Ph.D. Thesis by Bjørn Petrat-Melin October 2014 - AU...

Characterization of the in vitro digestion and bioactive potential of

bovine beta- and kappa-casein variants

Ph.D. Thesis by

Bjørn Petrat-Melin

October 2014

Department of Food Science

Science and Technology, Aarhus University

Preface

The work presented in this thesis was carried out as a PhD project at the Department of

Food Science during the period 2010 – 2014. The project was funded by Aarhus University

as a PhD scholarship, and by the Danish/Swedish Milk Genomics Initiative. As part of the

PhD project, a 3 month stay at the Laboratory for Protein Chemistry, Department of

Molecular Biology and Genetics, Aarhus University, was conducted under the supervision

of Associate Professor Jan Trige Rasmussen.

Main supervisor

Associate Professor Jette Feveile Young

Department of Food Science, Aarhus University

Co-supervisor

Professor Lotte Bach Larsen


Assessment committee

Senior Scientist Niels Oksbjerg (Chairman)


Professor Gerd Vegarud

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life

Sciences, Ås, Norway

Professor Georg Erhardt

Department of Animal Breeding and Genetics, Justus-Liebig University, Gießen, Germany

Summary

Milk is the exclusive source of nutrition for the newborn mammal, and in many western

societies dairy-based foods are a staple component in the adult’s diet as well. Milk proteins

are known to be a source of bioactive peptides, that may provide a health benefit to the

newborn, as well as to the adult. Bioactive peptides are liberated from their parent protein

by proteases, e.g. during processing, or by digestive enzymes in the gastroinestinal tract.

These peptides are known to exert a wide range of physiologically relevant effects, such as

antimicrobial, antithrombotic, antihypertensive, opioid, or immune-modulating effects.

The action of proteases is highly dependent on the specific amino acid sequence of

proteins, and therefore variation in sequence may affect the release of bioactive peptides.

The bovine casein (CN) cluster is strikingly polymorphic, which is reflected in the more

than 40 described CN variants carrying amino acid substitutions in one or more positions.

The present project attempts to establish a link between genetic variation, and nutrition

and health-related properties of bovine β-CN variants A1, A2, B, and I and κ-CN variants A,

B, and E.

All seven CN variants were subjected to in vitro gastrointestinal digestion (IVGD), using

pepsin and pancreatic enzymes in two separate steps. The digestion was characterized by

assessing the degree of hydrolysis (DH), digestibility, and fragmentation pattern.

Additionally, a peptide profile of the β-CN variants was obtained by using tandem mass

spectrometry. Based on the peptide profile three peptides were chosen for synthesizing:

the two V-β-casomorphin-9 (V-βCM-9), VYPFPGPIHN1 (β-CN A1 and B) and

VYPFPGPIPN (β-CN A2 and I), as well as the β-CN variant B derived tripeptide TER. All

CN hydrolysates and derived peptides were assessed for trolox equivalent antioxidant

capacity (TEAC) and angiotensin-1 converting enzyme (ACE) inhibitory capacity. In

addition, the intestinal transport of ACE inhibitory activity of β-CN hydrolysates and

peptides was evaluated using a Caco-2 two-chamber assay.

The key results from charaterization of the IVGD were, that the β-CNs were digested to

equal DH, but gel electrophoresis indicated a different cleavage pattern of β-CN variant B,

compared to variants A1, A2, and I. This was supported by the subsequent peptide profiling,

1 The single-letter amino acid codes are used throughout the summary.

which revealed several different cleavage sites between the variants. The most notable

difference was caused by the S122R substitution in the B variant, which resulted in a novel

trypsin cleavage site. The κ-CNs were digested to a higher DH by pepsin, but reached a

lower DH by the complete IVGD, compared to the β-CNs.

The TEAC of the β-CNs was increased approx. 1.5 fold by IVGD, but no apparent

differences between variants were observed. In contrast, the TEAC of the κ-CNs increased

4 to 9 fold. Furthermore, undigested κ-CN variant A was a significantly more efficient

antioxidant than the B and E variants, and after IVGD both the A and B variants showed a

significantly higher TEAC than the E variant. Of the two 0V-βCM-9 the TEAC of

VYPFPGPIPN was 20% lower than that of VYPFPGPIHN. This was likely caused by the

H67P substitution.

Weak ACE inhibition by all undigested β-CN, but not κ-CN variants was observed, which is

a possible consequence of the different amino acids in the C-terminal tetrapeptide of these

two types of CN. ACE inhibition by all CNs was increased significantly by pepsin digestion,

however only a modest effect resulted from further pancreatic enzyme digestion. This

suggests that the C-terminals present after hydrolysis by pepsin are favorable for ACE

inhibition. The digested κ-CN variants showed stronger ACE inhibition than the digested

β-CN variants. All three peptides were ACE inhibitors, in order of potency: TER =

VYPFPGPIHN > VYPFPGPIPN. This is the first time TER and VYPFPGPIHN are described

as ACE inhibitory peptides. The intestinal brush border caused an increase in ACE

inhibition by all β-CN hydrolysates, and affected the peptides differently, as evident from

Caco-2 experiments. No transport of ACE inhibitory activity could be detected.

In conclusion, these findings reveal that genetic variation manifested in amino acid

substitutions may contribute to differences in physiological responses, i.e. fragmentation

pattern and bioactive potential. This strengthens our knowledge of genetically contingent

differences in nutritional and health-related properties of bovine milk proteins. Thus,

perhaps supporting the future development of ingredients or food products for specific

applications or targeted consumer segments, by facilitating differentiation of raw materials

in the dairy industry.

Sammendrag

Mælk er det nyfødte pattedyrs eneste ernæringskilde, og i mange vestlige samfund er

mejeriprodukter ligeledes en fast bestanddel af den voksnes kost. Mælkeproteiner er en

velkendt kilde til bioaktive peptider, der kan være gavnlige for den nyfødtes, såvel som den

voksnes, helbred. Bioaktive peptider frigives fra deres oprindelige plads i proteiner af

proteaser, for eksempel under forarbejdning, eller af fordøjelsesenzymer i mave-tarm

kanalen. Disse peptider vides at kunne have fysiologisk relevante effekt, såsom

antimikrobiel, antitrombotisk, blodtrykssænkende, opioid, eller immun-modulerende.

Proteasers aktivitet er stærkt afhængig af proteiners specifikke aminosyresekvens, hvorfor

variation i sekvensen kan påvirke frigivelsen af bioaktive peptider. De bovine kasein (KN)

gener er påfaldende polymorfiske, hvilket er reflekteret i de mere end 40 beskrevne KN

varianter, der har aminosyreudskiftning i en eller flere positioner. Det indeværende projekt

forsøger at danne en kobling mellem genetisk variation i β-KN varianterne A1, A2, B og I,

samt κ-KN varianterne A, B og E og deres ernærings- og helbredsmæssige egenskaber.

Alle syv KN varianter blev behandlet i et in vitro fordøjelsessystem (IVF), hvor der

anvendtes pepsin og pankreasenzymer i to adskilte trin. Fordøjelsen blev karakteriseret

ved at bestemme fordøjelsesgraden (degree of hydrolysis, DH), fordøjeligheden og

fragmenteringsmønsteret. Desuden blev der lavet en peptidprofil af β-KN varianterne ved

anvendelse af tandem massespektrometri. På baggrund af peptidprofilen udvalgtes tre

peptider til syntetisering: de to V-β-kasomorfin-9 (V-βKM-9), VYPFPGPIHN2 (β-KN A1 og

B) og VYPFPGPIPN (β-KN A2 og I), samt tripeptidet TER afledt af β-KN variant B. Alle KN

hydrolysater og afledte peptider blev vurderet med hensyn til deres trolox ækvivalent

antioxidant kapacitet (TEAC) og angiotensin-1 konverterende enzym (ACE) inhiberende

kapacitet. Derudover blev transport over tarmepitelet estimeret for ACE inhibering

medieret af β-KN hydrolysat eller peptider, ved anvendelse af et Caco-2 to-kammer

system.

De væsentligste resultater fra karateriseringen af IVF var, at β-KN varianterne blev

fordøjet til samme DH, men gel-elektroforese indikerede et distinkt kløvningsmønster for

β-KN variant B, sammenlignet med A1, A2 og B varianterne. Dette blev underbygget af den

2 Et-bogstavkoderne for aminosyrer anvendes i sammendraget.

efterfølgende peptidprofilering, der afslørede flere afvigelser i kløvningsmønstrene for de

fire varianter. Den mest nævneværdige forskel skyldtes S122R udskiftningen i B varianten,

som resulterede i et unikt kløvningspunkt for trypsin. κ-KN varianterne blev fordøjet til en

højere DH af den fulde IVF, sammenlignet med β-KN varianterne.

β-KN varianternes TEAC blev øget ca. 1,5 gange af IVF, uden at udvise nogen forskelle

mellem varianter. I modsætning hertil blev κ-KN varianternes TEAC øget fire til ni gange.

Desuden var A-varianten af κ-KN en signifikant bedre antioxidant end B og E varianterne

før IVF, og efter IVF udviste både A og B varianterne signifikant højere TEAC end E

varianten. Af de to V-βKM-9 havde VYPFPGPIPN en 20% lavere TEAC end VYPFPGPIHN.

Dette tilskrives aminosyreudskiftningen H67P.

Alle de ufordøjede β-KN varianter var svagt ACE inhiberende, hvilket ikke var tilfældet for

κ-KN varianterne. Dette er en mulig konsekvens af de forskellige aminosyresekvenser i det

C-terminale tetrapeptid i de to typer af KN. Alle KN varianternes ACE inhibering blev øget

af fordøjelse med pepsin, men blot en beskeden effekt af yderligere fordøjelse med

pankreasenzymer blev observeret. Dette antyder at de tilstedeværende C-terminaler, efter

fordøjelse med pepsin, er favorable for ACE inhibering. De fordøjede κ-KN varianter

udviste stærkere ACE inhibering end de fordøjede β-KN varianter. Alle tre peptider var

ACE inhibitorer rangeret i styrke således: TER = VYPFPGPIHN > VYPFPGPIPN. Dette er

første gang TER og VYPFPGPIHN beskrives som ACE inhiberende peptider. Tarmepitelet

forårsagede en forøgelse af ACE inhiberingen fra alle hydrolysater af β-KN varianter, og

havde forskellige effekter på de tre peptider. Her forblev ACE inhibering fra VYPFPGPIHN

uændret, VYPFPGPIPN var forstærket og TER var reduceret, estimeret ved forsøg med

Caco-2 celler. Der kunne ikke detekteres transport af ACE inhiberende aktivitet.

Konkluderende viser disse resultater at genetisk variation manifesteret i aminosyre-

udskiftninger muligvis bidrager til forskellige fysiologiske respons, for eksempel

fragmenteringsmønster eller bioaktivt potentiale. Dette styrker vores viden om genetisk

betingede forskelle i ernærings- og helbredsmæssige egenskaber ved bovine

mælkeproteiner. Således understøttes potentielt fremtidig udvikling af ingredienser eller

fødevarer med særlige applikationer, eller målrettet specifikke forbrugersegmenter, ved at

facilitere differentiering af råvarer i mejeriindustrien.

List of papers

This thesis is based on three papers, that can be found in appendix C. They will be referred

to throughout the thesis as papers 1 – 3. In addition, one co-authorship is listed, which will

not be included or discussed in the thesis.

Included manuscripts

Paper 1

In vitro digestion of purified beta-casein variants A1, A2, B, and I: Effects

on antioxidant and angiotensin-converting enzyme inhibitory capacity

B. Petrat-Melin, P. Andersen, J. T. Rasmussen, N. A. Poulsen, L. B. Larsen, J. F.

Young

Journal of Dairy Science, 2014, accepted for publication.

Paper 2

In vitro gastrointestinal digestion of bovine β-casein variants A1, A2, B,

and I results in different bioactive peptides identified by LC-MS/MS

Bjørn Petrat-Melin, Thao T. Le, Hanne S. Møller, Lotte B. Larsen, Jette F. Young

Manuscript intended for submission to Molecular Nutrition and Food Research.

Paper 3

Purification and in vitro digestion of bovine kappa-casein variants A, B

and E: Effects on antioxidant and angiotensin-1 converting enzyme

inhibitory capacity

B. Petrat-Melin, G. H. Kristiansen, L. B. Larsen, J. F. Young

Manuscript intended for submission to Journal of Agricultural and Food Chemistry.

The author’s contribution to the papers

Paper 1

The author: designed the experiments with some of the co-authors; performed the

experimental work with some of the co-authors; analyzed the results with some of the

co-authors; wrote the paper.

Paper 2




Paper 3




Co-authorship

Fatty acids induce angiopoietin-like 4 secretion to the apical and

basolateral side of the intestinal tissue

Søren Drud Nielsen, Karoline Blaabjerg, Bashar Amer, Bjørn Petrat-Melin, Grith

Mortensen, Randi Jessen, Hanne D. Poulsen, Trine K. Dalsgaard, Jette F. Young

Manuscript intended for submission to Lipids

Conference contributions

During the PhD project the author attended different international conferences related to

the subject matter of the studies. Contributions to conferences were poster presentations,

and one oral presentation. The poster from the LMC Food in Front conference is not

included as part of the thesis, however the remaining are.

Phytanic acid induced Glucose uptake in Porcine Primary Myotubes may

be mediated by Glucose Transporter 4

Bjørn Melin Nielsen; Brita Ngum Che; Jette Feveile Young

Poster presented at LMC Food in Front, Denmark, May 2011

Antioxidant capacity of κ-casein variant A and its hydrolysates

B. M. Nielsen, H. Najbjerg, K. Jajcevic, P. Andersen, J. Stagsted, L. B. Larsen, J. F.

Young

Poster presented at the 9th International Symposium on Milk Genomics and Human

Health, Netherlands, 6-8 Oct 2012

Purification and antioxidant capacity of β-casein genetic variants and

their hydrolysates

B. M. Nielsen, , P. Andersen, L. B. Larsen, J. F. Young

Poster presented at the 2nd International Conference on Food Digestion, Spain, 6-8

Mar 2013

Characterizing the in vitro Digestion and ACE inhibitory Peptides of

Bovine β-casein Variants

Bjørn Petrat-Melin, Thao T. Le, Hanne S. Møller, Lotte B. Larsen, Jette F. Young

Poster presented at the 11th International Symposium on Milk Genomics and

Human Health, Denmark, 6-8 Oct 2014

Purification, in vitro Digestion and Bioactivity of β-casein Variants

Bjørn Petrat-Melin

Oral presentation at the IMGC Workshop – Tools an Possibilities for Optimized Milk,

Denmark, 9-10 Oct 2014

Acknowledgements

The thesis presented here was made possible by funding from Aarhus University, the

Danish Council for Strategic Research, the Danish Cattle Federation, and Arla Foods amba.

First and foremost, my greatest thank you goes to my supervisors, Associate professor

Jette Feveile Young and Professor Lotte Bach Larsen, for their invaluable scientific input,

fruitful discussions, and not least, for their expert proof-reading of manuscripts. But most

of all, I value their seemingly endless capacity for consideration and understanding.

Also, I acknowledge the scientific input from, and discussions with, Associate Professor

Jan Trige Rasmussen at the Laboratory for Protein Chemistry, Aarhus University, during

my stay there, as well as input during the drafting of paper 1. I also truly appreciate all of

the help, and bits of “lab wisdom”, from all the skilled technicians during my fumbling

around, Hanne S. Møller, Randi Jessen, and Gitte H. Kristiansen at the Department of

Food Science, and Anni Bojsen and Margit S. Rasmussen at the Laboratory for Protein

Chemistry. A huge thank you goes to Pernille Pedersen for preparing the β-casein samples,

and to Heidi Najbjerg for setting up the method for κ-casein purification, and Gitte H.

Kristiansen for preparing the samples. And thank you to the great bunch of people

involved with the Danish-Swedish Milk Genomics Initiative for input at our always

enjoyable project meetings.

I also owe a huge thanks to all my great colleagues at the Department of Food Science in

Foulum, for creating such a relaxed and inspiring working environment. Honorary

mentions go to Søren Drud Nielsen, Simon Limbrecht Mogensen, and Jean Robert Møller

for always being available for a talk, be it work-related or otherwise; and Thao T. Le for all

the pleasant chats, and for her relentless battling with the ion-trap. In addition to being a

great colleague, Martin Krøyer Rasmussen provided valuable proof-reading of this thesis,

when it was most needed.

Finally, thank you to my family for supporting me and believing in me all the way, even

when I didn’t. And above all, THANK YOU to my girls Thea and Anna, and to my wife Sara,

I know it has been tough on you sometimes, but to me you make everything worthwhile – I

love you all very much, and I’ll be home again now!

Abbreviations

ACE = angiotensin-1 converting enzyme

Amino acids = throughout this thesis the single letter codes for amino acids is used

(please see appendix B for specific codes)

AT1 = angiotensin 1

AT2 = angiotensin 2

ATP = adenosine-triphosphate

BP = blood pressure

CMP = caseinomacropeptide

DH = degree of hydrolysis

GI tract = gastrointestinal tract

GndHCl = guanidine hydrochloride

HPLC = high performance liquid chromatography

IC50 = concentration needed to reach half-maximal inhibition

IVGD = in vitro gastrointestinal digestion

LC = liquid chromatography

MALDI-TOF = matrix-assisted laser desorption ionization – time-of-flight

MS = mass spectrometry

OPA = o-phtaldialdehyde

PAGE = polyacrylamide gel electrophoresis

QSAR = quantitative structure-activity relationship

RAS = renin-angiotensin system

RS = reactive species

SDS = sodium dodecyl sulphate

SN-TCA = soluble nitrogen in thricloroacetic acid

TEAC = trolox equivalent antioxidant capacity

TEER = transepithelial electrical resistance

TNBS = trinitrobenzenesulfonic acid

βCM = β-casomorphin

Table of contents

Outline of Thesis ...................................................................................................................... 1

Chapter 1. Introduction and General Background ..................................................................2

Bioactive Peptides ......................................................................................................... 3

Casein Polymorphism ...................................................................................................4

1.3 Aims and Hypothesis ......................................................................................................4

Chapter 2. Purification and Characterization of Casein Fractions ........................................ 6

2.2 β-Casein .......................................................................................................................... 7

2.1.2 Determination of Protein Content by Extinction Coefficients of β-CN Fractions . 9

2.2 κ-Casein ........................................................................................................................ 11

2.3 Summary of Casein Purification................................................................................... 13

Chapter 3. Gastrointestinal Digestion of Proteins ................................................................ 14

3.1 In silico Digestion ......................................................................................................... 16

3.2 In Vitro Gastrointestinal Digestion Models ................................................................. 19

3.2.1 Optimizing a Model for Simulating Gastrointestinal Digestion .......................... 20

3.3 In Vitro Gastrointestinal Digestion of β- and κ-Casein Variants ............................... 22

3.3.1 Methods ................................................................................................................. 23

Degree of Hydrolysis .................................................................................................. 23

Sodium-Dodecyl Sulphate Polyacrylamide Gel Electrophoresis .............................. 26

Peptide Profiling by Liquid Chromatography – Electrospray Ionization Tandem Mass Spectrometry ..................................................................................................... 27

3.4 Degree of Hydrolysis and Fragmentation of β- and κ-Casein Variants ..................... 30

β-Casein...................................................................................................................... 30

κ-Casein ...................................................................................................................... 36

3.5 Summary of In Vitro Gastrointestinal Digestion of β- and κ-Casein variants .......... 38

Chapter 4. Oxidation, Oxidative Stress, Antioxidants and Peptides.................................... 40

4.1 Food-derived Antioxidative Peptides ........................................................................... 41

4.2.1 Methods ................................................................................................................. 42

Overview of antioxidant capacity assays ................................................................... 42

The 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) Decolorization Assay 44

4.3 Antioxidant Capacity of β- and κ-Casein Hydrolysates and Derived Peptides ........... 45

β-Casein....................................................................................................................... 45

κ-Casein ...................................................................................................................... 48

Synthesized Peptides VYPFPGPIHN, VYPFPGPIPN, and TER ................................ 50

4.4 Summary of Antioxidant Capacity of β- and κ-Casein Hydrolysates and Peptides .... 51

Chapter 5. Hypertension, the Renin-Angiotensin System and ACE ..................................... 52

5.1 Angiotensin-1 Converting Enzyme and Inhibitors ....................................................... 53

5.1.1 Methods .................................................................................................................. 56

Evaluation of ACE Inhibition Using a Fluorescent Substrate .................................... 57

5.1.2 ACE Inhibition by β- and κ-Casein Hydrolysates and Peptides ........................... 58

β-Casein and derived Peptides .................................................................................. 58

κ-Casein ...................................................................................................................... 60

5.2 Summary of ACE Inhibition by β- and κ-Casein Hydrolysates and Peptides ............ 62

5.3 The Intestinal Brush-Border and it’s Model System................................................... 62

5.3.1 Transport of ACE Inhibition and Resistance to Brush Border Peptidases .......... 64

β-Casein Hydrolysates ............................................................................................... 64

Synthesized β-Casein Derived Peptides ..................................................................... 65

5.4 Summary of ACE Inhibition at the Brush Border ....................................................... 66

Chapter 6. General Summary, Conclusions, and Future Perspectives ................................. 67

References .............................................................................................................................. 71

Appendix A –Supplementary Data for Methods .................................................................. 84

Data Analysis ..................................................................................................................... 84

In Vitro Gastrointestinal Digestion ................................................................................... 84

ACE Inhibition Assay ......................................................................................................... 86

IC50 of ACE Inhibitory Peptides ........................................................................................ 86

Caco-2 Cell Culture .............................................................................................................87

Appendix B – Single Letter Amino Acid Codes .................................................................... 89

Appendix C – Papers ............................................................................................................. 90

Paper 1 ........................................................................................................................ 90

Paper 2 ...................................................................................................................... 123

Paper 3 ...................................................................................................................... 148

1

Outline of Thesis

This thesis consists of six main chapters. Chapter 1 will provide a short introduction to the

overall concepts and ideas, as well as present the aims and hypothesis of the project.

Chapters 2 – 5 will cover the general topics of the studies:

2. Purification and characterization of β- and κ-CN variants

3. Characterization of the in vitro of digestion of β- and κ-CN variants

4. Antioxidant capacity of β- and κ-CN variants, hydrolysates, and derived peptides

5. ACE inhibtion by β- and κ-CN variants, hydrolysates, and derived peptides

Each of the four chapters will present additional theory and background for each presented

topic; discuss and present associated methodologies; and present obtained results and

discuss these. Chapter 6 presents the general conclusions of the studies, and provides

future research suggestions and perspectives. An overview of the study elements is shown

in Figure 1.

Figure 1. Overview of the study design

2

Chapter 1. Introduction and General Background

The fundamental function of milk is to serve as the exclusive source of nutrition for the

neonate mammal, and consequently it must contain all required nutrients to ensure the

growth and survival of the neonate. Dietary protein is one of these essential nutrients

because it supplies the body with amino acids that are used for growth and maintenance of

cells and tissues. This is exemplified by the severe consequences of protein energy

undernutrition, which leads to growth stunting and wasting (Onís et al., 1993). Not only is

protein a vital part of the diet, it is also paramount that the quality of the protein is

adequate, and not all protein is created equal. Among the 20 common amino acids the

body has the capacity to synthesize 12, however 8 are essential in the sense that they

cannot be synthesized by humans. Because of it’s essential role for the neonate the amino

acid profile of milk proteins is perfectly balanced for the mammalian physiology. Bovine

milk and dairy products are staple components of the typical western diet, supplying more

than 8% of dietary energy, and 19% of protein ingested by European consumers

(www.fao.org, accessed Sep. 29th 2014). Thus, milk proteins may have a vast influence on

the nutritional status of consumers. It is recognized that, in addition to serving as a source

of amino acids, milk proteins can have significant effects on health. Examples are the

antimicrobial lactoferrin, the immuno-modulating

osteopontin, and immunoglobulins (Denhardt and

Guo, 1993; Lönnerdal and Iyer, 1995; Nuijens et al.,

1996; Farrell et al., 2004). These proteins are all

among the whey proteins, however, the most

abundant proteins in bovine milk are the caseins

(CNs), constituting approx. 80% of the total protein

in milk (Farrell et al., 2004; Krisciunaite et al.,

2012). There are four types of CN: αs1-, αs2-, β-, and

κ-CN, and their relative amounts are shown in Table

1. The CNs were originally considered as merely a

macronutrient source, but during the most recent

decades, studies have indicated that there may be

considerable impact on health from CN derived

bioactive peptides.

Table 1. The relative content

of proteins in bovine milk1

Protein %2

αs1-casein 33,1

αs2-casein 8,2

β-casein 32,3

κ-casein 9,1

β-lactoglobulin 9,4

α-lactalbumin 3,2

Minor proteins3 4,7

1 Mean of values compiled from

(Farrell et al., 2004; Krisciunaite et

al., 2012). 2 Percentage of total protein in milk. 3 E.g.Serum albumin,

immunoglobulins, lactoferrin.

3

Bioactive Peptides

Bioactive peptides are encrypted within the sequence of proteins. They are released by the

action of proteolytic enzymes, and have an impact on cells or tissues of the body. Bioactive

peptides derived from CNs have been associated with a range of different health-related

effects, and several reviews have been published on this matter (Meisel, 1997; 1998; Clare

and Swaisgood, 2000; Meisel and FitzGerald, 2003; Silva and Malcata, 2005). An overview

of some the potential effects attributed to these peptides is given in Figure 2. It is clear that

there may be a great potential in bioactive peptides for optimizing nutrition-based

interventions in the prevention of lifestyle diseases, such as cardiovascular diseases, type-2

diabetes, hypertension, or obesity. Two well-described effects of milk peptides are

antioxidant capacity and angiotensin-1 converting enzyme (ACE) inhibition (FitzGerald

and Meisel, 2000; Pihlanto, 2006). Studies have shown that both these activities are highly

dependent on the amino acid composition, as well as the specific amino acid sequence of

the peptides (Cushman et al., 1973; Chen et al., 1998). Since proteolytic enzymes hydrolyze

peptide bonds according to their pre-determined specificity, a given combination of

protein and protease will result in a unique combination of peptides. Together, this has

consequences for bioactive potential. In one study it was found that the dipeptidyl

peptidase IV inhibitory activity of milk protein hydrolysates was significantly affected by

the choice of proteases (Lacroix and Li-Chan, 2012). Conversely, it may be argued that

changes in amino acid sequence could have a similar impact on bioactivity.

Figure 2. Overview of potential physiological effects accredited to casein derived bioactive peptides. Modified from (Silva and Malcata, 2005; Hartmann and Meisel, 2007).

4

Casein Polymorphism

The bovine CNs are highly polymorphic. Presently, 15 β-CN and 13 κ-CN variants with

amino acid substitutions have been described (Caroli et al., 2009; Gallinat et al., 2013).

This has previously been shown to be of relevance during gastrointestinal digestion. It was

reported that the opioid peptide β-casomorphin-7 (βCM-7) is released from β-CN variant

A1 during digestion, and not from variant A2 (Jinsmaa and Yoshikawa, 1999; Noni, 2008).

βCM-7 can act as a µ-opioid receptor agonist, stimulate intestinal mucous secretion, inhibit

ACE, slow gastric motility, and more (Brantl et al., 1981; Teschemacher et al., 1997;

Claustre et al., 2002; ul Haq et al., 2014). Furthermore, in vitro gastrointestinal digestion

(IVGD) of β-CN variants A1, A2, and B and κ-CN variants A, B, and E, using pepsin, trypsin

and chymotrypsin, resulted in peptides that were unique to each variant (Lisson et al.,

2013). This was later found to be of consequence for the allergenicity of the different

variants (Lisson et al., 2014). Thus, evidence is mounting that genetic variations

manifested in the protein sequence may affect the health-related properties of bovine CNs

upon gastrointestinal digestion. In order to study the specific digestion pattern, and

investigate the bioactive potential of different CNs, they should preferably by available in a

pure form. There are two general strategies to obtain pure β- or κ-CN variants, (1) they can

be separated from milk containing multiple variants, or (2) the total β- or κ-CN fractions

can be isolated from samples obtained from cows with a known CN haplotype. Because of

available genetic information (see below), the latter simpler approach was taken in this

studies.

1.3 Aims and Hypothesis

The work presented in this thesis was carried out under the framework of the Danish-

Swedish Milk Genomics Initiative. In the Danish part of that project milk samples from

the morning milking of more than 800 cows were collected, and subsequently analyzed for

macro-composition by infra-red scanning, and genotyped for the major milk proteins

(Jensen et al., 2012a; Poulsen et al., 2013). This allowed the identification of seven milk

samples containing pure variants of β- or κ-CN. The overall aim of this project was to

establish, whether genetic polymorphisms resulting in amino acid substitutions in bovine

β-CN variants A1, A2, B, and I and κ-CN variants A, B, and E may affect parameters of

5

relevance for human nutrition and physiology. The investigated parameters were:

digestibility, pattern of hydrolysis, and the influence of digestion on antioxidant and ACE

inhibitory capacity. Based on the above discussion it is hypothesized that

Amino acid substitutions affect in vitro gastrointestinal digestion of different

bovine β- and κ-CN variants

Antioxidant and ACE inhibitory capacity of bovine β- and κ-CN variants are

affected by digestion and acid amino substitutions

Different variants of β-CN give rise to peptides differing in amino acid sequence

and bioactivity upon in vitro gastrointestinal digestion

6

Figure 3. Schematic representation of the proposed structure of the casein (CN) micelle in milk. αs- and β-caseins (orange) interact electrostatically with calcium-phosphate nanoclusters (grey) to stabilize the interior network. Some β-CN (blue) interact through hydrophobic interactions to the other CNs. κ-CN is located on the surface of the micelle (green), with its hydrophilic caseinomacropeptide extended outward (black). (Adapted from Dalgleish and Corredig, 2012).

Chapter 2. Purification and Characterization of Casein Fractions

The natural environment of most proteins is extremely complex, e.g. blood, cellular

cytoplasm, or milk. Therefore, in order to study their specific behavior under defined

conditions, they need to be separated from all the other proteins and biomolecules that are

present in that environment. This is achieved by a series of physical or chemical processes,

taking advantage of the distinct physicochemical properties of the target protein, while

sufficiently retaining its native characteristics. This latter part is especially important for

proteins that fulfill a specific function that relies on their three-dimensional structure, e.g.

enzymes, receptors, or antibodies. However, the main function of the CNs is to serve as a

source of nutrition, and possibly bioactive peptides, for the neonate, and consequently the

structure of the individual CNs is relatively disordered (Horne, 1998; Horne, 2002).

However, the four CNs (αs1, αs2, β, and κ) are clustered together in the casein micelle,

which does have a unique macromolecular structure. There has been some debate over the

years regarding which model most accurately describes the structure of casein micelles. In

their recently published review of the different theories Dalgleish and Corredig (2012)

describe the structure as a network of αs- and β-CN molecules, that interact

electrostatically through their phosphoserine groups with calcium nano-clusters in the

7

interior of the micelle (Figure 3). κ-CN is located at the surface, where it’s N-terminal part

(para-κ-CN) interacts hydrophobically with the other CNs. The C-terminal part

(caseinomacropeptide, CMP) is extended outward from the surface of the micelle, resulting

in electrostatic repulsion between micelles (Dalgleish and Corredig, 2012).

The traditional method for separating the CNs is based on the work of Hipp et al., who

described their different solubility in aqueous solutions of urea at different concentrations

(Hipp et al., 1952). The use of urea can be problematic because of its equilibrium with

cyanate in aqueous solutions. Cyanate may cause carbamylation of K and R side-chains, as

well as N-terminals (Stark et al., 1960; Kollipara and Zahedi, 2013) (throughout this thesis

the single-letter amino acid codes are used, please see appendix B). These modifications

may hinder the action of trypsin during digestion, modify biological activity, or interfere

with peptide identification by LC-MS (Lippincott and Apostol, 1999; Mun and Golper,

2000). For these reasons a purification procedure devoid of urea was desirable.

2.2 β-Casein

Part of the β-CN located in the CN micelle does not participate in the electrostatic

interactions with calcium nano-clusters, rather, this β-CN pool is loosely bound to the

other CNs through hydrophobic interactions. These interactions are weakened when the

temperature is lowered to 4 °C, thereby allowing this pool of β-CN to dissociate from the

CN micelle (Creamer et al., 1977; O'Connell et al., 2003; Dalgleish and Corredig, 2012).

This property of β-CN was previously utilized in the development of a purification method

at the department of Food Science, Aarhus University. Four milk samples, containing the

pure β-CN variants A1, A2, B, and I, were identified and used in the following procedure

(described in detail in paper 1). In brief, the milk was thawed and maintained at 4 °C for 48

hours, and was subsequently ultracentrifuged to sediment the CN micelles containing the

αs-, κ-, and undissociated β-CN. The supernatant, which consisted of whey and the

dissociated β-CN, was then acidified to pH 5, which is the isoelectric point of β-CN.

Thereby, the β-CN precipitated and could then be retrieved by centrifugation at 1000 × g,

and freeze-dried for storage at -80 °C.

8

Figure 4. Bovine β-casein variants A1, A2, B, and I were purified from milk samples, obtained from cows that were homozygous at the β-casein locus, and analyzed by liquid chromatography combined with mass spectrometry. The molecular weight (Da) of the variants are indicated with the variant name in paranthesis. Horizontal brackets indicate retention times of different milk proteins. Data are normalized to the maximum value within each individual chromatogram. All variants were analyzed in triplicate. (Paper 1).

The protein composition of the purified β-CN was estimated by liquid chromatography

(LC), which was coupled to mass spectrometry (MS) for verifying the molecular weights

(MWs) of the individual variants. The chromatograms and determined MWs are shown in

Figure 4. The Chemstation software package (Agilent Technologies, US) was used to

integrate the areas under the curve of individual peaks. The position of the different milk

proteins are indicated in Figure 4, based on previous work using the same LC-MS system

(Jensen et al., 2012b). The areas were used to calculate the relative amounts of the milk

proteins in the purified fractions. The relative β-CN content was approx. 90% (Table 2),

indicating a good separation of β-CN from the other milk proteins. Thus, with this method

it is possible to purify β-CN without using urea, and to an adequately high degree of purity

for the intended use.

9

AA

AA i Protein

nProtein(%) 100%

N V C

Table 2. Relative content (%) of milk proteins in the isolated β-

casein variant preparations. Values were calculated as relative

peak areas within each chromotogram by LC. (Paper 1)

β-casein

variant β-casein κ-casein α-casein

Whey

proteins

A1 89.7 ± 1.4 2.3 ± 0.4 4.6 ± 1.7 3.5 ± 2.5

A2 93.2 ± 1.7 0.5 ± 0.3 1.0 ± 0.5 3.2 ± 2.6

B 89.0 ± 0.1 5.6 ± 1.4 3.4 ± 0.3 1.4 ± 1.1

I 90.3 ± 1.1 2.9 ± 0.7 4.8 ± 0.8 1.7 ± 0.2

All values are expressed as mean percentage of total peak area ±

SEM (n = 3).

2.1.2 Determination of Protein Content by Extinction Coefficients of β-CN

Fractions

It is necessary to know the absolute protein content of a preparation to be able to achieve

the desired concentration in experimental solutions. For this purpose quantitative amino

acid analysis was chosen, because it is recognized as a gold standard for protein

determination. The procedure used is described in detail in paper 1, and is modified from

Barkholt and Jensen (1989). In brief, the β-CN fractions were acid hydrolyzed in 6 M HCl

at 110 °C overnight, then reconstituted in 10 mM HCl followed by ion-exchange

chromatography for separation of the amino acids. Quantification of the individual amino

acids was achieved by post-column derivatization with o-phtaldialdehyde (OPA) and

fluorometric detection, and then comparing the signal to that of an amino acid standard

solution. Integration of the spectrum peak areas provided the amount of individual amino

acids in the sample. This was used to calculate the amount of the original protein using

equation (1)

(1)

where nAA is the molar amount of a specific amino acid, NAA is the number of this amino

acid in the protein sequence, Vi is the initial volume of sample, and CProtein is the

10

concentration of β-CN under the assumption of 100% protein content in the weighed

sample material. Calculations were based on the average of values obtained based on G

and A residues (paper 1). The protein content of the β-CN fractions was between 69% and

82%. Unfortunately, this method was unavailable in-house, and in addition, is rather time-

consuming. Therefore, a more convenient method for protein determination of purified β-

CN fractions was desirable. A fast, easy, and inexpensive way of determining the

concentration of protein in aqueous solutions is to simply measure their absorbance at a

wavelength of 280 nm. This method builds on the pioneering work of Edelhoch, who

determined the molar extinction coefficients at 280 nm (ε280) of W, Y, and C dissolved in 6

M guanidine hydrochloride (GndHCl) (Edelhoch, 1967). This was later elaborated on by

others (Gill and von Hippel, 1989; Pace et al., 1995). These values were used to construct

equation (2) for predicting ε280 of samples containing several different proteins in known

relative amounts

(2)

where #Trp(i) is the number of W residues in the i´th protein, #Tyr(i) is the number of Y

residues in the i´th protein, #Cys(i) is the number of C residues in the i´th protein, MW(i) is

the molecular weight of the i´th protein, and Frac(i) is the relative amount of the i´th

protein in the sample (from Table 2), and where i is αs-CN, β-CN, κ-CN, or whey proteins.

ε280 of the β-CN fractions was determined accordingly, and compared to the values

predicted using equations (1) and (2) (Table 3). The discrepancies between the measured

and the predicted values were comparable to those found in an extensive study of 80

different proteins (Pace et al., 1995). In that study ε280 was determined experimentally and

compared to the expected values, and deviations of up to 16% was found. A rearrangement

of equation (2) allows the calculation of β-CN’s contribution to ε280. Changes in the

deviations were observed, and β-CN variant B now deviated more than 15%. This may be

attributable to an error in the relative protein determination by LC, where the detection of

protein was carried out by measuring absorbance at 214 nm. Quantitative measurements

of protein at this wavelength are quite reliable, and mainly proportional to the peptide

back-bone structure. However, there can be some specific amino acid induced variation,

and protein structure may influence readings as well (Kuipers and Gruppen, 2007).

( i ) ( i ) ( i )

( i )

( i )

#Trp 5500 #Tyr 1490 #Cys 125PredictedAbs( 280 ) Frac

MW

11

Using known values for the total and relative CN content of the milk samples from which

the β-CN was purified (Jensen et al., 2012b), the approximate calculated yield of β-CN

purified here was between 5% and 20%. This is a somewhat modest yield, and certainly

much lower than the 30% and 88% previously reported by researchers using procedures

incorporating urea (Garnier et al., 1964; Cayot et al., 1992). The low yield is likely a

reflection of the fact, that the β-CN being retrieved using the method presented here,

consists only of the fraction that is loosely bound to other CNs in the micelle through

hydrophobic interactions, as discussed above.

2.2 κ-Casein

Efforts were made to also develop a method free of urea for the purification of pure κ-CN

variants, however, the attempts were unfortunately not successful. Instead, a more

traditional method was developed at the Department of Food Science, Aarhus University

(described in detail in paper 3). As evident from Figure 3, κ-CN is situated mostly on the

surface of the CN micelle, and consists of two distinct regions. The CMP region of κ-CN,

rom position 106-169, is characterized by having two phosphorylation and three

glycosylation sites. In the Danish Holstein-Friesian and Jersey breeds approx. 95% of total

κ-CN was phosphorylated and approx. 35% was glycosylated (Jensen et al., 2012a). These

post-translational modifications (PTMs) are charged and believed to increase the stability

of the CN micelle macrostructure through electrostatic repulsion at the surface (Horne,

1998; Dalgleish and Corredig, 2012). In contrast to αs- and β-CN, κ-CN does not have a

Table 3. Molar extinction coefficients (ε)1 of β-casein variants at λ = 280 nm, measured

in 6 M guanidine hydrochloride. Values are given as means ± SEM (n = 3). (Paper 1)

β-casein

variant

ε (fraction)2

measured % Dev3

ε (β-casein)4

measured % Dev3

A1 13398 ± 483 2.66 10547 ± 380 - 7.94

A2 12642 ± 1033 5.38 11238 ± 918 - 1.94

B 12142 ± 948 - 5.55 9659 ± 754 - 15.75

I 14191 ± 1154 10.93 11448 ± 931 - 0.04

1 M-1cm-1. 2 Total purified fractions. 3 Deviation from predicted values. 4 Molar extinction coefficients deriving from β-casein alone.

12

cluster of phosphoserine residues with which it may interact with calcium nano-clusters. A

consequence of this is that κ-CN, unlike αs- and β-CN, is soluble in the presence of calcium

ions. By slowly increasing the concentration of calcium ions at neutral pH it was possible

to precipitate much of the αs- and β-CN, and thereby obtain a κ-CN enriched solution.

With cation-exhange fast protein LC the enriched solutions were purified further, and a

representative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE,

see section 3.3.1) analysis of the obtained fractions is given in Figure 5. It is clear that the

samples had been enriched with κ-CN by the calcium precipitation step. The relative

location of bovine CNs in SDS-tricine gels has previously been shown (Pardo and

Natalucci, 2002). This was used to select the fractions in the present study estimated to

contain predominantly κ-CN. These were pooled, dialyzed and characterized further. In

contrast to the β-CN variants, LC-MS analysis of the selected κ-CN fractions did not

indicate the presence of contamination by other milk proteins (Figure 6). Moreover, in a

recently published report an anion exchange based procedure was used for the purification

of κ-CN variants, which resulted in some contamination of κ-CN variant E by αs2-CN

(Lisson et al., 2013). Thus, it appears that preceding ion exchange with calcium

precipitation may improve the purity of κ-CN fractions.

Figure 5. SDS-PAGE of fractions obtained by cation-exchange fast protein liquid chromatography. Molecular weight markers are indicated to the left, fraction numbers are at the top, arrows show the location of the different caseins, and the bracket indicates samples 23-38, which were pooled before further analysis.

Selected fractions

13

2.3 Summary of Casein Purification

The method for β-CN purification presented here, while not appropriate for large scale, is

suitable for small-scale applications and research purposes, and the absence of urea makes

it especially suitable for IVGD studies. In addition, a combination of LC assisted

determination of relative protein composition, and absorbance at 280 nm appears

applicable for a relatively fast estimation of absolute protein content in the purified β-CN

fractions. For κ-CN, the presented method may improve upon previously reported

methods by the incorporation of calcium precipitation. However, calcium precipitation did

not sufficiently enrich the samples with κ-CN, and thus chromatography was still needed,

and it was therefore not possible to avoid the use of urea.

Figure 6. Bovine κ-casein variants A, B, and E were purified from milk samples, obtained from cows that were homozygous at the κ-casein locus, and analyzed by liquid chromatography combined with mass spectrometry. The molecular weight (Da) of the variants are indicated with the variant name in paranthesis. Horizontal brackets indicate region of post-translationally modified κ-casein. Data are normalized to the maximum value within each individual chromatogram. All variants were analyzed in triplicate. (from Paper 3).

14

Chapter 3. Gastrointestinal Digestion of Proteins

The human digestive system consists overall of two systems working together to break

ingested foodstuffs into sufficiently low MW structures, which are readily absorbed into

the circulation. These two systems are the gastrointestinal tract (GI tract) and its

accessory glands (Figure 7). The GI tract is effectively a cavity that runs all the way

through the body, beginning at the mouth and progressing through the esophagus into the

stomach, and further on into the small intestine. The small intestine is followed by the

large intestine that ends in the anus, where undigested and unabsorbed material is

expelled as feces. The accessory glands are the salivary glands, the gastric glands, the liver,

the gallbladder and the pancreas, which together secrete enzymes and other compounds

that are needed in the digestion process. Foodstuff is first broken down mechanically in the

mouth by chewing, or mastication, which serves to mix the food with saliva containing

amylase, which initiates the hydrolysis of starch and provides lubrication. The food is then

moved into the stomach, where it encounters an acidic environment with a pH of approx.

2.0 (Kalantzi et al., 2006). The purpose of the low pH value in the stomach is threefold: (1)

reducing the survival of many foodborne pathogenic bacteria; (2) enabling the conversion

of pepsinogen into active pepsin, the proteolytic enzyme of the stomach; and (3)

facilitating the denaturation of proteins for more efficient proteolysis by pepsin.

Figure 7. Overview of the gastrointestinal tract and accessory glands. (from en.wikipedia.org/wiki/ Digestion).

15

Pepsin (EC 3.4.23.1) is secreted as the precursor pepsinogen by the chief cells located in

the gastric glands in the gastric pits of the stomach (Figure 8). Concomitantly, the parietal

cells release hydrochloric acid which creates a low pH environment, that facilitates the

partial unfolding and autocatalytic hydrolysis of pepsinogen into active pepsin (Stepanov

et al., 1973; Kageyama, 2002). The active pepsin will then hydrolyze internal peptide

bonds of ingested proteins, preferentially at sites of aromatic and hydrophobic amino

acids, such as Y, F and L (Inouye and Fruton, 1967). However, pepsin is also known to

cleave proteins at several other sites, i.e. the specificity of pepsin appears to be rather

broad and influenced by amino acids several positions away from the scissile bond (Keil,

1992; Rawlings et al., 2014). The protein, which is now cleaved into smaller fragments, will

then be transported through the pyloric sphincter into the first section of the small

intestine, called the duodenum. The gastric half-emptying time for solutions of both intact

and pre-hydrolyzed milk proteins was found to be around 20 minutes (Calbet and Holst,

2004). In the upper duodenum the gastric content encounters bile and pancreatic juice

secreted by the gallbladder and pancreas, respectively. These contain, among other things,

bicarbonate to neutralize the stomach acid, increasing the pH to approx. 6.5 (Kalantzi et

al., 2006). A range of digestive enzymes are also secreted at this point, to further digest

starch, lipids, and nucleic acids, as well as proteins and peptides. That proteins are a highly

diverse class of macromolecules is evidenced by the diverse set of proteases that are

present in the duodenum. These include trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1),

elastase (EC 3.4.21.36), carboxypeptidases (EC 3.4.17.x), and aminopeptidases (EC

3.4.11.x) (Grønborg et al., 2004; Bourlieu et al., 2014), that together will cleave many of

Figure 8. Gastric pit and gastric gland with different cell types and their location indicated. Mucous cells secrete protective mucus, the parietal cells secrete HCl, the chief cells secrete pepsinogen and the enteroendocrine, or G cells, secrete gastrin. (Frank Boumphrey, M.D., via commons.wikimedia.org/ wiki/File:Gastric_gland.png)

16

the 400 different peptide bonds encountered in a protein composed of the 20 common

amino acids. Trypsin will cleave C-terminally to K and R, chymotrypsin will cleave

predominantly C-terminally to aromatic and bulky amino acids, and elastase will cleave C-

terminally to A, V, and to some extent L (from the MEROPS peptidase database,

merops.sanger.ac.uk/index.shtml (Rawlings et al., 2014)). Aminopeptidase and

carboxypeptidase will cleave amino acids off the N- and C-terminals, respectively. The

luminal digestion by pancreatic proteases only accounts for part of the process of breaking

proteins into readily absorbable fragments, with the remaining digestion managed by

brush-border proteases. These appear to be active mainly against tri-, tetra-, and larger

peptides (Pauletti et al., 1996). Once the ingested proteins have encountered all these

proteases, they will mostly be in the form of free amino acids, or di- and tripeptides. Some

larger fragments can, however, be resistant to complete hydrolysis by gastrointestinal

proteases, including brush-border proteases (Picariello et al., 2010). The digestion of

protein can be investigated using different model systems, some of which will be discussed

in the following sections.

3.1 In silico Digestion

Computer software can be used to do in silico simulations of a number of processes. A

simple search for entries in the Web of Science database that contain the expression “in

silico” in the title returned over 5000 results (www.webofknowledge.com, accessed Oct 27,

2014). Digestion of proteins can be simulated using in silico approaches, and many

different programs have been developed to this end, in large part as tools used in mass

spectrometric proteomics, e.g. mMass (Strohalm et al., 2008) or MS-Digest (Baker and

Clauser, http://prospector.ucsf.edu). Such programs are based on algorithms developed on

the basis of experimental cleavages of known sequences, and otherwise vary in their level

of probabilistic complexity. Besides their use in traditional proteomics these tools can also

be utilized as a technique for computational modelling of gastrointestinal protein

digestion. However, to be useful in this regard there must be the possibility of choosing the

relevant proteolytic enzymes for the analysis to properly reflect in vivo digestion. In the

present study in silico digestion was used as a preliminary tool to aid the generation of

hypothesis.

17

The results of digesting β-CN variants A1, A2, B and I using two different online tools are

shown in Table 4. Some of the differences that are evident between the two approaches

probably reflect the fact that different enzymes were used. However, if only pepsin, trypsin

and chymotrypsin A are used to digest β-CN variant A1 in the Biopep tool, i.e. the same

enzymes as was used in PeptideCutter, one of the resulting peptides is 65PGPIHNSL70,

which is not seen among the output peptides of PeptideCutter. This implies that these two

programs must rely on distinct underlying algorithms for estimating enzyme cleavage sites

in a given input sequence. Most of the peptides in Table 4 are different for the two

programs in the regions of the protein polymorphisms. However, both programs reveal

differences between β-CN variants upon hydrolysis, indicating that the amino acid

substitutions in these variants may be of importance for in vitro and/or in vivo digestion.

There are also similarities to be found, as both programs result in the fragment 89QPEV92,

however it stems from the A1, A2 and B variants when using Biopep, and from the I variant

when using PeptideCutter. The B variant gives rise to the fragments 120TER122 and 123QSL125

Table 4. Peptides resulting from the in silico digestion of β-casein variants A1, A2, B and I using two different programs. In the Biopep program the enzymes pepsin, trypsin, chymotrypsin A, elastase and elastase II were chosen. In PeptideCutter the enzymes pepsin, trypsin and chymotrypsin (low specificity) were chosen. Only peptides stemming from regions of amino acid substitutions and not found in the digests of all four variants are shown. Positions of substitutions are in bold.

β-casein variant2

Program Position1 Sequence A1 A2 B I

Biopep enzyme(s) action3

65 - 66 PI x x

67 - 70 HNSL x x

67 - 70 PNSL x x

89 - 92 QPEV x x x

89 - 93 QPEVL x

120 - 125 TESQSL x x x

120 - 122 TER x

123 - 125 QSL x

PeptideCutter4 59 - 67 VYPFPGPIH x x

59 - 70 VYPFPGPIPNSL x x

68 - 70 NSL x x

89 - 93 QPEVM x x x

89 - 92 QPEV x

120 - 124 TESQS x x x

120 - 122 TER x

123 - 125 QSL x 1 Position of the peptide within the mature protein. 2 (x) indicates the presence of this peptide in the digest of this particular variant. 3 www.uwm.edu.pl/biochemia/index.php/en/biopep. 4 web.expasy.org/peptide_cutter.

18

from both programs, and are the only fragments where complete consensus between the

two is observed. An identical analysis was carried out for the κ-CN variants A, B, and E,

and here a different outcome is seen (Table 5). There are no identical peptides between the

two programs output of peptides from the regions with amino acid substitutions. In

addition, quite large fragments were resistant to digestion using PeptideCutter. If the

fragments from Biopep using the same enzymes as PeptideCutter are considered, there

was, however, almost agreement between the simulations (not shown). Weimann et al.

reported the generation of distinct peptides from κ-CN variants using the Biopep enzyme

tool (Weimann et al., 2009). Some of these peptides were shown to be ACE inhibitory, e.g.

the tripeptide 148ASP150 from the B and C variants. This peptide was a result of including

chymotrypsin C in that study.

Table 5. Peptides resulting from the in silico digestion of κ-casein variants A, B and E using two

different programs. In the Biopep program the enzymes pepsin, trypsin, chymotrypsin A, elastase

and elastase II were chosen. In PeptideCutter the enzymes pepsin, trypsin and chymotrypsin (low

specificity) were chosen. Only peptides stemming from regions of amino acid substitutions and

not found in the digests of all four variants are shown.

κ-casein

variant2

Program Position1 Sequence A B E

Biopep

enzyme(s)

action3

129 - 138 EPTSTPTTEA x x

129 - 138 EPTSTPTIEA x

147 - 152 EDSPEV x x

147 - 148 EA x

149 - 152 SPEV x

154 - 159 ESPPEI x x

154 - 155 EG x

156 - 159 PPEI x

PeptideCutter4 117 - 145 TEIPTINTIASGEPTSTPTTEAVESTVAT x x

117 - 145 TEIPTINTIASGEPTSTPTIEAVESTVAT x

147 - 169 EDSPEVIESPPEINTVQVTSTAV x

147 - 169 EASPEVIESPPEINTVQVTSTAV x

147 - 169 EDSPEVIEGPPEINTVQVTSTAV x

1 Position of the peptide within the mature protein. 2 (x) indicates the presence of this peptide in the digest of this particular variant. 3 www.uwm.edu.pl/biochemia/index.php/en/biopep. 4 web.expasy.org/peptide_cutter.

19

It is clear from these analysis of β- and κ-CN variants that there are obvious differences

between the algorithms employed by the different computer simulations. Nevertheless,

both in silico procedures resulted in peptides that were unique to some variants, while

being absent from others. This is a clear indication that even the few amino acid

substitutions seen in these CN variants may be of some relevance regarding their fate in

the digestive system, and possibly also for their bioactive potential. For example, κ-CN

derived peptides EA (B variant, Table 5) and EG (E variant, Table 5) were previously found

to be ACE inhibitory (Das and Soffer, 1975; Cheung et al., 1980). The βCMs derived from

positions 60-70 of β-CN are also characterized as bioactive peptides. It is therefore

appropriate to investigate the digestion of these CN variants further, using an in vitro

digestion model.

3.2 In Vitro Gastrointestinal Digestion Models

In vitro models aimed at simulating gastrointestinal digestion provide a useful tool for

investigating the breakdown of foodstuff, and the release of it’s components. They have the

advantage of avoiding the need for human subjects, which is often associated with

substantial expense and ethical concerns, as well as considerable variation between

individuals. On the other hand, a main disadvantage of in vitro digestion models is the

challenge associated with replicating the complexity of in vivo digestion, which is a highly

dynamic process with a continuous flow of various enzymes, substrates, and other

compounds (Calbet and Holst, 2004; Camilleri, 2006). The simplest type of in vitro

models are static one chamber systems, where each phase of digestion is carried out in a

single reaction chamber without changes in buffer composition. Digestion models of this

type usually consist of a gastric phase, where the food or food-components are incubated

with pepsin in an acidic environment, typically around pH 2, for anywhere from 15

minutes to several hours. The in vitro gastric digestion is then followed by a step that

attempts to mimic the duodenal environment. Here, pH is increased to around 7, and

various combinations of pancreatic and brush border enzymes, as well as bile salts and

phospholipids, can be added (Wickham et al., 2009; Dupont et al., 2010; Hur et al., 2011).

Conversely, there are digestion models of much higher complexity, that may consist of

several reaction chambers, and where the flux of substrates, enzymes, buffers, and fluid

20

Figure 9. SDS-PAGE of in vitro digested commercial casein. Minutes of digestion are indicated at the top. The casein was digested at 37 °C with pepsin for 60 minutes at pH 2.0, and then with pancreatic enzymes at pH 6.5 up to a total of 180 minutes.

flows are intricately controlled by computer algorithms, based on data gathered from in

vivo studies of the human digestion system (Minekus et al., 1995; Menard et al., 2014).

However, these complex systems require expensive equipment, and are generally not

suited for processing very small sample sizes. In addition, when the component under

investigation is separated from its native food matrix, e.g. β- or κ-CN isolated from milk,

the use of such elaborate methods seems excessive. For these reasons, a digestion model of

the simpler static type was chosen for use in the present studies, further described in

section 3.2.1 below.

3.2.1 Optimizing a Model for Simulating Gastrointestinal Digestion

The most essential component of an in vitro digestion system is, of course, the enzymes. In

the present system, where the substrate is pure protein, porcine gastrointestinal proteases

were used. The porcine and human gastrointestinal tracts and accessory glands are quite

similar, and therefore the use of porcine proteases is an acceptable substitute for human

enzymes (Swindle et al., 2012). For the gastric step of the digestion pepsin isolated from

the porcine gastric mucosa at an enzyme:CN ratio of 1:200 was used. The gastric digestion

was carried out at 37 °C and pH 2.0 in the presence of 35 mM NaCl, which is within the

normal range of the adult fasted stomach (Kalantzi et al., 2006; Bourlieu et al., 2014). The

21

approx. 20 minutes gastric half-emptying time means that after 60 minutes almost 90% of

ingested milk protein will have left the stomach (Calbet and Holst, 2004). In addition,

preliminary experiments with a commercial CN preparation (SigmaAldrich, US) revealed

that, in the present system, the hydrolysis of CN by pepsin does not appear to continue

past 30 minutes (Figure 9). In the duodenum the pH is approximately 6.5, which can be

achieved by addition of NaHCO3 to the IVGD reaction. For the duodenal digestion,

pancreatic enzymes (PE) were obtained by dissolving porcine pancreatin in MilliQ H2O.

This solution contained some undissolved material, that was removed by centrifugation,

and the supernatant contained the proteolytic activity. In order to add the same proteolytic

activity of PE as pepsin, the proteolytic activity was determined for both enzyme

preparations (described in detail in paper 1, see data in appendix A). Acidified bovine

hemoglobin was used as the substrate for pepsin (Anson and Mirsky, 1932), and bovine CN

was used for the pancreatic enzymes (Kunitz, 1947). In a recent extensive survey of the

litterature on infant and adult digestion the half-time for intestinal content was reported to

be approximately 160 minutes, and 2 hours has recently been recommended for the

duration of intestinal digestion (Bourlieu et al., 2014; Minekus et al., 2014). Judging from

Figure 10 the degree of hydrolysis (DH, see section 3.3.1) of CN digested with PE also

appears to no longer increase significantly at this point.

Figure 10. The degree of hydrolysis during in vitro digestion of a commercial casein preparation. The casein was digested at 37 °C with pepsin for 30 minutes at pH 2.0, and then with pancreatic enzymes at pH 6.5 up to a total of 150 minutes. Error bars are sem (n=3).

22

3.3 In Vitro Gastrointestinal Digestion of β- and κ-Casein Variants

Based on the above argumentation, the IVGD procedure employed in the present studies

was as described in paper 1. In brief, the CN was dissolved to 10 mg/mL in simulated

gastric fluid (35 mM NaCl, pH 2.0) and equilibrated to 37 °C, followed by addition of 0.05

mg/mL pepsin. Incubation with pepsin lasted 60 minutes, after which the pH was adjusted

to 6.5 with 54 mM NaHCO3. Then, PE were added to a proteolytic activity equivalent to

that of the added pepsin, and the incubation was continued for a further 120 minutes.

Samples were taken at 0 (undigested), 60 (60 minutes pepsin digested), 65 (60 minutes

pepsin + 5 minutes PE), and 180 minutes (60 minutes pepsin + 120 minutes PE). The

enzymes were inactivated by heat treatment at 90 °C for 20 minutes at the time of

sampling, and kept at -20 °C until further analysis (see appendix A for data on heat-

treatment).

Considering the amino acid substitutions in the four β-CN variants investigated here

(Table 6), some differences in digestion pattern is expected. It is already well-established

in the literature that the H67P substitution in variant A2 (and likely also I) has an impact

on the generation of the opioid βCM-7 (Jinsmaa and Yoshikawa, 1999; Noni, 2008). As

mentioned above, trypsin cleaves proteins C-terminally to K and R, and therefore the

S122R in the B variant will presumably lead to changes in hydrolysis in this region as well.

In addition to having the same H67P substitution as the A2 variant, the I variant has a

M93L substitution which may increase susceptibility to pepsin hydrolysis at this position,

because of pepsin’s cleavage preference for L in the P1 position (see section 3).

Table 6. Position of amino acid substitutions within the mature protein of variants of β-casein (adapted from paper 1)

Variant1

Position A1 A2 B I

67 H P P

93 M L

122 S R

1 A1 is the reference sequence (Caroli et al., 2009).

23

The amino acid substitutions in the κ-CN variants A, B, and E provide a less clear picture

(Table 7). As an example, in an early paper by Tang on pepsin specificity the amino acids

are divided into three groups classifying peptide bonds as either highly susceptible,

susceptible, or non-susceptible (Tang, 1963). The T136I and the S155G substitutions

indicate a small reduction in susceptibility, and the D148A indicates unchanged

susceptibility. However, the MEROPS database indicates unchanged and slightly increased

susceptibilities for the former and the two latter substitutions, respectively. Regarding PE

the situation is equally ambiguous when considering the concerted effects of the different

enzymes. Furthermore, all the κ-CN substitutions lie in the CMP region, and therefore

PTMs may influence the action of digestive enzymes as well.

Table 7. Position of amino acid substitutions within the mature protein of variants of κ-casein (adapted from paper 3)

Variant1

Position A B E

136 T I

148 D A

155 S G 1 A is the reference sequence (Caroli et al., 2009).

3.3.1 Methods

Degree of Hydrolysis

A useful metric for the characterization of protein digestion of is the DH. DH is a measure

of the fraction of peptide bonds present in a given protein that has been hydrolyzed, and is

given by equation (3)

(3)

tot

hDH(%) %

h 100

24

where h is the amount of peptide bonds that have been hydrolyzed, and htot is the total

amount of peptide bonds in the protein. There are several different methods available with

which the DH can be estimated. They rely on quite different mechanisms of action, and

there are advantages and disadvantages related to all of them. Some of the most commonly

used methods include the pH-stat, soluble nitrogen in trichloroacetic acid (SN-TCA),

trinitrobenzenesulfonic acid (TNBS), and OPA methods. In a recent review of methods for

determination of DH a recommendation for a standardized method was pursued.

However, the author concluded that the studies comparing methods were too ambiguous

and inadequate (Rutherfurd, 2010). Moreover, the system used to generate the

hydrolysates under investigation may also impose restrictions on the applicability of

different methods. A commonly used method is the pH-stat, which is based on the

principle of titration of the reaction mixture with a base to keep a constant pH value during

the hydrolysis. A drawback of this method is that the calculation of DH is based on a

somewhat complicated relationship with base consumption. Also, the degree of α-amino

group dissociation is affected by a number of factors, such as the size of peptides, and

which amino acids are located adjacent to the α-amino group (Adler-Nissen, 1986). In

addition, the reaction must take place at neutral or alkaline pH, rendering it unsuitable for

application in the present system, where the gastric step of the digestion takes place at pH

2. The SN-TCA method has been described as a measure of DH. It consists of two steps,

where in the first intact and undigested protein is precipitated by TCA addition. In the

second step the nitrogen in the supernatant after centrifugation is quantified. However,

this does not give a direct measure of the actual number of peptide bonds being

hydrolyzed, and the quantification of soluble nitrogen is dependent on the complete

precipitation of undigested protein. Indeed, it was shown that 70% of protein or peptides

of more than 10 kDa in size were not precipitated by addition of 10% TCA. For this reason,

and because it does not directly quantify the hydrolyzed peptide bonds, this method is also

not applicable. In contrast to the above methods, the TNBS and OPA methods directly

measure the amount of free amino groups in the reaction, by forming a derivative that can

be measured by either absorbance (TNBS) or fluorometric readings (OPA). The TNBS

method requires a derivatization step before measuring, while OPA does not, and as such

can be measured in real-time. It does however, require the presence of a thiol group for the

derivatization to occur. In addition, both these reagents will form a derivative with the ε-

amino group of K side chains, which must be accounted for in the calculations of DH. In

25

h[ NH ] [ NH ]

DH(%) %[ NH ] [ NH ]

2 2 0

2 2 0

100

1972 Udenfriend et al. described an, at the time, novel reagent that would also form an

easily quantifiable derivative with terminal amino groups (Udenfriend et al., 1972). This

reagent was 4-phenylspiro[furan-2(3H),1’-phthalan]-3,3’dione, known as fluorescamine.

Fluorescamine, as OPA, forms a fluorescent derivative upon reaction with free amino

groups, as shown in Figure 11. Fluorescamine reacts with other nucleophiles as well as

primary amines, such as alcohols, however, these reactions are reversible and do not

contribute significantly to fluorescence. Furthermore, the reaction with primary amines is

by far the fastest of the reactions taking place. A drawback is that fluorescamine does not

react with P, however, this is true of TNBS and OPA as well. In the present studies the

method used for determination of DH was a combination of TCA precipitation of

unhydrolyzed protein, and derivatization with fluorescamine. This was followed by

fluorometric measurement using excitation and emission wavelengths of 390 nm and 480

nm. The method is essentially as described previously (Larsen et al., 2004), and is also

described in more detail in paper 1.

The DH was calculated using equation (4)

(4)

where [-NH2] is the concentration of primary amine, with subscripts h, 0, and ∞

indicating: before digestion, at time of sampling, and at theroretical complete hydrolysis

into free amino acids, respectively. The latter was calculated using equation (5)

Figure 11. The derivatization of primary amine with fluorescamine. The product is a fluorophor with excitation at 390 nm and emission at 470 nm. (Drawn using the online tool MarvinSketch, www.chemaxon.com/marvin/sketch).

+ R-NH2

26

Lys CN

AA

( f ) C[ NH ]

MW

2

1

Ezv

f

(5)

where fLys is the fraction of K residues in the protein, CCN is the concentration of the

protein, and MWAA is the mean MW of amino acids in the protein. Using the DH it is

possible to calculate the mean length of the peptides in the hydrolysate (MLP) as

100%/DH.

Sodium-Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

Separation of proteins and their hydrolysates by electrophoresis in acrylamide gels is an

excellent method for visualization, and estimation of protein and peptide fragmentation

and mass. The velocity (v) of a protein or peptide during electrophoresis is proportional to

its charge (z), the strength of the electric field (E), and the frictional coefficient (f) (Stryer

et al., 2006), and this relationship is given by equation (6)

(6)

The strength of the electric field is directly controlled by the experimenter; the friction is

determined by the structure of the gel, and by the size and shape of the protein; and the

electric charge is an intrinsic property of the native protein itself. For analysis of proteins

and peptides in the 1 – 100 kDa range Tricine-polyacrylamide gels are typically used. This

type of gel is superior in its ability to achieve excellent separation, even below 20 kDa

(Schägger and Vonjagow, 1987). Sodium dodecyl sulphate (SDS) in the sample buffer will

bind to the backbone structure of proteins and peptides, resulting in unfolding of the

protein. Approximately one SDS binds per two amino acids in the protein, and the number

of charges from bound SDS is far greater than the intrinsic charge of most proteins, which

can therefore be considered negligible. Thus, the amount of bound SDS adds negative

charges roughly proportional to the MW of the protein (Stryer et al., 2006). In the present

studies dithioerythritol was included in the sample buffer as well, to reduce any di-sulfide

bonds, and Tricine-gels with a 10-20% acrylamide gradient were used. In order to estimate

27

Figure 12. Schematic representation of the electrospray ionization source. (Figure adapted from www.waters.com/waters/en_US/Common-Ionization/nav.htm?cid=10073251).

the MW of the observed protein bands polypeptide MW markers in the range 3.5 to 26.6

kDa were used. The procedure is described in detail in paper 1.

Peptide Profiling by Liquid Chromatography – Electrospray Ionization Tandem Mass

Spectrometry

In order to construct meaningful hypothesis regarding observations made during IVGD of

proteins it is immensely helpful to know the peptide composition of the hydrolysate in

question. Traditional methods of achieving this were based on a type of electrophoretic or

chromatographic separation, and subsequent sequencing of the resultant peptide fractions.

However, with the advent of highly sensitive and precise tandem mass spectrometers, the

identification and sequencing of peptides in complex mixtures has been made much easier

and faster. This type of analysis allows for a much higher throughput, and more precise

identification of proteins and peptides alike (Soares et al., 2012). The most common

method for analyzing samples of a complex mixture of peptides is by coupling LC to

tandem MS (LC-MS/MS). This combines a high resolution separation, by e.g. reversed

phase high performance LC (HPLC), with the MS/MS system. The basic principle of all MS

is that the compound of interest, in this case peptides, is ionized and subsequently

separated in an electric field. The movements of ions in an electric field are determined by

the charge and mass of the ion and the electric field strength. In MS/MS applications the

ions can undergo collision in or immediately after the mass selector, where they are

fragmented by collision with an inert gas, e.g. Helium or Argon, and then re-enter into a

28

mass selector (Wells and McLuckey, 2005). This provides a series of ions with distinct m/z

ratios from each so-called parent ion, from which the sequence of the peptide can be

deduced (Soares et al., 2012). The system used for the studies presented here consisted of a

reversed phase micro-HPLC coupled to an electrospray ionization (ESI) source and an ion

trap MS/MS. The HPLC utilized a hydrophobic C18 column that separates the peptides

according to hydrophobicity by elution with a gradient of acetonitrile (buffer B) in water

(buffer A). Both buffer A and B were acidified with 1% acetic acid to stabilize the column,

improve retention of carboxylic acid groups, and reduce peak trailing. ESI is a soft

ionization technique that is well suited for ionization of proteins and peptides. The

principle of ESI is based on pumping the effluent from the HPLC column through a small

stainless steel capillary needle carrying a large voltage (Figure 12). This will cause the

molecules to lose electrons, and thus become charged. They are then accelerated towards

the opening in the counterplate by the electric field between the needle and plate. This

results in formation of progressively smaller droplets due to the force of the electric field,

evaporation of solvent and ions, and electrostatic repulsion between ions within the

droplets (Awad et al., 2015). Ultimately, the individual ions are separated and enter into

the mass selector, in this case a quadrupole ion-trap, as shown in (Figure 13). The ion-trap

consists of two end-plate electrodes with AC current, and a circular electrode with DC

current. By controlling the magnitude and direction of the currents ions with specific m/z

Figure 13. Scematic representation of a quadrupole ion trap. A positively charged cloud of ions (red and pink). An electric field is generated between the endcap electrodes (a) and the ring electrode (b). The figure shows two states (1 and 2) of AC current. (Adapted from en.wikipedia.org/wiki/ Quadrupole_ion_trap).

29

ratios are selected to undergo collision induced dissociation (not shown), as described

above, or travel to the detector. The fragmentation of a peptide is shown in Figure 14. The

most common fragmentation generates b- and y-series ions by breakage of the peptide

bond. B ions extend from the N-terminus and z ions from the C-terminus. Peptides do

break at other positions as well generating the less common a, c, x, and z ion series

(Johnson et al., 1988; Soares et al., 2012). The end result is a series of m/z values deriving

from the ion series of individual parent ions. This data is analyzed by automated software.

In the present studies the MASCOT algorithm was employed on an in-house server

(Hirosawa et al., 1993). The server used a custom database of known milk proteins,

including genetic variants. For further details please see paper 2.

Figure 14. Peptide fragmentation during collision induced dissociation. See text for explanation. (from Soares et al. 2012).

30

3.4 Degree of Hydrolysis and Fragmentation of β- and κ-Casein Variants

β-Casein

The DH of β-CN variants A1, A2, B, and I was determined at different points during IVGD

using the fluorescamine method described above. The results are shown in Table 8. The

DH for the β-CN variants after 60 minutes of pepsin digestion was around 3%, and

increased to approx. 20% after 5 minutes of additional PE digestion. After the full 60

minutes pepsin and 120 minutes PE digestion the DH was approx. 50%. At none of the

three points during digestion was there a significant difference between individual β-CN

variants, but the differences between time points were highly significant for all variants.

The relatively low DH following the initial digestion of β-CN with pepsin may be a result of

some aggregation, as β-CN has a tendency to form aggregates at higher temperatures

(Payens and Vanmarkw, 1963). It was shown that at 30 °C the hydrophobicity of β-CN is

four times as high as at 4 °C, and that this is a major contributor to formation of β-CN

micelles (O'Connell et al., 2003). These micelles were estimated to consist of up to approx.

25 β-CN monomers. It is plausible that these structures sterically hinder pepsin’s access to

Table 8. In vitro gastrointestinal digestion of β-casein variants using pepsin and pancreatic enzymes1. (Data from paper 1)

β-casein variant tpep(min)2

tpan(min)3 DH (%)4 MLP5

A1 60 0 3.6 ±0.42a 27.6 ± 3.01

A2 60 0 3.2 ±0.20a 31.5 ± 2.20

B 60 0 3.6 ± 0.22a 28.0 ± 1.95

I 60 0 2.6 ±0.17a 37.6 ± 2.46

A1 60 5 20.4 ± 1.71b 4.9 ± 0.42

A2 60 5 21.5 ±2.47b 4.6 ± 0.55

B 60 5 20.2 ±0.89b 5.0 ± 0.20

I 60 5 19.4 ± 1.38b 5.2 ± 0.33

A1 60 120 55.0 ±5.99c 1.8 ± 0.21

A2 60 120 52.4 ±5.54c 1.9 ± 0.19

B 60 120 46.2 ±3.98c 2.2 ± 0.16

I 60 120 49.9 ±4.24c 2.0 ± 0.17

1 Results are shown as the mean ± SEM (n = 3 (2 for A1)). 2 tpep: reaction time with pepsin. 3 tpan: reaction time with pancreatic enzymes. 4 DH: degree of hydrolysis. Different letters denotes significant difference within each casein type (p < 0.001). 5 MLP (100%/DH): mean length of peptides.

31

cleavage sites. However, the SDS-PAGE analysis revealed that very little intact β-CN

remained after digestion with pepsin, which is comparable to previous observations

(Dupont et al., 2010). In the study by Dupont and co-workers two IVGD models were

compared: one mimicking adult digestion utilizing a pepsin:substrate ratio of 1:20, and

one mimicking infant digestion utilizing 1:160, i.e. comparable to what was used in the

present IVGD model. The pattern of peptides observed after pepsin digestion in Figure 15

closely resembles that presented by Dupont and co-workers in their infant model.

However, after 60 minutes pepsin digestion their peptide bands are quite faint, which is

not the case in Figure 15. The most likely explanation is that here 32 µg of protein was

loaded on to each lane, where Dupont et al. loaded only 10 µg. The relatively low resistance

of β-CN towards hydrolysis by pepsin may be explained by its somewhat disordered

structure. Indeed, the more structured β-lactoglobulin, and to some extent ovalbumin,

showed markedly greater resistance towards gastric digestion in the study by Dupont et al.

Unfortunately, in that study the DH of β-CN was not determined, preventing a direct

comparison, but judging by the very similar peptide band patterns, they are most likely

similar as well. It is interesting to note the apparent discrepancy between a seemingly high

digestibility, and the relatively low DH observed following pepsin digestion. This is,

however, in agreement with Figure 15, where it is clear that little intact β-CN is left at this

point in the digestion, but several large high-abundant fragments remain. This could be

explained by β-CN having regions that were more resistant to hydrolysis than others,

Figure 15. SDS-PAGE of β-casein variants A1, A2, B, and I at different stages of simulated gastrointestinal digestion. 0 = undigested, 60 = 60 minutes pepsin, 65 = 60 minutes pepsin + 5 minutes pancreatic enzymes, 180 = 60 minutes pepsin + 120 minutes pancreatic enzymes. Arrow indicates position of missing band. (From paper 1).

32

which was addressed by Dupont et al. They argued that the presence of distinctly

hydrophobic stretches, calculated at pH 3.0, would perhaps make these regions more

inaccessible to proteases. This is in accordance with the argument presented above, that

aggregation of β-CN may delay hydrolysis by pepsin. The continued IVGD with PE resulted

in a rapid disappearance of peptide bands, accompanied by significant increases in the DH

after both 5 and 120 minutes (Table 8). In a study examining peptide release from

complete CN during digestion with pancreatin the DH was determined (Su et al., 2012). In

that study the DH after 120 minutes digestion was 20%, and the highest DH reported was

28.5% after 24 hours of digestion. These values are markedly lower than those found in the

present study, even after 24 hours of digestion. A possible explanation may be that in the

study by Su et al. the complete CN fraction was digested, compared to just β-CN here.

However, in the results of preliminary digestion experiments during development of the

IVGD model presented here (Figure 10), which were performed on a similar CN

preparation, a DH above 40% was observed. Moreover, Su and co-workers did not include

a gastric step in their digestion model, which will certainly add to the DH. Alternatively,

the lower DH observed by Su et al. could be explained by their use of the pH-stat method

of determining the DH. This method was previously compared to the TNBS and OPA

methods. Here it was shown that the DH determined after digestion of whey protein with

Debitrase, was significantly lower by the pH-stat method, compared to both the TNBS and

OPA methods (Spellman et al., 2003). It was argued that this may be a result of hydrolysis

with the Debitrase, which contains high exopeptidase activity, leading to a high

concentration of amino acids and di- and tripeptides. The primary amines in these

molecules have higher pK values, which leads to an underestimation of the DH

(Rutherfurd, 2010). Similar to Debitrase, pancreatin contains exopeptidase activity

(Mullally et al., 1994). Another study determined the DH by the OPA method after

extensive hydrolysis of complete CN with trypsin, which corresponded more closely to the

final DH of approx. 50% of β-CN in the present study (Wang et al., 2013).

Besides the obviously high digestibility of β-CN across all variants in general, a closer

examination of the individual band-patterns revealed a striking difference after 65 minutes

of digestion (60 minutes pepsin + 5 minutes PE). As is indicated by the arrow in Figure 15,

a peptide band was absent from the B variant around the 4 kDa position. The amino acid

substitution that distinguishes the B variant from the A1, A2, and I variants is S122R (Table

33

6), as discussed in section 3.3. In order to determine if this substitution could explain the

absence of this peptide band, identification of the peptide from the bands of the other three

variants was pursued. Initially, in-gel trypsination followed by extraction was attempted,

following a procedure essentially as described by Jensen et al. (2012a). In brief, small gel-

plugs were excised from the gel, and after dehydration and washing steps they were

incubated with a trypsin solution. Subsequently, peptide fragments were extracted with

formic acid and acetonitrile, and spotted on to a stainless steel target plate for matrix-

assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, using α-Cyano-4-

hydroxycinnamic acid as matrix. Unfortunately, these attempts did not result in a positive

identification, despite making modifications to the extraction procedure, or omitting the

in-gel trypsination. Consequently, a more traditional approach was taken, namely

electroblotting of the gel followed by Edman sequencing, carried out at the Laboratory for

Protein Chemistry, Aarhus University. This procedure is described in paper 1. In short, an

SDS-PAGE gel of the A1 variant was electroblotted on to a polyvinylidine difluoride

membrane, followed by staining and excision of the band of interest. N-terminal amino

acid sequencing was carried out by automated Edmann sequencing. The method was

modified from a report by Matsudaira (1987). The result of this analysis was the eight

amino acid N-terminal sequence tag 106H-K-E-M-P-F-P-K-. From the SDS-PAGE gel, the

MW of the complete peptide was estimated to be roughly 4 kDa. A 4 kDa β-CN fragment

starting at position 106 would extend to around position 139, placing the site of the S122R

substitution squarely in the middle. Therefore, trypsin cleavage at R122 in the B variant is

a likely explanation for the absence of this peptide.

In addition to the peptide discussed above, there are other subtle differences between the

β-CN variants apparent in Figure 15, i.e. several bands differ in intensity between variants.

To obtain a deeper understanding of the nuances of digestion pattern variation, peptide

profiling of the hydrolysates was performed by LC-MS/MS. Table 9 shows all the identified

peptides that either contained or neighbored a site of amino acid substitution. In total,

three peptides were identified after the gastric step of digestion. They were cleaved at

peptide bonds containing L, T, and F in either the P1 or P1’ positions, which is in good

accordance with pepsins reported specificity (Tang, 1963; Rawlings et al., 2014). Three of

the six peptide bonds also had a P in one of the sites, and this is more surprising, though it

has been reported previously by Lisson et al. (Lisson et al., 2013), who also identified the

34

f[59-80] fragment. Although P is generally not considered conducive to pepsin cleavage,

the fact that it is observed among the few cleavage sites here, and in the study by Lisson et

al., may be explained by pepsin being a protease, with a relatively broad specificity. Keil

argued that less specific proteases are more influenced by amino acids further from the

scissile bond (Keil, 1992). The fragment f[81-93] supports the hypothesis that the M93L

substitution increases the likelihood of hydrolysis of the I variant by pepsin. After the

initial five minutes digestion with PE seven peptides were identified from regions of amino

acid substitutions, with four peptides being exclusive to the B variant, and one to the I

variant. PE action generates peptides with different terminal amino acids, and the action of

trypsin is clear with most of the peptides being generated by cleavage at K108 or K114, and

R122 residues. The peptides f[108-122] and f[114-122] confirms that cleavage does happen

at R122 in the B variant, and therefore substantiates that this is the reason for the missing

band in the SDS-PAGE analysis. In addition, the peptide f[114-139], identified in all

variants, shows that hydrolysis at L139 also happens, and thus f[106-139] is a plausible

candidate for the band identified by Edman sequencing. After 120 minutes digestion with

PE 14 peptides from regions of amino acid substitutions were identified. At this point, the

sites of cleavage were more diverse, with hydrolysis also taking place at Q, N, S, and I,

which was not seen in the previous steps. This is an indication that several proteases, in

addition to trypsin, are now active, including exopeptidases. The PE used were obtained

from pancreatin, which has been shown to contain high trypsin activity, as well as

chymotrypsin, elastase, and amino- and carboxypeptidase activities (Mullally et al., 1994).

The A1/B derived peptide peptide f[67-92] confirms that the H67P does indeed have a

potential impact on the generation of βCM-7, as previously shown (Jinsmaa and

Yoshikawa, 1999; Noni, 2008). Two βCM-9 like peptides were also observed, namely f[59-

68] derived from either the A1 and B or the A2 and I variants. Their sequences differ at the

penultimate position, as a consequence of the H67P substitution, and they have a V at

position 0, compared to the traditional βCMs. The A2-like V-βCM-9 has been shown to

possess both antioxidant and ACE inhibitory capacity (Eisele et al., 2013), which has not

been shown for the A1-like V-βCM-9. Both peptides were chosen to be synthesized for

further evaluation of the impact of the H67P substitution on these bioactivities (see

chapters 4 and 5). Two of the peptides identified from the digested B variant were f[114-

119] and f[123-132], stemming from the region surrounding the Ser122Arg substitution.

35

Table 9. Peptides from regions with amino acid substitutions identified by LC-MS/MS after in vitro digestion of bovine β-CN variants A1, A2, B, and I. (from Paper 2)

Digestion phasea

Digested variant Positionb Sequencec

Genetic variantsd

Gastric B 59-80 (L)VYPFPGPIHNSLPQNIPPLTQT(P) A1, B

I 81-93 (T)PVVVPPFLQPEVL(G) I

A1 120-138 (F)TESQSLTLTDVENLHLPLP(L) A1, A2, I

Duodenal 1 I 81-92 (T)PVVVPPFLQPEV(L) A1, A2, B, I

B 108-119 (K)EMPFPKYPVEPF(T) A1, A2, B, I

B 108-122 (K)EMPFPKYPVEPFTER(Q) B

B 114-122 (K)YPVEPFTER(Q) B

A1, A2 114-125 (K)YPVEPFTESQSL(T) A1, A2, I

A1, A2, I 114-139 (K)YPVEPFTESQSLTLTDVENLHLPLPL(L) A1, A2, I

B 114-139 (K)YPVEPFTERQSLTLTDVENLHLPLPL(L) B

Duodenal 2 I 53-68 (F)AQTQSLVYPFPGPIPN(S) A2, I

I 57-68 (Q)SLVYPFPGPIPN(S) A2, I

A1, B 59-68 (L)VYPFPGPIHN(S) A1, B

A2, I 59-68 (L)VYPFPGPIPN(S) A2, I

A1 59-92 (L)VYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEV(M) A1, B

A1, B 67-92 (I)HNSLPQNIPPLTQTPVVVPPFLQPEV(M) A1, B

I 69-92 (N)SLPQNIPPLTQTPVVVPPFLQPEV(L) A1, A2, B, I

A1, A2, B 73-92 (Q)NIPPLTQTPVVVPPFLQPEV(M) A1, A2, B, I

I 81-92 (T)PVVVPPFLQPEV(L) A1, A2, B, I

B 114-119 (K)YPVEPF(T) A1, A2, B, I

A1 114-124 (K)YPVEPFTESQS(L) A1, A2, I

A1, A2 120-132 (F)TESQSLTLTDVEN(L) A1, A2, I

A1 120-139 (F)TESQSLTLTDVENLHLPLPL(L) A1, A2, I

B 123-132 (R)QSLTLTDVEN(L) A1, A2, B, I

a) Gastric: 60 min pepsin, Duodenal 1: 60 min pepsin + 5 min pancreatic enzymes, Duodenal 2: 60 min pepsin + 120 min pancreatic enzymes. b) The position of the peptide within the mature β-casein amino acid sequence. c) Peptide amino acid sequence using one letter abbreviations. The residues in parenthesis shows the neighboring amino acids. d) Which of the four variants contain the sequence of the peptide within their native sequence.

36

However, there is a gap between them, namely the tripeptide TER (f[120-122]), which was

not detected in the LC- MS/MS system used here. Unfortunately, peptides of this size are

generally not positively identified in the employed system, and therefore, it was deemed

likely that this peptide could be present in the hydrolysate. It was synthesized alongside

the two V-βCM-9 (Schafer-N, DK), and included in the bioactivity assessments.

κ-Casein

Following IVGD of the κ-CN variants A, B, and E the DH was determined as described

above. The results are shown in Table 10. The gastric digestion step resulted in a DH of

approx. 5% for variant A and approx. 9% for variants B and E, however the difference was

not statistically significant. The D148A and S155G substitutions in the B and E variants,

respectively, will increase susceptibility to pepsin hydrolysis slightly (the MEROPS

database (Rawlings et al., 2014)). After an additional 5 minutes PE digestion the A and B

variants reached a DH of approx. 20%, but now the E variant was markedly less digested

with a DH more than 7% lower. This suggests that the E variant is somehow less

susceptible to hydrolysis by PE, and trypsin in particular, which appears to be the most

active of the PE at this point in the IVGD, as discussed above. This cannot be explained by

Table 10. In vitro gastrointestinal digestion of κ-casein variants using pepsin and pancreatic enzymes1. (Data from paper 3)

κ-casein variant tpep(min)2 tpan(min)3 DH (%)4 MLP5

A 60 0 4.9 ± 0.74a 21.5± 3.77

B 60 0 9.5 ± 1.40a 10.9± 1.46

E 60 0 8.4 ± 2.21a 13.6± 3.42

A 60 5 20.2± 4.05ab 5.3 ± 0.88

B 60 5 20.9± 4.25ab 5.1 ± 0.92

E 60 5 12.9 ± 2.11a 8.3 ± 1.62

A 60 120 43.3 ± 6.23c 2.4 ± 0.31

B 60 120 44.4 ± 3.14c 2.3 ± 0.17

E 60 120 35.7 ± 6.35bc 3.0 ± 0.57 1 Results are shown as the mean ± SEM (n = 3). 2 tpep: reaction time with pepsin. 3 tpan: reaction time with pancreatic enzymes. 4 DH: degree of hydrolysis. Different letters denotes significant difference within each casein type (p < 0.05). 5 MLP (100%/DH): mean length of peptides.

37

the S155G substitution, because no trypsin sites are found in this region. Furthermore, the

specificities of chymotrypsin and elastase would be virtually unaffected, and therefore, the

explanation must lie elsewhere (discussed further below). After 120 minutes of PE

digestion the difference between the DH of three variants was unchanged. Thus, the initial

resistance of the E variant towards PE hydrolysis seems to have only delayed the digestion

process, as the absolute increase in the DH between 5 and 120 minutes PE digestion is

comparable for all three variants. The B variant retained a slightly higher DH throughout

the IVGD, perhaps explained in part by elastase having a preference for hydrolysis at A

over D. The DH after full IVGD was markedly higher than reported by Su et al. (2012), and

the arguments for the β-CN digestion hold true here as well.

Examination of the SDS-PAGE analysis may provide further insight into the issues

discussed above (Figure 16). The lanes with the undigested κ-CN variants all have a single

strong band close to the 17 kDa MW marker, corresponding to the intact proteins. Above

that band all three variants have an obvious smear, and the A and B variants have three or

four relatively weaker bands below, stretching down to approximately 14 kDa. Only a

single band around 15 kDa is visible in this region of the B variant. The LC-MS analysis of

the κ-CN fractions following purification did not indicate contamination by other CNs, but

did reveal a rather high heterogeneity of the A and E variants, caused by PTMs. These

PTMs will result in small changes to the MWs of the proteins, and a concomitant shift in

electrophoretic mobility. However, PTMs may only explain a small part of the higher MW

smear in the gel. Aggregation behavior of κ-CN monomers has been described in some

detail previously (Vreeman et al., 1981; Ossowski et al., 2012), but can hardly explain these

observations, since the highest MW staining observed is positioned at approximately 30 to

35 kDa. Another explanation may be sought in the CMP from κ-CN. CMP can be generated

by the action of both chymosin and pepsin, but since no exogenous enzymes have been in

contact with the samples at this point, this is irrelevant. However, the aspartic protease

cathepsin D was previously shown to both be present in milk, and capable of liberating

CMP from purified κ-CN (Larsen et al., 1996). In addition, CMP has been reported to

aggregate extensively, both when released from κ-CN and as part of the intact protein in a

complex system that is influenced by pH during hydrolysis (Mikkelsen et al.). The fact that

there is only a small amount of low MW bands present in the B variant lane suggest that

PTMs are involved as well. All of these different factors come together to form a complex

38

system of interactions, that together may explain these observations. Inspection of the

lanes with the κ-CN variants following digestion with pepsin reveals a remarkably high

gastric digestibility of all three variants. There are a few peptide at 5 to 8 kDa, and a very

faint band at approx. 16 kDa for the E variant. The relatively little remaining total protein

at this point corresponds with the higher determined DH, which indicates a mean length of

peptides of 14 to 22 amino acids. Therefore, many of the peptides present in the

hydrolysate at this point will have eluted from the gel under the electrophoresis conditions

used. Looking closer at these lanes, there does appear to be slightly higher staining

intensity for the A variant, which agrees with its lower DH here. The lanes showing both

stages of duodenal digestion are all virtually devoid of peptides, leading to the conclusion

that, either the peptides left at this point are too small to remain in the gel, or the collection

of peptides is too heterogenous in size to generate clearly visible bands.

3.5 Summary of In Vitro Gastrointestinal Digestion of β- and κ-Casein

variants

IVGD of β-CN variants A1, A2, B, and I revealed that the amino acid substitutions in these

proteins do not affect the DH during digestion. However, the M93L substitution may

increase the likelihood of hydrolysis by pepsin at this position, as evidenced by the

identification of β-CN(I) f[81-93]. In addition, the S122R substitution in the B variant

clearly defined a novel trypsin cleavage site, demonstrated by Edman sequencing and LC-

Figure 16. SDS-PAGE of κ-casein variants A, B, and E at different stages of simulated gastrointestinal digestion. 0 = undigested, 60 = 60 minutes pepsin, 65 = 60 minutes pepsin + 5 minutes pancreatic enzymes, 180 = 60 minutes pepsin + 120 minutes pancreatic enzymes. (From paper 3).

39

MS/MS. Furthermore, two βCM-like peptides differing in amino acid sequence were

identified from the A1/B variants and the A2/I variants, and chosen for solid-phase

synthesization along with a putative tripeptide from the B variant. These peptides were

characterized for bioactivity (see chapters 4 and 5). Undigested κ-CN variants A, B, and E

were determined to be relatively uncontaminated by other milk proteins, but displayed a

high degree of heterogeneity in SDS-PAGE. This could be the combined result of PTMs,

indigenous proteases, and complex aggregation behavior. IVGD of κ-CN A, B, and E

revealed a slightly different picture, with regard to the DH, compared to β-CN. The A

variant was less digested by pepsin, compared to the B and E variants, whereas the E

variant was less digested by PE, compared to the A and B variants. SDS-PAGE analysis

provided some support to this as well. In general, all the investigated CNs were highly

digestible in the IVGD model employed here, which supports their main biological

function as an easily accessible source of nutrients for the newborn.

40

Chapter 4. Oxidation, Oxidative Stress, Antioxidants and Peptides

Reduction and oxidation reactions are ubiquitous in living cells, and play an essential role

in the generation of the energy-rich compound adenosine-triphosphate (ATP) through

aerobic respiration. This takes place in the electron transport chain in the mitochondria,

which basically converts the energy from molecules such as carbohydrates and fatty acids

into readily usable ATP. This happens by facilitating the transport of electrons from the

donor molecules to oxygen, which then reacts with protons to release H2O. This system is

not infallible however, occasionally resulting in the generation of superoxide ( O

2 ), which

is a highly reactive free radical. Other radicals are found in biological systems as well,

including additional oxygen radicals, nitrogen species and metal ions (Halliwell, 2011).

Together, these compounds are denoted reactive species (RS). As a consequence of their

high reactivity RS can potentially damage biomolecules, such as proteins, fatty acids, or

nucleic acids which may have detrimental effects on health. Exogenous factors, such as UV

radiation, toxins, diet, tobacco smoke, or obesity may also increase oxidation in the

organism (Church and Pryor, 1985; Aseervatham et al., 2013; Al-Gubory, 2014).

Conversely, oxidative pathways play important roles in several physiological functions

such as immune responses, phagocytosis, and regulation of vascular tone (Valko et al.,

2007). There are systems in place in cells to regulate the level of RS. Generally, they may

be considered as two systems: (1) the antioxidative enzymes; and (2) the non-enzymatic

antioxidants. Examples of cellular antioxidative enzymes are superoxide dismutase,

glutathione peroxidase, and catalase. Non-enzymatic antioxidants are exemplified by α-

tocopherol (vitamin E), ascorbic acid (vitamin C), and carotenoids (Valko et al., 2007). In

order for proper cellular function there must be a balance between RS and the

antioxidative systems, i.e. between pro-oxidants and antioxidants. If a disproportionate

amount of pro-oxidants are present in the cell or the organism, it ends up in a state of

oxidative stress. Oxidative stress is considered to be a contributing factor in the

pathophysiology of many ailments, such as neuro-degenerative disorders, rheumatoid

arthritis, cardiovascular disease, inflammatory bowel disease, ageing, and cancer (Finch,

2007; Rahman, 2007; Valko et al., 2007; Halliwell, 2012). The implication of the above is

that maintaining the pro-oxidant/antioxidant balance is crucial, a task which the cell or

organism is rather adept at handling itself. However, excessive exposure to environmental

factors acting as pro-oxidants may skew the balance towards oxidation.

41

Food products are derived from living organisms, and therefore the discussion of oxidation

holds for them as well. They contain all the constituents of the living tissue, including pro-

oxidants and the antioxidant systems to counter them. However, these systems are no

longer maintained, and therefore oxidation will start to overpower the capacity of the

antioxidants over time. In addition, food processing will increase oxidation by introducing

oxygen and increasing exposure to environmental factors, or by removing antioxidants.

Oxidation reduces the quality and shelf-life of food, as it leads to deterioration in taste and

flavor, mainly due to oxidation of lipids (Elias et al., 2008). Synthetic antioxidants are

often added to counteract this. Some popular synthetic antioxidants are butylated

hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and propyl gallate (PG) (Belitz et

al., 2009). However, these are suspected of being harmful, are under regulation, and are

thus becoming more undesired by costumers (Gülçin, 2012). Therefore, food

manufacturers are looking to other sources for antioxidants that will be more acceptable,

and in addition, make it easier to maintain a “clean label”.

4.1 Food-derived Antioxidative Peptides

Many different antioxidant compounds are found in food products, e.g. plant polyphenols,

carotenoids, ascorbic acid, amino acids, peptides, or proteins (Elias et al., 2008; Berger et

al., 2012). Of these, proteins and protein hydrolysates have been shown to delay lipid

oxidation when added to foods (Elias et al., 2008). Endogenous carnosine, a H-containing

dipeptide found in muscle food, has been shown to inhibit lipid peroxidation when added

to ground meat products (Decker and Crum, 1991; Calvert and Decker, 1992).

Furthermore, CN hydrolysates added to ground beef was shown to retard lipid

peroxidation (Diaz and Decker, 2004). In relation to health benefits of antioxidative

compounds the effects on health are less clear. It seems to be well established that

oxidative stress exacerbates many disease states, but it is less clear if an increased intake of

antioxidants will alleviate this effect (Valko et al., 2007; Halliwell, 2012). It is likely,

though, that intake of dietary antioxidants will have an effect on diseases of the GI tract,

such as inflammatory bowel diseases and colorectal cancer (Halliwell et al., 2000; Stone et

al., 2014). Milk contains both protein antioxidants and low MW antioxidants (Clausen et

al., 2009). In addition, CN peptides have been shown to possess antioxidative capacity

42

towards both enzymatic and non-enzymatic linoleic acid peroxidation (Rival et al., 2001),

and protect intestinal cells (Caco-2) against H2O2 induced oxidative insult (García-Nebot

et al., 2011). In a study using rats fed a vitamin E deficient diet, an antiperoxidative effect

of feeding a fermented milk product was established, and the active components behind

the effect were identified as lactic acid and β-lactoglobulin (Zommara et al., 1998).

Pihlanto has previously reviewed the research on milk-derived antioxidative peptides.

These peptides were described as being composed of 5 to 11 amino acids, including H and

the hydrophobic residues P, Y, and W (Pihlanto, 2006). It was also established that

antioxidative peptides could be generated by enzymatic hydrolysis with different enzymes,

or by fermentation. Moreover, studies have shown that both total CN and individual CNs

possess antioxidant capacity, which is increased by digestion with gastrointestinal

proteases (Gómez-Ruiz et al., 2008; Kumar et al., 2010; Mao et al., 2011; Salami et al.,

2011). Thus, the antioxidant capacity of β-CN variants A1, A2, B, and I, and κ-CN variants

A, B, and E, as it developed during IVGD, was investigated in the present studies. In

addition, the antioxidant capacity of the synthesized β-CN derived peptides VYPFPGPIHN,

VYPFPGPIPN, and TER was determined.

4.2.1 Methods

Overview of antioxidant capacity assays

The chemistry of oxidation and antioxidants is rather complex, and a comprehensive

discussion is beyond the scope of this thesis. A short introduction will be given in the

following. Antioxidant capacity of foods and food components has been determined using

various assays. A few of the more popular are the oxygen radical absorbance capacity

(ORAC), ferric ion reducing antioxidant parameter (FRAP), diphenyl-1-picrylhydrazyl

(DPPH) scavenging, and the trolox equivalent antioxidant capacity (TEAC) (Huang et al.,

2005; Moon and Shibamoto, 2009). These assays are based on different molecular

mechanisms, and thus assess slightly different aspects of antioxidant capacity. However,

the basic feature of antioxidants is the same if they are defined according to Halliwell and

Gutteridge, who stated that an antioxidant is “any substance that delays, prevents or

removes oxidative damage to a target molecule” (Halliwell, 2007). Two things are clear,

(1) antioxidants must reduce RS, and become oxidized species in the process, and (2)

43

Figure 17. Lipid autoxidation. (1) initiation, reactive species abstracts H from polyunsaturated fatty acid (PUFA). (2) the alkyl radical from step 1 rearranges and reacts with O2, forming a peroxyl radical.

O2-dependent lipid autoxidation propagates to neighboring PUFA.

(1)

(2)

antioxidants must have a structure that is capable of stabilizing their electron

configuration, lest they themselves become RS. The RS in biological systems can initiate a

radical chain reaction, often by oxidation of polyunsaturated fatty acids (PUFAs) (Elias et

al., 2008; Belitz et al., 2009). Figure 17 shows the basic steps of lipid autoxidation. RS can

abstract a labile hydrogen from a PUFA, creating a lipid alkyl radical, which can

subsequently react with O2 and create a lipid peroxyl radical. The peroxyl radical then

abstracts hydrogen from another PUFA, and a radical chain-reaction is started. The chain

can be terminated if two peroxyl radicals react with each other and form stable products,

or if a peroxyl radical reacts with an antioxidant. As mentioned, an antioxidant must be

capable of stabilizing the electron gained by interacting with the radical. For the majority

of low MW antioxidants this is accomplished by the antioxidant having a delocalized

system of electrons, or a set of conjugated double bonds. This is exemplified by α-

tocopherol (Figure 18), that has an aromatic ring in its structure, allowing any extra

electrons to delocalize in the system of conjugated molecular pi orbitals (McMurry, 2003).

There are two general mechanisms by which antioxidants can act as radical chain breaking

agents, (1) by hydrogen atom transfer (HAT), or (2) by electron transfer (ET). HAT based

assays directly measure the ability of an antioxidant to act as a chain-breaking agent,

44

whereas ET based assays measure the more general radical scavenging ability, or reducing

power, of the antioxidant . Out of the antioxidant assays mentioned above, the ORAC assay

is based on HAT and the FRAP, DPPH, and TEAC assays are based on ET (Huang et al.,

2005).

The 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) Decolorization Assay

The TEAC assay relates the ability of an antioxidant to the antioxidant capacity of the

synthetic vitamin E analog trolox. A commonly used method to achieve this is by the 2,2'-

azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) decolorization assay (Re et al.,

1999; Moon and Shibamoto, 2009). This assay has been widely used for its convenience,

and its applicability in hydrophobic as well as hydrophilic systems. The method is

described in detail in paper 1. The principle is based on the formation of the stable ABTS

radical by reaction with ammonium persulphate (Figure 19). This radical is strongly

colored, absorbs at 734 nm, and loses color by reaction with trolox or another antioxidant.

Thus, by measuring the extent of decolorization by a given compound at 734 nm, and

relating that to the decolorization achieved by trolox, the TEAC value of a compound can

be obtained. This single method of assessing antioxidant capacity was employed in the

present studies, where the main focus was not on determining the absolute biological

antioxidant capacity; rather than it was on determining any differences between the radical

scavenging ability of the different β- and κ-CN variants.

Figure 18. Chemical structure structure of α-tocopherol.

45

4.3 Antioxidant Capacity of β- and κ-Casein Hydrolysates and Derived

Peptides

β-Casein

The TEAC was determined for β-CN variants A1, A2, B, and I as it developed during IVGD.

Similar studies using CNs from various species have previously been reported, but none

have compared different β-CN variants (Gómez-Ruiz et al., 2008; Kumar et al., 2010;

Salami et al., 2011). In all of these previous studies digestion with different GI proteases, or

mixtures thereof, resulted in increased ABTS radical scavenging by both total and

individual CNs. The magnitude of the increased radical scavenging did, however, vary

between studies. In addition, Salami et al. showed that the effect of hydrolysis was also

influenced by choice of enzyme. They observed different responses in radical scavenging

after hydrolyzing camel milk CNs with pepsin, trypsin, or chymotrypsin alone, or all three

combined (Salami et al., 2011). Therefore, it was reasoned that digestion with GI proteases

could have different outcomes for the antioxidant capacity of the β-CN variants, that, as

shown above, display different peptide profiles at different stages of IVGD. The results are

+ (NH4)2S2O8 (ammonium persulphate)

Figure 19. Formation of the ABTS+⦁ radical by reaction with ammonium persulphate. (Modified from Moon and Shibamoto, 2009).

46

Figure 20. Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on Trolox equivalent antioxidant capacity (TEAC, µmoles trolox equivalents/mg protein) of β-casein variants. β-casein was incubated with the ABTS radical for 60 minutes at room temperature and the reduction in abs(734 nm) was measured and related to that obtained with Trolox. Bars represent mean values, error bars are SEM (n=3 (n=2 for A1 variant)). Asterisks denote significant difference from the undigested sample (***: p < 0.001). (from paper 1).

shown in Figure 20. There were significant global effects of digestion time (p < 0.001) and

variant (p < 0.01). TEAC increased significantly after 60 minutes pepsin followed by 5

minutes PE digestion, and a further increase was observed by 120 minutes of PE digestion.

The TEAC of the A1 and I variants appeared to respond more to IVGD than the A2 and B

variants, and overall showed a slightly higher radical scavenging ability (p < 0.05). In a

study by Kumar et al. the initial TEAC value for undigested β-CN was 0.62 µmoles

trolox/mg CN, which is very close to what is presented here (Kumar et al., 2010). In their

study the digestion was not performed as successive steps, but as either pepsin digestion or

PE digestion using Corolase PP, a commercial preparation containing different pancreatic

endo- and exopeptidases (Mullally et al., 1994). Proteolysis had a larger effect in their

study compared to here, with TEAC values of 2.4 and 2.6 µmoles trolox/mg CN after

pepsin and Corolase PP digestion, respectively, compared to approx. 1.3 µmoles trolox/mg

CN here. Conversely, Gómez-Ruiz and colleagues reported TEAC values for intact ovine β-

CN of approx. 1.0 mg/mL, and an increase to approx. 1.4 mg/mL after digestion with

pepsin followed by trypsin and chymotrypsin, which corresponds to 4.0 and 5.6 µmoles

trolox/mg CN (Gómez-Ruiz et al., 2008). Thus, their reported antioxidant capacity for

ovine β-CN was higher, but the change caused by digestion was comparable. Aliaga et al.

showed that the amino acids that can scavenge the ABTS radical were, in order of

47

reactivity, C >> W > Y > H > Coxidized (Aliaga and Lissi, 2000). This was confirmed by

Clausen et al. who added F at the low end of reactivity (Clausen et al., 2009). However,

ovine and bovine β-CNs are more than 90% identical, and the ovine type only has one

additional Y, compared to bovine, so this is unlikely to explain the large difference in TEAC

values. Salami et al. reported an increase between those discussed so far, following

digestion of camel β-CN with pepsin, trypsin, and chymotrypsin (Salami et al., 2011).

Camel β-CN contains one additional F and Y, but lacks one W, compared to bovine. Taken

together, these studies all show that digestion of β-CN causes an increase in ABTS radical

scavenging ability. The variation in the magnitude of the increases may perhaps be

explained by different experimental conditions, i.e. by choice of digestive enzymes, or by

other assay variations. Contrary to these observations, Clausen et al. reported no effect of

proteolysis with endoproteinase Lys-C and trypsin, and interestingly, a decrease in ABTS

radical scavenging upon denaturation of complete CN with GndHCl (Clausen et al., 2009).

The rationale behind their use of GndHCl was that the effect of denaturation would be

equivalent to digestion, because of the exposure of buried amino acids resulting from both

treatments. The observed decrease upon protein unfolding, together with the other

reported studies showing an increase after digestion, indicates that there is more than

solvent exposure of scavenging amino acids behind the observed antioxidant capacity. In

the study by Salami et al. the largest effect on β-CN was seen after hydrolysis with

chymotrypsin alone, followed by the combination of pepsin, trypsin, and chymotrypsin,

and then came pepsin alone, and finally trypsin alone. This suggests that hydrophobic

amino acids at the C-terminal of peptides are conducive to ABTS radical scavenging. It may

also explain why Clausen et al. saw no effect of hydrolysis in their study, where only tryptic

or equivalent fragments, with K or R at the C-terminal, would be generated. It is also

plausible that the immediate environment is of some importance for radical scavenging,

e.g. intramolecular transfer of radicals between amino acids could take place within a

protein or peptide, and thereby reinforce the stability of the radical (Butler et al., 1982;

Prutz et al., 1982; Elias et al., 2008). This reasoning may help to explain the small

differences in TEAC values between the β-CN variants reported here. Considering the

amino acid substitutions alone, the A1 and B variants would perhaps be expected to display

the highest TEAC values due to the presence of an additional H over a P. However, a study

showed that a synthetic 15 amino acid peptide containing 6 L residues resulted in more

than 50% higher ABTS radical scavenging compared to Hydroxy-P at the same 6 positions.

48

Figure 21. Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on Trolox equivalent antioxidant capacity (TEAC, µmoles trolox equivalents/mg protein) of a commercial κ-casein (Com) and variant A (AA). κ-casein was incubated with the ABTS radical for 60 minutes at room temperature and the reduction in abs(734 nm) was measured and related to that obtained with Trolox. Bars represent mean values, error bars are SEM (n=3).Different letters denote significant difference between means (p < 0.05).

This indicates that L, in addition to those reported by Aliaga et al. and Clausen et al., may

be able to scavenge the ABTS radical. Thus, the M93L substitution may indeed serve to

increase the TEAC value of the I variant, as observed. Moreover, the additional trypsin site

in the B variant would not serve to increase radical scavenging, according to the above

reasoning.

κ-Casein

TEAC was determined for κ-CN as well, as it developed during IVGD. In a preliminary

experiment a commercial κ-CN preparation was compared to the A variant, and the results

are shown in Figure 21. The TEAC values start around 0.2, and increase to around 0.4

µmoles trolox/mg CN during digestion, with the A variant having a significantly higher

value at all points of digestion. The commercial κ-CN preparation is expected to contain a

heterogenous mixture of variants. Therefore, this result suggested that the A variant would

have a greater radical scavenging potential than some other variants that may be present in

the commercial product. In addition, the TEAC of the A variant increased to it’s maximum

value already after digestion with pepsin. This was somewhat surprising because the

commercial κ-CN increased more gradually throughout digestion, as did the β-CN variants

49

presented above. Furthermore, the DH was determined and here the two κ-CN

preparations were more similar during digestion, although the A variant did show a

slightly higher DH at 60 minutes and 180 minutes, compared to the commercial κ-CN. The

results from the experiments with κ-CN variants A, B, and E are shown in Figure 22. There

were global significant effects of both digestion time and variant, as well as the interaction

between the two. The A variant had a significantly higher TEAC value before digestion than

the B and E variants. This was rather surprising, because none of the amino acids

substituted in the κ-CN variants were reported to scavenge the ABTS radical (Aliaga and

Lissi, 2000; Clausen et al., 2009). Conversely, both oxidant and pro-oxidant capacity of all

the substituted amino acids were reported using assays estimating lipid peroxidation and

hydrogenperoxide and hydroxyl radical scavenging (Martinez-Tome et al., 2001). It is

unclear whether these effects may be relevant in the present system. After pepsin digestion

the TEAC values were the same for all variants, and slightly higher than for the intact

proteins. A further increase was observed after 5 minutes digestion with PE, and only the E

variant did not increase significantly above the level seen after pepsin digestion. The

largest increase was demonstrated after the full 120 minutes digestion with PE, but again

the E variant did not increase above the level of the A and B variants from the preceding

Figure 22. Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on Trolox equivalent antioxidant capacity (TEAC, µmoles trolox equivalents/mg protein) of κ-casein variants. κ-casein was incubated with the ABTS radical for 60 minutes at room temperature and the reduction in abs(734 nm) was measured and related to that obtained with Trolox. Bars represent mean values, error bars are SEM (n=3).Different letters denote significant difference between means (p < 0.05). (from paper 3).

50

step. A possible reason is the lower DH of the E variant in the final steps of digestion

(Table 10 section 3.4). Caroli et al. listed the A and B variants as common, and the E

variant as rather common (Caroli et al., 2009). Therefore, it may be assumed that the

commercial κ-CN preparation contains some E variant, which may explain both the

slightly lower DH, and concomitantly lower TEAC, compared to the A variant. The TEAC

values reported here are markedly lower than those presented by Kumar et al. as well as

Gómez-Ruiz et al. (Gómez-Ruiz et al., 2008; Kumar et al., 2010), who reported

approximately 5 and 6 fold higher TEAC values after IVGD of κ-CN. However, the fold

increase from pre- to post-IVGD was similar between those two studies and the present

one, indicating that the differences in absolute TEAC values may be attributable to

interlaboratory variations. Comparing the β- and κ-CN variants it is clear in the data

presented here, that the ABTS radical scavenging capacity of κ-CN is lower, but conversely

responds more to IVGD, which is in line with Gómez-Ruiz et al. In the studies discussed

above the TEAC values after IVGD were nearly identical between β- and κ-CNs within each

study.

Synthesized Peptides VYPFPGPIHN, VYPFPGPIPN, and TER

Three peptides were chosen from the peptide profile of the β-CN variants for further

characterization by measuring their TEAC values. No ABTS radical scavenging was

detected for TER. Of the two V-βCM-9 peptides VYPFPGPIPN was previously reported as

antioxidative in the ABTS decolorization assay (Eisele et al., 2013). However, no reports on

the impact of the H67P substitution in the β-CN variants could be found. Figure 23 shows

that the A1-like V-βCM-9 has a significantly higher TEAC than its A2-like counterpart,

indicating that a H residue is favorable over a P at the penultimate position. This is in

accordance with H, and not P, having proven ABTS radical scavenging capacity (Aliaga and

Lissi, 2000; Clausen et al., 2009). However, the TEAC of A2-like V-βCM-9 is 20% lower

than that of A1-like. According to Clausen et al. the ABTS radical scavenging capacity of Y

is almost 2500 times that of H and F, so it seems unlikely that the H67P substitution alone

can explain the observed difference. The introduction of an additional P may affect the

peptide structure, because of the constrained conformation of the P sidechain, and

subsequently reduce the intramolecular radical stabilization.

51

4.4 Summary of Antioxidant Capacity of β- and κ-Casein Hydrolysates and

Peptides

The ABTS racical scavenging capacity of β-CN variants A1, A2, B, and I, and κ-CN variants

A, B, and E was determined before, and at three points during IVGD. The β-CN variants

showed small differences between variants, and the TEAC was significantly increased by

the end of digestion. The κ-CN variants showed more individual differences in TEAC, with

the A variant having a higher TEAC before digestion compared to the B and E variants, and

the E variant having a lower TEAC at the end of digestion, compared to the A and B

variants. Taken together, these results suggest that there are significant effects of amino

acid substitutions in these proteins, either directly on ABTS radical scavenging or by

indirect effects through the lower DH. The DH also clearly affected the TEAC, and

apparently κ-CN more than β-CN. The two synthesized V-βCM-9 were assessed as well,

and here it was shown that the H67P substitution significantly reduced the TEAC of the

decapeptide. The magnitude of the reduction was more than would be expected by the

substitution alone, and it is therefore suggested that some structural change of the peptide

takes place as well, which adds synergistically to the reduction in TEAC.

Figure 23. Trolox equivalent antioxidant capacity of two V-βCM-9 like peptides. Bars represent mean values of three independent determinations. Error bars are sem (n=3). Asterisk denotes significant difference between means (p < 0.001).

52

Chapter 5. Hypertension, the Renin-Angiotensin System and ACE

One of the main systems regulating blood pressure (BP) homeostasis in the body is the

renin-angiotensin system (RAS). The intricate details of its function and the knowledge

obtained through decades of research has been comprehensively reviewed (Oparil and

Haber, 1974; Peach, 1977; Reid et al., 1978; Paul et al., 2006). A brief description will be

given here, and an overview of pathways and interactions is given in Figure 24. When BP

becomes low renal perfusion is decreased, which stimulates the juxtaglomerular cells to

secrete the enzyme renin into circulation. Renin is highly specific for the L10-L11 bond at

the N-terminus of angiotensinogen, a glycoprotein that is produced by and secreted from

the liver. The product of renin’s action is the decapeptide angiotensin 1 (AT1). In a second

proteolytic step AT1 is C-terminally truncated into the active octapeptide angiotensin 2

(DRVYIHPF-(HL), AT2) by removal of the dipeptide HL. This second step is the result of

hydrolysis by angiotensin-1 converting enzyme (ACE, EC 3.4.15.1). ACE is a membrane-

bound protease found in high concentrations in pulmonary endothelium, but it has been

described in multiple tissues throughout the body (Paul et al., 2006). Initially, the main

effect of AT2 was considered to be stimulation of vasoconstriction leading to an increase in

BP. However, several other effects have been described as well. AT2 induces aldosterone

secretion from the adrenal cortex, and aldosterone then causes an increase in tubular

Figure 24. The renin-angiotensin system (RAS). The RAS is a system of enzymes and peptide hormones that act on different tissues in the body to maintain blood pressure homeostasis. See text for a detailed description. (Modified from en.wikipedia.org/wiki/Renin-angiotensin_system).

53

sodium and chloride reabsorption and associated water retention, leading to increased

blood volume, and consequently also a BP increase. AT2 also causes secretion of

vasopressin (antidiuretic hormone) from the pituitary gland, which has the effect of

increased water reabsorption. In addition to conversion of AT1 to AT2 ACE also deactivates

bradykinin by removal of a C-terminal dipeptide. Bradykinin is a vasodilator, so it’s

deactivation will also serve to elevate BP. The RAS is controlled mainly by a feedback loop,

where AT2 sensitizes the juxtaglomerular apparatus to the increased perfusion caused by

the elevated BP. Thereby, renin secretion is inhibited and BP remains within an

appropriate range. Hypertension presents a major global health challenge, and is

attributed to approx. 10 million deaths annually (Lim et al., 2012). The RAS offers several

possible drug targets for the treatment of hypertension.

5.1 Angiotensin-1 Converting Enzyme and Inhibitors

ACE is the target of many antihypertensive drugs, because of the many actions associated

with it’s action on AT1 and bradykinin, and inhibiting ACE has significant BP lowering

effects (Johnston et al., 1979; Simon et al., 1983; Brown and Vaughan, 1998). ACE was first

described in 1953 where the presence of an enzyme capable of converting hypertensin 1

(AT1) into hypertensin 2 (AT2) was observed in horse plasma (Skeggs et al., 1954). ACE is

a highly glycosylated zinc metalloprotease composed of two homologous domains

(Bernstein et al., 1989). It’s primary action is the removal of C-terminal dipeptides, and it

has been showed to take a variety of peptide substrates (Ondetti and Cushman, 1982). It

was described that the peptide venom from Bothrops jararaca, a south-american viper,

could inhibit the action of ACE, and this peptide became the inspiration for development of

the first synthetic ACE inhibitor Captopril (Ondetti et al., 1971; Hayashi and Camargo,

2005). Many other synthetic ACE inhibitors have since entered the market (Brown and

Vaughan, 1998).

There have been considerable efforts made to elucidate the structural requirements for

ACE inhibitory peptides (AIPs). Cheung et al. reported results from studies with synthetic

dipeptides, that indicated that aromatic amino acids and P were favorable at the C-

terminal position, and aliphatic branched sidechain amino acids were favorable at the

54

penultimate position (Cheung et al., 1980). Later, Wu et al. conducted thorough

quantitative structure-activity relationship (QSAR) studies of the requirements for di- and

tripeptides, as well as for tetra- to decapeptides (Wu et al., 2006a; Wu et al., 2006b). For

dipeptides they found that amino acids with bulky and aromatic side chains were favorable

for inhibition, which was similar to the findings of Cheung et al. For tripeptides they

reported that aromatic for the C-terminal, positively charged for the middle position, and

hydrophobic for the N-terminal amino acids, respectively, appeared to constitute the

sequences with the greatest inhibitory potential. For the longer peptides the conclusion

was, that the preferred amino acids for ACE inhibition, starting from the C-terminus, are Y

and C for the first position, H, W, and M for the second position, I, L, V, and M for the

third position, and W for the fourth position. In an early report by Cushman et al. snake

venom analogs up to ten amino acids long were tested for ACE inhibition, and it was

concluded that by far the most important determinant of potency was the C-terminal

tripeptide (Cushman et al., 1973). This was, however, somewhat refuted by the QSAR

study of Wu et al. on larger peptides, wherein it was established that the fourth amino acid

from the C-terminus significantly contributed to the predictive power of their model (Wu

et al., 2006a). Thus, it appears that the C-terminal tripeptide of a given AIP should be

considered decisive for inhibition, but that amino acids further from the C-terminus may

modulate the inhibitory potency as well. The proposed binding model of AIPs involves

interaction of the C-terminal tripeptide with the S1, S1’, and S2’ subsites of the ACE catalytic

site, which would compete with substrates such as AT1 and bradykinin. Moreover, it will

position the AIP such that it is in a favorable position to interact with the zinc-ion of the

catalytic site (Ondetti and Cushman, 1982). This was elucidated further by in silico docking

simulations based on the AIPs IPA, FP, and GKP (Pan et al., 2011). In their report it was

suggested that the interactions of AIPs with the ACE catalytic site included hydrogen-

bonds, hydrophilic, hydrophobic, and electrostatic interactions, as well as interaction with

the zinc-ion. Similar interactions were demonstrated by analysis of crystal structures of the

three synthetic ACE inhibitors lisinopril, captopril, and enalaprilat (Natesh et al., 2003;

Natesh et al., 2004). The above discussion illustrates that ACE is somewhat unspecific

towards binding of inhibitors, perhaps as a result of the many different modes of

interaction that are in play.

55

The discovery that ACE could be inhibited by peptides has spawned much research into

identifying possible candidate AIPs derived from food proteins, reviewed in (Ariyoshi,

1993; Iwaniak et al., 2014). Sources of such peptides are quite diverse, e.g. fig tree latex,

sardines, pork, chicken, flaxseed, egg, and pea (Maruyama et al., 1989; Ukeda et al., 1992;

Ariyoshi, 1993; Fujita et al., 2000; Kim et al., 2001; Vermeirssen et al., 2005; Escudero et

al., 2010; Marambe et al., 2011). An excellent source of food derived AIPs is milk proteins

(FitzGerald and Meisel, 2000). In particular, the CNs have been shown to release AIPs as a

result of hydrolysis during food processing or in vitro digestion (Saito et al., 2000; Kudoh

et al., 2001; Hernández-Ledesma et al., 2004a; Ruiz et al., 2004; Salami et al., 2011;

Moslehishad et al., 2013). Generation of any bioactive peptide is the combined result of

protein sequence and structure, as well as the specificity of the protease. Moreover, as the

above discussion shows, the sequence of the AIPs themselves ultimately determines their

inhibitory potential. ACE inhibitory capacity of whole CN or β-CN from camel was

significantly different if the digestion was done with pepsin, trypsin, chymotrypsin, or with

trypsin and chymotrypsin together (Salami et al., 2011). Considering the above discussion,

together with the results presented in sections 3.4 and 4.3, it may be hypothesized that the

different variants of β- and κ-CN differ in their potential for ACE inhibition. This was

investigated for 11 variants of κ-CN using an in silico IVGD approach (Weimann et al.,

2009). In that study, four potential AIPs were identified and chosen for synthesis and in

vitro evaluation of ACE inhibition. These peptides and their IC50 (concentration needed to

achieve half-maximal inhibition) for ACE inhibition are shown in Table 11. These AIPs

support the hypothesis stated above. In addition, there was an 11-fold difference in IC50 of

ASP and VSP, and a 2.4-fold difference between AHHP and ACHP, resulting from single

amino acid substitutions. Furthermore, Hernández-Ledesma et al. demonstrated that

IVGD had a significant impact on ACE inhibition by fermented milk products, human

milk, and infant formulas (Hernández-Ledesma et al., 2004b; a; Hernández-Ledesma et

al., 2007). Thus, the ACE inhibitory capacity of β-CN variants A1, A2, B, and I and κ-CN

variants A, B, and E as it developed during IVGD was investigated in the present studies.

In addition, the ACE inhibitory capacity of the synthesized β-CN derived peptides

VYPFPGPIHN, VYPFPGPIPN, and TER was determined.

56

5.1.1 Methods

Assays for determining enzyme inhibition are simply enzyme activity assays performed in

the presence of the inhibitor compound. In order to determine the activity of an enzyme

three things are needed: (1) the enzyme in a sufficiently pure form; (2) a substrate that

enables monitoring the progression of hydrolysis, either by measuring substrate

disappearance or product formation; (3) a suitable buffer system, that facilitates the action

of the enzyme. The traditional method for assessing ACE activity was described by

Cushman and Cheung in 1971. Their method was based on hydrolysis of the synthetic

peptide of hippuric acid (HA) and H and L (HHL). ACE will remove the HL dipeptide, and

thereby release HA. The HA must then be extracted into ethyl acetate, which yields approx.

90% extraction, including a small fraction of unhydrolyzed HHL. After drying, the HA is

spectrophotometrically quantified (Cushman and Cheung, 1971). Disadvantages of the

method are that it is time-consuming, and the extraction is incomplete. Consequently,

several modified versions have been published over the years, utilizing chromatographic

separation of HA, HL, and HHL, e.g. reversed-phase HPLC and capillary electrophoresis

(Meng et al., 1995; Zhang et al., 2000). However, these methods are still somewhat time-

consuming and require expensive equipment. Alternative methods have been suggested as

well, utilizing direct in-reaction derivatization of primary amines with TNBS and OPA,

followed by spectrophotometric quantification (Matsui et al., 1992; Chang et al., 2001).

Table 11. The IC50a

of suspected angiotensin-1 converting

enyme inhibitory peptides derived from in silico digestion

of κ-casein variants. (Data from Weimann et al., 2009)

Peptide Variant Positionb

IC50

(µM)c

ASP B, C 148 – 150 242.3

VSP F1 148 – 150 021.8

AHHP C 96 – 99 847.6

ACHP G2 96 – 99 360.7

(a) Concentration needed to reach half-maximal

inhibition.

(b) The position of the peptide within the mature κ-casein

amino acid sequence.

57

However, these derivatizing compounds will react non-specifically with any primary

amine. Yet another method was described recently, where a diagnostic assay for measuring

urine HA was adapted to use in a high-throughput microplate-based assay (Jimsheena and

Gowda, 2009). That method was based on adding pyridine and benzene sulfonyl chloride

to the reaction, where a yellow complex with HA will form, which can be

spectrophotometrically detected. The disadvantages of this method are the use of toxic

organic solvents, and the complex-formation is a highly exothermic reaction.

Evaluation of ACE Inhibition Using a Fluorescent Substrate

An elegant method was described by Sentandreu and Toldrá in 2006. This is a procedure

performed in a single step using o-aminobenzoylglycyl-p-nitrophenylalanylproline (Abz-G-

F(N02)-P) (Figure 25). This compound is quenched by intramolecular fluorescence

resonance energy transfer (FRET). Upon hydrolysis by ACE Abz-G-OH is released, which

can be detected fluorometrically using excitation and emission wavelengths of 340 and

400 nm, respectively. The reaction is carried out in a Tris or borate buffer, and the whole

procedure, including fluorometric reading, can be completed in considerably less time than

the traditional HHL based method. In addition, it eliminates the need for organic solvents,

and can be carried out in a single step reaction in microtiter plates. For the studies

presented here the fluorometric readings were measured kinetically (see appendix A). This

enables the use of Vmax for calculations of enzyme activity. The procedure is described in

detail in paper 1. For the assays with CN hydrolysates a single concentration of hydrolysate

giving less than approx. 80% inhibition was chosen. For the synthesized peptides a dilution

series was used, to facilitate calculation of IC50 by four-parameter logistic regression (see

appendix A).

+ ACE

Fluorogenic

Figure 25. The angiotensin-1 converting enzyme substrate o-aminobenzoylglycyl-p-nitrophenylalanylproline. Yields Abz-G-OH upon hydrolysis. Abz-G-OH is fluorescent with emission at 420 nm. (Modified from www.shop.bachem.com).

58

5.1.2 ACE Inhibition by β- and κ-Casein Hydrolysates and Peptides

β-Casein and derived Peptides

The method described above was used to evaluate the ACE inhibitory capacity of β-CN

variants A1, A2, B, and I as it developed during IVGD. The results are shown in Figure 26,

measured using an assay concentration of 0.1 mg/mL. The undigested β-CNs displayed

some ACE inhibition, which increased significantly for all variants after 60 minutes pepsin

digestion. There was another significant increase following 5 minutes digestion with PE,

and a smaller not significant increase after 120 minutes with PE. At 60 and 65 minutes of

digestion the A1 variant exhibited slightly higher ACE inhibition than the A2 variant (p <

0.05). What these data suggests is that digestion of β-CN with pepsin generates AIPs.

Pepsin hydrolyzes proteins preferably at Y, F, and L residues, which is in accord with the

favorable amino acids described earlier (Cheung et al., 1980; Wu et al., 2006a; Wu et al.,

2006b). It is interesting to note that further digestion with PE does not result in a more

pronounced increase, considering that the DH is markedly increased from approx. 3% to

approx. 50%, which is indicative of a large increase in peptide concentration. Reports have

shown that digestion of fermented milk fractions, infant formula, and human milk with

Figure 26 Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on angiotensin-converting enzyme (ACE) inhibitory capacity by β-casein variants (see legend) at 0.1 mg/mL. Captopril concentration was 20 nM. Bars represent mean values, error bars are SEM (n=3). Asterisks denote significant difference from the preceding timepoint (***: p < 0.001). Different letters within timepoint denote significant difference (p < 0.05). There was no overall effect of variant on ACE inhibition. (Modified from paper 1).

59

pepsin and PE generated ACE inhibitory capacity as well (Hernández-Ledesma et al.,

2004b; a; Hernández-Ledesma et al., 2007). In these studies the highest ACE inhibitory

capacity was obtained after pepsin digestion, and then a gradual decrease was observed

during the subsequent digestion with PE. As Wu et al. reported (2006a), there seems to be

some effect of amino acids further from the C-terminus. These results, together with those

presented here, suggest that the longer peptides present after pepsin digestion may be as

potent as the shorter peptides present after PE digestion. Fitzgerald and Meisel reviewed

the literature on AIPs derived from bovine milk proteins, wherein it is evident that there is

a large variation in IC50 values for di- and tripeptides as well as for AIPs of 5-10 amino

acids in length (FitzGerald and Meisel, 2000, and references therein). Examples include

IPP and YP with reported IC50 of 5 and 720 µM, respectively, and YKVPQL and KVLPVPQ

with reported IC50 of 22 and 1000 µM, respectively (Nakamura et al., 1995; Maeno et al.,

1996; Yamamoto et al., 1999). These examples illustrate the relative importance of specific

peptide sequences, and hence, of the physicochemical properties of AIPs.

Table 12. The IC50a

of angiotensin-1 converting enyme

inhibitory peptides derived from β-casein variants A1, A2, B,

and I. (Paper 2)


IC50

(µM)c SEM

TER B 120 – 122 90 a 8.8

VYPFPGPIHN A1, B 59 – 68 123 a 14.2

VYPFPGPIPN A2, I 59 – 68 656 b 7.6

(a) Concentration needed to reach half-maximal inhibition.

(b) The position of the peptide within the mature β-casein

amino acid sequence.

(c) Different letters within column denote statistically

significant difference (P < 0.001).

The three β-CN derived synthesized peptides were also assessed for ACE inhibitory

capacity, and their IC50 values are shown in Table 12. The novel AIP TER is the strongest

inhibitor of the three, and VYPFPGPIHN is slightly weaker, however, VYPFPGPIPN is 5-6

fold weaker than the two former peptides. It was perhaps surprising that TER proved to be

a moderately strong AIP, because it consists solely of charged and polar amino acids.

However, Escudero et al. identified the similar peptides ER and PER from the hydrolysates

60

of pork meat protein after IVGD with pepsin and pancreatin (Escudero et al., 2010). The

IC50 for these AIPs were determined to be 667 and >1000 µM for ER and PER,

respectively. Thus, it would seem as though the T residue at the N-terminus of TER is

favorable for ACE inhibition, although it does go somewhat against the results of Wu et al.

(2006b). A2-like βCM-9 (YPFPGPIPN) was found in 8 month ripened Gouda cheese, and

was reported to have an IC50 of 14.8 µM (Saito et al., 2000). Later, an IC50 of 325 was

reported for the A2-like V-βCM-9 (VYPFPGPIPN) (Eisele et al., 2013). Thus, a 22-fold

decrease was caused by the addition of an N-terminal V residue, again indicating that

amino acids longer from the binding pockets in the catalytic site of ACE also affect the

binding affinity of AIPs. The same peptide is shown herein as having an even higher IC50.

The H67 instead of P67 in A1-like V-βCM-9 markedly enhances ACE inhibition. The

natural substrate of ACE (AT1), is also a decapeptide with a H residue in the penultimate

position, and it was one of the preferred amino acids at this position according to Wu et al.

as well. The charge of the imidazole ring of the H residue thus facilitates stronger

interaction with the S1’ pocket of the catalytic site in ACE.

κ-Casein

The ACE inhibitory capacity of κ-CN variants A, B, and E, as it developed during IVGD was

measured, and the results are shown in Figure 27. The assay concentration used here was

0.05 mg/mL. In contrast to the β-CNs the undigested κ-CNs did not inhibit ACE. Because

it is the C-terminus of a peptide that interacts with ACE the explanation may be found

there. The last 9 amino acids in all the β-CN variants are VRGPFPIIV, and those of the κ-

CNs are TVQVTSTAV. Both sequences end with a V, but are otherwise quite distinct from

each other. The β-CN C-terminus is markedly more hydrophobic with a grand average of

hydropathicity index of 1.34, compared to 0.89 for the κ-CN terminus, calculated using the

online ProtParam tool on the expasy website (web.expasy.org/protparam, accessed

October 25, 2014). The four C-terminal amino acids of the β-CNs correspond favorably to

those suggested by Wu et al., while those of the κ-CNs are less in line with those

suggestions (Wu et al., 2006a). In addition, the ACE inhition at a concentration of 800 µM

of the dipeptide AV, corresponding to the C-terminus of κ-CN, was reported to be just 15%,

while the C-terminal nonapeptide VRGPFPIIV from β-CN had an IC50 of 630 µM

(Ichimura et al., 2003; Miguel et al., 2006). Upon initial digestion with pepsin there was a

61

large increase in ACE inhibition to approx. 60%, followed by smaller increases at the final

two time points, with a final inhibition just above 80%. A significant difference was

observed between 60 and 180 minutes of IVGD for the A variant, but not the B and E

variants. The evolution of ACE inhibition during IVGD of the κ-CNs shares many

characteristics with the β-CNs, and thus much of the same argumentation hold here as

well. However, ACE inhibition by the κ-CNs is higher than by the β-CNs, even though the

concentration was 50% lower, indicating that IVGD of κ-CN may result in more potent

AIPs than β-CN. Comparison of κ-CN variants revealed no effect of variant, neither overall

nor within single time points. This suggests that the amino acid substitutions in these

variants do not have a significant impact on ACE inhibitory potential. This does not

exclude the possibility of more or less potent AIPs being generated by IVGD, since the

effect of such variant-specific AIPs may be drowned out by the combined inhibition of all

AIPs in the hydrolysates.

Figure 27. Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on angiotensin-converting enzyme (ACE) inhibitory capacity by κ-casein variants (see legend) at 0.05 mg/mL. Bars represent mean values, error bars are SEM (n=3). Asterisk denote significant difference between bars (*: p < 0.05). All digested samples were significantly different from the undigested CNs. (Paper 3).

62

5.2 Summary of ACE Inhibition by β- and κ-Casein Hydrolysates and Peptides

The capacity of bovine CNs to generate ACE inhibitory capacity during both food

processing and IVGD has been extensively described. Here, the ACE inhibitory capacity of

β-CN variants A1, A2, B, and I and κ-CN variants A, B, and E was evaluated before, and at

three different points during IVGD. β-CN displayed some ACE inhibition before IVGD, and

κ-CN did not, which is a possible consequence of the specific C-terminal amino acids in the

two types of CN. For all investigated CN variants there was a pronounced effect of IVGD,

which was markedly larger for the κ-CNs than for the β-CNs, evidenced by the higher ACE

inhibition by the κ-CNs using only half the concentration of the β-CNs. Furthermore, it

appears that amino acid substitutions in these CNs have little or no impact on the total

ACE inhibitory capacity during IVGD. However, obvious differences were revealed when

the individual β-CN derived peptides were assessed. It is clear that for βCM-9 derived

peptides the H found in the A1 and B variants at the penultimate position confers greater

ACE inhibition than the P found in the A2, and I variants.

5.3 The Intestinal Brush-Border and it’s Model System

The ability of a peptide to inhibit ACE in an in vitro setting is one thing, but whether this

ability bears any physiological relevance is determined by other factors as well. If the AIPs

are generated during processing or other pre-treatment before ingestion, they must be

resistant to GI proteases to retain their activity. If they are generated by IVGD as herein, or

by digestion in the lumen of the GI in vivo, they will naturally be mostly resistant to further

hydrolysis here, but must still resist hydrolysis by the brush border peptidases (Zhou,

1994; Pauletti et al., 1996). The collection of brush border proteases consists mainly of

exopeptidases, of which there are several of both amino- and carboxypeptidases (Tobey et

al., 1985). Thus, there is the possibility that any in vitro established AIPs will lose their

activity before having a chance to exert their effects on the organism. Moreover, the AIPs

need to traverse the epithelial wall as well, either by active transport, facilitated diffusion,

or passive diffusion. An example of one of the main peptide transporters in the brush

border is peptide transporter 1 (PepT1), which has affinity for di- and tripeptides (Adibi,

1997). Passive diffusion can take place through the tight junctions that uphold the integrity

of the intestinal wall. This route, while believed to be mostly devoid of proteolytic activity,

63

does impose structural restrictions regarding size, charge, and hydrophilicity (Pauletti et

al., 1996). Using a metabolically stable hexapeptide, and different analogs thereof, Pauletti

et al. showed that the permeability of hydrophilic peptides across a Caco-2 cell monolayer

was enhanced by the addition of a positive charge. Conversely, as molecular size was

increased the effect of charge diminished (Pauletti et al., 1997). Thus, there are different

routes with different requirements for transport or permeation of potentially hypotensive

AIPs.

The human intestinal cell line Caco-2 has traditionally been used by the pharmaceutical

sciences to study the toxicity and absorption of drugs. The Caco-2 cell line is derived from

a human colon adenocarcinoma, but undergoes spontaneous enterocytic differentiation in

culture. The principle of this method was established and characterized by Hidalgo et al.

(Hidalgo et al., 1989). The Caco-2 cells are seeded on to a permeable membrane support

that constitutes the bottom of an insert in a standard cell culture well Figure 28. This

effectively creates a system of two fluid compartments separated only by a monolayer of

polarized intestinal cells, much like the organization of the in vivo intestinal wall. The

integrity of the cell monolayer can by assessed by measuring the transepithelial electrical

resistance (TEER) across the monolayer (Hidalgo et al., 1989; Hubatsch et al., 2007;

Günzel et al., 2010). This is a measure of the ionic permeability, or conductivity, and is

thus a proxy for the leakiness of the monolayer. The monolayer permeability can also be

evaluated by using different non-metabolizable non-internalized compounds, such as

phenol red, lucifer yellow, or [14C]mannitol (Jovov et al., 1991; Hubatsch et al., 2007). The

differentiation of the Caco-2 cells can be assessed by using different markers, e.g. gene

Apical chamber

Caco-2 cell monolayer

Basolateral chamber Permeable membrane

Figure 28. Schematic representation of the Caco-2 monolayer assay. The cells are seeded on a permeable membrane support in a small culture well insert, such that the apical and basolateral chambers are separated only by the cell monolayer.

64

Figure 29. Angiotensin-1 converting enzyme (ACE) inhibition by 0.17 mg/mL in vitro digested β-casein variants A1, A2, B, and I before and after 60 minutes incubation with a differentiated caco-2 monolayer. Bars represent mean values from three independent experiments performed in triplicate. Error bars are SEM. Asterisk denotes significant difference between bars of the same variant (p<0.05) (from paper 2).

expression of sucrose isomerase, alkaline phosphatase, or PepT1 (Natoli et al., 2011). In

situ activity measurements of the two former can also be used (Ferruzza et al., 2012).

During optimization of the assay for the present studies in situ alkaline phosphatase

activity, lucifer yellow permeability, and TEER measurements were used (see appendix A

for data). The method is described in further detail in paper 2.

5.3.1 Transport of ACE Inhibition and Resistance to Brush Border Peptidases

β-Casein Hydrolysates

ACE inhibitory capacity of the hydrolysates of β-CN variants A1, A2, B, and I was evaluated

before and after incubation with the Caco-2 monolayer for one hour. Samples taken from

the basolateral (BL) chamber at the same time were evaluated as well, but did not exhibit

ACE inhibition that was within the sensitivity of the ACE assay used here. After one hour

ACE inhibition had increased significantly for the A1, A2, and B variants (p<0.05), and

there was an increase for the I variant as well (p<0.1). This was perhaps somewhat

surprising because it was expected that there would be some transport, as well as

hydrolysis, of peptides, which would reduce ACE inhibition. However, in a study

examining the stability of whey protein hydrolysate towards incubation with both Caco-2

cell homogenate and rat intestinal acetone powder, an increase in inhibition was observed

65

(Vermeirssen et al., 2005). In that study, the IC50 value for the whey hydrolysate was

determined, and reported to be approx. 0.04 mg/mL before and approx. 0.13 mg/mL or

approx. 0.09 mg/mL after incubation with Caco-2 homogenate or rat intestinal acetone

powder, respectively. Those values indicate that the ACE inhibition by the whey protein

hydrolysate was comparable to that presented here for the β-CN variants. The most likely

explanation, as was suggested by Vermeirssen as well (2005), is that the peptidases in the

brush border further hydrolyzes the protein hydrolysates, resulting in an array of peptides

with higher ACE inhibitory capacity. As discussed above, there are several exopeptidases at

the brush border, and it is possible that ACE inhibitory dipeptides will be the product of

the action of some. For example, DPP-IV is a dipeptidyl aminopeptidase with a specificity

for removing dipeptides from N-terminals containing a P in the second position (Chen,

2006). Such dipeptides are potential AIPs, as discussed above in section 5.1.

Synthesized β-Casein Derived Peptides

The three β-CN derived peptides VYPFPGPIHN, VYPFPGPIPN, and TER were incubated

with the Caco-2 monolayer for 2 hours. Subsequently, ACE inhibition of the sample buffers

Figure 30. Angiotensin-1 converting enzyme (ACE) inhibition by 83 µM synthesized β-CN derived peptides before and after 120 minutes incubation with a differentiated caco-2 monolayer. Bars represent mean values from two independent experiments performed in triplicate. Error bars are SEM. Asterisk denotes significant difference between bars of the same peptide (p<0.05) (from paper 2).

66

from both the apical and the basolateral chamber was assessed at an assay concentration of

83 µM (Figure 30). A small non-significant increase was observed for VYPFPGPIHN,

suggesting that it was neither transported, nor hydrolyzed to any noticeable degree. In

contrast to this, ACE inhibition by VYPFPGPIPN was more than doubled, possibly due to

hydrolysis by dipeptidases in the brush border that resulted in ACE inhibitory dipeptides.

ACE inhibition by TER was reduced to less than half after incubation. This may be the

result of either hydrolysis and loss of activity; transport across the monolayer; or

internalization into the cells of the monolayer. Transport across the monolayer is unlikely,

because no ACE inhibition was detected in the basolateral chamber after incubation.

Internalization is probably the more likely explanation, because TER is a substrate for

PepT1, which is expressed in Caco-2 cells and transports di- and tripeptides (Adibi, 1997;

Sun et al., 2008; Natoli et al., 2011). There was no detectable ACE inhibition from the

basolateral chambers of the two decapeptides.

5.4 Summary of ACE Inhibition at the Brush Border

After incubating the β-CN hydrolysates and derived peptides with a Caco-2 monolayer, it is

clear that the brush border has an effect on the activity of AIPs. The hydrolysate of all four

β-CN variants responded with a modest increase in ACE inhibition after incubation,

although it was only a tendency for the I variant. The most likely explanation for this

observation is that brush border peptidases hydrolyzed some peptides, and thus generated

a set of peptides with greater ACE inhibitory capacity. When the pure peptides were

incubated with the monolayer the effect differed between all three peptides. This suggests

that whatever peptidase activity is present has some specificity that is influenced by the

sequences of these peptides. That no ACE inhibition could be detected for any of the

basolateral samples may be explained by either, (1) no or little transport of peptides; (2)

hydrolysis of AIPs during transport; (3) inadequate sensitivity of the ACE inhibition assay.

It was scheduled that all samples should be analyzed by LC-MS/MS in order to describe

the peptide composition , but unfortunately there was an equipment failure that could not

be corrected in time for the submission of the present thesis.

67

Chapter 6. General Summary, Conclusions, and Future Perspectives

The overall aim of the presented PhD project was to explore possible links between

genetically contingent variations in bovine CNs and properties related to human nutrition,

physiology, and health. The genotypes of more than 800 cows in the Danish part of the

Danish-Swedish Milk Genomics Initiative were examined to identify those that were

homozygous for either β- or κ-CN. These were used for purification of the genetic variants

used in the presented work. The purified CNs were the β-CN variants A1, A2, B, and I, and

the κ-CN variants A, B, and E. Overall, the presented results show that pure β- and κ-CN

variants can be isolated from milk from genotyped animals, and that single amino acid

substitutions have some impact on digestion and bioactive potential of these seven CN

variants. More specifically, it was shown that

Relatively pure (approx. 90%) β-CN can be isolated without

chromatography, and without using urea, in stead utilizing cold storage

and ultracentrifugation. However, the yield will be lower than by using

traditional chromatographic methods, because the isolated fraction of β-

CN is limited by the release of free β-CN from the CN micelles.

Calcium-precipitation of αs- and β-CN from the total CN fraction leads to

κ-CN enrichment of the remaining supernatant. This enables the isolation

of κ-CN from the supernatant to a high purity (>95%) by ion-exchange

chromatography.

LC-based analysis of protein composition, in combination with predicted

and measured absorbance at 280 nm, can be used to estimate absolute

protein content of purified β-CN fractions.

All CN variants are highly digestible, however, β-CN appears to contain

regions that may be slightly resistant to hydrolysis by pepsin. This was not

observed for κ-CN, and this resistance towards pepsin hydrolysis is

hypothesized to stem from β-CN’s tendency to aggregate at 37 °C.

Upon digestion all the β-CN variants reached a rather high DH of approx.

50%, with no difference observed between variants. However,

fragmentation patterns and peptide profiles were detected by SDS-PAGE

68

and LC-MS/MS, demonstrating that amino acid substitutions clearly affect

the hydrolysis by gastrointestinal proteases.

The κ-CN variants were more digested by pepsin than β-CN, evidenced by a

higher DH and smaller fragments observed by SDS-PAGE, but did not

obtain the high DH of β-CN by the end of IVGD. Additionally, the DH of

variant E was lower than those of variants A and B, possibly a result of

PTMs.

The antioxidant capacity (TEAC) of all studied CNs was significantly

affected by IVGD. The effect of digestion was larger on the κ-CN variants

than on the β-CN variants. The undigested κ-CN variants displayed

significantly different TAEC values (A > B = E), which had changed after

IVGD (A = B > E). The lower TEAC of variant E after IVGD may be an

indirect effect of the lower DH.

ACE inhibition was observed for the undigested β-CN variants, but not the

κ-CN variants, possibly due to the C-terminal residues of the intact

proteins having different affinities for binding to the catalytic site of ACE.

Inhibition by all CNs was significantly affected by IVGD, and small

differences between β-CN variants were observed at 60 and 65 minutes of

digestion, but by the end of IVGD ACE inhibition was the same for all

variants. In addition, the κ-CN hydrolysates appeared to more efficiently

inhibit ACE, than the β-CN hydrolysates.

Antioxidant capacities of the β-CN derived peptides were significantly

different, with no TEAC of the tripeptide TER, and a significantly higher

TEAC of the decapeptides VYPFPGPIHN, as compared to VYPFPGPIPN.

This was likely due to the penultimate H residue conferring additional

radical scavenging, which was possibly further enhanced by radical

stabilizing conformational changes in the peptide.

ACE inhibition by these peptides also differed significantly, with the order

of IC50 being TER = VYPFPGPIHN < VYPFPGPIPN. TER has, to the best of

the authors knowledge, not previously been described as an AIP. The

penultimate H residue confers significantly stronger ACE inhibition by the

69

longer peptides, which is in agreement with the reported structural

requirements for AIPs.

The proteases at the brush border of Caco-2 cells may hydrolyze the AIPs

of total hydrolysates from IVGD of the β-CN variants, and thereby generate

a more potent collection of peptides. ACE inhibitory activity of the

synthesized peptides was affected differently by the brush border, with no

change for VYPFPGPIHN, an increase for VYPFPGPIPN, and a decrease

for TER.

No transport of ACE inhibitory activity across a Caco-2 monolayer could be

detected in the present study.

Taken together, this thesis expands on the growing number of studies showing that there

are clear indications that genetic variation in bovine CNs impacts on the peptide profile

following digestion. It may also affect the antioxidative and ACE inhibitory capacity of the

resulting hydrolysates and peptides, and the behavior and stability of the peptides in the

intestine. Thus, it may be advisable to consider genetic variation, alongside other

technological and nutritional properties, in the development of new or improved dairy-

based food products.

Further studies in this area should elucidate the differences in fragmentation pattern of CN

variants upon IVGD. This could be achieved by

Improved peptide profiling by quantitative LC-MS/MS

Use of higher complexity IVGD models

IVGD of skim milk samples from cows with known CN haplotypes

Investigating the importance of PTMs for IVGD of CNs, particularly κ-CN

Furthermore, additional screening of in vitro bioactive potential could be carried out, e.g.

Enzyme inhibitory capacity, e.g. DPP-IV and renin.

Gut protective effects (regulation of mucus gene expression, or mucus

secretion).

70

Continued analysis of transport kinetics of CN hydrolysates across the

Caco-2 monolayer

NMR or MS based metabolomics using intestinal cell cultures, or ex vivo

intestinal tissue (e.g. Ussing chamber).

In conclusion, the present work shows that, in addition to technological consequences of

genetic variation in milk proteins, there are clearly indications of nutrition and health-

related consequences as well. New information is added to the field of potential biological

effects of the different genetic variants of the bovine CNs by hydrolysis with digestive

enzymes. It is obvious that specific amino acid changes could result in enhanced

bioactivities, however the physiological implications are still unclear, as in vivo effects are

notoriously difficult to predict based on in vitro data. Nevertheless, we are broadening our

knowledge - and we are just now beginning to scratch the surface of this exciting research

field.

71

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84

Appendix A –Supplementary Data for Methods

This appendix will provide some additional information concerning the methods used,

which can not be found in the attached papers (appendix B).

Data Analysis

All CN variants were purified from one individual sample each based on genotypic

information (see section 1.3), and subsequently validated by LC/ESI-MS in triplicate. All

other assays were carried out in two or three independent experiments. The statistical

software package R (version 3.0.2) was used to analyze differences between different

variants and treatments by two-way ANOVA. “Variant” and “digestion time” were used as

factors in the ANOVA. Individual comparisons were done using Tukey’s honest significant

difference post-hoc test, or individual paired t-test where applicable. Differences were

deemed statistically significant if p ≤ 0.05.

In Vitro Gastrointestinal Digestion

The proteolytic activity was determined for the pepsin and PE preparations used in the

IVGD as described in paper 1. One unit of proteolytic activity was defined herein as the

volume of enzyme preparation that will produce 1 mg trichloroacetic acid soluble BSA

equivalents per minute, as detected by the bicinchoninic acid kit according to the

manufacturers manual (Fischer Scientific, US). Figure A.1 shows the results from the

analysis of pepsin. The activity of the PE was assessed analogously. The linear regression

was used to calculate the specific activity.

85

To verify the heat-inactivation of pepsin and PE used during the IVGD procedure, the

activity assessment was carried out using both heat-treated and not heat-treated enzyme

preparations. The results are shown in Figure A.2. There was no detectable activity after

heat-treament.

Figure A.1. Increasing volumes of a pepsin preparation was incubated with acidified hemoglobin. Plotted against the concentration of trichloroacetic acid soluble BSA equivalents released per minute. The results are the mean values from three independent determinations carried out in duplicate.

Figure A.2. Proteolytic activity of pepsin (left) and pancreatic enzymes (right) before and after

heat-treatment at 90 °C for 20 minutes.

86

ACE Inhibition Assay

As mentioned in section 5.1.1 the ACE activity, and it’s inhibition was based on kinetic

readings. The slope of the increase in fluorescence units (arbitrary) was taken as the

relative activity. Uninhibited ACE was considered as 100% activity, and substrate without

enzyme was considered as 0% activity, or background. The synthetic ACE inhibitor

captopril was used as a reference inhibitor. Figure A.3 shows representative curves from

one experiment.

IC50 of ACE Inhibitory Peptides

Based on the inhibition percentages of the three β-CN derived peptides the IC50 was

calculated by four parameter logistic regression (Sebaugh, J. L., Pharmaceutical Statistics,

2011, 10, 128-134). The calculations were performed using the built-in function of the

scientific graphing and statistical software package SigmaPlot v. 11. Figure A.4 shows a

representative inhibition curve of a peptide, with the IC50 indicated.

Figure A.3. Angiotensin-1 converting enzyme was incubated with an internally quenched substrate for 30 minutes without inhibitor (square, diamond), with peptide (star), or with captopril (horizontal curves).

87

Caco-2 Cell Culture

To monitor the integrity of the caco-2 monolayer during incubation with samples, the

TEER values were measured before and at the end of treatment. This was done in order to

ensure that the different treatments did not have any detrimental effects on the integrity of

the monolayer, which would scew the results. The TEER values were calculated as

TEERstart = (Rinitial - Rblank) × A

TEERend = (Rend - Rblank) × A

TEERloss% = (1-(TEERend/TEERstart)) × 100%

where R is the resistance measured with chopstick electrodes connected to a volt-

ohmmeter, and A is the growth surface area. Subscripts indicate measuring R at the

beginning or end of incubation, and Rblank is the background resistance for a cell culture

insert with no cells. Any cell culture insert where there was a loss in TEER of more than

20% were not included in the further analysis. In addition, the permeability of the

fluorescent dye lucifer yellow (LY) was assessed during treatment. LY at 100 µM was added

to all experimental solutions and controls (Hidalgo et al. Gastroenterology, 1989, 96, 736-

749). After incubation, fluorescence of all samples was measured and converted to

Figure A.4. Example of inhibition curve for angiotensin-converting enzyme (ACE) inhibition by a synthesized peptide. The dashed line indicates the IC50 (mM).

88

concentration by using an LY standard. Samples were only included in the dataset if less

than 0.5% LY had permeated per hour. During assay optimization the activity of alkaline

phosphatase was monitored after 8, 15, and 22 days in culture, as a marker of

differentiation. The general principle of the assay is based on incubation with p-nitro-

phenol phosphate at slightly alkaline pH for 10 minutes, and quantification of the p-

nitrophenol generated in the reaction buffer, by comparing to a standard curve of p-

nitrophenol. The assay was carried out basically as described (Ferruzza et al. Toxicology in

Vitro, 2012, 26, 1247-1251). The increased activity of alkaline phosphatase is indicative of a

differentiated monolayer.

0

2

4

6

8

10

8 15 22

nm

ol p

NP/

cm2

/min

Days post confluence

ALP activity

Figure A.5. Alkaline phophatase activity of caco-2 monolayer after 8, 15, or 22 days in culture.

89

Appendix B – Single Letter Amino Acid Codes

A = Alanine

C = Cysteine

D = Aspartic acid

E = Glutamic acid

F = Phenylalanine

G = Glycine

H = Histidine

I = Isoleucine

K = Lysine

L = Leucine

M = Methionine

N = Asparagine

P = Proline

Q = Glutamine

R = Arginine

S = Serine

T = Threonine

V = Valine

W = Tryptophan

Y = Tyrosine

90

Appendix C – Papers

Paper 1

Manuscript accepted for publication in Journal of Dairy Science

In vitro digestion of purified beta-casein variants A1, A

2, B, and I: Effects on antioxidant and

angiotensin-converting enzyme inhibitory capacity

B. Petrat-Melin*, P. Andersen

*, J. T. Rasmussen

†, N. A. Poulsen

*, L. B. Larsen

*, J. F. Young

*1

* Department of Food Science, Aarhus University, 8830 Tjele, Denmark

† Department of Molecular Biology and Genetics – Molecular Nutrition, Aarhus University, 8000

Aarhus C, Denmark

1 Corresponding author: [email protected]

91

ABSTRACT

Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk, and hence

certain technological properties such as casein (CN) number and cheese yield. However, reports

show that such polymorphisms may also have an impact on the health-related properties of milk.

Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified

from bovine milk originating from cows homozygous for the variants A1, A

2, B, and I by a

combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-

CN was determined by HPLC, variants were verified by mass spectrometry, and the molar

extinction coefficients at λ = 280 nm were determined. β-CN from each of the variants was

subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-

converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at three different

stages of digestion, and related to that of the undigested samples. It is shown that neither molar

extinction coefficients nor their overall digestibility varied significantly between these four variants,

however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular,

after 60 minutes of pepsin followed by 5 minutes of pancreatic enzyme digestion one ≈ 4 kDa

peptide with the N-terminal sequence 106

H-K-E-M-P-F-P-K- was absent from β-CN variant B. This

is likely a consequence of the 122

Ser to 122

Arg substitution in this variant introducing a novel trypsin

cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a

significant increase in antioxidant capacity upon digestion, as measured by the trolox equivalent

antioxidant capacity assay. After 60 minutes of pepsin + 120 minutes of pancreatic enzymes

digestion, the accumulated increase in antioxidant capacity was ≈ 1.7 fold for the four β-CN

variants. ACE inhibitory capacity was also significantly increased by digestion, with the B variant

reaching the highest inhibitory capacity at the end of digestion (60 minutes of pepsin + 120 minutes

of pancreatic enzymes), possibly as a consequence of the observed alternative digestion pattern.

These results demonstrate that genetic polymorphisms have an impact on the digestion pattern and

bioactivity of milk proteins. Moreover, their capacity for radical scavenging and ACE inhibition is

affected by digestion.

Key words: Milk proteins, beta-casein, genetic polymorphism, bioactive peptide.

92

INTRODUCTION

Bioactive peptides are defined as protein fragments that interact with, or have an effect on, bodily

tissues or functions, and thus may influence health positively, and milk proteins are an excellent

source of such peptides (Meisel, 1998; Shah, 2000; Nagpal et al., 2011). The four caseins (CN), αs1,

αs2, β, and κ, constitute ≈ 80% of the protein in bovine milk. Casein derived peptides have been

shown to have a range of effects, such as antihypertensive, antithrombotic, antimicrobial, opioid,

immune modulating, and mineral binding (Silva and Malcata, 2005; Phelan et al., 2009), and are

therefore suitable candidates for the development of novel functional foods. Bioactive peptides are

encrypted within the primary structure of proteins, and may be released through various types of

enzymatic hydrolysis, i.e. the targeted action of microbial or plant derived enzymes; the action of

microbial enzymes during fermentation; or the action of digestive enzymes, either in vitro or in the

gastrointestinal tract (Calbet and Holst, 2004). The set of peptides that are generated from any given

protein depends on the specificity of the proteolytic enzymes and, consequently, on the structure of

the protein itself. Variations in primary structure may therefore influence the bioactive potential of

proteins, e.g. by altering enzyme cleavage sites, modifying protein structure, or by changing the

behavior of the liberated peptides (Kaminski et al., 2007; Caroli et al., 2009). These and other

variations can be the result of genetic polymorphisms, and may suggest that some variants of

proteins behave differently from others with regard to certain health effects (Kaminski et al., 2007).

About 40% of the CN in bovine milk is β-CN (Bobe et al., 1998), and until recently, at least 12

different variants carrying amino acid substitutions had been identified, designated A1, A

2, A

3, B, C,

D, E, F, G, H1, H

2, and I, with the most common being the A

1, A

2, and B variants, reviewed by

Caroli et al. (2009). The number of described variants was increased to 15 in 2013 by the novel

variants J, K, and L described by Gallinat et al. (2013), identified in Bos indicus breeds using DNA-

sequencing techniques. In a recent study involving approximately 800 Danish dairy cattle of the

breeds Danish Holstein and Danish Jersey the B variant was, however, found to be slightly less

common in Danish Holstein than the I variant (Poulsen et al., 2013).

Recently, it was demonstrated that the in vitro digestion of β-CN genetic variants A1,

A2, and B generated different arrays of peptides, and the authors suggested that these peptide

variations could have an impact on immunoglobulin-E binding activity (Lisson et al., 2013). β-CN

also exhibits antioxidative capacity (Pihlanto, 2006), and studies have shown that this capacity is

enhanced by digestion of the protein (Gómez-Ruiz et al., 2008; Kumar et al., 2010). In 2009,

93

Weimann et al. published the results of an in silico digestion study of κ-CN, wherein variations in

the generation of angiotensin-converting enzyme (ACE) inhibitory peptides from different genetic

κ-CN variants was characterized (Weimann et al., 2009). These effects are of relevance with regard

to ailments such as arthritis, neurodegenerative disease, cardiovascular disease, and cancer

(Halliwell, 2007; Valko et al., 2007). Together these studies indicate that genetic polymorphisms

may indeed influence the bioactive potential of proteins upon digestion.

In order to investigate the digestion and bioactive potential of β-CN variants and their

digestion products, the different variants need to be available in purified form. The basis for the

traditional method of separating β-CN from the other CN was described more than half a century

ago by Hipp et al. (1952). This method hinges on the different solubilities of the caseins in a urea

solution, and often involves some type of chromatography, as reviewed by Imafidon (1997). In the

last two decades methods have been developed, using reverse-phase HPLC (Bobe et al., 1998;

Bonfatti et al., 2008), that are highly effective and able to separate variants of β- and κ-CN, as well

as β-LG. However, these methods still use denaturing conditions that disrupt the native secondary

structure of the proteins. For some applications, such as in vitro digestion, the presence of urea can

be problematic as it can cause carbamylation of Lys and Arg side chains, as well as protein amino

terminals (Stark et al., 1960; Stark, 1965; Kollipara and Zahedi, 2013). It is therefore preferable to

avoid urea in the purification process, because carbamylation of proteins may lead to changes in the

pattern or extent of digestion, as well as changed chromatographic retention times. Moreover,

protein modifications induced during the purification procedure could have the potential to alter the

behavior of the purified variants relative to the native forms (Mun and Golper, 2000). If denaturing

is required in other downstream applications, an alternative chaotrope, like guanidine

hydrochloride, that does not cause protein modifications, could be considered.

The aim of the work presented here was to develop a method for purification of β-CN

from milk of cows homozygous for the genetic variants A1, A

2, B, and I, without the use of urea in

the purification protocol. Moreover, to the best of these authors knowledge, the molar extinction

coefficients of isolated β-CN or its variants have not been reported previously. Therefore, these

were determined in order to facilitate rapid protein quantification of the different variants purified

using the presented method. Susceptibility to digestive enzymes, and the bioactivity of peptides, is

largely determined by amino acid sequence. Consequently, the in vitro digestion pattern of each of

these four variants, as well as their bioactive potential in relation to antioxidative and ACE

inhibitory capacities upon in vitro digestion was investigated.

94

MATERIALS AND METHODS

Reagents and Chemicals

Angiotensin-converting enzyme (ACE, EC 3.4.15.1), pepsin (EC 3.4.23.1), pancreatin, bovine CN,

fluorescamine, coomassie brilliant blue G-250 (CBB G-250), ortho-phtaldialdehyde, amino acid

standards, Captopril, and 2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were all

from Sigma (St Louis, US). 2-aminobenzoylglycyl-p-nitrophenylalanyl-proline (ACE substrate)

was purchased from Bachem (Bubendorf, CH). Polypeptide molecular weight markers (3.5 to 26.6

kDa) were from Biorad Laboratories (Hercules, US). All other reagents and chemicals were of

analytical grade.

Purification of β-CN Variants

β-CN was purified from milk samples collected by the Danish-Swedish Milk Genomics Initiative as

previously described (Jensen et al., 2012a). In brief, morning milk from more than 800 individual

cows in mid-lactation was collected, analyzed for fat and protein composition by Milkoscan (Foss

Analytical, DK) and stored at -20 °C until use. Genotyping of all cows was carried out as described

earlier (Poulsen et al., 2013), enabling the identification of milk samples from cows that were

homozygous for the common β-CN genetic variants A1 and A

2, as well as the less frequent variants

B and I. The amino acid substitutions characterizing these variants are shown in Table 1. The frozen

milk samples were moved from -20 °C to 4 °C and kept there for 48 hours for thawing and

dissociation of β-CN from the CN micelle, and stirring was applied for the final 24 hours.

Skimming of the milk samples was then done by centrifugation at 2600 x g and 4 °C for 30 min,

and discarding the top fat-layer, except for the sample with the I variant which had been skimmed

prior to being frozen. The skimmed milk was then ultracentrifuged at 150,000 × g and 4 °C for 2 h,

using an Optima L-80XP Ultracentrifuge (Beckman Coulter Inc., CA), and a titanium Fixed-angle

70-Ti Rotor (angle 23°). This separated the soluble phase, containing the dissociated β-CN, from

the colloidal phase containing the remaining β-CN as well as αs1-, αs2-, and κ-CN . The β-CN was

then isoelectrically precipitated by addition of one tenth final volume 10% acetic acid, leaving for 5

min at room temperature (RT), and then adding one tenth final volume 1 M sodium acetate. The β-

CN was then recovered by centrifugation at 1000 x g and 4 °C for 10 min, and washed three times

in MilliQ H2O. After the final washing step the isolated β-CN was resuspended in MilliQ H2O and

95

lyophilized before storage at -80 °C until use. An overview of the study design and progression of

analysis is shown in figure 1.

Determination of Protein Concentration by Amino Acid Analysis

Amino acid analysis was chosen for protein determination for its long-standing reputation as being

the gold standard (Barkholt and Jensen, 1989). The purified, lyophilized β-CN variants were

dissolved to 0.125 mg/mL in 0.75 M guanidine hydrochloride (GndHCl), and dried by vacuum-

centrifugation. The protein samples were then acid hydrolyzed in 6 M HCl at 110 °C for 20 hours,

vacuum-centrifuged, and redissolved in 10 mM HCl. The amino acid composition was determined

essentially as described by Barkholt and Jensen (1989), using cation exchange chromotography

(CK10U column, 120 × 6 mm, Mitsubishi Chemical, JP), combined with post-column

derivatization with ortho-phtaldialdehyde after oxidation with hypochlorite. The amino acid

derivatives were then detected by an LC 1250 fluoroscence detector (GBC Scientific Equipment,

AU), and the resulting data obtained with the Clarity software package version 2.4 (DataApex, CZ).

The analysis was performed in triplicate from two independent assays, and the protein content

calculated thusly:

AA

AA i β

nprotein% 100%

N V C

where nAA is the molar amount of a specific amino acid, NAA is the number of this amino acid in the

protein sequence, Vi is the initial volume of sample, and Cβ is the concentration of β-CN under the

assumption of 100% protein content in the weighed sample material. Calculations were based on

the average of values obtained based on glycine and alanine residues.

Liquid Chromatography/Electrospray Ionization - Mass Spectrometry

Liquid chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS) was

used to confirm the identity and assess the purity of the isolated β-CN variants. In summary, the

lyophilized β-CN preparations were solubilized in 50 mM GndHCl to a concentration of 3 µg/µL,

and fresh dithioerythritol was added to 15 mM final concentration. The samples were filtered

through a 0.2 µm polytetrafluoroethylene filter (Mini-Uniprep, Whatman, US). Otherwise the

analysis was carried out as previously described (Jensen et al., 2012b). All systems were controlled

96

by ChemStation software, which was also used to obtain individual peak areas from the

chromotograms by integration. The areas were used to calculate the relative protein composition.

Determination of Molecular Extinction Coefficients of the Different β-CN Variants

Lyophilized β-CN was dissolved to 1 mg/mL in 6 M GndHCl, followed by absorption measurement

at 280 nm in a Cary 60 UV/Vis spectrophotometer (Agilent Technologies, US) using solvent as

blank. Their measured molar extinction coefficients were then calculated using the concentrations

obtained from the amino acid analysis, together with the protein composition obtained from the

LC/ESI-MS. The calculation of predicted absorbances was described earlier (Edelhoch, 1967; Gill

and von Hippel, 1989), and here used to create the following equation:

( i ) ( i ) ( i )

( i )

( i )

#Trp 5500 #Tyr 1490 #Cys 125PredictedAbs( 280 ) Frac

MW

where #Trp(i) is the number of Trp residues in the i´th protein, #Tyr(i) is the number of Tyr residues

in the i´th protein, #Cys(i) is the number of Cys residues in the i´th protein, MW(i) is the molecular

weight of the i´th protein, and Frac(i) is the relative amount of the i´th protein in the sample, where i

= (αs1, αs2, β, κ, whey proteins). The numeric factors for Trp, Tyr, and Cys are the extinction

coefficients for these specific amino acids, as determined by Edelhoch (1967). The equation can be

used to extract the molar extinction coefficient deriving from individual milk proteins by dividing

protein i's predicted extinction coefficient by the total predicted extinction coefficient, and

multiplying the obtained fraction by the measured extinction coefficient.

Digestive Enzyme Activities

In order to add equal and physiological (Kalantzi et al., 2006) proteolytic activities in the gastric

and intestinal steps of the digestion, the proteolytic activities of both enzyme preparations were

determined. Pepsin from porcine gastric mucosa was dissolved to 10 mg/mL in 0.1 M phosphate

buffer (PB) that had been adjusted to pH 2.0 with 2 M HCl. The porcine pancreatic extract

pancreatin was suspended to 10 mg/mL in 0.1 M PB (pH 6.5), and vortexed periodically at RT for

30 min, followed by centrifugation at 10,000 g and 4 °C for 20 minutes to precipitate undissolved

material and obtain a water-soluble proteolytic fraction. The supernatant was used for determination

of enzyme activity in the preparation. Proteolytic activity was determined analogously for pepsin

97

and pancreatin extract using 2% acidified bovine hemoglobin (Hb) (Anson and Mirsky, 1932) and

1% bovine CN, respectively, as substrates. Enzyme solutions were incubated at 37 °C with substrate

in a 1 to 9 ratio for 10 min. Then, an equal volume 10% trichloroacetic acid (TCA) was added to all

reactions to stop the enzymatic activity. Solutions were precipitated for 20 minutes at RT, and

centrifuged at 8000 g and RT for 5 minutes. One unit of proteolytic activity was defined as the

volume of enzyme solution producing 1 mg TCA soluble BSA equivalents per minute, as detected

by the bicinchoninic acid assay kit (Thermo Scientific, US) according to the manufacturer manual

under the defined assay conditions.

In vitro Gastrointestinal Digestion

The following procedure, based on previous studies (Calbet and Holst, 2004; Dupont et al., 2010;

Ulleberg et al., 2011), was developed for the present study.

β-Casein solutions. β-CN was dissolved with agitation and gentle heating (50 °C) as 8

mg/mL in simulated gastric fluid (30 mM NaCl) with 0.1 mM NaOH. Following complete

dissolution the pH was adjusted to 2.0 with 2 M HCl. CN solutions were made fresh for each digest.

Digestion. CN solutions were equilibrated to 37 °C before the addition of pepsin at 25

U/g β-CN. The reaction was allowed to run for 60 minutes before adding 54 mM NaHCO3, which

brought the pH to 6.5 and inactivated pepsin (Piper and Fenton, 1965). Then, pancreatic enzyme

solution was added at 25 U/g β-CN and the reaction was continued for an additional 120 minutes.

All samples were heat inactivated at 90 °C for 10 minutes and kept at -20 °C until use.

SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

All samples were diluted five times in an SDS sample buffer (1 M Tris, 1% SDS, 2 mM

dithioerythritol, 20% glycerol, 0.05% CBB G-250) and 32 µg protein per lane was loaded to precast

criterion 10% to 20% Tris-Tricine gels (Biorad, US). Tris-tricine gels were used as they are more

suitable for separation of low molecular weight peptides. The gels were run in a Tris-Tricine

running buffer (100 mM Tris, pH 8.3, 100 mM Tricine, 0.1% SDS), then kept in fixing buffer (50%

ethanol, 8% phosphoric acid) overnight followed by staining with CBB (0.02% CBB G-250, 2%

phosphoric acid (85 %), 5% aluminium sulphate, 10% ethanol) for at least 2 hours (Kang et al.,

2002). Gel images were captured using a UVP Multispectral Imaging System (BioSpectrum,

US/UK).

98

Peptide Identification by Electroblotting and Edman Sequencing

Peptide fragments were subjected to N-terminal amino acid sequencing by cutting bands of interest

from a polyvinylidine difluoride (PVDF) membrane electroblot of an unstained SDS-PAGE gel

(Matsudaira, 1987). Bands were visualized by staining the membrane for 1 minute in 0.1% CBB in

40% methanol, 10% acetic acid and destaining in 40% methanol. N-terminal amino acid sequences

were established by automated Edmann degradation using a Procise Protein Sequencer (Applied

Biosystems, Foster City, CA).

Degree of Hydrolysis by Quantification of N-terminal Amines with Fluorescamine

The degree of hydrolysis (DH) was determined by the method described by Larsen et al. (2004),

which is fast and convenient. In brief, hydrolysates were mixed 1:1 with 24% TCA and precipitated

on ice for at least 30 minutes followed by centrifugation at 14,000 g at 4 °C for 20 minutes. The

supernatants were then used for the fluorescamine primary amine analysis (Udenfriend et al., 1972).

Standard (L-leucine) or sample (30 µL) with TCA was mixed with 900 µL 0.1 M sodium

tetraborate buffer (pH 8.0), followed by addition of 300 µL 0.2 mg/mL fluorescamine in dry

acetone. Fluorescence was measured using excitation and emission wavelengths of 390 nm and 480

nm, respectively. The degree of hydrolysis can then be calculated as follows:

2 h 2 0

2 2 0

[ NH ] [ NH ]DH 100

[ NH ] [ NH ]

where [-NH2] is equal to the concentration of primary amines in the hydrolysed (h) or unhydrolysed

(0) samples, and [-NH2]∞ is equal to the theoretical maximal primary amine concentration assuming

total digestion to free amino acids. [-NH2]∞ was calculated as

( )[ ]

lys CN

2

AA

1 f CNH

MW

where flys is the fraction of Lysine residues in the CN, CCN is the CN concentration, and MWAA is

the mean molecular weight of amino acids in the CN.

2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) Decolorization assay

99

The ABTS decolorization assay is widely usd to determine antioxidant capacity of food constituents

(Moon and Shibamoto, 2009), and was used to determine the antioxidant capacity of the β-CN

variants and their hydrolysates, essentially as described elsewhere (Re et al., 1999). Excess ABTS

(18.7 mM) was mixed with ammonium persulphate (8.8 mM) and allowed to react overnight at RT

for the formation of the ABTS+∙

radical. The stock solution was then diluted with 75 mM PB (pH

7.4) to give an assay absorbance at 734 nm of ≈ 0.7. β-CN samples were diluted to 0.1 mg/mL with

75 mM PB (pH 7.4). A volume of 50 µL sample or the water-soluble vitamin E derivative Trolox in

75 mM PB (pH 7.4) was mixed with 200 µL ABTS+∙

working solution, and incubated for 60

minutes at RT in the dark before measuring absorbance at 734 nm on a Synergy 2 Microplate reader

(BioTek Instruments Inc., US). The extent of decolorization enables the calculation of the trolox

equivalent antioxidant capacity (TEAC).

Angiotensin-Converting Enzyme Inhibition

The internally quenched fluorescent tripeptide 2-aminobenzoylglycyl-p-nitrophenylalanyl-proline

(ACE substrate) was used to assess the capacity of the β-CN hydrolysates for ACE inhibition. The

method was modified from Sentandreu and Toldra (2006). Briefly, 50 µL ACE stock solution (0.5

U/mL, 0.05 M sodium tetra-borate, 1 M NaCl, 2 mg/mL bovine serum albumin, 50% (v/v) glycerol,

pH 8.2) was diluted 50 times in 0.05 M sodium tetra-borate buffer with 1 M NaCl (BB, pH 8.2), and

50 µL were mixed with 50 µL sample or control in a microtiter plate and incubated for 10 minutes

at 37 °C. Then, 200 µL ACE substrate solution (187.5 µM in BB) was added to each well and

fluorescence readings were initiated immediately, using the excitation and emission wavelengths of

350 and 420 nm, respectively, in a Synergy 2 Microplate reader (BioTek Instruments Inc.).

Readings were taken every minute for 25 minutes.

Data analysis

The β-CN variants were purified from one individual sample each based on genotypic information

(see methods), and subsequently validated by LC/ESI-MS in triplicate. All other assays were

carried out in three independent experiments, unless otherwise stated. The statistical software

package R (version 3.0.2) was used to analyze differences between different variants and

treatments by two-way ANOVA. “Variant” and “digestion time” were used as factors in the

ANOVA. Individual comparisons were done using Tukey’s honest significant difference post-hoc

test. Differences were deemed statistically significant if p ≤ 0.05.

100

RESULTS

Purification of β-CN Variants

The four bovine β-CN variants A1, A

2, B, and I were successfully purified using a combination of

cold storage, ultracentrifugation, and acid precipitation of the obtained supernatant. The LC/ESI-

MS analysis of the precipitated β-CN variants confirmed their homozygousity and genotypes by

having only a single major β-CN peak with the expected masses of 24,018 Da, 23,977 Da, 24,087

Da, and 23,959 Da for variants A1, A

2, B, and I, respectively (all representing forms with 5

phosphorylations) (Figure 2). Using the milk composition data by Milkoscan, combined with

protein quantification by amino acid analysis and assuming 40% of the CN is β-CN, the yield of β-

CN from the purification was calculated. The yields of the four isolated β-CN variants A1, A

2, B,

and I were estimated to be between 5% and 20% of total β-CN originally present in the milk

samples, and the purity relative to total protein determined by LC/ESI-MS varied from 89.2% to

93.2% (Table 2).

Molar Extinction Coefficients of β-CN Variants

The absorption at λ = 280 was measured for the purified fractions of β-CN variants dissolved in 6 M

GndHCl (Table 3). The relative protein composition had been determined by LC/ESI-MS, using the

protein detection wavelength of 214 nm. This was used together with the protein concentration

determined by amino acid analysis, and molecular weight, to calculate the molar extinction

coefficients (ε). For the whole fractions ε-values of 12142 to 14191 M-1

cm-1

were obtained,

deviating less than 11% from the predicted values, based on the known amino acid sequences of

each variant (Table 2). Using the relative composition of milk proteins in each of the purified

preparations, the contribution to ε from β-CN was estimated to be in the range from 9659 to 11448

M-1

cm-1

. These values deviated from the predicted values for these variants by 0% to 16 %. No

significant differences between variants were found in the data presented here.

In vitro Gastrointestinal Digestion

A two-step in vitro gastrointestinal digestion using pepsin and pancreatic enzymes was employed to

estimate the digestibility and digestion pattern of the β-CN variants. The effect of digestion on

101

antioxidant capacity and ACE inhibitory capacity of the hydrolysates was subsequently assessed

(see below). Based on the determined specific proteolytic activities of the enzyme solutions, the

amount of enzyme used in the in vitro digestion corresponded approximately to an

enzyme:substrate ratio of 1:200 in the reaction. The DH for the β-CN variants after 60 minutes of

pepsin digestion (Table 4) was around 3%. After five minutes of digestion with pancreatic enzymes

the DH increased to around 20%, and after 120 minutes of pancreatic enzyme digestion, it was near

50% for all β-CN variants, with the order of DH being A1 > A

2 > I > B. DH can be converted to a

mean length of peptides by 100%/DH (Table 4), which provides a more intuitive measure of

hydrolysis. Following digestion the hydrolysates were analyzed by SDS-PAGE (a representative gel

image is shown in Figure 3). The lanes with the intact β-CN variants show roughly the same pattern

with one clear large band around 25 kDa, corresponding roughly to the molecular weight of β-CN.

In addition, for all four variants a fainter band estimated to approximately 50 kDa is visible, likely a

result of dimerised β-CN, as well as several more or less faint bands and smears. After 60 minutes

of pepsin digestion little undigested β-CN remained, and several bands of lower molecular weight

had appeared for all variants. The addition of pancreatic enzymes changed the digestion pattern

between the β-CN variants, as the B variant was missing a low molecular weight band around 4

kDa, compared to the A1, A

2, and I variants after 5 minutes. After 60 minutes of pepsin + 120

minutes of pancreatic enzyme digestion no bands were detectable, indicating that all peptides had

eluted from the gel under the conditions used here. The band corresponding to the missing band in

the B variant was electroblotted from the A1 variant onto a PVDF membrane, and subsequently the

peptide fragments were sequenced by Edman degradation. The strongest N-terminal sequence tag

deduced from the resulting chromatograms from eight degradation cycles was 106

H-K-E-M-P-F-P-

K-.

Antioxidant Capacity

The ABTS decolorization assay was used to determine the TEAC values for the β-CN variants

(Figure 4). This method has been used frequently for assessing the antioxidant capacity of food

constituents in vitro (Moon and Shibamoto, 2009). By incubating the stable ABTS+∙

radical with the

synthetic vitamin E analog Trolox, or with the β-CN variants and their hydrolysates, the TEAC

values for the β-CN variants were obtained. There was a significant overall effect of digestion time

(p < 0.001), and of variant (p < 0.01). All four β-CN variants had TEAC values slightly below 0.8

µmoles trolox equivalents/mg protein when undigested, and a small non-significant increase upon

102

60 minutes of pepsin digestion was observed. After 5 minutes of digestion with pancreatic enzymes

the overall TEAC values increased further, compared to the undigested proteins (p < 0.001). At the

end of digestion an additional increase was seen (p < 0.001). Following digestion, there was an

accumulated increase in TEAC for variants A1, A

2, B, and I of 67%, 59%, 59%, and 75%,

respectively. However, these differences were not reflected in a significant interaction effect

between variant and digestion time. Throughout the course of digestion the A1 and I variants

showed progressively higher TEAC values than the A2 variant (p < 0.05) and the B variant (p <

0.1).

Angiotensin-Converting Enzyme Inhibition

ACE inhibition was determined using a synthetic internally quenched fluorescent substrate, and the

results are expressed as 100% minus the residual ACE activity after incubation with the different

samples (Figure 5). The synthetic specific ACE inhibitor captopril at a concentration of 20 nM

inhibited nearly all enzyme activity (98.9% inhibition). For β-CN at an assay concentration of 0.1

mg/mL there was some inhibition from the undigested protein for all four variants (9.3% to 14.3

%), with the A2 and I variants having the highest inhibition. After 60 minutes of digestion with

pepsin the inhibition increased significantly to between 44% and 54% (p < 0.001), with the A1 and

B variants being the most inhibitory. After further digestion for 5 minutes with pancreatic enzymes,

ACE inhibition increased to between 58% and 64% (p < 0.001), and the inhibition levels converged

somewhat. At the end of pancreatic enzyme digestion another smaller increase in inhibition was

seen, which appeared to be most pronounced for the B variant. However, there was no overall

significant effect of neither variant nor its interaction with time.

DISCUSSION

The research field of milk genomics attempts to link variation at the genome level to variation in

nutritional, technological, and health-related properties of milk. So far, the focus has been mainly

on productional, compositional, and technological phenotypes, such as milk yield, casein number,

and coagulation behavior (Martin et al., 2002; Poulsen et al., 2013), and less on nutritional and

health-related properties, although the latter have been gaining interest in recent years (Kaminski et

al., 2007; Caroli et al., 2009; Lisson and Erhardt, 2011). In the work presented here the A1, A

2, B,

and I variants of β-CN from milk samples of cows that were homozygous at the β-CN locus have

103

been purified using a relatively simple, non-denaturing method. Thawing and storing the milk

samples at 4 °C favors the monomeric state of β-CN, which has a strong tendency to form

polydisperse micellar structures at higher temperatures, as shown by Payens and Van Markwijk

(1963), and more recently by O’Connel and colleagues (2003). The hydrophobic stretch in the

primary structure of β-CN facilitates its association with the other CN through hydrophobic

interactions, which are disrupted by lowering the temperature to 4 °C. Stirring was applied as an

attempt to affect the calcium balance towards the dissociated state, and thereby further enhance the

dissociation of β-CN into the soluble phase, as discussed by Dalgleish and Law (1989). The

LC/ESI-MS analysis showed that the β-CN samples were around 90% pure using the reported

method, albeit with a more modest yield (≈ 12%) than Garnier et al. (1964) and Cayot et al. (1992),

who reported yields of ≈ 30% and ≈ 88%, repectively, using urea-based procedures. These results

do indicate that the present method can be used for semi-preparative scale purification of β-CN for

in vitro digestion or bioactivity studies, and limits the risk of modifications with a potential to

influence further analysis. In addition, there were no apparent consequences of skimming the milk

before rather than after freezing, as judged by protein integrity from LC/ESI-MS, SDS-PAGE, and

DH analysis.

As expected, the ε values for the four variants did not differ significantly, because

molar extinction coefficients are largely determined by the number of Tyr, Trp, and Cys residues in

a protein (Edelhoch, 1967; Pace et al., 1995), and all 4 variants have the same number of these

residues, i.e. four Tyr, one Trp, and no Cys. In the study by Pace et al (1995) the molar extinction

coefficients at 280 nm of 80 different proteins were measured and compared to their respective

predicted values. In general, deviations of less than ± 5% were seen, with a small number deviating

± 10% to 16%, which is comparable to the deviations presented here. For the β-CN fractions, the I

variant deviated the most, however, when only considering the contribution to the absorption from

β-CN the B variant deviated most. This may, in part, be ascribed to the estimation of milk protein

composition of the fractions analysed by LC using absorbance at 214 nm, which, although being

quite reliable, can exhibit some deviation (Kuipers and Gruppen, 2007). Predicted molar extinction

coefficients based on LC/ESI-MS analysis of protein composition can thus be used to determine the

total protein content of purified β-CN samples, thereby avoiding the use of more time-consuming

assessments, such as amino acid analysis. In addition, for compositional analysis of raw materials at

the dairy, it is relevant to know that the response factor for β-CN absorbance at 280 nm does not

differ between variants.

104

A wide range of in vitro digestion models exist, varying greatly in complexity and

equipment requirements, and the choice of model depends largely on the end-points of interest, as

well as the compound or food components under investigation (Wickham et al., 2009; Hur et al.,

2011). In the present study, a single isolated protein was under investigation, and for this relatively

simple substrate a low complexity method was developed, and the enzymes utilized were gastric

and intestinal proteases, i.e. pepsin and pancreatic enzymes, respectively. It has previously been

shown that casein, whey, or their hydrolysates all have similar rates of gastric emptying with half-

times between 18 and 21.5 minutes (Calbet and Holst, 2004). Based on this, a digestion time of 60

minutes for pepsin was chosen for the static system used here. Furthermore, preliminary

experiments (data not shown) indicated plateauing of pancreatic enzyme digestion after 90 to 120

minutes, and therefore 120 minutes for this step was employed. In the present study, a low DH after

pepsin digestion was observed, but SDS-PAGE revealed that little intact β-CN was present at this

point. This is in good accordance with Dupont et al. (2010), who observed almost complete

disappearance of intact β-CN after 60 minutes of pepsin digestion. The pattern of low molecular

weight bands from our study is similar to that of Dupont and coworkers, even though their pepsin to

β-CN ratio was approximately 10-fold higher than what is presented here. Dupont et al. (2010) also

observed a comparable DH when using a pepsin concentration closer to what is presented here,

which was done in an attempt to closer mimic infant digestion, albeit their observed pattern of

peptides differed slightly from what was seen in the present study. Herein, after 5 minutes of

pancreatic enzyme digestion a rapid increase in DH was determined, and by 120 minutes all

detectable protein bands disappeared. Pancreatin contains several different proteolytic activities, i.e.

trypsin, chymotrypsin, elastase, carboxypeptidase A, and dipeptidase (Mullally et al., 1994), thus,

even though the added level of enzyme activity was comparable to that of pepsin, the protein will be

hydrolyzed at multiple sites simultaneously, which is likely the underlying reason for the rapid

progression of DH. Dupont et al. (2010) did not, under their specific assay conditions, observe the

complete disappearance of protein bands after duodenal digestion. Perhaps this was a consequence

of the shorter digestion time used (60 minutes), but likely more important, because only two

proteases were used, namely trypsin and chymotrypsin. In another study on in vitro digestion of

CN, the DH after 24 h of digestion with a pancreatin:CN ratio of 1:100 only reached 28.5% (Su et

al., 2012), where our results show considerably higher DH after just 2 h of pancreatic enzyme

digestion. Su et al. (2012) did not use pepsin, they used complete pancreatin, not an extract, and

also digested the total CN fraction. Taken together, this might explain the differences in DH

105

reached. In addition, β-CN variant B also exhibited an altered pattern of digestion evident from gel

electrophoresis, which revealed that an approximately 4 kDa peptide, corresponding to about 35

amino acids, was missing compared to the A1, A

2, and I variants. Edman sequencing of this peptide

from β-CN variant A1

identified the 8 N-terminal amino acids as 106

H-K-E-M-P-F-P-K-. The

sequence of β-CN between position 106 and 35 amino acids towards the C-terminal covers position

122, where a Ser is replaced by an Arg in the B variant introducing a novel trypsin cleavage site.

This is the probable cause of the missing peptide, as the products of trypsin cleavage at this site

would produce two peptides of sufficiently small size to elute from the gel completely.

A range of proteins, derived from a variety of plant foods, as well as animal based

foods, such as milk, are known to harbor peptides with antioxidant capacity in their sequence, for

reference see Pihlanto (2006) and Sarmadi and Ismael (2010). The results presented here show that

the intact as well as the digested β-CN variants possess similar antioxidant capacity towards the

ABTS radical, but with a significantly higher radical quenching capacity following in vitro

digestion. This has previously been shown for both combined and individual CN types (Gómez-

Ruiz et al., 2008; Kumar et al., 2010). The effect of digesting β-CN on antioxidant capacity in our

observations (≈ 1.7 fold) were moderately larger than those presented by Gómez-Ruiz et al. (2008),

who analyzed ovine β-CN. However, Kumar et al. (2010) observed a notable greater increase (≈ 4.0

fold). These differences may be a result of different digestion procedures. It was previously noted

that His residues, and their position relative to the N-terminal, may be of importance for the

antioxidative capacity of peptides (Chen et al., 1998). Furthermore, a study investigating the

reactions of individual amino acids with the ABTS radical showed that His together with Cys, Trp,

and Tyr possesses quenching capacity (Aliaga and Lissi, 2000). There are 9 of these amino acids in

the A1 and B variants, whereas there are only 8 in the A

2 and I variants, because the His at position

67 is replaced by Pro. As shown in the present study, there appears to be subtle differences in the

digestion pattern of the variants that may in part be caused by this substitution. Jinsmaa and

Yoshikawa (1999) clearly indicated that there were very different susceptibilities towards

hydrolysis with gastrointestinal enzymes at position 67 of β-CN variants. Therefore, one could

expect that, with respect to TEAC values, the A1 and B variants would be similar, and hence, also

the A2 and I variants. This could, however, not be confirmed here. This may be because not only the

total number of ABTS scavenging amino acids and the DH determine the antioxidant capacity, but

also the position of these amino acids within individual peptides. Further studies are needed to

elucidate this question.

106

ACE inhibitory capacity has been widely reported for β-CN (FitzGerald and Meisel,

2000), and depends on the specific amino acids sequence of candidate peptides. Studies have

revealed that the C-terminal amino acids most strongly associated with ACE inhibitory capacity are

hydrophobic and aromatic residues, such as Trp, Phe, and the branched chain amino acids (Wu et

al., 2006). This is in agreement with Cheung et al. (1980). However, herein also Pro was found to

be favourable at a C-terminal position. FitzGerald and Meisel (2000) also point out that Arg and

Lys at the C-terminal position appear favourable for ACE inhibition. The amino acid substitutions

in β-CN variants A2, B, and I are all potentially activity-altering substitutions, according to the

findings in the mentioned studies. As expected, a marked increase in ACE inhibition was seen upon

initial digestion, which affected the A1 (5.8 fold) and B (5.4 fold) variants to a greater degree than

the A2 (3.0 fold) and the I (2.6 fold) variants. This could be a consequence of the

67His residue

being more permissive than the 67

Pro towards pepsin cleavage after the 66

Ile residue. Cleavage here

would create a peptide with Ile in the C-terminal position. The effect of continued digestion was

much less dramatic, and after 60 minutes of pepsin followed by 120 minutes, compared to only 5

minutes, pancreatic enzyme digestion, none of the variants displayed a significant increase in

inhibition. Nevertheless, the B variant did appear to increase slightly more than the other variants.

Maybe a consequence of the 122

Ser to 122

Arg substitution, as a peptide with a C-terminal Arg may

have ACE inhibitory capacity, as discussed above. Clearly, more work is needed in order to fully

understand the development of ACE inhibitory capacity during gastrointestinal digestion of β-CN

variants.

In summary, the results presented here demonstrate that amino acid substitutions,

caused by genetic polymorphisms at the β-CN locus, do indeed have an impact on the manner in

which these proteins are processed by the gastrointestinal system. Variations in the array of peptides

and bioactive capacities of the β-CN studied here suggest potential health-related implications of

underlying genetic variation, which should be studied further, but show perspective for targeting

raw materials for specific food products.

ACKNOWLEDGEMENTS

The authors would like to extend their gratitude to Hanne S. Møller for operating the LC/ESI-MS,

to Margit S. Rasmussen for invaluable assistance with the amino acid analysis, and to Anni Boisen

for carrying out the Edman sequencing.

107

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113

Table 1. Position of amino acid substitutions

within the mature protein of variants of β-casein

Variant1

Position A1 A

2 B I

67 His Pro Pro

93 Met Leu

122 Ser Arg

1 A

1 is the reference sequence (Caroli et al., 2009).

114

Table 2. Relative content of milk proteins in the isolated β-

casein variant preparations. Values were calculated as relative

peak areas within each chromotogram by LC/ESI-MS

β-casein

variant β-casein κ-casein α-casein

Whey

proteins

A1 89.7 ± 1.4 2.3 ± 0.4 4.6 ± 1.7 3.5 ± 2.5

A2 93.2 ± 1.7 0.5 ± 0.3 1.0 ± 0.5 3.2 ± 2.6

B 89.0 ± 0.1 5.6 ± 1.4 3.4 ± 0.3 1.4 ± 1.1

I 90.3 ± 1.1 2.9 ± 0.7 4.8 ± 0.8 1.7 ± 0.2

All values are expressed as mean percentage of total peak area

± SEM (n = 3).

115

Table 3. Molar extinction coefficients (ε)1 of β-casein variants at λ = 280 nm,

measured in 6 M guanidine hydrochloride. Values are given as means ± SEM (n = 3)

β-casein

variant

ε (fraction)2

measured % Dev3

ε (β-casein)4

measured

%

Dev3

A1 13398 ± 483 2.66 10547 ± 380 - 7.94

A2 12642 ± 1033 5.38 11238 ± 918 - 1.94

B 12142 ± 948 - 5.55 9659 ± 754 - 15.75

I 14191 ± 1154 10.93 11448 ± 931 - 0.04

1 M

-1cm

-1.

2 Total purified fractions.

3 Deviation from predicted values.

4 Molar extinction coefficients deriving from β-casein alone.

116

Table 4. In vitro gastrointestinal digestion of β-casein variants using

pepsin and pancreatic enzymes1

β-casein

variant tpep(min)2 tpan(min)

3 DH (%)

4 MLP

5

A1

60 0 3.6 ± 0.42

a

27.6 ±

3.01

A2

60 0 3.2 ± 0.20

a

31.5 ±

2.20

B 60

0 3.6 ± 0.22a

28.0 ±

1.95

I 60

0 2.6 ± 0.17a

37.6 ±

2.46

A1 60 5 20.4 ± 1.71

b 4.9 ± 0.42

A2 60 5 21.5 ± 2.47

b 4.6 ± 0.55

B 60 5 20.2 ± 0.89b 5.0 ± 0.20

I 60 5 19.4 ± 1.38b 5.2 ± 0.33

A1 60 120 55.0 ± 5.99

c 1.8 ± 0.21

A2 60 120 52.4 ± 5.54

c 1.9 ± 0.19

B 60 120 46.2 ± 3.98c 2.2 ± 0.16

I 60 120 49.9 ± 4.24c 2.0 ± 0.17

1 Results are shown as the mean ± SEM (n = 3 (2 for A

1)).

2 tpep: reaction time with pepsin.

3 tpan: reaction time with pancreatic enzymes.

4 DH: degree of hydrolysis. Different letters denotes significant

difference (p < 0.001).

5 MLP (100% / DH): mean length of peptides.

117

Petrat-Melin

Figure 1.

118

Petrat-Melin

Figure 2.

119

Petrat-Melin

Figure 3.

120

Petrat-Melin

Figure 4.

121

Petrat-Melin

Figure 5.

122

Figure 1. Overview of experimental design.

Figure 2. Bovine β-casein variants A1, A

2, B, and I were purified from milk samples, obtained from

cows that were homozygous at the β-casein locus, and analyzed by liquid chromatography

combined with mass spectrometry. The molecular weight (Da) of the variants are indicated with the

variant name in paranthesis. Horizontal brackets indicate retention times of different milk proteins.

Data are normalized to the maximum value within each individual chromatogram. All variants were

analyzed in triplicate.

Figure 3. Representative SDS-PAGE gel of β-casein variants subjected to in vitro gastrointestinal

digestion with pepsin and pancreatic enzymes. At the top is indicated the variant present in each set

of four lanes, and the numbers indicate the digestion time, with 0: undigested. 60: 60 minutes of

pepsin. 65: 60 minutes of pepsin + 5 minutes of pancreatic enzymes. 180: 60 minutes of pepsin +

120 minutes of pancreatic enzymes. Molecular weight markers are indicated to the left (kDa).

Figure 4. Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on Trolox

equivalent antioxidant capacity (TEAC, µmoles trolox equivalents/mg protein) of β-casein variants.

β-casein was incubated with the ABTS radical for 60 minutes at room temperature and the

reduction in abs(734 nm) was measured and related to that obtained with Trolox. Bars represent

mean values, error bars are SEM (n=3 (n=2 for A1 variant)). Asterisks denote significant difference

from the undigested sample (***: p < 0.001).

Figure 5. Effect of in vitro gastrointestinal digestion with pepsin and pancreatic enzymes on

angiotensin-converting enzyme (ACE) inhibitory capacity of β-casein variants (see legend) at 0.1

mg/mL. Captopril concentration was 20 nM. Bars represent mean values, error bars are SEM (n=3).

Asterisks denote significant difference from the undigested sample (***: p < 0.001). There was no

overall effect of variant on ACE inhibition.

123

Paper 2

Manuscript intended for submission to Molecular Nutrition and Food Research

In vitro gastrointestinal digestion of bovine β-casein variants A1, A2, B, and I

results in different bioactive peptides identified by LC-MS/MS

Bjørn Petrat-Melin, Thao T. Le, Hanne S. Møller, Jette F. Young*, Lotte B. Larsen

Department of Food Science, Aarhus University, 8830 Tjele, Denmark

* Corresponding author: [email protected], Tel: +45 87158051, Fax: +45 87154891

Abbreviations used: CN = casein, ACE = angiotensin-1 converting enzyme, FBS = fetal

bovine serum, ABTS = 2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), LY = lucifer

yellow, MES = 2-(N-morpholino)ethanesulfonic acid, TEAC = trolox equivalent

antioxidant capacity, RT = room temperature, PB = phosphate buffer, BB = sodium-

tetraborate buffer, IC50 = concentration needed to reach half-maximal inhibition, GM =

complete growth medium, DMEM = Dulbecco’s modified Eagle medium, TEER =

transepithelial electrical resistance, HBSS = Hank’s balanced salt solution, βCM = β-

casomorphin, DPP-IV = dipeptidyl peptidase IV, PepT1 = peptide transporter 1.

Keywords: angiotensin-1 converting enzyme, beta-casein, bioactive peptide, genetic

variant.

124

ABSTRACT

Amino acid substitutions in bovine β-casein may affect their fragmentation pattern and

bioactive potential after simulated gastrointestinal digestion. The β-casein variants A1, A2,

B, and I were digested and a peptide profile of the hydrolysates was obtained by LC-

MS/MS. The hydrolysates and the peptides VYPFPGPIHN (variants A1 and B),

VYPFPGPIPN (variants A2 and I), and TER (variant B) were further characterized for

bioactivity. Antioxidant capacity was not detectable for TER, however, it was significantly

higher for VYPFPGPIHN than for VYPFPGPIPN. TER and VYPFPGPIHN showed

significantly higher angiotensin-1 converting enzyme (ACE) inhibition than VYPFPGPIPN

(IC50 of 90, 123, and 656 µM, respectively). ACE inhibition by both hydrolysates and

peptides was also determined before and after incubation with intestinal cells. The

incubation increased ACE inhibition slightly and equally for the hydrolysates of all four

variants. Conversely, ACE inhibition of VYPFPGPIHN was not affected, but a 2-fold

increase for VYPFPGPIPN, and a 2-fold decrease for TER was seen. In conclusion, protein

sequence variation results in the generation of peptides which differ in antioxidant and

ACE inhibitory capacity, and are affected differently by intestinal cells. Therefore, genetic

polymorphisms in β-casein may be a relevant determinant of bioactive behavior.

125

1. INTRODUCTION

The sole source of nutrition for the newborn mammal is milk from the mammary glands of

its mother. This milk must provide sufficient energy and building blocks to support

optimal growth and development, and above all must provide protection to increase the

neonate’s chances of survival. Consequently, there has been enormous positive selection

for individuals producing milk containing a variety of protective compounds. We therefore

see today that mammalian milk has become an extremely complex biofluid, that contains a

vast and diverse array of bioactive molecules, such as hormones, fatty acids,

immunoglobulins, carbohydrates, and bioactive peptides [1, 2]. Bioactive peptides are

protein fragments that are encrypted within the sequence of a parent protein, and can be

liberated by the action of proteolytic enzymes, e.g. in the controlled processing of raw

materials, or in the gastrointestinal tract [3]. The four caseins (CN) αs1-, αs1-, β-, and κ-CN,

constitute approximately 80% of the protein in bovine milk, and of this approximately 40%

is β-CN [4]. Presently, 15 genetic variants of bovine β-CN carrying amino acid substitutions

have been described [5, 6]. Bovine β-CN has been shown to be an outstanding source of

bioactive peptides that exert potentially physiologically relevant effects, such as immune

modulation, mineral binding, opioid agonism, thrombin inhibition, antioxidant capacity,

and angiotensin-1 converting enzyme (ACE) inhibition [7, 8]. Oxidative stress may be

linked to ailments such as neurodegenerative disorders and cancer [9]. Therefore,

antioxidative food derived peptides may be of interest for their potential ability to

neutralize free radicals. However, knowledge about the consequences of genetic variation

on CN derived antioxidative peptides is lacking. ACE is an integral part of the renin-

angiotensin system for the control of blood pressure homeostasis, where its function is to

generate angiotensin-2 by removal of the C-terminal dipeptide from angiotensin-1.

Angiotensin-2 is a potent vasoconstrictor, and inhibition of ACE will therefore have a

blood pressure lowering effect, and many antihypertensive drugs, such as Captopril,

Enalapril, and Lisinopril, are ACE inhibitors. The World Health Organisation assessed the

global prevalence of hypertension to ≈40% for adults 25 years or older [10], which is

estimated to cause close to ten million deaths annually [11]. This has lead to much research

into ACE inhibitory effects of food constituents during recent decades [12], and a large

number of milk-derived ACE inhibitory peptides have been identified, including many

from β-CN [13]. The inhibitory capacity of a peptide is determined by its sequence, and the

amino acid residues at the C-terminal in particular. Cheung et al. published the results of

126

one of the first systematic investigations into this particular subject more than three

decades ago [14]. Since then, many studies have followed, showing that ACE inhibitory

peptides can possess quite diverse structural characteristics. Most of these peptides have

been catalogued in online databases, such as Biopep [15] and ACEpepDB [16]. From this, it

follows that modifications to the amino acid sequence of proteins could affect their

potential for generation of ACE inhibitory peptides. Such modifications can be the result of

genetic polymorphisms manifested in amino acid substitutions, which have been shown to

have consequences for the digestion pattern of β-CN variants [17, 18]. In addition,

Weimann et al. published the results of an in silico digestion study, where it was argued

that gastrointestinal digestion of κ-CN variants would lead to the generation of variant-

specific ACE inhibitory peptides [19]. Besides peptide variation, it is also essential to

consider the stability of any bioactive peptides when exposed to the intestinal

environment. Both animal and human intervention studies with different allegedly anti-

hypertensive foods, hydrolysates, or peptides have been reported [20, 21]. Some of these

provide evidence for the survival and intestinal uptake of ACE inhibitory compounds, and

even systemic blood pressure lowering effects.

The aim of the studies presented here was to investigate the impact of genetic

variants of β-CN on the peptides generated in an in vitro gastrointestinal digestion system.

This was done by tandem mass spectrometric analysis of the in vitro digested β-CN

variants A1, A2, B, and I. Three peptides, with suspected bioactivity, derived from different

variants of β-CN, were synthesized and assessed for antioxidant capacity, and together

with the hydrolysates from the digestion, they were assessed for ACE inhibition as well.

Furthermore, the stability of any ACE inhibitory capacity towards exposure to the human

intestinal Caco-2 cell line was investigated.

2. MATERIALS AND METHODS

2.1. Materials

Caco-2 cells were from American Type Tissue Collection (HTB-37, ATTC, US). DMEM,

fetal bovine serum (FBS), and trypsin-EDTA was from Gibco (Naerum, DK).

Penicillin/streptomycin, non-essential amino acids, PBS, porcine pepsin, porcine

pancreatin, sodium tetra-borate, BSA, ACE, Hank’s buffered salt solution (HBSS), HEPES,

2-(N-morpholino)ethanesulfonic acid (MES), Captopril, 2,2’-Azinobis-(3-

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ethylbenzothiazoline-6-sulfonic acid) (ABTS), and lucifer yellow (LY) were all from Sigma

Aldrich (St. Louis, MO, US). 2-aminobenzoylglycyl-p-nitrophenylalanyl-proline was from

Bachem (Bubendorf, CH). The peptides VYPFPGPIHN, VYPFPGPIPN, and TER were

synthesized by Schafer-N (Copenhagen, DK).

2.2. Purification of β-casein variants A1, A2, B, and I

β-CN variants A1, A2, B, and I were purified from milk of cows homozygous for these

genetic variants using a previously described method [18]. In brief, the frozen milk

samples from genotyped cows [22] were thawed and kept at 4 °C for 48 h, and skimmed by

centrifugation at 2600 g for 30 minutes. This was followed by ultracentrifugation at

150,000 g and 4 °C for 2 h, whereafter β-CN could be acid precipitated from the

supernatant. Finally, the samples were lyophilized and stored at -80 °C until use. The

purity of the β-CN was assessed to ≈ 90% by LC and the variants were verified by MS. For

data see Petrat-Melin et al. [18].

2.3. In vitro gastrointestinal digestion of β-casein variants

The purified β-CN variants were subjected to in vitro gastrointestinal digestion as

previously described [18]. Briefly, the digestion consisted of a two-step static system with a

simulated gastric phase using porcine pepsin at pH 2.0 for 60 minutes, followed by a

simulated duodenal phase. In the duodenal phase the pH was increased to 6.5 by addition

of 55 mM NaHCO3, and digestion was carried out for 120 minutes with a water-soluble

proteolytic extract of porcine pancreatin. Equal enzyme activities were used for both steps,

corresponding to a w/w ratio of enzyme to CN of approximately 1 to 200.

2.4. Peptide profiling of hydrolysates by LC-MS/MS

The β-CN hydrolysates were diluted to 2 mg/mL in MilliQ H2O, ultrafiltrated using an

Amicon 10 kDa molecular weight cut-off spinfilter (Millipore, Cork, IE) and directly

injected to an LC-ESI-MS/MS. The LC system consisted of a 1200 series capillary pump

and autosampler (Agilent Technologies, Waldbronn, DE) fitted with a Jupiter C18 300-Å

micro-column (Phenomenex, Værløse, DK) of dimensions 150 mm x 0.5 mm with a

particle size of 5μm. The gradient of solvent A (1% acetic acid) and solvent B (80%

acetonitrile, 1% acetic acid) was as follows: 2% B over 5 minutes increasing to 100% B over

25 minutes at a flow rate of 10 µL/minute and keeping at 100% B for 10 minutes. The

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column was operated at 20 °C. The LC system was coupled to an HCT Ultra ion-trap

tandem mass spectrometer equipped with an electrospray ion source (Bruker Daltonik,

Bremen, DE). The MS was run in positive mode with an m/z range from 50 to 3000.

Tandem MS spectra were recorded and searched against a custom Mascot database

(Matrix Science, MA, US) of known genetic variants of bovine milk proteins. The search

was performed with no specific enzyme cleavage sites, an MS mass tolerance of 0.1%, and

an MS/MS mass tolerance of 0.5 Da. Peptide hits above the Mascot score significance

threshold (P = 0.05) were accepted, and spectra from peptide hits of interest that were

below threshold were manually analyzed using DataAnalysis version 4.0, Biotools version

3.1 (Bruker Daltonik, Bremen, DE), and the FindPept tool on the Expasy web-server [23].

2.5. 2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) Decolorization

assay

Trolox antioxidant capacity (TEAC) was determined by the ABTS decolorization assay,

essentially as described elsewhere [24]. ABTS (18.7 mM) was allowed to react with

ammonium persulphate (8.8 mM) overnight at room temperature (RT), for generation of

the stable ABTS+∙ radical. The stock solution was diluted with 75 mM phosphate buffer

(PB, pH 7.5) to give an assay absorbance at 734 nm of ≈0.8. Samples or trolox standards,

appropriately diluted with PB, were then mixed 1:4 with the ABTS+∙ working solution, and

incubated for 60 minutes at RT in the dark before absorbance reading in a Synergy2

microplate reader (BioTek Instruments Inc., VT, US). The percentage of decolorization

relative to that of trolox was used to calculate the TEAC values for the samples.

2.6. Angiotensin-1 converting enzyme inhibitory capacity

The internally quenched fluorescent tripeptide 2-aminobenzoylglycyl-p-nitrophenylalanyl-

proline (ACE substrate) was used to assess the capacity of the β-CN hydrolysates and

derived peptides for ACE inhibition. The method was modified from Sentandreu and

Toldra [25]. Briefly, ACE stock solution (0.5 U/mL, 0.05 M sodium tetra-borate, 1 M NaCl,

2 mg/mL bovine serum albumin, 50 % (v/v) glycerol, pH 8.2) was diluted 50 times in 0.05

M sodium tetra-borate buffer with 1 M NaCl (BB, pH 8.2), and 50 µL were mixed with 50

µL sample, negative control (20 nM captopril), or positive control (BB) in a microtiter

plate and incubated for 10 minutes at 37 °C. Then, 200 µL ACE substrate solution (187.5

µM in BB) was added to each well and fluorescence readings were initiated immediately,

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using excitation and emmission wavelengths of 350 and 420 nm, respectively, in a Synergy

2 Microplate reader (BioTek Instruments Inc. VT, US). Readings were taken every minute

for 30 minutes. and inhibition was calculated as the reduction in the slope of flourescence

readings relative to control. IC50 (concentration needed to reach half maximal inhibition)

values were calculated using SigmaPlot version 11 (Systat Software, DE).

2.7. Cell culture

The caco-2 human intestinal cancer derived cell line was cultured in complete growth

medium (GM, DMEM supplemented with 10% fetal bovine serum, 100 U/mL penicillin,

0.1 mg/mL streptomycin, 1% non-essential amino acids) in 75 cm2 cell culture flasks at 37

°C, 95% air and 5% CO2 in a humidified incubator. GM was changed three times weekly.

Subculturing was performed at 50-60% confluence by washing twice with PBS, and

incubating with 1.25 mg/mL trypsin and 0.1 mg/mL EDTA for 5-10 minutes until

detachment was observed. GM, which contains FBS with trypsin inhibitor, was added to

stop the reaction, and the cell suspension was centrifuged at 200 g for 4 minutes and RT.

The supernatant was discarded and the cell pellet resuspended in fresh GM for re-seeding.

2.8. Stability of β-CN derived angiotensin-1 converting enzyme inhibitory

capacity upon exposure to caco-2 monolayer

Caco-2 cells were seeded at a density of 6.5×104 cells/cm2 on polycarbonate cell culture

inserts with a pore size of 0.4 µm and a growth surface area of 0.6 cm2 (Merck-Millipore,

Darmstadt, DE) and kept in standard 24-well cell culture plates. They were allowed to

grow and differentiate into a tight monolayer for 20-25 days, with GM changed three times

weekly. The integrity of the monolayers was assessed by measuring the trans-epithelial

electrical resistance (TEER) with an ERS-2 Volt-ohm meter equipped with chopstick

electrodes (Merck-Millipore, Darmstadt, DE). The TEER values were calculated as

(Ωmonolayer - Ωblank) × 0.6 cm2, and only inserts with values above 200 Ω×cm2, measured at

37 °C, were used for experiments. Either β-CN hydrolysates from the in vitro digestion, or

β-CN derived synthesized peptides VYPFPGPIHN (variants A1 and B), VYPFPGPIPN

(variants A2 and B), and TER (variant B), were assessed for stability towards incubation

with the intestinal cell monolayer, as described in the following. First, the inserts were

washed in HEPES-buffered HBSS (HHBSS, pH 7.4) at 37 °C for 10 minutes while

measuring initial TEER values. Then, the inserts were incubated with samples dissolved in

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MES-buffered HBSS (MHBSS, pH 6.5) in the apical chamber (AP), and with HHBSS in the

basolateral chamber (BL). The initial concentration of the β-CN hydrolysates was 1 mg/mL

and that of the peptides was 500 µM. LY was added at 100 µM to all samples and controls

for assessment of monolayer integrity during the assay. The incubation was carried out at

37 °C for 60 minutes (hydrolysates) or 120 minutes (peptides) on a rocker table set to 30

rpm, and TEER values were measured again at the end of incubation. Only data from

inserts where less than 1 % of the initial LY had leaked into the BL, and the loss in TEER

value was less than 25 %, was used. Experimental solutions from all chambers were

aspirated at the end of incubation, and kept at -20 °C until further analysis.

2.9. Data Analysis

The β-CN variants A1, A2, B,and I were purified from one homozygous cow each, and the

identity of each variant was verified by LC-MS in triplicate. The in vitro digestion was

carried out as three independent experiments, and subsequent assays for each digest were

performed in triplicate. Activity assays for the synthesized peptides were carried out as

three independent assays performed at least in duplicate. The incubation experiment with

the peptides was carried out as two independent assays in triplicate. Statistical analysis

was accomplished using the statistical software package R version 3.0.2. Data was analyzed

using the “aov” function of the “stats” package for R, which is an ANOVA that takes a

general linear model as input. Variant, digestion time, and the interaction of the variant

and digestion time factors were used in the analysis of data from experiements with the

digested β-CN variants. Peptide was used as factor for experiments with the synthesized

peptides. Statistical significance of differences between factor levels was tested using the

Tukey HSD post hoc test. P-values ≤ 0.05 were taken to indicate statistically significant

differences.

3. RESULTS

3.1. Peptide Profiling of β-CN Hydrolysates by LC-ESI-MS/MS

The β-CN variants A1, A2, B, and I were digested using a two-step in vitro gastrointestinal

digestion procedure. Peptide profiling of the hydrolysates was carried out by LC-MS/MS.

The peptides shown in Table 1 represent significant hits from the Mascot database search,

or manually analyzed spectra, an example of which is shown in Figure 1. Only peptides

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from areas of amino acid substitutions were of interest for the present study. Three

peptides, one from each of the regions with amino acid substitution, were identified after

the gastric phase of digestion. They were cleaved by pepsin at positions with L, T, or F

residues in either the P1 or the P1’ sites. Seven peptides were identified after 5 minutes of

duodenal digestion, covering two of the substituted regions. These peptides were cleaved at

sites that were similar to those of the gastric phase, as well as sites with K and R in the P1

position. Following 120 minutes of duodenal digestion, 14 peptides of interest were

identified, covering all three regions of amino acid substitutions. In addition to the above

described cleavage sites, also sites with Q, N, V, and S at the P1 or P1’ sites were seen,

indicating the action of a more diverse set of proteaes in this phase of the digestion. The

sensitivity of the employed LC-MS/MS system could not succesfully identify peptides

below approximately six amino acids. However, from the B variant the peptides f[114-119]

and f[123-132] were identified. The gap between these fragments, f[120-122], contains the

sequence TER. This peptide, as well as the peptides VYPFPGPIHN and VYPFPGPIPN,

f[59-68] from variants A1/B and A2/I, repectively, was selected for further analysis of

bioactivity.

3.2. Antioxidative Capacity of Peptides TER, VYPFPGPIHN, and

VYPFPGPIPN

The ABTS decolorization assay was used to determine the TEAC values for three β-CN

derived synthesized peptides. The tripeptide TER did not excibit any antioxidant capacity

in the employed system (data not shown). However, the two decapeptides, VYPFPGPIHN

and VYPFPGPIPN, had significantly different TEAC values (Figure 2), with a 21%

reduction as a consequence of the His to Pro substitution at position 67 (P < 0.001).

3.3. ACE inhibitory Capacity of Synthesized Peptides

The ACE inhibitory capacity was determined using an internally quenched fluorescent

tripeptide that gains fluorescense upon cleavage, and measuring the fluorescense increase

every minute for 30 minutes. The slopes of the measured fluorescense against time were

linear during the assay, with R-squared above 0.99 for all but the smallest slopes, that were

close to constant. All three synthesized peptides displayed ACE inhibition (Table 2).

However, there were significant differences in their IC50 values, with TER and

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VYPFPGPIHN being the most potent inhibitors, and VYPFPGPIPN showing less inhibitory

capacity.

3.4. ACE Inhibitory Capacity of Hydrolysates and Peptides Exposed to Caco-

2 Cell Monolayer

ACE inhibition of in vitro digested β-CN variants A1, A2, B, and I was determined before

and after 120 minutes of exposure to a differentiated monolayer of caco-2 intestinal cells

at an assay concentration of 0.17 mg/mL. A global significant average increase of 6.7% (sd

= 2.3%) in ACE inhibition was observed after 60 minutes of exposure to intestinal cells,

compared to control (Figure 3) (P < 0.05). There was no significant difference between the

magnitudes of increase in ACE inhibition by the digests of all tested β-CN variants.

Samples from the basolateral chambers of the caco-2 monolayer displayed no detectable

ACE inhibition in the assay used here, indicative of little or no transport of inhibitory

peptides. Solutions of the three synthesized β-CN derived peptides VYPFPGPIHN,

VYPFPGPIPN, and TER were also assessed for ACE inhibition before and after 120

minutes of incubation with the caco-2 cell monolayer, at an assay concentration of 83 µM.

The observed changes in inhibition were significantly different between the three peptides

(P < 0.01), with no change for VYPFPGPIHN (P > 0.05), an 81% increase for VYPFPGPIPN

(P < 0.05), and a 62% reduction in inhibition for TER (P < 0.001) (Figure 4). Like the

hydrolysates, no ACE inhibition was detected in the samples from the basolateral

chambers of the caco-2 monolayers.

4. DISCUSSION

The main function of gastrointestinal proteases is to hydrolyze the peptide bonds of

ingested protein, so the fragments become progressively smaller, and eventually small

enough to be absorbed across the small intestinal wall [26]. There are both endo- and

exoproteases secreted to the gastrointestinal tract [27]. The potential of a protease for

cleaving a specific peptide bond in a substrate is determined largely by the amino acid

residues in the vicinity of the peptide bond [28]. The main protease in the stomach is

pepsin, which can hydrolyze peptide bonds of quite variable composition, but seems to

have somewhat of a preference for bonds with L or F in the P1 and P1’ positions. However,

it will cleave at several other amino acid combinations as well, such as peptide bonds

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involving A, V, T, D, and E, residues [29, 30], and its specificity is affected by amino acids

further from the peptide bond as well [28]. Presented here is the in vitro digestion of β-CN

variants A1, A2, B, and I, where pepsin was used in the gastric step of the digestion. Three

peptides from the regions of amino acid substitution were identified using LC-MS/MS,

with two of them positioned adjacent to each other. The peptide f[58-80] from β-CN

variant A1/B was also identified by Lisson et al. after a similar digestion of β-CN variants

[17]. Another was unique for one variant, namely β-CN f[81-93] from the I variant. All

three peptides were hydrolyzed at sites containing L, F, and T, which corresponds well with

the above, however, more surprising, G and P were found at three out of the five unique

sites as well. Examining the specificity matrix on the MEROPS database of proteases,

which is based on 417 different cleavages by pepsin, it can be seen that V residues are often

found a few positions downstream of the cleaved site [31]. This is valid for the 80T-81P

cleavage in β-CN variant I, which has a three-V stretch in positions 82-84, and this could

have increased the affinity of pepsin towards this particular site. The L in the 138P-139L

bond most likely helped facilitate this cleavage, as L was found in 51 out of the 417

analyzed cleavages in the MEROPS specificity matrix. Following five minutes of duodenal

digestion with pancreatic enzymes more peptides were identified, with some cleavages by

pepsin obviously still present, but also cleavage C-terminally to K and R was observed. This

is indicative of the action of trypsin, which is a highly specific protease cleaving almost

exclusively at the C-terminal side of K and R residues [32]. The C-terminal of two of the

peptides is 122R from the B variant, which provides evidence that the S to R substitution

does indeed generate a cleavage site for trypsin that is unique to the B variant. Of the seven

different fragments identified at this stage of digestion, β-CN f[108-119] was also identified

by Lisson et al. [17]. After 120 minutes of duodenal digestion 14 peptides were identified,

with all three regions of amino acid substitutions represented. The N-terminals of these

peptides were more diverse than observed in the early duodenal phase, showing hydrolysis

at sites with N, Q, I, S, and M in addition to the above mentioned. The underlying cause of

the more diverse set of cleavage sites observed at this point is likely, that the pancreatin

proteolytic extract used here contains a number of different proteases, i.e. trypsin,

chymotrypsin, elastase, carboxypeptidase A, and dipeptidase [33]. Three of the 14 peptides

were also reported by Lisson et al., including the homologues 59VYPFPGPIHN68 from

variants A1 and B and 59VYPFPGPIPN68 from variants A2 and I. These two peptides are

very similar to β-casomorphin-9 (βCM-9), a member of the family of bioactive β-CN

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derived peptides called β-casomorphins, that have have been extensively characterized, as

reviewed by Ul Haq et al.[34]. βCMs are 4-11 amino acid peptides starting at position 60 of

β-CN, and the longer (8-11 amino acids) can be classified as A1-like or A2-like based on the

presence of H or P, respectively, at position 67 in the parent protein. Numerous

bioactivities have been reported for βCMs, e.g. opioid agonism, immune modulation,

antioxidative capacity, stimulation of mucus secretion, and ACE inhibition [34-36]. That

βCMs can possess antioxidant capacity was demonstrated by Eisele et al., when they

reported a TEAC value for A2-like V-βCM-9 of 1.95 [37], which is more than four-fold what

is reported in the present study. However, the TEAC values are within the same order of

magnitude, and is possibly a result of slight differences in methodology. Eisele et al.

incubated at 30 °C for 15 minutes, compared to room temperature (≈ 22 °C) for 60

minutes. In the present study, the TEAC value for A1-like V-βCM-9 was determined in

order to reveal whether the H to P substitution had an effect on the TEAC value, and a

moderate, but significant, reduction in TEAC was observed. This may be ascribed to H

being an amino acid with known weak antioxidative capacity towards the ABTS+∙ radical

[38].

Saito et al. reported a potent ACE inhibitory effect of A2-like βMC-9 with an IC50 of

14.8 µM [36], which was followed up by Eisele et al. who reported that the addition of a V

residue at the N-terminal reduced ACE inhibition 22-fold [37]. In the present study an IC50

of 656 µM was found for A2-like V-βCM-9 (Table 2), which was approximately twice that

found by Eisele et al. Thus, a rather profound effect of a minor change to the N-terminal of

this particular peptide was seen. However, the 67H to 67P substitution may also have an

impact on ACE inhibition. It was found here that the His rather than a Pro residue in the

penultimate position, reduces the IC50 more than fivefold, making A1-like V-βCM-9 a

significantly stronger ACE inhibitor than A2-like V-βCM-9. This is in accordance with Wu

et al. who found H to be one of the more favorable amino acids at the penultimate position,

for ACE inhibition [39]. As discussed above, the 122S to 122R substitution in β-CN variant B

results in a novel trypsin cleavage site. The consequence is an alternative digestion pattern

in this region of the B variant, compared to the A1, A2, and I variants. In addition, cleavage

at 119F-120T was observed, indicating the possible presence of the tripeptide 120TER122. This

peptide was found to moderately inhibit ACE activity, with an IC50 somewhat lower than

that of A1-like V-βCM-9, though this was not significant. Together, these results suggest

that variants A1 and B of β-CN harbor greater potential for ACE inhibitory capacity at the

135

particular regions of amino acid substitutions. Also, a novel ACE inhibitory tripeptide may

be exclusively generated by digestion of the B variant.

In order to exhibit any physiological effect on blood pressure, the inhibitory peptides

need to enter the blood stream. Thus, peptides liberated from ingested proteins during

gastrointestinal digestion must resist degradation by intestinal brush border peptidases. In

the present study the retention of ACE inhibitory capacity following incubation with

intestinal cells was assessed for total β-CN hydrolysates, as well as the three synthesized

peptides discussed above. Generally, for the hydrolysates a small increase in ACE

inhibition was detected following incubation with caco-2 cells. Conversely, Vermeirssen et

al. evaluated the stability of pea and whey protein hydrolysates towards caco-2

homogenates, and observed a decrease in ACE inhibition after one hour of incubation [40].

Possible explanations for the discrepancy of results may be found in the different

experimental setups employed. Vermeirssen et al. used caco-2 homogenates versus the

intact cell monolayer used here. A cell homogenate contains intracellular proteases that

could degrade the active peptides, whereas peptides exposed to the intact cell monolayer is

only exposed to a range of brush border peptidases [41]. In the present study, a moderate

hydrolysis of the peptides in the β-CN hydrolysates may have generated an altered

collection of peptides with an enhanced ACE inhibitory profile. In addition, different

protein hydrolysates were investigated in the two studies, and the caco-2 derived

proteolytic activity may have opposite effects on pea and whey protein hydrolysates,

compared to the β-CN investigated here. ACE inhibitory capacity was not transported

across the cell monolayer to a sufficiently high degree, if at all, to be detectable in the assay

used here. This is in accordance with Vermeirssen et al. who did not detect any ACE

inhibition in the basolateral chamber after incubation with caco-2 monolayers either [40].

Two hours incubation of the synthesized peptides A1- and A2-like V-βCM-9 and β-CN

variant B derived TER with the caco-2 monoloayer had significantly different outcomes for

each peptide. This indicates different susceptibilities towards brush-border peptidases,

with A2-like V-βCM-9 possibly undergoing conversion into a more active form, and A1-like

V-βCM-9 being resistant to hydrolysis. In addition, no significant uptake or transport of

these two peptides was noted, as evidenced by no apparent loss of activity apically, and no

appearance of activity in the basolateral chamber. Nevertheless, it was earlier shown that

the hexapeptide LHLPLP (β-CN f[133-138]) was both transported and converted into

HLPLP by caco-2 cells. Transport of βCM-7 (YPFPGPI) across a caco-2 monolayer has also

136

been demonstrated, but this peptide was also extensively hydrolyzed by pipeptidyl

peptidase IV (DPP-IV) [42]. DPP-IV hydrolyzes βCM-7 because it favors peptide substrates

with a P residue in the second position from the N-terminal, from which it cleaves a

dipeptide [43]. The added V residue at the N-terminal of V-βCM-9 likely prevents the

action of DPP-IV. The peptide TER displayed the highest pre-incubation ACE inhibition,

which was reduced to less than half post-incubation, demonstrating a different fate from

the two V-βCM-9 peptides. Several explanations for the loss of TER induced ACE

inhibition from the apical chamber are plausible. TER may be transported paracellularly

across the caco-2 monolayer, or it could be transported actively across the apical

membrane by peptide transporter 1 (PepT1), which use both di- and tripeptides as

substrates [44], and then being transported across the basolateral membrane by a different

peptide transporter. This is, however, unlikely to account for the entire loss of inhibition.

Another explanation is the uptake by PepT1 followed by intracellular hydrolysis by

peptidases in the cytosol, or hydrolysis by brush-border enzymes on the apical membrane

of the caco-2 cells. It is clear that more work is needed to clarify these issues, e.g. further

characterization of apical and basolateral samples and transport pathways.

In summary, the present study shows that genetic polymorphisms result in different

sets of peptides being generated during in vitro gastrointestinal digestion of β-CN variants

A1, A2, B, and I. As a result, peptides with varying antioxidative and ACE inhibitory

capacities were observed, including the possibly novel ACE inhibitory peptide TER. In

addition, contrasting stabilities towards caco-2 cells were shown, resulting in both

increases and decreases in ACE inhibition. However, no apparent transport of ACE

inhibition across the caco-2 monolayer was observed for any of the variants’s hydrolysates

or the synthesized peptides. Taken together, this demonstrates that protein variation at the

sequence level influences both gastrointestinal digestion pattern and the bioactive

potential of generated peptides.

ACKNOWLEDGEMENTS

The authors declare no conflict of interest.

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141

Figure 1. Manually analyzed tandem MS spectrum from hydrolysate of β-casein variant A1

digested 60 minutes with pepsin followed by 120 minutes with pancreatic enzymes. The

analysis was carried out using DataAnalysis, Biotools, and FindPept (see methods).

Figure 2. The trolox equivalent antioxidant capacity (TEAC) of the β-casein derived

decapeptides VYPFPGPIHN and VYPFPGPIPN was determined using the 2,2’-azinobis-(3-

ethylbenzothiazoline-6-sulfonic acid) decolorization assay. The results are shown as the

mean of three independent experiments measured in duplicate. Error bars are SEM, and

asterisk denotes statistically significant difference (P < 0.001).

Figure 3. Angiotensin-1 converting enzyme (ACE) inhibition of 0.17 mg/mL in vitro

digested β-casein variants A1, A2, B, and I before (black) and after (gray) 60 minutes

incubation with a differentiated caco-2 monolayer. Bars represent mean values from three

independent experiments performed in triplicate. Error bars are SEM. Asterisks denote

statistically significant difference (P < 0.05).

Figure 4. Angiotensin-1 converting enzyme (ACE) inhibition of 83 µM β-casein derived

peptides before (black) and after (gray) 120 minutes incubation with a differentiated caco-

2 monolayer. Bars represent mean values from two independent experiments performed in

triplicate. Error bars are SEM, and different letters denote statistically significant

difference (P < 0.05).

142

Table 1. Peptides from regions with amino acid substitutions identified by LC-MS/MS after in vitro digestion of

bovine β-CN variants A1, A

2, B, and I

Digestion

phasea

Digested

variant Positionb Sequence

c

Genetic

variantsd

Gastric: B 59-80 (L)VYPFPGPIHNSLPQNIPPLTQT(P) A1, B

I 81-93 (T)PVVVPPFLQPEVL(G) I

A1 120-138 (F)TESQSLTLTDVENLHLPLP(L) A

1, A

2, I

Duodenal 1: I 81-92 (T)PVVVPPFLQPEV(L) A1, A

2, B, I

B 108-119 (K)EMPFPKYPVEPF(T) A1, A

2, B, I

B 108-122 (K)EMPFPKYPVEPFTER(Q) B

B 114-122 (K)YPVEPFTER(Q) B

A1, A

2 114-125 (K)YPVEPFTESQSL(T) A

1, A

2, I

A1, A

2, I 114-139 (K)YPVEPFTESQSLTLTDVENLHLPLPL(L) A

1, A

2, I

B 114-139 (K)YPVEPFTERQSLTLTDVENLHLPLPL(L) B

Duodenal 2: I 53-68 (F)AQTQSLVYPFPGPIPN(S) A2, I

I 57-68 (Q)SLVYPFPGPIPN(S) A2, I

A1, B 59-68 (L)VYPFPGPIHN(S) A

1, B

A2, I 59-68 (L)VYPFPGPIPN(S) A

2, I

A1 59-92 (L)VYPFPGPIHNSLPQNIPPLTQTPVVVPPFLQPEV(M) A

1, B

A1, B 67-92 (I)HNSLPQNIPPLTQTPVVVPPFLQPEV(M) A

1, B

I 69-92 (N)SLPQNIPPLTQTPVVVPPFLQPEV(L) A1, A

2, B, I

A1, A

2, B 73-92 (Q)NIPPLTQTPVVVPPFLQPEV(M) A

1, A

2, B, I

I 81-92 (T)PVVVPPFLQPEV(L) A1, A

2, B, I

B 114-119 (K)YPVEPF(T) A1, A

2, B, I

A1 114-124 (K)YPVEPFTESQS(L) A

1, A

2, I

A1, A

2 120-132 (F)TESQSLTLTDVEN(L) A

1, A

2, I

A1 120-139 (F)TESQSLTLTDVENLHLPLPL(L) A

1, A

2, I

B 123-132 (R)QSLTLTDVEN(L) A1, A

2, B, I

(a) Gastric: 60 minutes pepsin, Duodenal 1: 60 minutes pepsin + 5 minutes pancreatic enzymes, Duodenal 2: 60

minutes pepsin + 120 minutes pancreatic enzymes.

(b) The position of the peptide within the mature β-casein amino acid sequence.

(c) Peptide amino acid sequence. The residues in parenthesis shows the neighboring amino acids.

(d) Shows which of the four variants contain the sequence of the peptide within their native sequence.

143

Table 2. The IC50a of angiotensin-1 converting enyme inhibitory

peptides derived from β-casein variants


IC50

(µM)c SEM

TER B 120 – 122 090 a 08.8

VYPFPGPIHN A1, B 59 – 68 123 a 14.2

VYPFPGPIPN A2, II 59 – 68 656 b 07.6

(a) Concentration needed to reach half-maximal inhibition.

(b) The position of the peptide within the mature β-casein amino acid

sequence.

(c) Different letters within column denote statistically significant

difference (P < 0.001).

144

Figure 1

145

Figure 2

146

Figure 3

147

Figure 4

148

Paper 3

Manuscript intended for submission to Journal of Agricultural and Food Chemistry

Purification and in vitro digestion of bovine kappa-casein variants A, B and E:

Effects on antioxidant and angiotensin-1 converting enzyme inhibitory

capacity

B. Petrat-Melin, G. H. Kristensen, L. B. Larsen, J. F. Young*

Department of Food Science, Aarhus University, Blichers Allé 20, DK-8830 Tjele

* Corresponding author: [email protected], Tel: +45 87158051, Fax: +45 87154891

ABSTRACT

149

INTRODUCTION

Traditionally, research concerning protein variations in bovine milk has aimed at

attributes with an impact on the technological characteristics of milk [1-4]. However, it is

now generally accepted that a number of health-related effects of dairy consumption may

be influenced by genetic polymorphisms in milk proteins [5-7]. Mutations in the milk

protein coding genes result in approximately 50 protein variants carrying amino acid

substitutions in one, two, or three positions within the mature polypeptide chain [5]. The

20 amino acids are characterized by having vastly different physico-chemical properties,

and consequently, substituting one for another could change the behavior of a protein, or

may alter its susceptibility towards enzymatic hydrolysis by proteolytic enzymes during

food processing or digestion. Changes in the position or kinetics of proteolysis could result

in the generation of unique protein fragments, that may have new or altered physiological

effects, i.e. the collection of potential bioactive peptides that are released from the parent

proteins is modified. The caseins (CN) in milk are known to be an excellent source of

bioactive peptides affecting a wide range of physiological systems, such as the immune

system, cardio-vascular health, nutrient uptake, intestinal motility, enteroendocrine cells,

and more [8, 9]. Reports have been published recently, describing the results of both in

silico and in vitro studies attempting to elucidate the importance of protein variation for

health-related outcomes [10-13]. These publications emphasize the fact that the amino acid

substitutions in bovine CN does affect their digestion pattern, and consequently their

bioactive potential. However, much more work in this area is needed, as it is becoming

increasingly clear that diet is a major determinant for the general health status of

populations. It has been estimated that hypertension was the cause of more than 9 million

deaths globally in 2010 [14]. An anti-hypertensive effect has been linked to many CN

derived bioactive peptides, including a few κ-CN derived [15], and also in vivo effects of

intact milk proteins, milk protein hydrolysates, as well as specific peptides have been

shown [16]. Oxidative stress is also believed to have some influence on health status [17,

18], and evidence for inherent antioxidant capacity of κ-CN has been published, as well as

its evolution during digestion [13, 19, 20]. From the above it is evident that investigations

into the influence of CN variation on the generation of bioactive peptides is warranted.

A proteolytic process that all ingested CN undergoes is the hydrolysis by

gastrointestinal enzymes during transit through the gastrointestinal (GI) tract.

150

MATERIALS AND METHODS

Reagents and Chemicals

Angiotensin-converting enzyme (ACE, EC 3.4.15.1), pepsin (EC 3.4.23.1), pancreatin,

bovine CN, fluorescamine, coomassie brilliant blue G-250 (CBB G-250), Captopril, and

2,2’-Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dithioerythritol (DTE)

were all from Sigma (St Louis, USA). 2-aminobenzoylglycyl-p-nitrophenylalanyl-proline

(ACE substrate) was purchased from Bachem (Bubendorf, CH). Polypeptide molecular

weight markers (3.5 to 26.6 kDa) were from Biorad Laboratories (Hercules, USA). Spectra

Multicolor broad range (10 kD to 260 kD) marker was from Thermo Scientific (Schwerte,

DE). All other reagents and chemicals were of analytical grade.

Purification of κ-CN Variants

The κ-CN variants A, B, and E were purified from bovine milk from cows previously

genotyped to be homozygous at the κ-CN locus, as part of the Danish-Swedish Milk

Genomics initiative [3]. In breif, morning milk was collected from cows in mid-lactation

and stored at -20 °C until use. The milk was skimmed by centrifugation for 30 minutes at

2600 g and 4 °C in 50 mL centrifuge tubes, and the fat-layer on top was discarded. The CN

fraction was separated from the whey fraction by acid precipitation. The pH was adjusted

to 4.6 by addition of 10% acetic acid (up to 10% of the milk sample volume), and letting the

CN precipitate for 2 minutes at room temperature (RT). Then 1 M sodium acetate was

added (same volume as acetic acid), and the samples were centrifuged for 10 minutes at

1500 g and 4 °C. The whey fraction was carefully decanted and the CN pellet was

resuspended and washed twice in MilliQ H2O with centrifugation for 5 minutes at 1500 g

and 4 °C. κ-CN differs from αs1-, αs2-, and β-CN by not being calcium sensitive, i.e. κ-CN is

soluble in the presence of calcium ions [21]. This was utilized to obtain a κ-CN enriched

sample by first dissolving the CN pellet in a volume of 37 °C MilliQ H2O equal to the

starting milk sample volume, and carefully adjusting the pH to between 7.0 and 7.5 by

addition of 1 M NaOH. DTE was added to a final concentration of 10 mM, and stirring was

applied for 20 minutes. Then, 5 M CaCl was added drop-wise to a final concentration of

0.25 M, followed by stirring for 30 minutes at 37 °C. The samples were then centrifuged for

30 minutes at 1500 g and 4 °C. The obtained supernatant, which was enriched with κ-CN,

was now dialyzed against buffer A (50 mM sodium acetate, 4 M urea, 1 mM DTE) until the

151

conductivity was equal to that of buffer A alone. The proteins in the dialyzed sample was

then separated by ion exchange fast protein liquid chromatography (FPLC) on an ÄKTA

FPLC equipped with a Frag-900 fraction collector (Amersham Pharmacia Biotech AB, SE).

The system was operated by the UNICORNTM software package. The samples were filtrated

through a 0.45 µm syringe filter before being loaded onto an XK 26/20 100 mL column

(GE Healthcare, SE) packed with Poros HS-50 resin (Applied Biosystems, USA). After

equilibrating the column in buffer A, a stepwise elution with a flow-rate of 5 mL/minute

was carried out with the following steps of buffer B (buffer A with 2 M NaCl) in buffer A:

0.1 column volume (CV), 0% - 5%; 2.4 CV, 5%; 0.1 CV, 5% - 7%; 2.4 CV, 7%; 0.1 CV, 7% -

10%; 2.4 CV, 10%; 0.1 CV, 10% - 15%; 0.1 CV, 15%; 0.1 CV, 15% - 20%; 2.4 CV, 20%; 0.1 CV,

20% - 100%. Inline absorbance at 280 nm was used for detection of protein. Fractions of

14 mL were collected during the step-wise elution and analyzed by SDS-PAGE. The protein

samples were mixed in a 1:1 ratio (or 1:2 depending on protein concentration) with

Laemmli sample buffer (20 mM Tris, 2% SDS, 20% Glycerol and Pyronin Y), all samples

were reduced with 10 mM DTE. They were then loaded on to precast Tris-HCl Criterion

Any kD gels (8% - 16% acrylamide, Biorad, SE), and electrophoresis was performed at 200

V for 40 to 60 minutes. A Spectra Multicolor broad range (10 kDa to 260 kDa) marker was

used (Thermo Scientific, DE). The proteins were fixed in the gel in a fixing buffer (50%

Ethanol, 8% Phosphoric acid) for 2 to 3 hours, and subsequently dyed with Colloidal

Coomasie Blue staining (0.02% CBB G-250, 2% Phosphoric acid (85%), 5% Aluminium

sulphate, 10% Ethanol) for at least 2 hours. Fractions containing κ-CN were desalted by

dialyzing twice against 10 mM ammonium bicarbonate using a 6 kDa to 8 kDa molecular

weight cut-off membrane (Millipore, USA), followed by lyophilization. All samples were

stored at -80 °C until further analysis.

Protein Determination of Purified κ-CN Samples

The protein content of the κ-CN fractions was determined by the bicinchoninic acid (BCA)

assay (Thermo Scientific, DE), according to the manufacturers instructions. In brief, the

working solution containing Cu2+ ions and BCA was added to a microtiter plate and mixed

with sample or bovine serum albumine standards by placing on a shaker board for 30

seconds. Incubation was carried out for 30 minutes at 37 °C. Proteins in an alkaline

environment will chelate and reduce Cu2+ to Cu+, which in turn forms a strongly colored

152

complex with BCA. The color formation was detected by measuring absorbance at 562 nm

in a Synergy 2 Microplate reader (BioTek Instruments Inc., USA).

Liquid Chromatography/Electrospray Ionization - Mass Spectrometry

Liquid chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-

MS) was used to confirm the identity and assess the purity of the isolated κ-CN variants.

In summary, the lyophilized κ-CN preparations were solubilized in 3 mM GndHCl to a

concentration of 3 µg/µL, and fresh dithioerythritol was added to 15 mM final

concentration. The samples were filtered through a 0.2 µm polytetrafluoroethylene filter

(Mini-Uniprep, Whatman, USA). Otherwise the analysis was carried out as previously

described [22]. All systems were controlled by ChemStation software, which was also used

to obtain individual peak areas from the chromotograms by integration. The areas were

used to calculate the relative protein composition.

In Vitro Gastrointestinal Digestion

The κ-CN variants were digested using a two-step simulated gastrointestinal digestion

procedure mimicking the gastric and duodenal phases of digestion, as previously described

[13]. In short, porcine pepsin at pH 2.0 was used for the gastric phase, and a water-soluble

extract of pancreatin at pH 6.5 was used for the duodenal phase. Prior to digestion the

proteolytic activity of the enzymes had been determined by quantifying the amount of TCA

soluble product formed per minute, using acidified bovine hemoglobin and bovine CN at

pH 6.5 for pepsin and pancreatic enzymes, respectively. The digestion was performed by

dissolving the κ-CN variants in simulated gastric fluid (35 mM NaCl, pH 2.0), and

digesting with 20 U pepsin/g CN (approximately w:w enzyme:CN of 1:200) for 60 minutes

at 37 °C. Then, the pH was increased to 6.5 by addition of 54 mM NaHCO3, and the

digestion was continued with 20 U pancreatic enzymes/g CN for 120 minutes at 37 °C.

Samples were taken after 0 and 60 minutes of pepsin digestion, and after a further 5 or 120

minutes pancreatic enzyme digestion. All samples were heated to 90 °C for 10 minutes for

inactivation of proteolytic activity, and stored at -20 °C until further analysis.

Extent and pattern of Protein Fragmentation

Degree of Hydrolysis. The fraction of peptide bonds in the κ-CN variants

that had been hydrolyzed during simulated digestion was estimated by quantifying the N-

153

terminals in the samples. This was achieved by the Fluorescamine assay as described

elsewhere [23]. Briefly, the samples were precipitated in 12% TCA on ice, and the

supernatant was mixed with a sodium tetraborate buffer (pH 8.0), and fluorescamine in

dry acetone was added to a final concentration of 0.05 mg/mL. Fluorescamine reacts with

primary amines, i.e. N-terminals and Lys sidechains, and forms a fluorescent compound

that can be detected using an exitation wavelength of 390 nm and an emission wavelength

of 480 nm [24]. The degree of hydrolysis (DH) can then then be calculated as

2 h 2 0

2 2 0

[ NH ] [ NH ]DH 100

[ NH ] [ NH ]

where [-NH2] is equal to the concentration of primary amines in the hydrolysed (h) or

unhydrolysed (0) samples, and [-NH2]∞ is equal to the theoretical maximal primary amine

concentration assuming total digestion to free amino acids. [-NH2]∞ was calculated as

( )[ ]

lys CN

2

AA

1 f CNH

MW

where flys is the fraction of Lysine residues in the CN, CCN is the CN concentration, and

MWAA is the mean molecular weight of amino acids in the CN.

SDS-PAGE. The fragmentation pattern of the κ-CN variants was investigated

by gel electrophoresis. The digested samples were mixed 1 to 4 with sample buffer (1 M

Tris, 1% SDS, 2 mM dithioerythritol, 20% glycerol, 0.05% CBB G-250) for a concentration

of 2 mg/mL. Each lane of a precast criterion 10% to 20% Tris-tricine gel (Biorad, USA) was

loaded with 40 µg protein, that was separated using 100 V and a run-time of 60 to 90

minutes in Tris-tricine running buffer (100 mM Tris, pH 8.3, 100 mM Tricine, 0.1% SDS).

Tris-tricine gels were chosen for their suitability for separating low molecular weight

peptides. After electrophoresis the gels were kept in fix buffer (50% ethanol, 8%

phosphoric acid), followed by staining with CBB (0.02% CBB G-250, 2% phosphoric acid

(85 %), 5% aluminium sulphate, 10% ethanol) overnight. Gel images were captured using a

UVP Multispectral Imaging System (BioSpectrum, US/UK).

154

Trolox Equivalent Antioxidant Capacity (TEAC)

The ABTS decolorization assay was used to evaluate the antioxidant capacity of the κ-CN

variants during digestion. This assay has been used extensively to estimate the

antioxidative capacity of food constituents [25], and was carried out essentially as

described [26]. ABTS at 18.7 mM was mixed with 8.8 mM ammonium persulphate and

incubated overnight at RT for formation of the stable ABTS+∙ radical. A working solution

was prepared by diluting the stock solution approximately 100 times, until an assay

absorbance of 0.7 at 765 nm was achieved. The κ-CN samples (50 mL) were mixed with

ABTS+∙ working solution (200 µL) in a microtiter plate, and incubated in the dark at RT for

60 minutes before measuring absorbance at 765 nm in a Synergy 2 Microplate reader

(Biotek Instruments Inc., USA). The loss of absorbance caused by the κ-CN samples was

related to that of the synthetic vitamin E analog Trolox for calculation of the TEAC values.

Angiotensin-1 Converting Enzyme Inhibition

The capacity of the κ-CN variants and their hydrolysates to inhibit the activity of ACE was

evaluated using the internally quenched fluorescent tripeptide 2-aminobenzoylglycyl-p-

nitrophenylalanyl-proline (ACE substrate), basically as described [27]. The samples were

diluted to give an assay concentration of 0.05 mg/mL in 50 mM sodium tetra-borate buffer

with 1 M NaCL (BB, pH 8.2) and 50 µL were incubated with 50 µL ACE (0.01 U/mL) for

10 minutes at 37 °C. Then, 200 µL ACE substrate solution was added (187.5 µM in BB),

and fluorescense readings were taken every minute for 30 minutes in a Synergy 2

Microplate reader (Biotek Instruments Inc. USA) using excitation wavelength 350 nm and

emission wavelength 420 nm. The slope of the fluorescense from an uninhibited control

was defind as 100% activity, and the blank control as 0%.

Data Analysis

The κ-CN variants were purified from one individual sample each based on genotypic

information (see methods), and subsequently validated by LC/ESI-MS in duplicate. All

other assays were carried out in three independent experiments, unless otherwise stated.

The statistical software package R (version 3.0.2) was used to analyze differences between

different variants and treatments by two-way ANOVA. “Variant” and “digestion time” were

used as factors in the ANOVA. Individual comparisons were done using Tukey’s honest

155

significant difference post-hoc test. Differences were deemed statistically significant if p ≤

0.05.

RESULTS

Purification of κ-CN Variants

Variants A, B, and E of bovine κ-CN were purified by calcium precipitation followed by ion-

exchange FPLC in a urea-based buffer. Milk samples containing the pure variants had been

predicted based on data from previous genotyping of the dairy cows (data not shown, see

methods). The identity of the variants was verified, and the protein composition was

determined by LC/ESI-MS (Figure 1). The A and E variants had almost identical retention

times in the system used here, but were distinguishable by their masses of 19032 Da and

19002 Da for A and E, respectively. The B and E variants had nearly identical masses of

19000 Da and 19002 Da, respectively, but were distinguishable by their different retention

times, with the B variant eluting 5 minutes later than the E variant. For the A variant, and

the E variant in particular, there were groups of unseparated peaks just before the main κ-

CN peak, which is known to represent glycosylated forms of κ-CN [22]. The added glycans

effectively caused a reduction in elution times. On the downstream side of the main κ-CN

peaks essentially no protein was detectable for any of the variants, indicating that a very

good separation of κ-CN from the other CN had been achieved during the purification

process.

In Vitro Digestion of κ-CN Variants

To investigate the digestion pattern and digestibility of the A, B, and E variants of κ-CN

they were subjected to a simulated gastrointestinal digestion system. The digestion

consisted of two phases, a gastric and a duodenal phase. The fragmentation pattern was

visualized by gel electrophoresis, and a representative gel image can be seen in figure 2. In

the three lanes with undigested CN a strong band is seen just above the 17 kDa marker,

which is the intact κ-CN that has a molecular weight of approximately 19 kDa. Above these

bands a smear is visible, and below are a few fainter bands, three to four for the A and E

variants, and a single one for the B variant. In the lanes with the κ-CN after 60 minutes

pepsin digestion no intact κ-CN is visible, the faint bands from the undigested lanes have

almost completely disappeared, and new smeared bands have appeared around the 6.5

156

kDa marker. After an additional 5 minutes of digestion with pancreatic enzymes only a

single very faint band is visible at the position of the 6.5 kDa marker, and after 120 minutes

of pancreatic enzyme digestion no κ-CN fragments were left from any of the three variants.

The DH was determined to give an indication of the extent to which the three

κ-CN variants were digested in the present system, and the results are shown in figue 3.

There was a significant overall effect of time, but not of variant, and there was no

significant interaction effect of time and variant. The DH after 60 minutes pepsin digestion

was greatest for the B and E variants, however, the difference to the A variant was not

significant. After 5 minutes digestion with pancreatic enzymes it was the A and B variants

that showed the highest DH, having increased by 15% and 11%, respectively, while the E

variant only increased by 4.5%. After 120 minutes pancreatic enzyme digestion a DH of

almost 45% was reached for the A and B variants, and the DH for the E variant was ≈10%

lower. However, for all three variants the increase in DH from 5 to 120 minutes of

pancreatic enzyme digestion was ≈23%. Thus, the E variant seemed to reach a lower final

DH as a consequence of the lower increase after the initial 5 minutes of pancreatic enzyme

digestion.

Trolox Equivalent Antioxidant Capacity of in Vitro Digested κ-Casein

Variants

The ABTS decolorization assay was used to determine the TEAC values for the three κ-CN

variants A, B, and E, before and after different points of in vitro digestion (Figure 4). There

was a significant overall effect of both variant and digestion time, as well as a significant

interaction between variant and digestion time. Individual evaluations revealed that the

undigested A variant displayed two to three times as high TEAC value as the B and E

variants (0.17 vs. 0.05 and 0.08, respectively). However, after 60 minutes pepsin digestion

all three variants reached similar levels between 0.22 and 0.24. The first 5 minutes of

pancreatic enzyme digestion resulted in a further increase to between 0.30 and 0.37. The

individual increase for each variant was only significant for the A and B variants. All three

variants exhibited a substantial increase in TEAC after 120 minutes of pancreatic enzyme

digestion, however, the level of variants A and B was significantly higher than that of the E

variant, which was statistically indistinguishable from the A and B variants after 5 minutes

pancreatic enzyme digestion. The total fold increase in TEAC for the three different

variants was 3.4, 9.6, and 5.4 for A, B, and E, respectively.

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Angiotensin-1 Converting Enzyme Inhibitory Capacity of in Vitro Digested κ-

Casein Variants

The capacity of the κ-CN variants A, B, and E for ACE inhibition was determined both

before and after different stages of digestion (Figure 5). There was a significant overall

effect of time, but not of variant or the interaction of time and variant. The undigested κ-

CN variants A and B had no significant effect on ACE. Following 60 minutes pepsin

digestion ACE inhibition had increased to ≈62% for the A and E variants, and 68% for the

E variant. After a further digestion for 5 minutes with pancreatic enzymes ACE inhibition

was around 74% for all three variants, and after 120 minutes pancreatic enzymes digestion

the inhibition increased slightly to about between 81% and 84% for the three variants.

Individual comparisons revealed no statistically significant difference between the

undigested variants, and no statistically significant difference between any of the digested

variants, regardless of digestion time. The IC50 values for the κ-CN variants was

determined at the end of the in vitro digestion (Figure 6), and was determined to lie

between 0.39 and 0.48 mg/mL, with no significant difference between variants.

DISCUSSION

This study is one of the first to investigate the consequences of genetic polymorphisms in

bovine κ-CN on biologically relevant endpoints, by characterizing the pattern of in vitro

digestion pattern, and the effects on antioxidant capacity and ACE inhibitory capacity of

variants A, B, and E. A prerequisite for such studies is the ability to obtain the pure

variants from the CN fraction of milk. There are generally two approaches to achieving

this, which is either separating the individual κ-CN variants in milk samples containing a

mixed phenotype, or isolating the total κ-CN fraction from milk samples of cows with

defined homozygous κ-CN alleles. The former approach has been validated for the

separation of genetic variants of the major proteins in milk, including variants of κ-CN,

using reversed-phase HPLC [28, 29]. However, by this approach the A and E variants of κ-

CN were not clearly separated [28]. Utilizing an approach based on genotyping of the dairy

cows it is possible to obtain pure κ-CN variants by more traditional separation methods, as

shown here as well as by Lisson et al. [6], based on ion-exchange chromatography. Lisson

et al. did, however, observe some contamination of κ-CN variant E by αs2-CN (2013). The

method used for separating κ-CN in the present study included a calcium-precipitation

158

step prior to the ion-exchange chromatography. This resulted in a more efficient

separation of the three variants A, B, and E of κ-CN from the other CN. Interestingly, the

LC-MS analysis indicated the presence of a larger proportion of glycosylated κ-CN of the A

and E variants, compared to the B variant. A small part of this difference may be explained

by the Thr to Ile substitution at position 136 in the B variant, because this position has

previously been shown to be a potential glycosylation site [30].

The results from the in vitro digestion of κ-CN variants A, B, and E described

here show that κ-CN is a highly digestible protein, as is β-CN, compared with e.g. whey

proteins [13, 31, 32], as no intact protein was observed already after the gastric step of

digestion with pepsin. This is likely due to the rather unstructured conformation of κ-CN

[33], which leaves it more accessible to digestive proteases. It also underscores the fact that

κ-CN, along with the other CN, serves as the main source of amino acids for the suckling

calf, and as such should be readily digestible. Not only is virtually all of the κ-CN digested,

it is digested to a rather high DH, again indicative of a very digestible protein, seemingly

devoid of regions that are resistant to gastrointestinal proteases. Comparison of the three

investigated variants reveals only insignificant differences in their susceptibility to

digestion by pancreatic enzymes, with the E variant being somewhat less digested during

this phase of the digestion. The Ser to Gly substitution at position 155 in the E variant

could explain some, but likely not all, of this observation. The two amino acids

downstream of position 155 are Pro residues, which may cause a slightly different

conformation of the peptide bond in the presence of Gly rather than Ser at position 155.

However, the E variant was also the most heavily glycosylated of the three variants, and the

glycosylation moieties may have sterically hindered the action of the protease at some

potential cleavage sites.

The antioxidant capacity of in vitro digested κ-CN variants A, B, and E was

also estimated using the ABTS decolorization assay for determining TEAC values. This

assay was chosen because it lends itself well to use in food systems [25]. The higher

antioxidant capacity observed for the intact A variant, compared to the B and E variants,

was surprising, because none of the substituted amino acids are reported as having ABTS

reducing capacity. However, both Asp and Ser were reported to protect against hydrogen

peroxide-induced oxidative damage to deoxyribose, and Thr to act as a pro-oxidant in this

system [34]. In the same report, Asp, Ser, and Thr were shown to inhibit lipid

peroxidation, and have scavenging activity towards hypochloric acid. This indicates some

159

inherent antioxidant capacity in these three amino acids, albeit in other assay systems than

used here. Hence, this may explain some of the different antioxidant capacities of the three

κ-CN variants. The antioxidant capacity increased for all variants during digestion,

however the E variant showed a significantly lower TEAC value by the end of digestion.

The most likely explanation for this is that the E variant was also the one with the lowest

DH at this point, and DH was shown to affect TEAC both here and by others [19, 20].

Further studies are warranted to increase the knowledge of the effects of specific

variations, such as amino acid substitutions and degree of glycosylation on antioxidant

capacity of κ-CN variants.

An evaluation of the digested κ-CN variants ACE inhibitory capacity was also

performed. Prevously, Weimann et al. indicated the generation of unique peptides from

different variants, after an silico digestion analysis using a combination of gastrointestinal

proteases [10]. In that study putative ACE inhibitory peptides were synthesized and

evaluated for inhibition, and four peptides were found to inhibit ACE. One of these four

peptides, Ala-Ser-Pro, was derived from the B variant as a consequence of the Asp to Ala

substitution at position 148, and was reported to have an IC50 of 242.3 µM. Until now,

many ACE inhibitory peptides derived from bovine CN have been reported, however, most

of these stem from α- and β-CN [15, 35, 36]. In the present study effects of in vitro

digestion of κ-CN variants A, B, and E, on ACE inhibition was evaluated. The results

showed that none of the three intact variants displayed ACE inhibition, but rather

surprisingly seemed to slightly increase ACE activity, i.e. displayed negative inhibition.

This effect was small, only significant for the E variant, and perhaps an artefact in the

assay. The effect of digestion was substantial already after the gastric phase, and there was

no further development following the duodenal digestion phase. The IC50 of the three

variants was determined for the hydrolysates at the end-point of the in vitro digestion, and

was found to be approximately 0.04 to 0.05 mg/mL, which is comparable to the IC50 of pea

and whey protein digests, as previously shown [37]. The ACE inhibition was determined

during digestion using 0.1 mg/mL final assay concentration, and therefore may have been

too high to reveal small differences between the variants. This is because inhibition curves

tend to level off as inhibition approaches 100%, which was the case here. Consequently,

further evaluations at lower concentrations are warranted in order to elucidate this point.

CONCLUSION

160

In the present study it was shown that purification of κ-CN of defined phenotype is a

convenient and effective method of obtaining pure genetic variants for research purposes.

In addition, the inclusion of a calcium precipitation step preceding ion-exchange

chromatography abolished contamination by other milk proteins during purification. In

vitro digestion of κ-CN variants A, B, and E revealed potential differences in susceptibility

towards cleavage by gastrointestinal proteases, which may be a consequence of both amino

acid substitutions and varying levels of glycosylation in the samples used here. These

questions should be further addressed using mass spectrometric peptide profiling of the

digested κ-CN. The bioactivities antioxidant capacity and ACE inhibition was greatly

increased by in vitro digestion, but only antioxidant capacity showed a difference between

the three investigated variants. Together, these results demonstrate that genetic

polymorphisms in κ-CN have an impact on biologically relevant endpoints.

ACKNOWLEDGEMENTS

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Table 1. Position of amino acid substitutions

within the mature protein of variants of κ-

casein

Variant1

Position A B E

136 Thr Ile

148 Asp Ala

155 Ser Gly

1 A is the reference sequence (Caroli et al.,

2009).

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Petrat-Melin

Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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Figure 6.

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Figure 1. Representative chromatogram from LC/ESI-MS of purified κ-casein variants A,

B, and E. Proteins were detected at 214 nm and the molecular mass of the main peak from

each variant is indicated. The spectra were standardized to the maximum value within

each variant.

Figure 2. Gel electrophoresis of in vitro gastrointestinally digested κ-casein variants A, B,

and E. At the top of the gel the variants and digestion times are given. 0 = undigested, 60 =

60 minutes pepsin digestion, 65 = 60 minutes pepsin followed by 5 minutes pancreatic

enzyme digestion, 180 = 60 minutes pepsin followed by 120 minutes pancreatic enzyme

digestion. Pepsin digestion was done at pH 2.0, and pancreatic enzyme digestion was done

at pH 6.5. Positions of the molecular weigt markers are indicated to the left. The samples

were reduced with 2 mM dithioerythritol, and the protein bands were visualized by

staining with Coomassie Brilliant Blue.

Figure 3. The degree of hydrolysis after in vitro digestion of κ-casein variants A, B, and E.

The caseins were digested for 60 minutes with pepsin at pH 2.0 (60), and for an additional

5 (60+5) or 120 (60+120) minutes with pancreatic enzymes at pH 6.5. The bars represent

mean values of three independent experiments and error bars are sem (n=3). Different

letters indicate statistically significant difference between means (P<0.05).

Figure 4. The trolox equivalent antioxidant capacity (TEAC) of κ-casein variants A, B, and

E, before and after different stages of in vitro digestion using the 2,2'-azino-bis(3-

ethylbenzothiazoline-6-sulphonic acid) decolorization assay. 0: undigested, 60: 60

minutes pepsin, 60+5: 60 pepsin followed by 5 minutes pancreatic enzymes, 60+120: 60

pepsin followed by 120 minutes pancreatic enzymes. The bars represent mean values from

three independent experiments, and the error bars are sem (n=3). Different letters indicate

statistically significant difference between means (P<0.05).

Figure 5. Angiotensin-1 converting enzyme (ACE) inhibition of κ-casein variants A, B,

and E before and after different stages of in vitro digestion. ACE inhibition was monitored

using an internally quenched fluorescent tripeptide that gained fluorescense upon

hydrolysis by ACE. 0: undigested, 60: 60 minutes pepsin, 60+5: 60 pepsin followed by 5

minutes pancreatic enzymes, 60+120: 60 pepsin followed by 120 minutes pancreatic

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enzymes. The bars represent mean values from three independent experiments, and the

error bars are sem (n=3). Different letters indicate statistically significant difference

between means (P<0.05).

Figure 6. Angiotensin-1 converting enzyme (ACE) inhibition of κ-casein variants A, B,

and E after in vitro digestion at 37 °C with pepsin for 60 minutes at pH 2.0, followed by

digestion with pancreatic enzymes for 120 minutes at pH 6.5. Inhibition is expressed as the

concentration in mg/mL needed to reduce ACE activity to half-maximal (IC50). The bars

represent mean values of three independent experiments, and error bars are sem (n=3).

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