and 120573-lactorphin onintestinal mucin secretion and gene expression in human gobletcellsrdquo Food Research International vol 54 no 1 pp 1287ndash12912013

[69] K S Han A Deglaire R Sengupta and P J Moughan ldquoHydro-lyzed casein influences intestinal mucin gene expression in theratrdquo Journal of Agricultural and Food Chemistry vol 56 no 14pp 5572ndash5576 2008

[70] R C Sprong A J Schonewille and R van der Meer ldquoDietarycheese whey protein protects rats against mild dextran sulfatesodium-induced colitis role of mucin and microbiotardquo Journalof Dairy Science vol 93 no 4 pp 1364ndash1371 2010

[71] M Faure C Mettraux D Moennoz et al ldquoSpecific amino acidsincrease mucin synthesis and microbiota in dextran sulfatesodium-treated ratsrdquo Journal of Nutrition vol 136 no 6 pp1558ndash1564 2006

[72] G A Castro J E Carvalho S V Tinti A Possenti and V CSgarbieri ldquoAnti-ulcerogenic effect of a whey protein isolate andcollagen hydrolysates against ethanol ulcerative lesions on oraladministration to ratsrdquo Journal of Medicinal Food vol 13 no 1pp 83ndash90 2010

[73] L FHMezzaroba J E Carvalho AN Ponezi et al ldquoAntiulcer-ative properties of bovine 120572-lactalbuminrdquo International DairyJournal vol 16 no 9 pp 1005ndash1012 2006

[74] S S Comstock E A Reznikov N Contractor and S M Dono-van ldquoDietary bovine lactoferrin alters mucosal and systemicimmune cell responses in neonatal pigletsrdquo Journal of Nutritionvol 144 no 4 pp 525ndash532 2014

[75] Z Tang Y Yin Y Zhang et al ldquoEffects of dietary supplemen-tation with an expressed fusion peptide bovine lactoferricin-lactoferrampin on performance immune function and intesti-nal mucosal morphology in piglets weaned at age 21 drdquo BritishJournal of Nutrition vol 101 no 7 pp 998ndash1005 2009

[76] G A Biziulevicius V Zukaite T Normantiene G Biziulevici-ene and I G Arestov ldquoNon-specific immunity-enhancingeffects of tryptic casein hydrolysate versus Fermosorb fortreatmentprophylaxis of newborn calf colibacillosisrdquo FEMSImmunology and Medical Microbiology vol 39 no 2 pp 155ndash161 2003

[77] H Otani K Nakano and T Kawahara ldquoStimulatory effect ofa dietary casein phosphopeptide preparation on the mucosalIgA response of mice to orally ingested lipopolysaccharidefrom Salmonella typhimuriumrdquo Bioscience Biotechnology andBiochemistry vol 67 no 4 pp 729ndash735 2003

[78] G Vinderola C Matar and G Perdigon ldquoMilk fermentationproducts of L helveticus R389 activate calcineurin as a signal topromote gut mucosal immunityrdquo BMC Immunology vol 8 no19 pp 19ndash28 2007

[79] H Kitamura and H Otani ldquoFecal IgA levels in healthy personswho ingested cakes with or without bovine casein phosphopep-tidesrdquoMilchwissenschaft vol 57 no 11-12 pp 611ndash614 2002

[80] D E W Chatterton D N Nguyen S B Bering and P TSangild ldquoAnti-inflammatory mechanisms of bioactive milkproteins in the intestine of newbornsrdquoThe International Journalof Biochemistry ampCell Biology vol 45 no 8 pp 1730ndash1747 2013

[81] F S De Medina A Daddaoua P Requena et al ldquoNew insightsinto the immunological effects of food bioactive peptides inanimal models of intestinal inflammationrdquo Proceedings of theNutrition Society vol 69 no 3 pp 454ndash462 2010

[82] G T Sousa F S Lira J C Rosa et al ldquoDietary whey proteinlessens several risk factors for metabolic diseases a reviewrdquoLipids in Health and Disease vol 11 article 67 2012

[83] A S Gad Y A Khadrawy A A El-Nekeety S RMohamed NS Hassan and M A Abdel-Wahhab ldquoAntioxidant activity andhepatoprotective effects of whey protein and Spirulina in ratsrdquoNutrition vol 27 no 5 pp 582ndash589 2011

[84] E M Hamad S H Taha A-G I Abou Dawood M ZSitohy andMAbdel-Hamid ldquoProtective effect ofwhey proteinsagainst nonalcoholic fatty liver in ratsrdquo Lipids in Health andDisease vol 10 article 57 2011

[85] T Chitapanarux P Tienboon S Pojchamarnwiputh and DLeelarungrayub ldquoOpen-labeled pilot study of cysteine-richwhey protein isolate supplementation for nonalcoholic steato-hepatitis patientsrdquo Journal of Gastroenterology and Hepatologyvol 24 no 6 pp 1045ndash1050 2009

[86] J E de Aguilar-Nascimento B R Prado Silveira and D BDock-Nascimento ldquoEarly enteral nutrition with whey proteinor casein in elderly patients with acute ischemic stroke adouble-blind randomized trialrdquo Nutrition vol 27 no 4 pp440ndash444 2011

[87] R Lopez-Posadas P Requena R Gonzalez et al ldquoBovine gly-comacropeptide has intestinal antiinflammatory effects in ratswith dextran sulfate-induced colitisrdquo Journal of Nutrition vol140 no 11 pp 2014ndash2019 2010

[88] P Requena A Daddaoua E Martınez-Plata et al ldquoBovine gly-comacropeptide ameliorates experimental rat ileitis by mech-anisms involving downregulation of interleukin 17rdquo BritishJournal of Pharmacology vol 154 no 4 pp 825ndash832 2008

[89] M B E Turbay A deMoreno de LeBlanc G Perdigon G S deGiori and EMHebert ldquo120573-Casein hydrolysate generated by thecell envelope-associated proteinase of Lactobacillus delbrueckiissp lactis CRL 581 protects against trinitrobenzene sulfonicacid-induced colitis in micerdquo Journal of Dairy Science vol 95no 3 pp 1108ndash1118 2012


[90] R E Pratley and A Salsali ldquoInhibition of DPP-4 a newtherapeutic approach for the treatment of type 2 diabetesrdquoCurrent Medical Research and Opinion vol 23 no 4 pp 919ndash931 2007

[91] T Reihner ldquoType 2 diabetes in children and adolescentsrdquoWorldJournal of Diabetes vol 4 no 6 pp 270ndash281 2013

[92] K M Hirahatake J L Slavin K C Maki and S H AdamsldquoAssociations between dairy foods diabetes and metabolichealth potential mechanisms and future directionsrdquo Metab-olismClinical andExperimental vol 63 no 5 pp 618ndash627 2014

[93] D J Drucker andM A Nauck ldquoThe incretin system glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4inhibitors in type 2 diabetesrdquoThe Lancet vol 368 no 9548 pp1696ndash1705 2006

[94] P V Bharatam D S Patel L Adane AMittal and S SundriyalldquoModeling and informatics and designing anti-diabetic agentsrdquoCurrent Pharmaceutical Design vol 13 no 34 pp 3518ndash35302007

[95] M A Nauck N Kleine C Oslashrskov J J Holst B Willms andW Creutzfeldt ldquoNormalization of fasting hyperglycaemia byexogenous glucagon-like peptide 1 (7ndash36 amide) in Type 2 (non-insulin-dependent) diabetic patientsrdquo Diabetologia vol 36 no8 pp 741ndash744 1993

[96] G P Fadini and A Avogaro ldquoCardiovascular effects of DPP-4inhibition beyond GLP-1rdquo Vascular Pharmacology vol 55 no1ndash3 pp 10ndash16 2011

[97] P T Gunnarsson M S Winzell C F Deacon et al ldquoGlucose-induced incretin hormone release and inactivation are differ-ently modulated by oral fat and protein inmicerdquo Endocrinologyvol 147 no 7 pp 3173ndash3180 2006

[98] B L Petersen L S Ward E D Bastian A L Jenkins J Camp-bell and V Vuksan ldquoA whey protein supplement decreasespost-prandial glycemiardquo Nutrition Journal vol 8 article 472009

[99] G T D Sousa F S Lira J C Rosa et al ldquoDietary whey proteinlessens several risk factors for metabolic diseases a reviewrdquoLipids in Health and Disease vol 11 article 67 2012

[100] T Akhavan B L Luhovyy P H Brown C E Cho and GH Anderson ldquoEffect of premeal consumption of whey proteinand its hydrolysate on food intake and postmeal glycemia andinsulin responses in young adultsrdquo American Journal of ClinicalNutrition vol 91 no 4 pp 966ndash975 2010

[101] C Gaudel A B Nongonierma S Maher et al ldquoA wheyprotein hydrolysate promotes insulinotropic activity in a clonalpancreatic 120573-cell line and enhances glycemic function in obobmicerdquo Journal of Nutrition vol 143 no 7 pp 1109ndash1114 2013

[102] A B Nongonierma and R J Fitzgerald ldquoDipeptidyl peptidaseIV inhibitory and antioxidative properties of milk protein-derived dipeptides and hydrolysatesrdquo Peptides vol 39 no 1 pp157ndash163 2013

[103] S T Silveira D Martınez-Maqueda I Recio and B Hernan-dez-Ledesma ldquoDipeptidyl peptidase-IV inhibitory peptidesgenerated by tryptic hydrolysis of a whey protein concentraterich in 120573-lactoglobulinrdquo Food Chemistry vol 141 no 2 pp1072ndash1077 2013

[104] G Tulipano V Sibilia A M Caroli and D Cocchi ldquoWheyproteins as source of dipeptidyl dipeptidase IV (dipeptidylpeptidase-4) inhibitorsrdquo Peptides vol 32 no 4 pp 835ndash8382011

[105] M Uchida Y Ohshiba and O Mogami ldquoNovel dipeptidylpeptidase-4-inhibiting peptide derived from 120573-lactoglobulinrdquo

Journal of Pharmacological Sciences vol 117 no 1 pp 63ndash662011

[106] H Uenishi T Kabuki Y Seto A Serizawa and H Naka-jima ldquoIsolation and identification of casein-derived dipeptidyl-peptidase 4 (DPP-4)-inhibitory peptide LPQNIPPL fromgouda-type cheese and its effect on plasma glucose in ratsrdquoInternational Dairy Journal vol 22 no 1 pp 24ndash30 2012

[107] I M E Lacroix and E C Y Li-Chan ldquoEvaluation of the poten-tial of dietary proteins as precursors of dipeptidyl peptidase(DPP)-IV inhibitors by an in silico approachrdquo Journal of Func-tional Foods vol 4 no 2 pp 403ndash422 2012

[108] A B Nongonierma C Mooney D C Shields and R J Fitz-gerald ldquoIn silico approaches to predict the potential of milkprotein-derived peptides as dipeptidyl peptidase IV (DPP-IV)inhibitorsrdquo Peptides vol 57 pp 43ndash51 2014

[109] F Bray and B Moslashller ldquoPredicting the future burden of cancerrdquoNature Reviews Cancer vol 6 no 1 pp 63ndash74 2006

[110] M M Manson ldquoCancer preventionmdashthe potential for diet tomodulate molecular signallingrdquo Trends in Molecular Medicinevol 9 no 1 pp 11ndash18 2003

[111] T M de Kok S G van Breda and M M Manson ldquoMecha-nisms of combined action of different chemopreventive dietarycompoundsrdquo European Journal of Nutrition vol 47 no 2supplement pp 51ndash59 2008

[112] E G DeMejia and V P Dia ldquoThe role of nutraceutical proteinsand peptides in apoptosis angiogenesis and metastasis ofcancer cellsrdquo Cancer and Metastasis Reviews vol 29 no 3 pp511ndash528 2010

[113] R K Kanwar and J R Kanwar ldquoImmunomodulatory lacto-ferrin in the regulation of apoptosis modulatory proteins incancerrdquo Protein amp Peptide Letters vol 20 no 4 pp 450ndash4582013

[114] S J Furlong J S Mader and D W Hoskin ldquoBovine lacto-ferricin induces caspase-independent apoptosis in human B-lymphoma cells and extends the survival of immune-deficientmice bearing B-lymphoma xenograftsrdquo Experimental andMolecular Pathology vol 88 no 3 pp 371ndash375 2010

[115] G Pepe G C Tenore R Mastrocinque P Stusio and P Cam-piglia ldquoPotential anticarcinogenic peptides from bovine milkrdquoJournal of Amino Acids vol 2013 Article ID 939804 7 pages2013

[116] J S Mader D Smyth J Marshall and D W Hoskin ldquoBovinelactoferricin inhibits basic fibroblast growth factor- and vas-cular endothelial growth factor165-induced angiogenesis bycompeting for heparin-like binding sites on endothelial cellsrdquoTheAmerican Journal of Pathology vol 169 no 5 pp 1753ndash17662006

[117] Y C Yoo S Watanabe R Watanabe K Hata K I Shimazakiand I Azuma ldquoBovine lactoferrin and lactoferricin a peptidederived from bovine lactoferrin inhibit tumor metastasis inmicerdquo Japanese Journal of Cancer Research vol 88 no 2 pp184ndash190 1997

[118] L Tone Eliassen G Berge B Sveinbjoslashrnsson J S SvendsenL H Vorland and Oslash Rekdal ldquoEvidence for a direct antitu-mor mechanism of action of bovine lactoferricinrdquo AnticancerResearch vol 22 no 5 pp 2703ndash2710 2002

[119] L T Eliassen G Berge A Leknessund et al ldquoThe antimicrobialpeptide Lactoferricin B is cytotoxic to neuroblastoma cellsin vitro and inhibits xenograft growth in vivordquo InternationalJournal of Cancer vol 119 no 3 pp 493ndash500 2006


[120] C R Brinkmann S Thiel and D E Otzen ldquoProtein-fattyacid complexes biochemistry biophysics and functionrdquo FEBSJournal vol 280 no 8 pp 1733ndash1749 2013

[121] A-K Mossberg Y Hou M Svensson B Holmqvist and CSvanborg ldquoHAMLET treatment delays bladder cancer develop-mentrdquoThe Journal of Urology vol 183 no 4 pp 1590ndash1597 2010

[122] S Perego S Cosentino A Fiorilli G Tettamanti and AFerraretto ldquoCasein phosphopeptides modulate proliferationand apoptosis in HT-29 cell line through their interactionwith voltage-operated L-type calcium channelsrdquo Journal ofNutritional Biochemistry vol 23 no 7 pp 808ndash816 2012

[123] S Perego A Zabeo E Marasco et al ldquoCasein phosphopeptidesmodulate calcium uptake and apoptosis in Caco2 cells throughtheir interaction with the TRPV6 calcium channelrdquo Journal ofFunctional Foods vol 5 no 2 pp 847ndash857 2013

[124] A Hatzoglou E Bakogeorgou C Hatzoglou P-MMartin andE Castanas ldquoAntiproliferative and receptor binding propertiesof 120572- and 120573-casomorphins in the T47D human breast cancercell linerdquo European Journal of Pharmacology vol 310 no 2-3pp 217ndash223 1996

[125] R Maneckjee R Biswas and B K Vonderhaar ldquoBinding ofopioids to humanMCF-7 breast cancer cells and their effects ongrowthrdquo Cancer Research vol 50 no 8 pp 2234ndash2238 1990

[126] V Packard Human Milk and Infant Formula ElsevierAcademic Press New York NY USA 2012

[127] A M Zivkovic and D Barile ldquoBovine milk as a source offunctional oligosaccharides for improving human healthrdquoAdvances in Nutrition vol 2 no 3 pp 284ndash289 2011

[128] D Barile and R A Rastall ldquoHuman milk and related oligosac-charides as prebioticsrdquo Current Opinion in Biotechnology vol24 no 2 pp 214ndash219 2013

[129] M Lange D C Dallas A le Parc J M L N de Moura Belland D Barile ldquoHuman nutrition determining functional prop-erties ans sources of recently identified food componentsoligosaccharides glycolipids glycoproteins and peptidesrdquo inEncyclopedia of Agriculture and Food Systems N van Alfen Ed461 p 441 Elsevier New York NY USA 2014

[130] G Boehm and G Moro ldquoStructural and functional aspects ofprebiotics used in infant nutritionrdquo Journal of Nutrition vol 138no 9 pp 1818Sndash1828S 2008

[131] G R Gibson and M B Roberfroid ldquoDietary modulation ofthe human colonic microbiota introducing the concept ofprebioticsrdquo Journal of Nutrition vol 125 no 6 pp 1401ndash14121995

[132] T Urashima E Taufik K Fukuda and S Asakuma ldquoRecentadvances in studies on milk oligosaccharides of cows andother domestic farm animalsrdquo Bioscience Biotechnology andBiochemistry vol 77 no 3 pp 455ndash466 2013

[133] D Barile M Marotta C Chu et al ldquoNeutral and acidic oligo-saccharides in Holstein-Friesian colostrum during the first3 days of lactation measured by high performance liquidchromatography on a microfluidic chip and time-of-flight massspectrometryrdquo Journal of Dairy Science vol 93 no 9 pp 3940ndash3949 2010

[134] K Marino J A Lane J L Abrahams et al ldquoMethod for milkoligosaccharide profiling by 2-aminobenzamide labeling andhydrophilic interaction chromatographyrdquo Glycobiology vol 21no 10 pp 1317ndash1330 2011

[135] R W Veh J-C Michalski A P Corfield M Sander-WewerD Gies and R Schauer ldquoNew chromatographic system for the

rapid analysis and preparation of colostrum sialyloligosaccha-ridesrdquo Journal of Chromatography A vol 212 no 3 pp 313ndash3221981

[136] M Meyrand D C Dallas H Caillat F Bouvier P Martin andD Barile ldquoComparison of milk oligosaccharides between goatswith and without the genetic ability to synthesize 120572s1-caseinrdquoSmall Ruminant Research vol 113 no 2-3 pp 411ndash420 2013

[137] A Martinez-Ferez S Rudloff A Guadix et al ldquoGoatsrsquo milk as anatural source of lactose-derived oligosaccharides isolation bymembrane technologyrdquo International Dairy Journal vol 16 no2 pp 173ndash181 2006

[138] D Barile N Tao C B Lebrilla J-D Coisson M Arlorio andJ B German ldquoPermeate from cheese whey ultrafiltration is asource of milk oligosaccharidesrdquo International Dairy Journalvol 19 no 9 pp 524ndash530 2009

[139] M Roberfroid ldquoPrebiotics the concept revisitedrdquo The Journalof Nutrition vol 137 supplement 2 no 3 pp 830Sndash837S 2007

[140] Z-T Yu C Chen and D S Newburg ldquoUtilization of majorfucosylated and sialylated human milk oligosaccharides byisolated human gut microbesrdquo Glycobiology vol 23 no 11 pp1281ndash1292 2013

[141] G AWeiss C Chassard andTHennet ldquoSelective proliferationof intestinal Barnesiella under fucosyllactose supplementationin micerdquo British Journal of Nutrition vol 111 no 9 pp 1602ndash1610 2014

[142] D L Oliveira A Costabile R A Wilbey A S Grandison L CDuarte and L B Roseiro ldquoIn vitro evaluation of the fermenta-tion properties and potential prebiotic activity of caprine cheesewhey oligosaccharides in batch culture systemsrdquoBioFactors vol38 no 6 pp 440ndash449 2012

[143] C Nagler-Anderson ldquoMan the barrier Strategic defences in theintestinal mucosardquoNature Reviews Immunology vol 1 no 1 pp59ndash67 2001

[144] D S Newburg and W A Walker ldquoProtection of the neonateby the innate immune system of developing gut and of humanmilkrdquo Pediatric Research vol 61 no 1 pp 2ndash8 2007

[145] H H Tong M A McIver L M Fisher and T F DeMarialdquoEffect of lacto-N-neotetraose asialoganglioside-GM1 and neu-raminidase on adherence of otitis media-associated serotypesof Streptococcus pneumoniae to chinchilla tracheal epitheliumrdquoMicrobial Pathogenesis vol 26 no 2 pp 111ndash119 1999

[146] A L Morrow G M Ruiz-Palacios M Altaye et al ldquoHumanmilk oligosaccharides are associated with protection againstdiarrhea in breast-fed infantsrdquo Journal of Pediatrics vol 145 no3 pp 297ndash303 2004

[147] PM Simon P L Goode AMobasseri andD Zopf ldquoInhibitionof Helicobacter pylori binding to gastrointestinal epithelialcells by sialic acid-containing oligosaccharidesrdquo Infection andImmunity vol 65 no 2 pp 750ndash757 1997

[148] A Imberty Y M Chabre and R Roy ldquoGlycomimetics and gly-codendrimers as high affinity microbial anti-adhesinsrdquo Chem-istry vol 14 no 25 pp 7490ndash7499 2008

[149] M X Maldonado-Gomez H Lee D Barile M Lu and R WHutkins ldquoAdherence inhibition of enteric pathogens to epithe-lial cells by bovine colostrum fractionsrdquo International DairyJournal vol 40 pp 24ndash32 2015

[150] J Hakkarainen M Toivanen A Leinonen et al ldquoHuman andbovine milk oligossaccharides inhibitNeisseria meningitidis piliattachment in vitrordquo Journal of Nutrition vol 135 no 10 pp2445ndash2448 2005


[151] D S Newburg GM Ruiz-Palacios andA LMorrow ldquoHumanmilk glycans protect infants against enteric pathogensrdquo AnnualReview of Nutrition vol 25 no 1 pp 37ndash58 2005

[152] C F Manthey C A Autran L Eckmann and L Bode ldquoHumanmilk oligosaccharides protect against enteropathogenic Escher-ichia coli attachment in vitro and EPEC colonization in sucklingmicerdquo Journal of Pediatric Gastroenterology and Nutrition vol58 no 2 pp 165ndash168 2014

[153] A Daddaoua V Puerta P Requena et al ldquoGoat milk oligosac-charides are anti-inflammatory in rats with hapten-inducedcolitisrdquo Journal of Nutrition vol 136 no 3 pp 672ndash676 2006

[154] F Lara-Villoslada E Debras A Nieto et al ldquoOligosaccharidesisolated from goat milk reduce intestinal inflammation in arat model of dextran sodium sulfate-induced colitisrdquo ClinicalNutrition vol 25 no 3 pp 477ndash488 2006

[155] C Ashley W H Johnston C L Harris S I Stolz J LWampler and C L Berseth ldquoGrowth and tolerance of infantsfed formula supplemented with polydextrose (PDX) andorgalactooligosaccharides (GOS) double-blind randomizedcontrolled trialrdquo Nutrition Journal vol 11 no 1 article 38 2012

[156] S Fanaro B Marten R Bagna et al ldquoGalacto-oligosaccharidesare bifidogenic and safe at weaning a double-blind randomizedmulticenter studyrdquo Journal of Pediatric Gastroenterology andNutrition vol 48 no 1 pp 82ndash88 2009

[157] M E Davis A Rao S Gauthier Y Pouliot L Gourley and AAllain ldquoPreparing an aqueous solution of whey protein isolateand a proteolytic enzyme holding solution under conditionseffective to partially hydrolyze whey protein isolate to providea hydrolysate having increased ACE-suppressing activity inmammalsrdquo US Patent 6998259 2006

[158] I Recio I Lopez-Exposito A Quiros et al ldquoBioactive pep-tides identified in enzymatic hydrolyzates of milk caseins andmethod of obtaining samerdquo WO Patent 131586 2006

[159] O Tossavainen T Suomalainen J Sahlstein and A M Maki-nen ldquoProcess for producing a product containing antihyperten-sive tripeptidesrdquo US Patent 6972282 B1 2005

[160] E Damiens I Yazidi J Mazurier I Duthile G Spik and YBoilly-Marer ldquoLactoferrin inhibits G1 cyclin-dependent kinasesduring growth arrest of human breast carcinoma cellsrdquo Journalof Cellular Biochemistry vol 74 no 3 pp 486ndash498 1999

[161] X Sun R Jiang A Przepiorski S Reddy K P Palmano andG W Krissansen ldquolsquoIron-saturatedrsquo bovine lactoferrin improvesthe chemotherapeutic effects of tamoxifen in the treatment ofbasal-like breast cancer in micerdquo BMC Cancer vol 12 article591 2012

[162] B Fang M Zhang M Tian L Jiang H Y Guo and F Z RenldquoBovine lactoferrin binds oleic acid to form an anti-tumor com-plex similar to HAMLETrdquo Biochimica et Biophysica ActamdashMolecular andCell Biology of Lipids vol 1841 no 4 pp 535ndash5432014

[163] J S Mader J Salsman D M Conrad and D W HoskinldquoBovine lactoferricin selectively induces apoptosis in humanleukemia and carcinoma cell linesrdquoMolecular CancerTherapeu-tics vol 4 no 4 pp 612ndash624 2005

[164] H M Habib W H Ibrahim R Schneider-Stock and H MHassan ldquoCamel milk lactoferrin reduces the proliferation ofcolorectal cancer cells and exerts antioxidant and DNA damageinhibitory activitiesrdquo Food Chemistry vol 141 no 1 pp 148ndash1522013

[165] J R Kanwar G Mahidhara and R K Kanwar ldquoNovel alginate-enclosed chitosan-calcium phosphate-loaded iron-saturated

bovine lactoferrin nanocarriers for oral delivery in colon cancertherapyrdquo Nanomedicine vol 7 no 10 pp 1521ndash1550 2012

[166] C Freiburghaus B Janicke H Lindmark-Mansson S MOredsson and M A Paulsson ldquoLactoferricin treatmentdecreases the rate of cell proliferation of a human colon cancercell linerdquo Journal of Dairy Science vol 92 no 6 pp 2477ndash24842009

[167] C Freiburghaus H Lindmark-Mansson M Paulsson and SOredsson ldquoReduction of ultraviolet light-induced DNA dam-age in human colon cancer cells treated with a lactoferrin-derived peptiderdquo Journal of Dairy Science vol 95 no 10 pp5552ndash5560 2012

[168] N Zemann P Klein E Wetzel F Huettinger and M Huet-tinger ldquoLactoferrin induces growth arrest and nuclear accumu-lation of Smad-2 inHeLa cellsrdquoBiochimie vol 92 no 7 pp 880ndash884 2010

[169] A Varadhachary J S Wolf K Petrak et al ldquoOral lactoferrininhibits growth of established tumors and potentiates conven-tional chemotherapyrdquo International Journal of Cancer vol 111no 3 pp 398ndash403 2004

[170] Y-C Yoo R Watanabe Y Koike et al ldquoApoptosis in humanleukemic cells induced by lactoferricin a bovine milk protein-derived peptide involvement of reactive oxygen speciesrdquo Bio-chemical and Biophysical Research Communications vol 237 no3 pp 624ndash628 1997

[171] J S Mader A Richardson J Salsman et al ldquoBovine lacto-ferricin causes apoptosis in Jurkat T-leukemia cells by sequen-tial permeabilization of the cell membrane and targeting ofmitochondriardquo Experimental Cell Research vol 313 no 12 pp2634ndash2650 2007

[172] T-N Zhang and N Liu ldquoEffect of bovine lactoferricin on DNAmethyltransferase 1 levels in Jurkat T-leukemia cellsrdquo Journal ofDairy Science vol 93 no 9 pp 3925ndash3930 2010

[173] G Berge L T Eliassen K A Camilio K Bartnes BSveinbjoslashrnsson and Oslash Rekdal ldquoTherapeutic vaccinationagainst a murine lymphoma by intratumoral injection of a cat-ionic anticancer peptiderdquoCancer Immunology Immunotherapyvol 59 no 8 pp 1285ndash1294 2010

[174] Y T TungH L ChenCC Yen et al ldquoBovine lactoferrin inhib-its lung cancer growth through suppression of both inflam-mation and expression of vascular endothelial growth factorrdquoJournal of Dairy Science vol 96 no 4 pp 2095ndash2106 2013

[175] M Deng W Zhang H Tang et al ldquoLactotransferrin acts as atumor suppressor in nasopharyngeal carcinoma by repressingAKT through multiple mechanismsrdquo Oncogene vol 32 no 36pp 4273ndash4283 2013

[176] T Sakai Y Banno Y Kato Y Nozawa and M KawaguchildquoPepsin-digested bovine lactoferrin induces apoptotic cell deathwith JNKSAPK activation in oral cancer cellsrdquo Journal ofPharmacological Sciences vol 98 no 1 pp 41ndash48 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Commercial milk products containing peptides with proven antihypertensive activity

Commercial name Obtention process Protein source Active sequence(s) Publicationnumber [reference]

Peptide C12 Hydrolysis with trypsin 120572s1-Casein FFVAPFPEVFGK JP62270533 [31]Biozate Hydrolysis with trypsin Whey proteins Whey peptides US6998259 [157]

Lowpept Hydrolysis with pepsin 120572s1-Casein RYLGY AYFYPEL WO2005012355[158]

Calpis Fermentation 120573-Casein VPP IPP US5449661A[37 38]

Evolus Fermentation 120573-Casein VPP IPP US6972282 [159]

shown to be less susceptible to diseases (ie diarrhea andrespiratory diseases) than those that were not breastfed Thisprotective effect which was previously attributed to humanmilk antibodies is today strongly correlatedwith the presenceof complex oligosaccharides (OS) the third most abundantcomponent of human milk [10] Human milk is composedof OS in concentrations varying according to different stagesof lactation 20ndash23 gL in colostrum and 12ndash14 gL in maturemilk [11] being even more abundant than proteins (12 gL)[12] Human milk oligosaccharides (HMO) are complexsugars having 3 to 20 monosaccharide units [13] that are notdigestible by human enzymes [14] These compounds haveimportant functions related to the newbornrsquos developmentand health at local and systemic levels including prebioticprobifidogenic effects and antiadherence of pathogenic bac-teria [15] brain development [16] and immunomodulatoryproperties [17] among others

In the last fifty years chronic disorders have become theleading cause of morbidity and mortality in industrializedcountries with increasing incidence also observed in devel-oping countries Chronic disorders include cardiovascularand neurological diseases stroke cancers immune disor-der and chronic respiratory disease obesity diabetes andmetabolic syndrome [18] In Europe 87 of all deaths occurdue to chronic diseases and the number of people affectedis expected to rise considerably over the next few decadesThe majority of chronic diseases are caused by risk factorswhich are mostly preventable Diet and lifestyle are twoenvironmental factors that strongly affect these diseases thusmodifications of these habits are becoming a new strategy fordisease preventiontreatment

The aim of this paper is to review the recent literature onthe physiological effects of proteins peptides and oligosac-charides with special emphasis on animal and human trialsOther aspects such as the limited availability of in vivo studiesdemonstrating the biological activities of OS frombovine andcaprine milk and the current challenges associated with therecovery and commercial production of these compoundshave also been addressed

2 Impact of Milk Proteins and Peptides onthe 21st Century Diseases

21 Milk-Derived Peptides against Cardiovascular DiseasesCardiovascular diseases (CVD) have become the leading

cause of morbidity and mortality worldwide representingan important medical and public health issue [19] Althoughearlier studies associated the consumption of wholemilk withhigher incidence of CVD it has been demonstrated that milkcontains a plethora of bioactive substances which may con-tribute to the prevention of most of the risk factors of CVD[20] Recently bioactive milk peptides have gained interestbecause of their notable antihypertensive antioxidant anti-inflammatory and hypocholesterolaemic effects In this sec-tion the most current scientific information regarding invitro and in vivo studies on the role of milk proteins-derivedpeptides on CVD is summarized and discussed

211 Milk Peptides with Antihypertensive Activity Epidemi-ological studies suggest that the dietary intake of milk anddairy foods is related to decreased risk of hypertension [21]In addition to their high mineral content (eg calciumpotassium and magnesium) that can lower blood pressure[22] other milk components such as proteins and theirhydrolyzed products have been also linked to the antihy-pertensive effect of milk and dairy products Angiotensin-converting enzyme (ACE) is a multifunctional enzyme thatacts as one of the main regulators of blood pressureThus ACE inhibition is currently considered as one of thebest strategies for hypertension treatment Most biologicallyactive peptides generated from milk proteins have demon-strated ACE inhibitory activity In the last two decadesantihypertensive effects of some of these peptides have beenevaluated in spontaneously hypertensive rats (SHR) andhypertensive humans and the peptide sequences doses andmaximum decreases of systolic blood pressure (SBP) havebeen summarized in several reviews [23ndash25]The hydrolyzateobtained by the action of pepsin on casein containing the120572s1-casein-derived peptides RYLGY and AYFYPEL has beenpatented and commercialized under the name of Lowpeptby its antihypertensive properties demonstrated in bothSHR [26] and hypertensive humans [27] (Table 1) Pepsinhas been also used to hydrolyze whey protein lactoferrinwith the release of peptides containing ACE activity andACE-dependent vasoconstriction inhibitory properties [28]Antihypertensive effects in SHR after short-term and long-term treatments have been also observed for those peptides[29 30] Trypsin is another gastrointestinal enzyme used torelease the antihypertensive peptide 120572s1-casein peptide f(23ndash34) from casein during the manufacture of the commercial



























Table2Ch

emop

reventivep

ropertieso

flactoferrin

andits

deriv


againstcancerd

emon

strated

bycellcultu

reexperim

entsandanim

alsm

odels

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Breastcancer

Hum

anlactoferrin

MDA-

MB-231cells

Inhibitio

nof

cellgrow

thCellcyclearrest

[160]

Bovine

lactoferrin

4T1x

enograftBa

lbcmice

Improvem

ento

ftam

oxifenchem

opreventivee

ffects

Dow

nregulationof

proinfl

ammatorycytokines

[161]

Bovine

lactoferrin

-oleicacid

complex

MCF

-7cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[162]

Bovine

lactoferric

inMCF

-7T

-47D

and

MDA-

MB-435

cells

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]

Colon

cancer

Camellactoferrin

HCT

-116

cells

Inhibitio

nof

cellproliferatio

nAntioxidant

activ

ityInhibitio

nof

DNAdamage

[164

]

Bovine

lactoferrin

Caco-2

xeno

graft

mou

semod

elInhibitio

nof

tumor

grow

th[165]

Bovine

lactoferrin

-oleicacid

complex

HT-29

cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[166]

C26cells

Cytotoxica

ctivity

[118]

Caco-2

cells

Inhibitio

nof

cellproliferatio

n[166]

Cell

cycle

arrestby

downregulationof

cyclinE1

Bovine

lactoferric


co-2

cells

Redu

ctionof

DNAdamage

Cell

cycle

arrestby

downregulationof

cyclinE1

[167]

Colo-35

andHT-29

cells

Cytotoxica

ctivity

indu

ctionof

apop

tosis

[163]

Cervicalcancer

Bovine

lactoferrin

HeLac

ells

Inhibitio

nof

cellgrow

thIndu

ctionof

nucle

araccumulationof

Smad-2

[168]

Fibrosarcoma

Bovine

lactoferric

inMethAcells

Cytotoxica

ctivity

Tumor

cellmem

braned

isrup

tion

[118]

Headandneck

cancer

Hum

anlactoferrin

Squamou

scarcino

maO

12tumor

bearingmice

Redu

ctionof

tumor

Immun

omod

ulatoryeffects

[169]

Hepatocarcino

ma

Bovine

lactoferrin

-oleicacid

complex

HepG2cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[162]


Table2Con

tinued

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Leuk

emia

Bovine

lactoferric

in

THP-1h

uman

mon

ocyticleuk

emic

cells

Indu

ctionof

apop

tosis

Activ

ationof

ROSgeneratio

nandCa

2+M

g2+ -depend

ent

endo

nucle

ases

[170]

JurkatTleuk

emiacells

Indu

ctionof

apop

tosis

bytriggerin

gmito

chon

drial

swellin

gandreleaseo

fcytochrom

ecIndu

ctionof

cellmem

branep

ermeabilization

[163171]

Activ

ationof

ROSgeneratio


activ

ityRe

ductionof

DNAmethyltransfe

rasese

xpression

[172]

Lymph

oma

RajiandRa

mos


oma

cells

Indu

ctionof

apop

tosis

Stim

ulationof

DNAfragmentatio

nchromatin

cond

ensatio

nandnu

clear

disin

tegration

[114]

Ramos

B-lymph

omac

ellsxeno

graft

sin

SCID

beige

mice

Extensionof

survivalof

mice

[114]

Bovine

lactoferric

inA20

celllymph

omas

insyngeneic

Balbcmice

Tumor


nof

thetum

ors

Indu

ctionof

long

-term

specificc

ellularimmun

ity[173]

B16-BL

6melanom

aand

L5178Y

-ML2

5lymph

omac

ellsmetastasis

mod

elsin

syngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Lung

cancer

Bovine

lactoferrin

A549cells

Transgenicmiceo

verexpressing

hVEG

F-A165

Dow

nregulationof

proinfl

ammatorycytokines

Supp

ressionof

tumor

developm

ent

[174]

Melanom

aBo

vine

lactoferric

inB16F

10cells

Cytotoxica

ctivity

[118]

Spon

taneou

sB16-BL6

metastasis

mod

elsinsyngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Nasop

haryngeal

carcinom

aHum

anlactoferrin

5ndash8FC

NE2

and

HONE1

cells

Xeno

graft

Balbcmice

Supp

ressionof

tumorigenesisthroug

hinhibitio

nof

the

AKT

pathway

[175]

Neuroblastoma

Bovine

lactoferric

in

Hum

anMYC

N-amplified

and

non-MYC

N-amplified

neurob

lasto

ma

cells

Cytotoxica

ctivity

Destabilizationof

thec

ytop

lasm

icmem

brane

Activ

ationof


[119]

SH-SY-5Y

neurob

lasto

max

enografts

innu

derats

Redu

ctionof

thetum

orgrow

th[119]

Oralcancer

Bovine

lactoferric

inOralsqu

amou

scarcino

maS

AScell

Indu

ctionof

apop

tosis

Cleavage

ofcaspase-3andpo

ly-ADPrib

osep

olym

erase

Phosph

orylationof


kinase

andc-JunN-te

rminalkinasestre

ssactiv

ated

protein

kinase

[176]

Ovaria

ncancer

Bovine

lactoferric

inSkov3andCa

ov3

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]




















4 Future Prospects








[140]

Mice HMO (21015840-FL and31015840-FL)







EPEC
















Acknowledgments



References

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Table2Ch

emop

reventivep

ropertieso

flactoferrin

andits

deriv


againstcancerd

emon

strated

bycellcultu

reexperim

entsandanim

alsm

odels

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Breastcancer

Hum

anlactoferrin

MDA-

MB-231cells

Inhibitio

nof

cellgrow

thCellcyclearrest

[160]

Bovine

lactoferrin

4T1x

enograftBa

lbcmice

Improvem

ento

ftam

oxifenchem

opreventivee

ffects

Dow

nregulationof

proinfl

ammatorycytokines

[161]

Bovine

lactoferrin

-oleicacid

complex

MCF

-7cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[162]

Bovine

lactoferric

inMCF

-7T

-47D

and

MDA-

MB-435

cells

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]

Colon

cancer

Camellactoferrin

HCT

-116

cells

Inhibitio

nof

cellproliferatio

nAntioxidant

activ

ityInhibitio

nof

DNAdamage

[164

]

Bovine

lactoferrin

Caco-2

xeno

graft

mou

semod

elInhibitio

nof

tumor

grow

th[165]

Bovine

lactoferrin

-oleicacid

complex

HT-29

cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[166]

C26cells

Cytotoxica

ctivity

[118]

Caco-2

cells

Inhibitio

nof

cellproliferatio

n[166]

Cell

cycle

arrestby

downregulationof

cyclinE1

Bovine

lactoferric


co-2

cells

Redu

ctionof

DNAdamage

Cell

cycle

arrestby

downregulationof

cyclinE1

[167]

Colo-35

andHT-29

cells

Cytotoxica

ctivity

indu

ctionof

apop

tosis

[163]

Cervicalcancer

Bovine

lactoferrin

HeLac

ells

Inhibitio

nof

cellgrow

thIndu

ctionof

nucle

araccumulationof

Smad-2

[168]

Fibrosarcoma

Bovine

lactoferric

inMethAcells

Cytotoxica

ctivity

Tumor

cellmem

braned

isrup

tion

[118]

Headandneck

cancer

Hum

anlactoferrin

Squamou

scarcino

maO

12tumor

bearingmice

Redu

ctionof

tumor

Immun

omod

ulatoryeffects

[169]

Hepatocarcino

ma

Bovine

lactoferrin

-oleicacid

complex

HepG2cells

Inhibitio

nof

proliferatio

nIndu

ctionof

apop

tosis

[162]


Table2Con

tinued

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Leuk

emia

Bovine

lactoferric

in

THP-1h

uman

mon

ocyticleuk

emic

cells

Indu

ctionof

apop

tosis

Activ

ationof

ROSgeneratio

nandCa

2+M

g2+ -depend

ent

endo

nucle

ases

[170]

JurkatTleuk

emiacells

Indu

ctionof

apop

tosis

bytriggerin

gmito

chon

drial

swellin

gandreleaseo

fcytochrom

ecIndu

ctionof

cellmem

branep

ermeabilization

[163171]

Activ

ationof

ROSgeneratio


activ

ityRe

ductionof

DNAmethyltransfe

rasese

xpression

[172]

Lymph

oma

RajiandRa

mos


oma

cells

Indu

ctionof

apop

tosis

Stim

ulationof

DNAfragmentatio

nchromatin

cond

ensatio

nandnu

clear

disin

tegration

[114]

Ramos

B-lymph

omac

ellsxeno

graft

sin

SCID

beige

mice

Extensionof

survivalof

mice

[114]

Bovine

lactoferric

inA20

celllymph

omas

insyngeneic

Balbcmice

Tumor


nof

thetum

ors

Indu

ctionof

long

-term

specificc

ellularimmun

ity[173]

B16-BL

6melanom

aand

L5178Y

-ML2

5lymph

omac

ellsmetastasis

mod

elsin

syngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Lung

cancer

Bovine

lactoferrin

A549cells

Transgenicmiceo

verexpressing

hVEG

F-A165

Dow

nregulationof

proinfl

ammatorycytokines

Supp

ressionof

tumor

developm

ent

[174]

Melanom

aBo

vine

lactoferric

inB16F

10cells

Cytotoxica

ctivity

[118]

Spon

taneou

sB16-BL6

metastasis

mod

elsinsyngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Nasop

haryngeal

carcinom

aHum

anlactoferrin

5ndash8FC

NE2

and

HONE1

cells

Xeno

graft

Balbcmice

Supp

ressionof

tumorigenesisthroug

hinhibitio

nof

the

AKT

pathway

[175]

Neuroblastoma

Bovine

lactoferric

in

Hum

anMYC

N-amplified

and

non-MYC

N-amplified

neurob

lasto

ma

cells

Cytotoxica

ctivity

Destabilizationof

thec

ytop

lasm

icmem

brane

Activ

ationof


[119]

SH-SY-5Y

neurob

lasto

max

enografts

innu

derats

Redu

ctionof

thetum

orgrow

th[119]

Oralcancer

Bovine

lactoferric

inOralsqu

amou

scarcino

maS

AScell

Indu

ctionof

apop

tosis

Cleavage

ofcaspase-3andpo

ly-ADPrib

osep

olym

erase

Phosph

orylationof


kinase

andc-JunN-te

rminalkinasestre

ssactiv

ated

protein

kinase

[176]

Ovaria

ncancer

Bovine

lactoferric

inSkov3andCa

ov3

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]




















4 Future Prospects








[140]

Mice HMO (21015840-FL and31015840-FL)







EPEC
















Acknowledgments



References

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Con

tinued

Type

ofcancer

Animal

speciesprotein-

peptide

Celllineanim

almod

elEff

ectsmechanism

sofaction

Reference

Leuk

emia

Bovine

lactoferric

in

THP-1h

uman

mon

ocyticleuk

emic

cells

Indu

ctionof

apop

tosis

Activ

ationof

ROSgeneratio

nandCa

2+M

g2+ -depend

ent

endo

nucle

ases

[170]

JurkatTleuk

emiacells

Indu

ctionof

apop

tosis

bytriggerin

gmito

chon

drial

swellin

gandreleaseo

fcytochrom

ecIndu

ctionof

cellmem

branep

ermeabilization

[163171]

Activ

ationof

ROSgeneratio


activ

ityRe

ductionof

DNAmethyltransfe

rasese

xpression

[172]

Lymph

oma

RajiandRa

mos


oma

cells

Indu

ctionof

apop

tosis

Stim

ulationof

DNAfragmentatio

nchromatin

cond

ensatio

nandnu

clear

disin

tegration

[114]

Ramos

B-lymph

omac

ellsxeno

graft

sin

SCID

beige

mice

Extensionof

survivalof

mice

[114]

Bovine

lactoferric

inA20

celllymph

omas

insyngeneic

Balbcmice

Tumor


nof

thetum

ors

Indu

ctionof

long

-term

specificc

ellularimmun

ity[173]

B16-BL

6melanom

aand

L5178Y

-ML2

5lymph

omac

ellsmetastasis

mod

elsin

syngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Lung

cancer

Bovine

lactoferrin

A549cells

Transgenicmiceo

verexpressing

hVEG

F-A165

Dow

nregulationof

proinfl

ammatorycytokines

Supp

ressionof

tumor

developm

ent

[174]

Melanom

aBo

vine

lactoferric

inB16F

10cells

Cytotoxica

ctivity

[118]

Spon

taneou

sB16-BL6

metastasis

mod

elsinsyngeneicm

ice

Inhibitio

nof

tumor

metastasis

inlung

[117]

Nasop

haryngeal

carcinom

aHum

anlactoferrin

5ndash8FC

NE2

and

HONE1

cells

Xeno

graft

Balbcmice

Supp

ressionof

tumorigenesisthroug

hinhibitio

nof

the

AKT

pathway

[175]

Neuroblastoma

Bovine

lactoferric

in

Hum

anMYC

N-amplified

and

non-MYC

N-amplified

neurob

lasto

ma

cells

Cytotoxica

ctivity

Destabilizationof

thec

ytop

lasm

icmem

brane

Activ

ationof


[119]

SH-SY-5Y

neurob

lasto

max

enografts

innu

derats

Redu

ctionof

thetum

orgrow

th[119]

Oralcancer

Bovine

lactoferric

inOralsqu

amou

scarcino

maS

AScell

Indu

ctionof

apop

tosis

Cleavage

ofcaspase-3andpo

ly-ADPrib

osep

olym

erase

Phosph

orylationof


kinase

andc-JunN-te

rminalkinasestre

ssactiv

ated

protein

kinase

[176]

Ovaria

ncancer

Bovine

lactoferric

inSkov3andCa

ov3

Cytotoxica

ctivity

Indu

ctionof

apop

tosis

[163]




















4 Future Prospects








[140]

Mice HMO (21015840-FL and31015840-FL)







EPEC
















Acknowledgments



References

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















4 Future Prospects








[140]

Mice HMO (21015840-FL and31015840-FL)







EPEC
















Acknowledgments



References

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







[140]

Mice HMO (21015840-FL and31015840-FL)







EPEC
















Acknowledgments



References

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


References

































































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Review Article Milk Proteins, Peptides, and...

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