Faculty of Bioscience Engineering

Academic year 2010 – 2011

MILK AND PEA PROTEIN HYDROLYSATES WITH POTENTIAL TO

ACTIVATE CCK1 RECEPTOR

NADIN AL SHUKOR Promoters: Prof. dr. John Van Camp

Prof. dr. Guy Smagghe

Tutor: ir. Dorien Staljanssens

Master’s dissertation submitted in partial fulfillment of the requirements for the

degree of Master of Science in Nutrition and Rural Development, Main subject: Human Nutrition

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COPYRIGHT

“All rights reserved. The author and the promoters permit the use of this Master’s Dissertation

for consulting purposes and copying of parts for personal use. However, any other use falls

under the limitations of copyright regulations, particularly the stringent obligation to explicitly

mention the source when citing parts out of this Master’s dissertation.”

Ghent University, August 2011

Promoter Promoter

Prof. dr. ir. John Van Camp Prof. dr. ir. Guy Smagghe

Tutor The Author

ir. Dorien Staljanssens Nadin Al Shukor

ii

ACKNOWLEDGMENT

First of all, I would like especially to thank Prof. dr. ir. John Van Camp and Prof. dr. ir. Guy

Smagghe for giving me an opportunity to undertake my dissertation under their supervision

and I am really very grateful to them for their advice, suggestions and inspirations and

invaluable advice, without which this manuscript would not have been completed.

I warmly would like to thank ir. Dorien Staljanssens for her patience, her encouragement,

advice, availability, support, and help in the framework of this work. I also appreciate the

friendly behaviors of the staff members of the Agrozoology Laboratory at the Faculty of

Bioscience Engineering. My profound gratitude goes also to ir. Anne-Marie De Winter, our

master programme coordinator and Marian Mareen for their support and help during my

master’s studies.

Thanks to my mother for everything, it is true that your body had left our life but your soul is

still alive and it goes with me everywhere without it i could not live.

Thanks my father, you are who taught me patience and gave me whole support and helped me

in achieving my dreams. To you my great father I dedicate this work.

And to you all

� My lovely partner who was and is still always beside me: My husband

� The smile of my life: My son

� My unique friends: My brothers

� My country: Syria

� My sponsor: Damascus University

� And to all my relatives and my friends

� Last but not least thanks Belgium, especially Flanders

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LIST OF ABBREVIATIONS

BSA: Bovine Serum Albumin

CCK: Cholecystokinin

CCK1R: CCK receptor-1

CCK-8S: Sulfated Cholecystokinin Octapeptide

CHO-CCK1R cells: Chinese Hamster Ovary cells Expressing CCK1R

CHO cells: Chinese Hamster Ovary cells

CNS: Central Nervous System

DH: Degree of Hydrolysis (%)

FBS: Fetal Bovin Serum

GID: Gastrointestinal Digestion Enzymes

GLP-1: Glucagon-Like Peptide-1

GPCR: G-Protein Coupled Receptor

LGM: Lorglumide

NPY: Neuropeptide Y

OXM: Oxyntomodulin

PYY: Peptide YY

TNBS: Trinitrobenzenesulphonic Acid

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TABLE OF CONTENTS

ACKNOWLEDGEMENT

CHAPTER 1: INTRODUTION .......................................................................... - 1 -

CHAPTER 2: LITERATURE REVIEW ............................................................. - 2 -

2.1. Obesity and overweight .................................................................................................................... - 2 -

2.2. Satiety hormones ............................................................................................................................... - 3 -

2.3. Cholecystokinin ................................................................................................................................. - 5 -

2.4. Cholecystokinin receptors ................................................................................................................ - 7 -

2.4.1. Introduction ................................................................................................................................. - 7 -

2.4.2. Functions ..................................................................................................................................... - 8 -

2.4.3. Receptor structure and ligands interactions ............................................................................... - 10 -

2.5. Bioactive Peptides ............................................................................................................................ - 12 -

2.5.1. Enzymatic hydrolysis: ............................................................................................................... - 13 -

2.5.2. Microbial fermentation: ............................................................................................................. - 13 -

2.6. Milk and pea protein structure ...................................................................................................... - 14 -

CHAPTER 3: MATERIALS AND METHODS ................................................. - 16 -

3.1. Cell lines and products .................................................................................................................... - 16 -

3.2. Cell culture ....................................................................................................................................... - 16 -

3.3. Protein hydrolysates ........................................................................................................................ - 16 -

3.3.1. Hydrolysis of the pea and whey proteins with alcalase and promod enzymes .......................... - 17 -

3.3.2. Hydrolysis of purified whey and casein prteins with peptidase................................................. - 17 -

3.3.3. Gastrointestinal digestion .......................................................................................................... - 17 -

3.4. Cell-based bioassay to screen for CCK1R activity ....................................................................... - 17 -

3.6. Determination of degree of hydrolysis ........................................................................................... - 19 -

3.7. Data analysis .................................................................................................................................... - 19 -

3.8. Statistics ........................................................................................................................................... - 20 -

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CHAPTER 4: RESULTS ................................................................................ - 21 -

4.1. Measurement cellular response to the natural ligand CCK-8S with the plate reader and the confocal

microscopy .............................................................................................................................................. - 21 -

4.2. Measurement of agonist and antagonist effects on the cell population level .............................. - 22 -

4.3. Do milk and pea proteins have potency to act directly on the CCK1 receptor? ........................ - 24 -

4.3.1. Aim of the study ........................................................................................................................ - 24 -

4.3.2. Effect of different whey and pea protein hydrolysates on CCK1R activation by a plate reader - 24 -

4.3.3. Effect of enzymes and hydrolysis time on peptide length ......................................................... - 26 -

4.3.4. The correlation between the % response and the peptide length ............................................... - 27 -

4.3.5. Comparison of the results obtained by Tecan with those of a confocal microscopy ................. - 28 -

4.3.6. Comparison of the results obtained by confocal scanning microscopy with and without lorglumide- 29 -

4.3.7. Evaluation of the effect of different purified protein hydrolysates on the cellular response by a plate

reader ................................................................................................................................................... - 31 -

4.3.8. Effect of hydrolysis of casein and whey purified proteins with peptidase/gastrointestinal digestion

enzymes on the peptide length ............................................................................................................ - 33 -

4.3.9. Comparison of the results obtained by Tecan with those of the confocal microscopy .............. - 33 -

CHAPTER 5: DISCUSSION .......................................................................... - 37 -

CHAPTER 6: CONCLUSION ......................................................................... - 41 -

REFERENCES ............................................................................................... - 42 -

ANNEXES Annex 1: Supplementary Tables

Supplementary Table 1: ANOVA: Fixed effects, main effect and interactions

Supplementary Table 2: Two-Sample T-Test and confidence interval for whey sample and pea sample

Supplementary Table 3: Two-Sample T-Test and confidence interval for alcalase sample and promod

sample

Supplementary Table 4: Tukey multiple comparisons

Annex 2: Supplementary Figures

Supplementary Figure 1: The net response induced by increasing concentrations (0.005 – 3 mg/ml) of whey

protein hydrolysed with promod/alcalase enzymes for 1, 3 and 6h in five repeated experiments

Supplementary Figure 2: The net response induced by increasing concentrations (0.005 – 3 mg/ml) of pea

protein hydrolysed with promod/alcalase enzymes for 1, 3 and 6h in five repeated experiments

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Supplementary Figure 3: The fluorescence kinetics responses of the CHO-CCK1R to 1 nM CCK and those

of CHO-CCK1R and CHO-K1 cells to whey hydrolysed with alcalase for 3h of the 3g/l concentration

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LIST OF FIGURES

Figure 1: Model represents different signals that influence food intake………………………3

Figure 2: Primary sequence of the most predominant mammalian forms of CCK: CCK-58,

CCK-33, and CCK-8…………………………………………………………………………...6

Figure 3: Simplified view of signaling pathway of the CCK1R via the Gq type of G-protein

coupled receptor after binding with an agonist………………………………………………..9

Figure 4: Representation of the CCK1 receptor and its agonist binding sites……………….12

Figure 5: Dose-dependent CCK1R-mediated cellular response obtained with a plate

reader………………………………………………………………………………………….21

Figure 6: Dose-dependent CCK1R-mediated cellular response obtained with a confocal

microscopy……………………………………………………………………………………22

Figure 7: Representative dose–response curves for JMV180 and lorglumide monitored with a

plate reader…………………………………………………………………………………....23

Figure 8: Effect of different pea and whey protein hydrolysates on the net cellular response:

3g/l pea and whey……………………………………………………………………….........25

Figure 9: Mean peptide length of different pea and whey hydrolysates……………………..27

Figure 10: The correlation between peptide length and the % of the maximum response…..28

Figure 11: Representative of the fluorescence kinetics curves induced by whey hydrolysed

with alcalase for 3h and measured with the microscope………………………………….......29

Figure 12: Representative of the fluorescence kinetics curves induced by pea and whey

proteins hydrolysed with promod measured with the microscope with and without lorglumide.

……………………………………………………………………………………………..30-31

Figure 13: Comparison between the net responses induced by 3g/l of purified casein

hydrolysates and the net response resulting from the non hydrolysed casein….......................32

Figure 14: The net response induced by 3g/l of purified casein and whey hydrolysates

obtained with the microscope…………………………………...…………………………….34

Figure 15: Fluorescence kinetics induced by 3g/l of purified casein hydrolysates measured

with two platforms…………………………………………………………………………….36

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LIST OF TABLES

Table 1: Maximum response induced by increasing concentrations of whey hydrolysates

expressed as a percentage of the maximum response caused by 1 nM CCK………………4.3.2

Table 2: Maximum response induced by increasing concentrations of pea hydrolysates

expressed as a percentage of the maximum response caused by 1 nM CCK………………4.3.2

Table 3: DH degree (%) of pea and whey hydrolysed by alcalase/promod for 1 / 3 and 6

hours………………………………………………………………………………………..4.3.3

Table 4: DH degree (%) and mean length of peptides derived from hydrolysis of casein and

whey purified proteins with peptidase/GID enzymes………..……………………….……4.3.8

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CHAPTER 1: INTRODUCTION

Cholecystokinin is a hormone and neuropeptide released from the endocrine cells in the

intestinal mucosa upon ingestion of food. CCK induces satiety and regulates many

physiological processes like gall bladder contraction, gastrointestinal motility, pancreas

secretion, gastric acid secretion, panic, and anxiety. CCK effects are mediated by two

receptors, CCK1 and CCK2 receptor. CCK1 receptor is mainly expressed in the

gastrointestinal tract, and CCK2 receptor is mainly expressed in the brain.

CCKRs are G protein-coupled receptors, which during activation elicit an inositol

trisphosphate (IP3)-induced calcium release from the endoplasmic reticulum. This

intracellular Ca+2-flux is a measure for the activation of the receptor and can be visualized

with fluorescent sensor dyes.

In this study we hypothesized that milk and pea protein hydrolysates activate the CCK1

receptor. To test this hypothesis, receptor activation induced by agonist binding followed by

an intracellular calcium increase was monitored by using a fluorescent sensor dye. Changes

in intracellular Ca+2 concentrations were monitored by measuring changes in fluorescence

signals with a plate reader and a confocal microscopy. Response curves were generated for the

natural agonist CCK-8S, the full antagonist lorglumide, pea and whey protein hydrolysates,

the non hydrolysed whey and casein proteins, and the purified whey and casein protein

hydrolysates. The non hydrolysed pea was non-soluble which made it difficult to be tested.

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CHAPTER 2: LITERATURE REVIEW

2.1. Obesity and overweight

The incidence of obesity and overweight incidences has increased rapidly in recent years.

Globally, there are more than 1 billion overweight adults, at least 300 million of them obese.

The main causes of this obesity and overweight are increased consumption of energy-dense

foods high in saturated fats and sugars, and reduced physical activity. These factors have led

obesity rates to rise three times or more since 1980 in some areas of North America, the

United Kingdom, Eastern Europe, the Middle East, the Pacific Islands, Australia and China.

The obesity epidemic is not restricted to industrialized societies; it also increases in

developing countries (WHO, 2010).

Obesity and overweight pose a major risk for serious diet-related chronic diseases like

diabetes type 2, cardiovascular diseases and hypertension (Yun, 2010), which leads as a

consequence to an increase in social and financial burdens. It is believed that solution for

obesity and overweight in humans could be body weight maintenance after body weight loss.

In this concept we are facing two difficulties: weight loss is always difficult to achieve

through lifestyle changes. Second, in most cases body weight maintenance is rather difficult

after body weight loss.

Anti-obesity drug treatments are currently licensed, such as orlistat. Using these drugs alone,

modest weight loss is achieved with several side effects. Therefore, there is the necessity to

identify and define more potent and crucial pharmacological targets. Over the last ten years

new hormones such as leptin and gherlin were discovered. This together with a better

understanding of previously identified hormones such as cholecystokinin, pancreatic

polypeptide, peptide YY and glucagon-like peptide 1 have resulted in a good overview on

control of satiety and thereby going more insight into the mechanism of regulation energy

balance (Huda et al., 2006). Research specifically aimed at prevention and treatment of

obesity is accumulating. In the context of these researches, many studies are focusing on high

protein diets since they have the potential to act on different metabolic targets that could

regulate body weight after weight loss (Westerterp-Plantenga M S, 2006).

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2.2. Satiety hormones

The gut is an active enteroendocrine organ which mediates appetite and nutrient absorption

through its physical structures and the secretion of an array of regulatory peptides from the

endocrine cells lining the intestinal epithelium (Larsen et al., 2003) as illustrated in Figure 1.

Many gut peptides have been identified as key peptides in appetite management. With this

regard, the most studied are cholecystokinin (CCK), pancreatic polypeptide, peptide YY,

glucagon-like peptide-1 (GLP-1), oxyntomodulin and ghrelin. With the exception of ghrelin,

these hormones act to increase satiety and decrease food intake (Huda et al., 2006).

Figure 1: Model represents different signals that influence food intake. Source: Stephen C et

al., 2008.

Legend: During meals, signals such as CCK, GLP-1 that arise from the gut (stomach and

intestine) trigger nerve impulses in sensory nerves traveling to the hindbrain. These satiation

signals synapse with neurons in the nucleus of the solitary tract (NTS) where they influence

meal 9size. Ghrelin from the stomach acts on the vagus nerve and stimulates neurons in the

ARC directly. Signals related to body fat content such as leptin and insulin circulate in the

blood to the brain. They pass through the blood-brain barrier and interact with neurons in the

region of the ARC. Within the brain, neural circuits integrate information from the NTS and

several hypothalamic nuclei determine food intake and energy expenditure (Schwartz et al.,

2000).

Peptide YY is a 36-amino-acid linear peptide and is a member of the PP fold peptide family

(Tatemoto, 1982; Taylor, 1985). It is secreted from the endocrine L cells of the small and

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large bowel, (Adrian et al., 1985). Peptide YY effects on the gastrointestinal system are

reflected by an inhibition of secretion of fluid and electrolyte in the small bowel and postpone

intestinal meal transport (Taylor, 1993). Plasma PYY levels increase within 30 min of

nutrients reaching the gut and are suppressed in the fasted state. This could suggest neural

regulation within the gut as most PYY is released from the distal small intestine and colon in

advance of the arrival of nutrients to this region of the intestine (Anini Y, 1999). Effects on

appetite have been shown in rodents and humans. In human, intravenous PYY3-36 infusions

in healthy subjects led to a 33% drop of energy intake, reduction in food intake time and

hunger score. These effects lasted about 12h after the infusion (Batterham et al., 2002). A

study of Batterham et al. (2006) on obese and lean males was done to identify the role of PYY

in protein mediated satiety. In this study isocaloric meals high in one macronutrient were

used: (high-protein, high-fat, or high-carbohydrate). A remarkable increase was noticed in

plasma PYY level in both groups as a result of the high-protein meal. The other observation

that came out of this study was the greatest reduction in hunger in both normal and obese

subjects that was caused by the high protein diet (Batterham et al., 2006). This result was

consistent with previous studies (Porrini et al., 1997; Latner & Schwartz, 1999; Lejeune et al.,

2006).

Glucagon-like peptide 1 is expressed in the L-cells of the small and large intestine, and in

neurons in the nucleus of tractus solitarius (NTS). It plays a potential role in suppression of

meal-induced gastric acid and pancreatic juice secretion and slows down gastric emptying

(Schjoldager et al., 1989; Wettergren et al., 1993). Although there is a contradicting data in

humans about GLP-1 effects on regulation of food intake, a meta-analysis study on lean and

overweight subjects suggested that reduction of energy intake by 11.7% is achieved by

peripheral GLP-1 infusion in a dose-dependent manner (Verdich et al., 2001). Glucagon-like

peptide 1 is released after 5 to 30 min of food ingestion. It is secreted in response to

carbohydrates and fats intake, but also proteins and amino acids have an effect on release of

GLP-1 (Elliott et al., 1993; Herrmann et al., 1995). Lejeune et al (2006) showed that this

hormone is one of the anorexigenic hormones (hormones that increase satiety and decrease

food intake) that mediate protein-induced satiety during a high protein diet over a period that

lasts for several days (Lejeune et al., 2006).

Oxyntomodulin is a 37-amino-acid peptide, produced by proglucagon processing in the gut

and brain (Bataille et al., 1982; Holst, 1999). It comes to expression in the central nervous

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system (CNS) and the L cells of the intestine and the pancreas (Holst, 1999). Experiments in

rodents showed that peripheral OXM injections caused a reduction in food intake (Dakin et

al., 2004). In human subjects, intravenous administration of OXM inhibits gastric acid

secretion and gastric emptying, which in its turn leads to reduction of appetite and promotion

of satiety (Schjoldager et al., 1989). Oxyntomodulin is rapidly released after food intake from

the same particular endocrine cells of the distal gut that release PYY and GLP-1 (Bottcher et

al., 1984; Varndell et al., 1985). Levels increase within 30 min and continue rising for several

hours ( Hornnes et al., 1980; Ghatei et al., 1983). Fatty acids in the gut lumen produced by

hydrolysis of fat trigger particularly this release (Read et al., 1984; Stanley et al., 2004).

From all gut hormones, ghrelin is well known as the only endogenous peripheral hormone that

causes hunger and lead to an increase in food intake (Huda et al., 2006). Ghrelin leads to an

increase in gastric acid secretion and gastric motility (Date et al., 2000; Mori et al., 2000;

Asakawa et al., 2001). Nevertheless, this is not confirmed by all studies (Sibilia et al., 2002).

In humans ghrelin is potentially important for controlling appetite. Administration of ghrelin

in humans results in an 28% increase in energy consumed from a free-choice buffet and rises

hunger scores (Wren et al., 2001). Normally, before meals ghrelin levels increase while they

drop to very low levels between 60 and 120 min after eating. This was in agreement in many

studies done in humans and in rodents (Tschop et al., 2000; Ariyasu et al., 2001; Tschop et

al., 2001). Although the exact mechanisms by which the post-meal ghrelin level is suppressed

are not determined, ghrelin could be suppressed in response to carbohydrate-rich meals, rather

than protein- or fat-rich meals. However, this might be a secondary consequence to a high

insulin increase with a carbohydrate-rich meal (Erdmann et al., 2004).

2.3. Cholecystokinin

Cholecystokinin (CCK) is a hormone present in the brain and the gut. The ingestion of

nutrients into the small intestine stimulates CCK release from the entire endocrine cells (I

cell). This cell has a triangular shape with the apical surfaces oriented toward the intestinal

lumen and secretory granules containing CCK concentrated around the base. This shape helps

intestinal nutrients to stimulate I cells to release their contents into the blood and surrounding

tissue. In the brain, it is widely distributed, and it functions as a neurotransmitter. CCK plays

a role in diverse behaviors and states as anxiety, sexual behavior, learning, and memory and

spontaneous activity (Timothy H, 2004). Different molecular forms of CCK are identified to

be present in the intestine, other neural tissues and blood (Reeve et al., 1994). CCK and

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Gastrin have the same carboxyl terminal pentapeptide (-Gly-Trp-Asp-Met-Phe-NH2) (Tracy

& Gregory, 1964; Mutt & Jorpes, 1968).

Different molecular forms of CCK with a range of 4 to 83 amino acids were recognized in

human tissue and blood (Liddle et al., 1984; Paloheimo & Rehfeld, 1994). All these

molecular forms derive from the same precursor (single CCK gene ) and differ from each

other as a result of posttranslational processing (Reeve et al., 1994). Small and large

molecular forms of CCK have similar biological activities. Some studies suggested that the

most predominant forms in human tissue and blood are CCK-33 and CCK-8 (Liddle et al.,

1985; Rehfeld, 1998).

Figure 2: Primary sequence of the most predominant mammalian forms of CCK: CCK-58,

CCK-33, and CCK-8. Source: Wank, 1995.

In the past, bioassays for studying and measuring circulating forms of CCK CCK have not

been sensitive enough (Johnson & McDermott, 1973; Marshall et al., 1978). Crossreactivity

with gastrinlike substances hampered the ability of radioimmunoassay to recognize CCK from

gastrin (Calam et al., 1982; Walsh et al., 1982). As well as, the antibodies that recognize one

particular molecular form of CCK might not be able to identify another molecular form.

Liddle and Goldfine et al have developed a sensitive and specific bioassay for measuring

human plasma CCK. This method is based on the response of the rat pancreatic acini to CCK

that trigger amylase release. According to Liddle and others, cholecystokinin is thought to

have a very short half-life of 1– 2 min. During10 to 30 min from starting a meal, CCK levels

elevate, then go down gradually and it can take about 3 to 5 h to come back to basal levels

(Liddle et al., 1985).

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An important point to mention is that CCK could be involved in the functional expression of

other gut hormones. A recent study has proven that CCK can inhibit the stimulatory effect of

peripheral ghrelin on food intake and neuronal activation in the hypothalamic the arcuate

nucleus (ARC) through dampening increased ARC neuronal activity. In this study, rodents

were given peripherally and separately CCK and ghrelin injections and compared with the effect

of giving CCK and gastrin injection together. The results showed that giving injections together

abolished the ghrelin induced food intake effect (Kobelt et al., 2005). Other studies showed

that central administration of both insulin and leptin led to an increased sensitivity to

peripheral CCK in rodents. This might suggest that either leptin or insulin work

synergistically with CCK in inducing satiety and regulation of food intake (Riedy et al., 1995;

Matson et al., 1997).

The main triggers for CCK secretion in the small intestine are dietary protein and fat, whereas

carbohydrates are considered to have a lesser stimulating effect (Liddle et al., 1985). Many

studies reported that protein is not only more satiating than carbohydrate, but also more

satiating than fat, and this effect on food intake is not just related to its energy content

(Trigazis et al., 1997; Peters et al., 2001). In the past, the mechanism by which proteins

inhibit food intake was attributed to changes in plasma and brain amino acids. Based on

studies in rats, it was noticed that plasma and especially brain amino acid concentrations

elevate relatively after protein intake ingestion (Anderson et al., 1994; Choi et al., 2000).

Therefore, it could be assumed that satiety signals initiated by protein consumption start in the

gastrointestinal tract. Peptides generated from dietary protein stimulate secretion of satiety

hormones such as cholecystokinin (Nishi et al., 2001; Darcel et al., 2005), which contributes

to satiety signals and, thereby food intake suppression (Reidelberger, 1994).

2.4. Cholecystokinin receptors

2.4.1. Introduction

Two types of CCK receptors have been recognized in normal tissue of human; these are

classified as the CCK1 receptor, previously called CCKA receptor and the CCK2 receptor

previously named CCKB receptor. These two receptors have been pharmacologically

distinguished on the basis of their affinity to the agonists CCK and gastrin that share the same

COOH-terminal pentapeptide amide sequence but differ in sulfation at the sixth (gastrin) and

seventh (CCK) tyrosyl residue. CCK1 and CCK2 receptors both belong to a specific family of

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G Protein-Coupled Receptors (GPCR) though they differ in terms of molecular structure,

distribution, and affinity for CCK. CCK1 receptor is primarily distributed in the

gastrointestinal tract , whereas CCK2 receptor is widely distributed in central nervous system

(Noble et al., 1999).

The CCK1 receptor is more selective since it needs the carboxyl-terminal heptapeptide-amide

of CCK with a sulfated tyrosyl residue, whereas the CCK2 receptor requires only the

carboxyl-terminal tetrapeptide for high-affinity binding and potent biological activity.

CCK2R could bind gastrin as it has the same C-terminaltetrapeptide amide as CCK (Dufresne

et al., 2006) and it is also called the gastrin-receptor. It was proven that the sulfated

octapeptide (CCK-8: Asp-Tyr (SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) has the highest

affinity for the CCK1receptor (Figure 2). Other natural molecular variants of CCK such as

CCK-33, CCK-39, and CCK-58 have similar affinity compared to CCK-8 to bind to the

CCK1 receptor (Solomon et al., 1984; Reeve et al., 2002).

2.4.2. Functions

CCK is well known to be a crucial regulatory peptide in the pancreas that triggers secretion of

digestive enzymes. CCK-mediated enzyme secretion is believed to be stimulated by the

interaction of CCK with CCK1 receptors present on the pancreatic acinar cells. After agonist

binding, signaling via G-protein-coupled receptors happens as it is represented in Figure 3.

Binding of the agonist to G-protein-coupled receptors results in activation of phospholipase C

(PLC). Phospholipase C (PLC) hydrolyzes phosphatidyl inositol 4, 5 biphosphate into inositol

trisphosphate (IP3) and diacylglycerol (DAG). Inositol trisphosphate (IP3) stimulates

intracellular Ca+2 release which acts together with diacylglycerol (DAG) to activate protein

kinase (PKC). PKC mediates phosphorylation of other proteins in a cell type specific manner

which leads to various cellular responses (Noble et al., 1999; Dufresne et al., 2006) .

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Figure 3: Simplified view of signaling pathway of the CCK1R via the Gq type of G-protein

coupled receptor after binding with an agonist. Source: Blenau & Baumann, 2001.

It is well stated that CCK-mediated intestinal feedback is mediated by activation of the vagal

afferent pathway. Vagal afferents express CCK1Rs. The peripheral endings of these afferent

neurons are present in the wall of the GI tract, both in the mucosal and muscle layers (Moran

et al., 1990; Moran & Kinzig, 2004). The activation of the terminals of these vagus nerves

stimulates cells in the brain which are on their turn responsible for controlling gastrointestinal

functions (Moran et al., 2001; Moran & Kinzig, 2004). Control the GI functions through an

interaction of CCK with CCK1R includes delayed gastric emptying, release of somatostatin

from D cells of the gastric mucosa which lead to inhibition of gastric acid secretion,

stimulation of gall bladder contraction, relaxation of the sphincter of Oddi, and slows down

gastrointestinal motility and stimulation of pancreatic exocrine secretion. Finally, it also leads

to satiety regulation.

Binding CCK to CCK2 receptor can exert different biological activities in the human body. It

can stimulate memory and learning processes, nociception, panic and anxiety, gastric acid

secretion and endocrine pancreas secretion (Noble et al., 1999; De Tullio et al., 2000;

Dufresne et al., 2006; Peter et al., 2006; Berna & Jensen, 2007; Rehfeld et al., 2007).

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2.4.3. Receptor structure and ligands interactions

CCK receptors are GPCRs that have seven typical transmembrane (TM) domains

(Kolakowski, 1994) as is illustrated in Figure 4. Interesting features of these CCK receptors

are sites of glycosylation on external loop and tail regions, and a conserved disulfide bond

that links between the first and the second extracellular loop regions (Hadac et al., 1996).

Another disulfide intradomain bond has been seen within the CCK1R amino terminus

(Pellegrini & Mierke, 1999; Ding et al., 2003).

Nowadays, a large set of data concerning binding sites of cholecystokinin receptors is

available, providing a nice view of the binding mode of natural and synthetic ligands to their

cognate receptors. Essentially four complementary approaches (site-directed mutagenesis,

photoaffinity labeling, NMR-NOE transfer, and three-dimensional modeling) were used for

providing these data. The binding site of CCK1 receptor to CCK has a special interest because

of the higher selectivity of this receptor for sulfated versus nonsulfated CCK and for sulfated

CCK versus gastrin. CCK1 receptor binds sulfated CCK with (500- to 1,000-fold) higher

affinity than the non-sulfated analogs of CCK (Gigoux et al., 1998; Gigoux et al., 1999).

Many structure-activity relationship studies on synthetic CCK analogs have referred to the

critical importance of sulfated tyrosine at position 2 in CCK-8 for binding to CCK1 receptor,

since its removal results in a 500 fold drop in affinity (Huang et al., 1989; Dufresne et al.,

1996). Both amino acids Met and Arg in the second extracellular loop of the CCK1R are

involved in the two interactions that account for this high selectivity of the CCK1 receptor for

sulfated CCK. Introducing a mutation of Arg in the second extracellular loop of the CCK1

receptor led to considerable decreases in the affinity and potency to CCK (Gigoux et al.,

1998; Gigoux et al., 1999).

A study using site-directed mutagenesis of evolutionarily conserved amino acids identified

two amino acids of the human CCK1Receptor that interact with the N-terminal moiety of

CCK. The two amino acids are Trp-39 and Gln-40 which located at the top of transmembrane

helix I. Those two amino acids when mutated to other amino acids of the same chemical

function, resulting in a loss in affinity of the receptor for the full agonists [Thr,Nle] CCK-9

and CCK-8. The loss in affinity for [Thr,Nle]CCK-9 of the mutant receptors did not result in a

loss in biological efficacy of [Thr,Nle]CCK-9 at the receptors as seen by identical production

of inositol phosphates. However, at the same time, similar loss in the biological potency

accompanied the decrease in affinity as revealed by parallel shifts in the dose-response curves

- 11 -

for inositol phosphate production (Kennedy et al., 1997). Methionine 195 of the CCK-1R was

identifed as a putative amino acid in interaction with the aromatic ring of the sulfated tyrosine

of CCK using three dimensional modeling (Gigoux et al., 1998). By dynamics-based docking

of CCK in a refined three-dimensional model of CCK-1R using two amino acids Arg-336 and

Asn-333 of CCK-1R were identified to interact with the Asp carboxylate and the C-terminal

amide of CCK, respectively (Gigoux et al., 1999). Again using the same approach mentioned

above, a network of hydrophobic interaction appeares to involve the COOH-terminal

tetrapeptide of CCK between TM helices III, V, VI, and VII (Escrieut et al., 2002; Archer-

Lahlou et al., 2005).

Multi-dimensional NMR studies have also been used in the development of a CCK1R binding

model. In these models important interactions happen between: Tyr27 with CCK1R residues

W39, P41, I45, and Y48; Met28 with receptor residues W39, P41, and P351; Met31 and

receptor residues A334, A337, and P351; and Asp32 with receptor residue R336. All the NMR

studies have used the non-sulfated CCK ligand and as mentioned previously, non-sulfated

CCK has the lowest affinity for CCK1R. Therefore, it could be that the experimental

constraints from these studies are not relevant to the high affinity CCK ligand complexes

(Pellegrini & Mierke, 1999; Giragossian & Mierke, 2001).

With the use of photoaffinity labeling, two hits in the CCK1 receptor were recognized. The

first was Trp39 at the top of TM I (the first transmembrane domain) and the second was

His347 within the third extracellular loop. Following this approach, a binding model of CCK

to the CCK1R was proposed in which the NH2-terminal moiety was in interaction with the

third extracellular loop of the receptor and the carboxyl terminus of CCK and the tyrosine

sulfate were in contact with Trp39 and Arg197, respectively ( Ji et al., 1997; Hadac et al.,

1998) (Figure 4).

- 12 -

Figure 4: Representation of the CCK1 receptor and its agonist binding sites. Source: Archer-

Lahlou et al., 2005.

Legend: Top, view of the CCK1R·CCK binding complex. ICL: intracellular loop, CL:

extracellular loop, and TM: transmembrane domain. Bottom, representation of the three-

dimensional model of CCK1R. In green are the amino acid side chains of CCK1R binding

site, while the ligand is colored in orange.

2.5. Bioactive Peptides

Bioactive peptides are specific protein fragments that can be shown to have an effect on body

functions and may ultimately influence health (Kitts & Weiler, 2003). These peptides are

inactive within the original protein, but once released, they act as regulatory compounds with

biological activities is based on the intrinsic amino acid composition and sequence (Meisel,

1997). The size of active sequences may vary from 2 to 20 amino acid residues, and many

peptides have multi-functional properties. Biologically active peptides basically can be

- 13 -

liberated from precursor proteins in one of the following ways: (a) enzymatic hydrolysis (b)

microbial fermentation (Korhonen, 2009).

2.5.1. Enzymatic hydrolysis

The most common way to produce bioactive peptides from protein is through enzymatic

hydrolysis. During digestion, cleavage of bioactive peptides from milk proteins occurs in the

gastrointestinal tract of the milk-consuming individual by pepsin and pancreatic enzymes

(trypsin and chymotrypsin ) (Pihlanto, 2006). Many of the known bioactive peptides like

ACE-inhibitory peptides and mineral-binding phosphopeptides or caseinophosphopeptides

(CPPs) for instance have been produced in vitro using pepsin. Other proteolytic enzymes,

such as Alcalase and Thermolysin, have also been used to release bioactive peptides from

different proteins (McDonagh D, 1998; Pihlanto- Leppala et al., 2000; Vermeirssen et al.,

2004; Roufik S, 2006; Da Costaa Elizabete Lourenço, 2007).

2.5.2. Microbial fermentation

Formation of different bioactive peptides during fermentation of dairy products through

microbial proteolysis is well reported (Matar, 2003; Fitzgerald. RJ, 2006). Examples of the

starter cultures which are used in dairy productions are Lactobacillus helveticus,

Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus plantarum, Lactobacillus

acidophilus, Lactococcus lactis, Streptococcus thermophilus. (Gomez-Ruiz, 2002; Fuglsang,

2003; Gobbetti, 2004; Donkora.Osaana N, 2007; Virtanen et al., 2007).

Several studies reported that food-born peptides stimulate cholecystokinin release. An in vivo

study was done to investigate dietary peptides induce satiety via cholecystokinin and

peripheral opioid receptors. First, this study emphasized the importance of protein digestion in

producing peptides that are functional in the stimulation of satiety. Next, it showed that casein

and soy protein hydrolysates induced satiety was inhibited by blocking CCK1R. This means

that the mechanism by which these proteins and their hydrolysates is mediated via CCK1R

(Pupovac & Anderson, 2002). Another study done by Nishi and Hara et al. has also

demonstrated that specific peptides from soybean suppress food intake by direct action on rat

small intestinal mucosal cells by stimulating cholecystokinin secretion (Nishi et al., 2003).

Milk and pea protein have received a growing attention in term of their importance on body

weight management. Some peptides found in these food items have positive effects in the area

- 14 -

of satiety. The mechanisms by which these peptides exert their effect on food intake via the

gut include slowing stomach emptying, perhaps via opioid receptors, and direct or indirect

stimulation of gut hormone receptors like cholecystokinin (Anderson & Moore, 2004). An in

vitro study showed that short-chain peptides from soy, potato, casein, pea and whey

hydrolysates were able to stimulate the release of cholecystokinin from enteroendocrine cells.

In addition to CCK release, some of these hydrolysates (soy, potato and casein) were directly

able to stimulate CCK1R-expressing cells which gave the opportunity to these hydrolysates to

be a target of functional food ingredients with satiating effects (Foltz et al., 2008).

Generally speaking, some peptides, released during gastrointestinal digestion or food

processing, play an interesting role in metabolic regulation with hormone-like activity,

suggesting their important use in the nutraceutical and functional food sector for disease risk

reduction and health promotion. Nevertheless, many challenges must be solved to help the

sustainability of this sector. One of these challenges is that some health benefits of protein and

peptide nutraceuticals were based on in vitro studies while not enough clinical trials were

conducted (Moller et al., 2008). Other problems are difficulties with large scale production,

bioavailability in the gastrointestinal tract, and safety issues which include the absence of

toxicity, cytotoxicity and allergenicity (Murray & FitzGerald, 2007). Despite all these

challenges, form a short overview of the current situation including the remarkable increase in

the prevalence of obesity and other related diseases and focusing more on the biological

activities of food proteins (Tripathi V, 2006; Yalcin, 2006; Hartmann R, 2007), it could be

expected that more experimental research and more bioactive peptide-based products will be

developed (Korhonen & Pihlanto, 2003).

2.6. Milk and pea protein structure

Milk proteins consist of 80% casein and 20% whey. Casein is organized in micelles which are

designed by nature to stabilize and transport essential nutrients, mainly protein and calcium

for the neonate (DeKruif, 2003). The micelles are composed of the main four caseins: as1-

casein (as1-CN), as2-CN, b-CN, and k-CN). Casein proteins are well known to be a precursor

for many bioactive peptides (Fox, 2003; Swaisgood, 2003).

The major whey proteins are beta-lactoglobulin, alpha-lactalbumin and bovine serum albumin

which account for 70-80% of the total whey proteins in the bovine milk, and other minor

proteins such as lysozyme and lactoferrin (Wong et al., 1996; Fox, 2003). Beta-lactoglobulin,

- 15 -

the most prevalent protein in whey is a globular protein. It associates to form an octamer

between pH 3.5 and 5.2, at low temperatures. Between pH 5.2 and 7.5, including the pH of

milk, beta-lactoglobulin tends to be found as a dimer. At very high and low pH, beta-

lactoglobulin exists as a monomer. Alpha-lactalbumin is a calcium metalloprotein and it is

stabilized against heat denaturation and aggregation by calcium. The amino acid sequence of

this protein is very similar to that of lysozyme. Bovine serum albumin (BSA) from bovine

milk is identical to the blood serum molecule. It acts as a carrier for insoluble fatty acids,

which protect the molecule against heat. Some minor whey proteins may have biological

functions. For example, lysozyme is considered a significant component of the antibacterial

system of milk, and lactoferrin which has an antimicrobial, antiviral and immunomodulatory

function (Shah, 2000; Steijns, 2001). Over the last decade, researchers have demonstrated that

bioactive peptides released from whey and casein proteins possess very important biological

functionalities, including antihypertensive, antioxidative, immunomodulatory, opioid, and

mineral-carrying activities (Meisel.H, 1998; Korhonen & Pihlanto, 2003; FitzGerald et al.,

2004).

Pea protein is mainly composed of water soluble proteins: globulins, which are also soluble in

salt solutions, and albumins. The insoluble protein is poorly characterized up to now

(Gueguen, 2000). Pea globulins are composed of two main families, legumin and vicilin,

which belong respectively to the 11S and 7S seed storage protein classes. Legumin is

homologous to soybean glycinin. The monomer is constructed from two subunits, the acidic α

and basic β polypeptides, linked by disulfide bonds. The β subunits constitute the

hydrophobic heart of the polymeric protein, while the more hydrophilic α subunits are situated

at the outside. 22 different α polypeptides and 11 β polypeptides have been identified. Vicilin

is characterised by a trimeric structure, like bean phaseolin and soybean conglycinin. The

trimeric structure of vicilin does not contain cysteine and therefore is only stabilised by weak

interactions. Pea vicilins are very complex structures in term of their subunit composition

(Gibbs et al., 1989). Albumins mainly comprise proteins with a biological function in the

seed, like plant defense proteins, enzymes and enzyme inhibitors. This protein fraction is

characterized by a high lysine and sulphur amino acid content and it is very heterogeneous. It

contains PA1 albumin, a sulphur-rich protein dimer including the two polypeptides PA1a and

PA1b and the larger dimer PA2 albumin including two similar polypeptides PA2a and PA2b

(Croy et al., 1984). Because of its good functional properties in food applications and the high

- 16 -

nutritional value, pea protein and its products are considered a rich source of biologically

active components that may exert beneficial health and therapeutic effects (Roya, 2010).

CHAPTER 3: MATERIALS AND METHODS

3.1. Cell lines and products

CHO (Chinese Hamster Ovary) cells expressing the rat CCK1R (CHO-CCK1R) cells were

prepared by Prof. Peter Willems (Smeets et al., 1996) and native CHO-K1 cells were

provided by Prof. Georges Leclercq (Ghent University Hospital, Department of Clinic

Biology, Microbiology and Immunology, Ghent Belgium). Ham’s F12 medium (1:1)

(DMEM-F12), Advanced Dulbecco’s modified Eagle’s medium, fetal bovine serum (FBS),

Fluo-4AM, Pluronic F-127, geneticin (G-418 antibiotic) and Hank’s buffered salt solution

(HBSS) were purchased from Invitrogen (Paisley, UK). Alpha, beta and kappa-casein, alpha-

lactalbumin and beta-lactoglubilin, bovine serum albumin (BSA), HEPES, probenecid,

lorglumide ((±)-4-[(3, 4-dichlorobenzoyl)amino]-5-(dipentylamino)-5-oxopentanoic acid

sodium salt; CR-1409), peptidase and gastrointestinal digestion enzymes (pepsin, trypsine,

and chemotrypsin), alpha, beta, kappa-casein, alpha-lactalbumin, and beta-lactoglubulin were

purchased from Sigma-Aldrich (St.-Louis, MO). Sulfated cholecystokinin octapeptide (CCK-

8S) was purchased from Bachem (Weil am Rhein, Germany), JMV-180 (Boc-Tyr(SO3H)-

Nle-Gly-Trp-Nle-Asp-2-phenylethylester) from Research Inc. (Barnegat, NJ) and clear

bottom black 96-well plates from Greiner (Frickenhausen, Germany). Pea protein

hydrolysates, whey protein hydrolysates, non hydrolysed pea, non hydrolysed casein and non

hydrolysed whey were kindly provided by Professor Van Aamerongen (Wageningen

University).

3.2. Cell culture

CHO-CCK1R and CHO-K1 cells were grown in an incubator at 5% CO2 and 37°C in

advanced DMEM-F12 supplemented with 10% FBS and 1% L-glutamine, 1% streptomycin

and penicillin. CHO-CCK1R cells medium was supplemented with 10 µl/ml geneticin to keep

a stable transfected culture.

3.3. Protein hydrolysates

Hydrolysis is a process in which a certain molecule during reaction with water is broken into

parts. Enzymes are often used to catalyze this process.

- 17 -

3.3.1. Hydrolysis of the pea and whey proteins with alcalase and promod enzymes

Both enzymes promod 278 P and alcalase were used for hydrolysis pea and whey proteins.

Protein solutions (5% (w/w)) were dissolved in demineralized water (DQ) and stirred. A bath

system was used for adjusting the temperature to 60° and 1M (NaOH) and (HCl) were used

for adjusting the pH to 7 for promod 278P and to 8 for alcalase. Samples were taken on

certain time points (1, 2, 4, 8, 10, 20, 40, 60, 180, 360 minutes after applying the enzymes

(2% enzyme respectively to protein fraction (w/w)), then inactivation at 80°C for 15 minutes

was done in a water bath. Subsequently the samples were centrifuged for 20min at 5000g, and

the supernatants were freeze-dried.

3.3.2. Hydrolysis of purified whey and casein proteins with peptidase

Hydrolysates from alpha, beta, kappa-casein, alpha-lactalbumin, and beta-lactoglubulin were

prepared by dissolving in distilled water at a ratio of 4g of the sample to 100 ml water and pH

was adjusted to 7. Next, peptidase was added in a ratio of 1 g enzyme per 250 g of the protein

sample. Samples were placed in a warm water bath for 2h and later on sample solutions were

heated for 15min at 80°C to deactivate the enzyme.

3.3.3. Gastrointestinal digestion

Samples from alpha, beta, kappa-casein, alpha-lactalbumin, and beta-lactoglubulin were used

with gastrointestinal digestion enzymes to prepare protein hydrolysates. After dissolving the

protein samples in distilled water in the same ratio as mentioned above (3.3.2), pH was

adapted to 2 by addition of 0.1 M HCl. Next, pepsin was added and the solutions were placed

for 2 hours in a water bath at 37°C. Trypsine and chemotrypisne were added after adjusting

the pH to 6.5 by using 0.1M NaOH. Then, the solutions were kept in a warm water bath for

2.5 hours and the enzyme activity was stopped by heating for 15 min at 80°C. Finally,

samples were freeze-dried.

3.4. Cell-based bioassay to screen for CCK1R activity

Activation of the CCK1 receptor leads to a rapid increase in intracellular calcium

concentration (within 0–30 s). The change in intracellular Ca+2 concentrations can be

monitored with a fluorescent dye. The change in florescence is a measure for the activation of

- 18 -

the receptor. The increase in intracellular Ca+2 concentrations was determined by a method

that was reported by Foltz et al. (Foltz et al., 2008). The fluorescent probe that was used in

this assay is the hydrophobic Fluo-4AM, a cell-permeant acetoxymethyl (AM) ester, which is

hydrolyzed by cellular esterases and becomes fluorescent upon Ca2+-binding. One half of a

96-well plate is seeded with CHO-CCK1R cells and the other half with CHO-K1 cells at

40,000 cells per well. Cells were incubated at 5% and 37°C CO2 for 20-24 h to allow

attachment. After that, the medium was removed and 50 µl of DMEM-F12 supplemented with

4 µM Fluo-4AM, 0.02% (w/v) of the surfactant pluronic, 4.55 mg/ml BSA, and 1.6 mM of the

anion transport inhibitor probenecid was added to the wells for 1 h at 19°C, as was

determined as the ideal dye loading temperature in preliminary experiments. Subsequently,

the wells were washed twice with 150 µl of HBSS supplemented with 2.5 mM probenecid, 20

mM HEPES and 1mg/ml BSA. Finally 100 µl of modified HBSS was added to the wells 20

min before the start of the experiment.

Two platforms were used: a fluorescence plate reader and a confocal microscope.

In the first method, a microtiterplate reader (infinite pro 200 (Tecan, Männedorf, Switzerland)

multimode plate reader with automated injection system) was handled using i-control™

software. Excitation and emission wavelengths were set to 480 nm and 520 nm respectively,

using Quad4 monochromators™ technology.

In the second setup, a Nikon A1R confocal laser scanning microscopy system (Nikon

Instruments Inc., Melville, NY) was used, mounted on a Nikon Ti-E inverted epifluorescence

microscope and equipped with a microscope incubator, Perfect Focus System and resonant

scanner. Multiwell plates were screened with a Plan Fluor 40 x/0.75 dry objective at full field

of view (636 µm x 636 µm), resulting in a pixel size of 1.24 µm x 1.24 µm. Fluo-4AM was

excited using a 488 nm multi-line Ar laser and fluorescence was detected through a 525/50

nm bandpass filter.

A similar measurement protocol was used for both setups. After washing, the plates were

immediately placed in the plate reader to equilibrate at the measurement temperature of 31°C,

which was determined as the best measuring temperature. Measurement started 30 min after

the start of washing the wells which were measured separately. Fluorescence was acquired at

2.5 fps with the plate reader and 3 fps with the microscope. First, the basal fluorescence of a

well was measured for 6 s after which 100 µl of 2nm CCK, 50 µM lorglumide, 0.01, 0.05,

0.25, and 1.2 mg/ml of some protein hydrolysates samples and 6mg/ml of all hydrolysates

- 19 -

(diluted in modified HBSS) were added and subsequently measurements were continued for

another 34 s. Contrary to the automatic addition of the sample in the plate reader, an

electronic pipette was used to perform sample addition with the confocal microscopy. Each

sample concentration was measured in 5 wells (technical replicates) for both cell types.

3.6. Determination of degree of hydrolysis

The degree of hydrolysis represents the percentage of peptide bonds hydrolyzed during

hydrolysis of protein (Adler-Nissen, 1976). The degree of hydrolysis is calculated using

hydrolysis equivalents (h), the number of peptide bonds cleaved during hydrolysis, expressed

as eq/kg protein or meq/g protein. Hydrolysis equivalents are assayed by measuring the

increase in free amino groups that are generated by the protein hydrolysis.

To assess the amount of amino groups before and after protein hydrolysis, a

spectrophotometric reaction technique with TNBS (Trinitrobenzenesulfonic acid hydrate) has

been used. TNBS reacts with amino groups in their unprotonated state, yielding a yellow

product whose absorbance was measured at 340 nm. 0.21 M Sodium phosphate as buffer was

prepared and pH was set at 8.2. TNBS stock (5% solution) and 1% SDS were prepared. A

standard curve was prepared using ± 3 mM leucine in 1% SDS and 20x, 10x, 4x en 2x

dilutions. Samples were prepared by dilution in 1% SDS to a concentration of 0.5 mg/ml in

duplo and 15 µl of each sample (leucine standard or hydrolysed proteins or non hydrolysed

sample) was pipetted in a microwell, followed by 45 µl of the sodium phosphate buffer. The

plate was covered with aluminum foil after addition of 45 µl 0.05% TNBS and then it was

incubated for 60 minutes in a 50° C stove. 90 µl 0.1 N HCl per well was added to stop the

reaction before reading the absorbance at 340 nm with the plate reader.

3.7. Data analysis

For calculation of response curves the following procedures reported by Staljanssens et al.

which was adapted from a method previously reported by Foltz et al. were applied

(Staljanssens et al., 2011). Fluorescence measurements Fi were normalized to the basal

fluorescence level before sample addition, which corresponds to the average fluorescence in

the first 6 s of the recording (F0) to avoid differences due to variations in cell density and dye

concentration. For correction of background fluorescence and non-specific responses, average

normalized fluorescence values of 5 technical replicates from CHO-CCK1R cells were

corrected by subtracting fluorescence values from CHO-K1 cells per time point and per

condition. The relative fluorescence plotted as a function of time. Net response was calculated

- 20 -

as the sum area below the curve. All net responses were expressed as a percentage of the

maximum net response caused by 1 nM CCK-8S.

3.8. Statistics

Differences in the potency of the whey and pea protein hydrolysates to increase the

intracellular Ca+2 (by activation of CCK1R) were analyzed using ANOVA fixed effects

followed by 2-samples t-test. Other results were only analyzed using 2-samples t-test.

Differences were only considered to be statistically significant if p values were less than 0.05.

- 21 -

CHAPTER 4: RESULTS

The experiments in part 4.1 and 4.2 were partially carried out in cooperation with master

student Annelies Billiet. In addition, extra data from the lab were received to obtain the

following Figures: 5, 6 and 7.

4.1. Measurement cellular response to the natural ligand CCK-8S with the plate reader

and the confocal microscopy

CHO-K1 cells were used in the assay as negative controls. CHO-K1 cells did not show a

significant difference in the cellular response when they were treated with CCK-8S compared

to cells treated with buffer. First, the plate reader was used to monitor the cellular response to

the natural ligand CCK-8S in CHO-CCK1R cells. The change in fluorescence was monitored

in time for increasing concentrations of CCK-8S (0.001 nM–1 nM) (Figure 5a). From RF

curves (Figure 5a), strong dose-dependent kinetics were seen in terms of time point and height

of the maximum RF. A significant increase in signal was shown with increasing

concentrations of CCK-8S. Dose-response curves were derived from calculating the net

response versus CCK-8S concentration (Figure 5b)

Figure 5: Dose-dependent CCK1R-mediated cellular response obtained with a plate reader.

Legend: (a) Kinetics of relative fluorescence (RF) of CHO-CCK1R cells in response to

increasing concentrations of CCK-8S (0.001–1 nM), where the curves present the mean of

results from 5 wells. (b) Dose–response curve for CCK-8S based on 4 experiments,

expressed as a percentage of the maximum cellular response induced by 1 nM CCK-8S.

The same kinetic experiments handled with the plate reader to measure the cellular response

to CCK-8S were used with the confocal scanning microscopy, as well. The population-

average response was monitored on whole images. One single area was acquired at full field

- 22 -

of view (636 µm × 636 µm) for each well, corresponding to 100–150 cells, and measuring the

average pixel intensity was done over the entire image (Figure 6a). Figure (6b and c)

illustrates the high similarity between the RF curves and CCK-8S dose–response curve

obtained from the confocal microscopy with those from the plate reader (Figure 5a and b).

Figure 6: Dose-dependent CCK1R-mediated cellular response obtained with a confocal

microscopy.

Legend: (a) Montage from confocal microscopy. This figure illustrates the increase in the

fluorescence response in a CCK dose dependent manner. We can see that the higher

concentrations of CCK resulted in faster and higher increases in fluorescence. (b) Kinetics of

relative fluorescence (RF) of CHO-CCK1R cells to increasing concentrations of CCK-8S

(0.001–1 nM). (c) Dose–response curve for CCK-8S expressed as a percentage of the

maximum net response induced by 1 nM CCK-8S.

4.2. Measurement of agonist and antagonist effects on the cell population level

The effect of the partial agonist, JMV-180, was determined using the plate reader. It was

proven previously that both CCK-8 and JMV-180 stimulate PPI hydrolysis in CHO-CCK1R

inducing Ca+2 increase by similar mechanisms. In addition, U-73122, the antagonist of

phospholipase C-mediated events was able to block the Ca+2 signaling stimulated by both

- 23 -

CCK-8 and JMV-180 (Yule DI, 1993). Different concentrations of JMV180 were used and all

net responses were calculated as a percentage of the 1nM CCK-8S induced response. A dose

response curve was established based on the net responses (Figure 7a). The maximum

response that could be stimulated by JMV-180 was 38 ± 8% SEM compared to 1 nM CCK-

8S.

(a) (b)

Figure 7: Representative dose–response curves for JMV180 and lorglumide monitored with a

plate reader.

Legend: (a) Representative dose–response curve for JMV180 monitored with a plate reader

based on 4 experiments in which the measurements for each concentration were repeated 5

times, expressed as a percentage of the maximum net response caused by 1 nM CCK-8S. (b)

dose–response curve for lorglumide monitored with a plate reader based on 4 experiments.

The antagonist lorglumide was tested for its potential to inhibit a CCK-8S-induced response

with the plate reader. It is well known that lorglumide which is produced by a chemical

manipulation of proglumide (Peter et al., 2006) is a selective and full antagonist of the

CCK1R that blocks the receptor sites for cholecystokinin. It is 2300 times more selective for

CCK1R than for CCK2R (Berna & Jensen, 2007). An IC50 value of 0.13 µM was reported by

Makovec et al (Makovec et al., 1986) for this antagonist and this value was 15 times lower

compared to the IC50 reported by Staljanssens et al. (Staljanssens et al., 2011), which was

probably because of the use of a different experimental design. The inhibiting effect of the

antagonist lorglumide was calculated as 100 % minus the net response. From these results, a

dose-response curve for the percentage of inhibition versus lorglumide concentrations was

established. Increasing concentrations of lorglumide were used and those concentrations

illustrated a dose-dependent inhibition of the response caused by 1 nM CCK-8S as shown in

Figure 7b. A full inhibition of 1 nM CCK-8S was seen at 50 µM of lorglumide.

- 24 -

4.3. Do milk and pea proteins have potency to act directly on the CCK1 receptor?

4.3.1. Aim of the study

The aim of the current study was to determine the effect of different milk and pea protein

hydrolysates on CCK1R activation. The potency of these hydrolysates to activate CCK1R was

assessed by measuring the intracellular Ca2+-flux in a cell based bioassay. The cellular

response to protein hydrolysates was measured in a plate reader and a confocal microscopy.

4.3.2. Effect of different whey and pea protein hydrolysates on CCK1R activation by a

plate reader

Increasing concentrations of protein hydrolysates (0.005 g/l – 3g/l) for 1, 3 and 6h were used.

Experiments were repeated 5 times for whey and pea hydrolysates. Non hydrolysed whey was

also tested in 3 repeated experiments. The non hydrolysed pea was non-soluble which made it

difficult to be tested. First observation was that all tested protein hydrolysates resulted in a

higher increase in the net cellular response in comparison to the non hydrolysed whey protein

that resulted in a mean net cellular response around 6 ± 3% SEM at 3g/l concentration.

Another finding is that a dose dependent increase in the net cellular response was seen at

higher concentrations for all tested hydrolysates (Table 1 and Table 2).

Repeated experiments for each protein hydrolysate type showed a high variability between the

results and higher response of 3 g/ l concentration (Supplementary Figure 1 and

Table 1: Maximum response induced by increasing concentrations of whey hydrolysates expressed as a percentage

of the maximum response caused by 1 nM CCK. Figures are reported as the mean ± SEM (n = 5).

Maximum response induced by whey protein hydrolysed with alcalase / promod enzymes for 1 / 3 and 6h

Hydrolysate whey + alcalase (1h) whey + alcalase (3h) whey + alcalase (6h) whey + promod (1h) whey + promod (3h) whey + promod (6h)

concentration (g/l)

3 56 ± 10 64 ± 12 40 ± 11 41 ± 12 36 ± 10 43 ± 5

0.6 16.6 ± 2.6 13.4 ± 2.9 11 ± 4 15 ± 2.3 10.1 ± 2.7 13.6 ± 3.5

0.125 8.7 ± 1.2 2.4 ± 3.9 5.7 ± 5. 5 4.7 ± 4.7 3.8 ± 2 7 ± 4

0.025 4.8 ± 1.7 2.7 ± 2.5 3.2 ± 3. 2 0.5 ± 2.2 0.2 ± 1.7 5.3 ± 4

0.005 4.2 ± 3.8 1.5 ± 1.2 2.2 ± 3 2 ± 3.1 0.5 ± 1.7 3.4 ± 2.5

Table 2: Maximum response induced by increasing concentrations of pea hydrolysates expressed as a percentage of the maximum response induced by 1 nM CCK. Figures represent the mean ± SEM (n = 5).

Maximum response induced by pea protein hydrolysed with alcalase / promod enzymes for 1 / 3 and 6h

Hydrolysate pea + alcalase (1h) pea + alcalase (3h) pea + alcalase (6h) pea + promod (1h) pea + promod (3h) pea + promod (6h)

concentration (g/l)

3 44 ± 10 32 ± 14 40 ± 12 31 ± 6 40 ± 9 8 ± 6

0.6 3 ± 6 2.8 ± 1.4 10.9 ± 2.8 2.3 ± 1.1 7. 9 ± 3.6 5.2 ± 2.6

0.125 3 ± 4 3.1 ± 1.5 6.9 ± 4. 7 0.44 ± 0.2 3.3 ± 5.2 7 ± 5.7

0.025 0.7 ± 3 1.6 ± 0.8 4.4 ± 2. 5 0.94 ± 0.5 1.2 ± 1.2 5.4 ± 4

0.005 1.5 ± 2.2 3.3 ± 1.7 2.6 ± 1. 5 0.44 ± 0.2 2.2 ± 2.2 15 ± 8.8

- 25 -

Supplementary Figure 2). Therefore, we calculated the mean of the 5 repeated experiments for

each type of hydrolysate for only the 3 g / l concentration and placed the whole results in one

bar diagram (Figure 8).

Effect of different pea and whey protein hydrolysates on the net

cellular response: 3g/l pea and whey

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

1h 3h 6h

hydrolysis time

% m

ax resp

onse

pea + alcalase

pea + promod

whey + alcalase

whey + promod

Figure 8: The mean net response induced by 3 g/ l of whey and pea hydrolysates measured

with a plate reader and expressed as a percentage of the maximum net response induced by 1

nM CCK. Figures present the mean ± SEM (n = 5)

Figure 8 indicates that whey protein hydrolysates obtained with the use of alcalase for 1 and

3h tended to induce more increase in the net cellular response as they resulted in mean net

cellular responses of 56 ± 10% SEM, 64 ± 12% SEM, respectively. The net cellular response

resulted from the other 1, 3h pea and whey hydrolysates ranged from 31 ± 6% SEM in case of

the 1h pea hydrolysate with promod to 44 ± 10% SEM for the 1h pea hydrolysate with

alcalase. The pea and whey protein hydrolysates obtained with the use of both enzymes for 6h

resulted in a net cellular response around 40% apart from the 6h pea hydrolysate with promod

which led to lesser response.

Statistical analysis showed that there is no interaction between the three factors (protein,

enzyme and time) and they do not affect each other positively or even negatively. However,

the interesting finding was that there was a significant difference between the results obtained

by whey protein hydrolysates and pea protein hydrolysates in the potency to induce the

cellular response (p-value < 0.05). Moreover, result obtained from whey protein hydrolyzed

with alcalase was significantly different from that obtained from whey hydrolysed with

- 26 -

promod. As well as, a significant difference was noticed between results from pea hydrolysed

with alcalase and that of pea hydrolysed with promod. In contrast, no significant differences

were found between the results in term of hydrolysis time (Supplementary Table 1).

Comparisons between the two proteins (whey/pea) and the two enzymes (alcalase/promod)

were made using 2- Samples T-test. It can be seen from the data in Supplementary Table 2

that whey hydrolysates reported significantly more net response than pea hydrolysates. In

addition, the data in Supplementary Table 3 indicate that the response from hydrolysates with

alcalase was significantly higher than the response caused by hydrolysates with promod.

Next, when only pea with promod was considered, statistical analysis showed that there is a

significant effect of hydrolysis time on hydrolysate activities (p-value < 0.05). The net

response induced by the 6h pea hydrolysate with promod was 8 ± 6% SEM compared to 40 ±

9% SEM at 3h and 31 ± 6% SEM at 1h.

Because of the first findings that showed no effect of hydrolysis time on the activity of whey

and pea hydrolysates in terms of the induced net response, whey and pea hydrolysates with

shorter hydrolysis time (1, 2, 4, 6, 8, 10, 20, 40 minutes) were also tested. Results from one

experiment in which the measurements were repeated 5 times at 3g per l concentration were

almost comparable with the results for the hydrolysates with longer hydrolysis time in terms

of the high variability between the results and range of net response. However, carrying out

one experiment is not enough to make a valid conclusion.

4.3.3. Effect of enzymes and hydrolysis time on peptide length

First observation form Table 3 is that the degree of protein hydrolysis increased with the

increase of exposure time of protein to hydrolysis enzymes. Comparing the degree of

hydrolysis for all tested hydrolysates mentioned in (4.3.2) showed that hydrolysis of both

whey and pea proteins with alcalase for 1, 3 and 6h resulted in higher degrees of hydrolysis

for both proteins compared to when promod was used. The highest DH shown with alcalase

was 19.6 ± 0.4% SD, whereas the highest DH shown with promod was 10.7 ± 0.9% (Table 3).

Comparing the DH of both whey and pea protein hydrolysates with alcalase, it was seen that

the higher degrees of hydrolysis were found for whey protein which resulted in shorter

peptides (Figure 9). At the same time, DH of whey protein hydrolysed with promod was

higher than that of pea protein hydrolysed with promod (Table 3).

- 27 -

Table 3: DH degree (%) of pea and whey hydrolysed by alcalase / promod

for 1 / 3 and 6 hours. Figures are the mean ± SD (n = 2).

hydrolysis time pea + alcalase pea + promod whey + alcalase whey + promod

1h 14.2 ± 1 7.16 ± 0.9 16.3 ± 1.2 8.5 ± 1.1

3h 16.6 ± 2.5 8.3 ± 0.7 19.1 ± 1.7 9.9 ± 1.3

6h 17.8 ± 3.5 8.6 ± 0.7 19.6 ± 0.4 10.7 ± 0.9

0

5

10

15

20

25

1h 3h 6h

me

an p

ep

tid

e le

ngt

h

Hydrolysis time

Mean peptide length of different pea and whey hydrolysates

pea + alcalase

pea + promod

whey + alcalase

whey + promod

Figure 9: Representative of the average peptide length for whey and pea proteins hydrolysed

by alcalase / promod for 1, 3 and 6h. Figures represent the mean ± SD (n = 2).

4.3.4. The correlation between the % response and the peptide length

From Figure10 we can see that alcalase hydrolysates from whey of 5 ± 2 SD amino acids

sequence resulted in a net response ranging between 40% and 64%. Concerning alcalase

hydrolysates from pea, peptides with 7 ± 1 SD residues in length induced a response of

around 40%. The lowest net response among all hydrolysates was seen for peptides with 12

amino acid residues long derived from pea hydrolysed with promod for 6h, whereas peptides

from whey with the same enzyme and with the same length resulted in much a higher

response. In general, peptides with 5 ± 2 SD to 12 ± 1 SD amino acids sequence but from

different hydrolysates yielded almost the same responses (40%). As a result and based on the

statistical analysis that showed there is no significant effect of hydrolysis time on the induced

response (Supplementary Table 1) we could assume there is no correlation between the

induced response and peptide length. ANOVA followed by Tukey test was done to compare

between the responses induced by the different treatments (pea with promod, pea with

alcalase, whey with promod and whey with alcalase). The only significant difference was

seen between the response induced by pea hydrolysed with promod and the response resulting

from whey hydrolysed with alcalase (Supplementary Table 4). Therefore, differences between

- 28 -

the cellular responses induced by different protein hydrolysates seem to be linked to the kind

of protein and the enzyme used for hydrolysis process.

Figure 10: The correlation between peptide length and the % of the maximum response.

Legend: This figure shows the relation between the peptide length of whey and pea

hydrolysates and the % response induced by these hydrolysates (n = 5 for the net response and

n = 2 for the peptide length).

4.3.5. Comparison of the results obtained by Tecan with those of a confocal microscopy

Further experiments to whey hydrolysed with alcalase for 3h were done with confocal

microscopy to confirm the results obtained with the plate reader. With the plate reader, 3 g per

l of this hydrolysate resulted in a net response of 64 ± 12% SEM (based on the mean of

repeated 5 experiments) of the 1 nM CCK-8S induced maximum response. This value

decreased to 4.25 ± 2% SEM (the mean of 2 repeated experiments) when confocal

microscopy was used. Relative fluorescence kinetics from the plate reader induced by this

protein hydrolysate showed a significant difference (p < 0.05) between the integrated

response area of the CHO-CCK1R cells caused by protein hydrolysate and that of the CHO-

K1 cells in all repeated experiments. On the contrary, the microscope confirmed that in one

experiment and did not in the other repeated experiment (Figure 11a and b).

- 29 -

(a) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

No

rma

lize

d f

luo

resc

en

ce

1 nM CCK

CHO-CCK1R

CHO-K1

(b) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

No

rma

lize

d f

luo

resc

en

ce

1 nM CCK

CHO-CCK1R

CHO-K1

Figure 11: Representative of the fluorescence kinetics curves induced by whey hydrolysed

with alcalase for 3h and measured with the microscope.

Legend: (11a and 11b) represent the 3h whey hydrolysate with alcalase induced fluorescence

kinetics resulting from two repeated experiments. The green line is the response of CHO-

CCK1R to 1 nM CCK, whereas the blue and the red lines present the response of CHO-

CCK1R and CHO-K1 cells to whey hydrolysate, respectively.

By looking at the shape of the fluorescence kinetics curves induced by whey hydrolyzed with

alcalase for 3h obtained with the plate reader (Supplementary Figure 3), we can see that the

curves from CHO-CCK1R cells parallel to those from CHO-K1 cells and the higher initial

rise in the curves of CHO-CCK1R cells seen in the five repetitions might be the reason for the

significant difference between the results of both cells. This suggests that the high net

responses seen in the plate reader might be not true positive results.

4.3.6. Comparison of the results obtained by confocal scanning microscopy with and

without lorglumide

Extra control experiments for some hydrolysates (3h pea and 1h whey hydrolysates with

promod) were also done with confocal microscopy. These experiments were carried out with

and without the CCK1R antagonist lorglumide to confirm the specificity and to test its

potential inhibitory effect on the protein hydrolysate induced response. Figure 12a and 12b

illustrate the significant difference between both CHO-CCK1R and the CHO-K1 cells

responses to both pea and whey hydrolysates.

Using lorglumide caused a significant difference between the protein hydrolysates induced

responses in the CHO-CCK1R cells and those responses obtained with the CHO-CCK1R cells

in the absence of lorglumide. On the other hand, no difference was observed in the net cellular

p= 0.2, 1.30% ± 2.21%

p= 4e-4, 7.21% ± 2.96%

- 30 -

responses with and without lorglumide which could be attributed to the significant effect of

lorglumide noticed on the response of CHO-K1 cells. In spite of the significant effect of

lorglumide on CHO-CCK1R cells response induced by pea hydrolysed with promod, the

shape of the curves of CHO-CCK1R with and without lorglumide looks similar (Figure 12a).

This leads to consider this example is not a good example of the real inhibitory effect of

lorglumide. In contrast, the significant effect of lorgumide on the response in CHO-CCK1R

cells induced by whey hydrolysate coincided with a change in the shape of the curve to

similar shape of that of CHO-K1 cells (Figure 12b), indicating to a possibility of direct

interaction of whey hydrolysate with CCK1R. However, these results are concluded from

conducting one experiment in the confocal microscopy for both hydrolysates. Therefore, more

experiments have to be carried out to validate these results.

(a) 10 g/l pea + promod 3h

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40

Time (s)

fi/f

0

1 nM CCK CHO-CCK1R

CHO-CCK1R

CHO-CCK1R + LGM

CHO-K1

CHO-K1 + LGM

CHO-CCK1R VS CHO-K1: P= 0.006

CHO-CCK1R VS CHO-CCK1R+LGM: P=

0.009

CHO-K1 VS CHO-K1+LGM: P=0.0002

CHO-CCK1R+LGM VS CHO-K1: P=0.9

- 31 -

(b) 10 g/l whey + promod 1h

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40

Time (s)

fi/f

0

1 nM CCK CHO-CCK1R

CHO-CCK1R

CHO-CCK1R + LGM

CHO-K1

CHO-K1 + LGM

Figure 12: Representative of the fluorescence kinetics curves induced by pea and whey

proteins hydrolysed with promod measured with the microscope with and without lorglumide.

Legend: (12a and 12b) represent the fluorescence kinetics responses induced by 10g/l of pea

hydrolysed with promod for 3h and whey hydrolysed with promod for 1h, respectively with

and without lorglumide. The green line is the response of CHO-CCK1R to 1 nM CCK. The

full blue and the red lines present the response of CHO-CCK1R and CHO-K1 cells,

respectively to protein hydrolysate without lorglumide, whereas the dashed blue and the red

lines represent the response of CHO-CCK1R and CHO-K1 cells to protein hydrolysate in the

presence of lorglumide.

4.3.7. Evaluation of the effect of different purified protein hydrolysates on the cellular

response by a plate reader

Discrepancy between the results of the plate reader and the microscope might be due to the

complex nature of the studied hydrolysates. Therefore, less complex protein hydrolysates

from alpha-lactalbumin, beta-lactoglobulin, alpha, beta, and kappa-casein with

peptidase/gastrointestinal digestion enzymes were tested for their potency to activate CCK1R.

3 g/l concentration of these hydrolysates was used and the net cellular response was

calculated. First, these hydrolysates were screened by a plate reader. Two repeated

experiments on the purified whey proteins hydrolysed with peptidase/GID enzymes showed

high discrepancy between the results for each hydrolysate except in case of alpha-lactalbumin

CHO-CCK1R VS CHO-K1: P= 5E-2

CHO-CCK1R VS CHO-CCK1R+LGM: P=

1E-2

CHO-K1 VS CHO-K1+LGM: P=0.01

CHO-CCK1R+LGM VS CHO-K1: P=0.16

- 32 -

with peptidase. The calculated average net response of the last hydrolysates was 9 ± 1% SD

(based on two experiments). Concerning purified casein proteins hydrolysed with

gastrointestinal digestion enzymes, results from kappa casein hydrolysate were more

compatible compared to results from alpha and kappa-casein. By contrast, results from

purified casein proteins hydrolysed with peptidase were much better and they all resulted in

an average net response around 10%. An experiment was carried out with the plate reader to

see if the non hydrolysed casein has any activity. Non hydrolysed casein was able to induce

an increase in the net response by 25 ± 11% SD. However, the result from the intact casein is

based on only one experiment in the plate reader and not confirmed with microscope, as well.

Therefore, this result is not enough to make a real and correct conclusion. Figure 13 compares

the net response induced by the intact casein and those induced by the purified casein protein

hydrolysates.

Comparison between the net response induced by 3g/l of

purified casein hydrolysates and the net resonse resulting from

the non hydrolysed casein

-10.00%

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

non hy

drolys

ed cas

ein

alph

a-ca

sein

+pep

tidas

e

beta

-cas

ein+

pept

idas

e

kapp

a-ca

sein

+pep

tidas

e

alph

a-ca

sein

+GID

beta

-cas

ein+

GID

kapp

a-ca

sein

+GID

% o

f m

axim

um

resp

onse

Figure 13: Comparison between the net responses induced by 3g/l of purified casein

hydrolysates and the net response resulting from the non hydrolysed casein.

Legend: The figure represents the net response induced by non hydrolysed casein, alpha,

beta-casein and kappa-casein hydrolysed by (peptidase/gastrointestinal digestion enzymes).

Figures present the mean ± SD (n = 2) for all samples apart from the non hydrolysed casein (n

= 1).

- 33 -

4.3.8. Effect of hydrolysis of casein and whey purified proteins with peptidase/

gastrointestinal digestion enzymes on the peptide length

Table 4 shows the degree of hydrolysis and the peptide chain length for the purified casein

and whey proteins after hydrolysis with peptides / GID enzymes. From this table it can be

noticed that peptidase was not able to cut both purified whey and casein proteins to produce

peptides with short sequence of amino acids. As we can see the lowest average peptide length

was in case of beta-lactoglobulin 46 amino acids sequence (DH = 2.13%). Conversely, the

degree of hydrolysis for the same proteins hydrolysed by gastrointestinal digestion enzymes

was from 6.97% to 8.54% with average peptide chain length ranging from 11 to 14 amino

acid residues except in the case of beta-lactoglobulin which gave a negative degree of

hydrolysis (might be due to low solubility).

4.3.9. Comparison of the results obtained by Tecan with those of the confocal

microscopy

To validate the results obtained by the plate reader, experiments for some purified protein

hydrolysates were done with confocal microscopy. Figure 14 represents the net responses

induced by alpha and beta-casein with peptidase, kappa-casein, alpha-lactalbumin and beta-

lactoglobulin with gastrointestinal digestion enzymes. Here also, we can recognize some

variability between results of both methods. The net responses induced by casein hyrolysates

in the plate reader as seen in Figure 13 were 12 ± 0.4% SD, 16 ± 8% SD and 8 ± 2% SD,

respectively. When the microscope was used, these values decreased to 6 ± 4% and 4 ± 3%

for alpha-and beta-casein hydrolysates, respectively and increased to 11 ± 7% for the kappa-

casein hydrolysate (Figure 14). Alpha-lactalbumin and beta-lactoglobulin with gastrointestinal

digestion enzymes did not show any net response with confocal microscopy.

Table 4: DH degree (%) and mean length of peptides derived from hydrolysis of casein and whey purified proteins

with peptidase / GID enzymes. Figures present the mean (n=2) gastrointestinal digestion enzymes peptidase (2h)

substrate % DH mean peptide length SD % DH mean peptide length SD

alpha-casein 8.54% 12 0.65% 0.99% 100 1.21%beta-casein 7.16% 14 0.20% 0.14% 721 0.33%kappa-casein 8.77% 11 0.33% 0.14% 721 0.85%alpha-lactalbumin 6.97% 14 0.67% 0.43% 232 0.12%beta-lactoglobulin -1.57% -6369 0.94% 2.13% 46 2.95%

- 34 -

The net response induced by 3g/l of purified casein and whey

hydrolysates obtained with the confocal microscope

-10.00%

0.00%

10.00%

20.00%

30.00%

alph

a- cas

ein+

pep

tidas

e

bet

a- cas

ein+

pep

tida

se

kap

pa- c

as+ GID

alph

a-lact

albu

min+

GID

beta

- lac

toglob

ulin

+ GID

% o

f m

axim

um

resp

onse

Figure 14: The net response induced by 3g/l of purified casein and whey hydrolysates

obtained with the microscope.

Legend: Representative of the net responses induced by some purified casein and whey

hydrolysates and expressed as a percentage of the maximum response induced by 1 nM CCK

(Figures report the mean of 2 repeated experiments ± SD).

Comparison between the response resulting from the CHO-CCK1R cells and that of the CHO-

K1 cells induced by 3 g/l of alpha, beta and kappa-casein hydolysates obtained by both the

plate reader and the microscope was made using T-tests. Despite the small net responses

induced by these hydrolysates, the statistical test revealed that the differences between the

responses from CHO-CCK1R and CHO-K1 cells were statistically significant with both

methods (Figure 15). However, not all experiments of these different hydrolysates represented

good shaped curves and similar in both measuring techniques. Kappa-casein hydrolyzed with

GID enzymes induced kinetics curves from the plate reader and the microscope represent

good examples of well shaped and similar curves obtained with both platforms, indicating to a

real effect to this hydrolysate on the cellular response.

- 35 -

CONFOCAL MICROSCOPY PLATE READER

alpha-casein + peptidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

fi/f0

1 nM CCK

CHO-CCK1R

CHO-K1

alpha-casein + pepidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (s)

fi/f

0

1 nM CCK

CHO-CCK1R

CHO-K1

alpha-casein + petidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

fi/f0

1 nM CCK

CHO-CCK1R

CHO-K1

alpha-casein + pepidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (s)

fi/f

0

1 nM CCK

CHO-CCK1R

CHO-K1

beta-casein + peptidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

fi/f0

1 nM CCK

CHO-CCK1R

CHO-K1

beta-casein + pepidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (s)

fi/f0

1 nM CCK

CHO-CCK1R

CHO-K1

beta-casein + petidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

fi/

f0

1 nM CCK

CHO-CCK1R

CHO-K1

beta-casein + pepidase (2h)

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (s)

fi/

f0

1 nM CCK

CHO-CCK1R

CHO-K1

p=0.02, 3.11% ± 2.54%

p=0.003, 1.59% ± 0.8%

p= 0.01, 9.21% ± 6.81%

p=9E-5, 9.59% ± 24% p= 0.002, 6.63 % ± 4 %

p=0.0008, 12.64 % ± 5. 4 %

p= 3e-5, 12 % ± 3.38 %

p= 0.025, 22.24 % ± 18 %

- 36 -

кappa-casein + GID

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

fi/f

0

1 nM CCK

CHO-CCK1R

CHO-K1

kappa-casein + GID

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (s)

fi/

f0

1nM CCK

CHO-CCK1R

CHO-K1

кappa-casein + GID

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40

Time (s)

fi/

f0

1 nM CCK

CHO-CCK1R

CHO-K1

kappa-casein + GID

0

0.5

1

1.5

2

2.5

3

0 10 20 30 40 50

Time (s)

fi/

f0

1nM CCK

CHO-CCK1R

CHO-K1

Figure 15: Fluorescence kinetics induced by 3g/l of purified casein hydrolysates measured

with two platforms.

Legend: From the figure above we can see on the left side the kinetics curves for all repeated

experiments in the microscope (two figures for each hydrolysate type resulting from two

repetitions), whereas the right side represents the results from the plate reader. The green lines

represent the response induced by 1 nM CCK. Protein hydrolysates induced responses in both

CHO-CCK1R and CHO-K1 cells are plotted by blue and red lines, respectively.

p= 0.04, 6% ± 5% p= 0.007, 9.34 % ± 5%

p= 0.001, 6 % ± 2 %

p= 0.0001, 15.74 % ± 7 %

- 37 -

CHAPTER 5: DISCUSSION

Satiety is regulated partially by intestinal peptide secretion, of which cholecysistokinin is well

known (Gibbs et al., 1973; Gibbs & Smith, 1982). Cholecystokinin effect on food intake

suppression is mediated via activation of its receptor (Schwartz & Moran, 1998). Selected

protein hydrolysates have been demonstrated to have a satiety effect via increasing CCK

secretion (Liddle, 2000). Others such as soy hydrolysates were recently demonstrated to act in

a dual mode on satiety signaling via stimulation of CCK secretion and direct activation of its

receptor, as well (Foltz et al., 2008). In this work we have directly studied the ability of some

selected protein hydrolysates to bind to CCK1R and stimulate it to elicit an intracellular

calcium response. Chinese hamster ovary cells expressing rat CCK1 receptor and the native

CHO-K1 cells were used to test our hypothesis. The increase in the intracellular Ca+2

monitored with fluorescent probe was considered as a measure of CCK1R activation.

Quantification of the intracellular Ca+2 increases was done with a multiwell fluorescence plate

reader and verified for some hydrolysates by another detection method, namely confocal

microscopy.

For validation of the model used, the activity of the natural ligand CCK-8S on its receptor was

tested with both platforms. The similarity between both methods results in terms of dose

response and the relative fluorescence curves confirmed the validity of the use of this model

to screen for ligands that could stimulate CCK1R. JMV180 as a partial agonist of CCK was

also tested for its potency to activate CCK1R. The maximum cellular response resulting from

using JMV-180 compared to 1 nM CCK-8S came in consent with already proven results

about its partial potency to activate CCK1R. This partial activation of CCK1R might be due

to different chemical structures of the two ligands and the inability of JMV-180 to interact

with some amino acids of the CCK1R binding sites that as considered as major keys in

CCK1R activation (Archer-Lahlou et al., 2005). Validation of the results from CCK-8S was

done with lorglumide, the full CCK1R antagonist. This allowed us to benefit from the

inhibitory effect of this antagonist to determine the specificity of the results from protein

hydrolysates in term of its relevancy for CCK1R activation.

Turning now to protein hydrolysates samples, different concentrations from whey and pea

hydrolysed with alcalase/promod enzymes for different times were tested. Results from these

hydrolysates showed that no selectivity was found when these different hydrolysates were

- 38 -

tested. They all resulted in an increase in the fluorescence signals but with different potency.

This might be due to having all these hydrolysates the active fractions that could induce a

cellular response or it could be linked to non specific responses seen with all hydrolysates.

These results seem in disagreement with previous results reported by Foltz et al. that showed

no direct effect of the commercial pea and whey hydrolysates on maximum cellular response

(Foltz et al., 2008). If we considered what we obtained is a real response, the reason behind

this difference could be the kind of enzymes used since in commercial hydrolysates

preparations mixtures of different types of enzymes with various specificities are often used

or the method of hydrolysis.

It is well known that the degree of hydrolysis depends on hydrolysis conditions, such as type

and specificity of the enzymes, substrate and time of reaction (Szymkiewicz et al., 2003).

During the hydrolysis process, the average peptide chain length for all whey and pea

hydrolysates decreased over time but without having a significant effect on the hydrolysates

induced response. If we eliminate the non specific response that could belong to other

receptors activation or to auto-fluorescence of the samples, this suggests that peptides with

different length might share the active fractions. It is well known that the natural hormone

with the highest affinity for CCK1R is the sulfated octapeptide (CCK-8S). Nevertheless, some

studies reported that other natural molecular forms of CCK such as CCK33, CCK39, and

CCK-58 have quite similar affinity in binding to CCK1R and stimulation of regulatory

processes compared to that of CCK-8S (Solomon et al., 1984; Reeve et al., 2002; Wu et al.,

2008). Therefore, peptides with different length might have similar effects on CCK1R

activation.

By looking at the activity of both enzymes used for hydrolysis we can see that alcalase-

generated hydrolysates showed shorter average peptide length compared to promod generated

hydrolysates. This could be due to the probability of having the former enzyme different

proteinases with different specificities (Sukan & Andrews, 1982). Moreover, both enzymes

reported higher hydrolysis efficiency on whey protein in comparison to pea protein. This

could be linked to having whey protein more specific cleavage sites for hydrolysis with these

enzymes or it might be related to other factors affect hydrolysis of pea protein. Szymkiewicz

et al. found that vasilin fraction of 30 kD in pea hydrolysate with alcalase disappeared after 90

min of hydrolysis, whereas vicilin fraction with the molecular weight of 20 kD was the most

resistant to the hydrolytic activity of Alcalase. Contrary to pea hydrolysate, the presence of

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peptides with molecular weight lower than 14.2 kD were seen in whey hydrolysed with

alcalase (Szymkiewicz et al., 2003). We already said that time of hydrolysis did not affect the

induced response. Therefore, if we assume that the increase in the fluorescence signal related

to a real response, then the difference between the whey and pea hydrolysates induced

response could be linked to enzyme specificities and the protein-enzyme combination.

Actually, repetition of the experiments for all whey and pea hydrolysates with the plate reader

showed high variability between the results of each hydrolysate type. This variability between

the results from replicates may be in some parts due to non complete solubility of these

hydrolysates, or to passage-number-related effects on cells growth rates and signaling

(Briske-Anderson et al., 1997; Esquenet et al., 1997; ). However, when the confocal

microscopy was used to validate the results obtained by the plate reader, high discrepancy

between both platforms results was observed. In fact, the way of measuring the fluorescent

signal in both methods might be considered somehow technically different. With the confocal

microscopy, a selected section is used to measure the fluorescence signal, whereas the whole

well is considered with the plate reader. This might lead to some variability between both

methods results. It is already mentioned above that compatibility between results of both

measuring methods was observed when experiments were carried out on the natural ligand

CCK-8S. In this study, we tested a much more complex system of food protein hydrolysates

from which a strong auto-fluorescence coming from molecules might result in an

overestimation of the results or even to generate completely false positive results with the

plate reader.

In general, the response of CHO-CCK-1R cells to most tested hydrolysates with the plate

reader was significantly higher compared to response from the native CHO-K1 cells.

Nevertheless, most of these hydrolysates did not induce good shaped fluorescence kinetics

curves that may suggest real and true positive responses. In most cases fluorescence kinetics

curves from CHO-CCK1R were parallel and very similar in the shape to those from CHO-K1

cells with only higher initial rise in the fluorescence signals for CHO-CCK1R curves.

Therefore, the discrepancy between the results from a plate reader and results from the

microscope together with the non good shaped curves resulted from the former technique

propose a high possibility of false positive results obtained with the plate reader. Actually,

one of the tested hydrolysates, namely promod generated hydrolysate from whey showed a

very small net response with the microscope but with good shaped kinetics curves. The

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response of CHO-K1 induced by this hydrolysate was flat and similar to the response of this

cell type to 1 nM CCK. In addition, the inhibitory effect of lorglumide to the response of

CHO-CCK1R cells was significant and coincided with a shift of CHO-CCK1R cells curve to

a flat curve. This may refer to a specific response induced by this hydrolysate that was

significantly inhibited by lorglumide (Makovec et al., 1985; Gonzalez-Puga et al., 2005).

However, the results from the microscope are based on only one experiment, whereas 5

repeated experiments were done with the plate reader. This suggests more replicates with the

microscope have to be conducted to obtain a real and valid conclusion.

We already mentioned that the discrepancy between the results of both methods used in

measuring the whey and pea protein hydrolysates induced response could be due to the nature

of these proteins since within each protein is a complex mixture of proteins. Therefore,

subfractions of whey protein such as alpha-lactalbumin and beta-lactoglobulin hydrolysed

with peptidase/gastrointestinal digestions enzymes were tested and compared with other

subfractions from casein (alpha, beta, and kappa-casein hydrolysed with same enzymes). The

inconsistency between both measuring techniques results observed for purified whey

hydrolysates might refer to non-specific background signals were obtained with the plate

reader. Indeed, results from the microscope could be more specific because of its efficiency to

reject out of focus fluorescence signals since the image comes from a section of the well and

not the whole well (Amos & White, 2003; Astner & Ulrich, 2010). Based on this, it might not

be correct to consider the responses seen for the purified whey hydrolysates only with the

plate reader in some experiments as actual positive cellular responses resulting from CCK1R

activation.

Contrary to the purified whey hydrolysates, alpha and beta-casein hydrolysates demonstrated

the presence of some effects on the cellular response after confirmation in one from two

experiments with the microscope. Since we do not have enough evidence to accept or reject

these results, more experiments should be conducted with the microscope before coming to a

reliable conclusion on the effect of these hydrolysates. The interesting finding was the

compatible results obtained with the plate reader and the microscope on kappa-casein

hydrolysate with good shaped curves. This might suggest a true positive response induced by

this hydrolysate. However, to make a valid conclusion and to confirm the specificity of this

ligand to CCK1R, experiments with the full antagonist lorglumide have to be executed.

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CHAPTER 6: CONCLUSION

This study was an attempt to search for bioactive fractions from milk and pea protein

hydrolysates with potency to activate CCK1R, the receptor that has been recognized to

mediate transition of a satiety signal to the brain (Moran et al., 1997). Despite the rather high

net responses obtained with the plate reader for most of these hydrolysates, results from the

microscope showed a highly overestimation of the responses induced by some of these

hydrolysates obtained with the plate reader. Furthermore, and unfortunately in most cases the

increase in the fluorescence signals measured with the plate reader was absent when the

confocal microscope was used. This strongly refers to false positive results were obtained

with the former measuring method. However, non compatible number of repeated

experiments was carried out with both methods. Therefore, carrying out more experiments

with the confocal microscopy is necessary, particularly for hydrolysates that showed some

responses coincided with good shaped curves from both measuring techniques. This could

lead to a better and more accurate conclusion on the probability of using these hydrolysates as

helpful agents for CCK1R activation.

Last but not least, it might be possible to conclude that the plate reader is not really well

suited to study complex molecules with potential to activate CCK1 receptor compared to

simpler molecules since we have obtained high discrepancy between the results from this

technique and the microscope.

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ANNEXES Annex 1: Supplementary Tables

Supplementary Table 1: ANOVA: Fixed effects, main effects and ineractions

Type111

Sum of sq

Protein 1 0.26666 0.26666 5.4155 0.0244

Enzyme 1 0.23134 0.23134 4.6981 0.0354

Time 2 0.13483 0.06741 1.3691 0.2644

Protein:enzyme 1 0.00024 0.00024 0.0048 0.9446

Protein:time 2 0.01079 0.00539 0.1095 0.8964

Enzyme:time 2 0.00439 0.00219 0.0446 0.9563

Protein:enyme:t

ime

2 0.30289 0.15144 0.0755 0.0557

P Value Source of

variation

Df Mean Sq F Value

whey 28 0.469

pea 30 0.327

0.02 56 [0.021 , 0.263]

Supplementary Table 2: Two-Sample T-Test and Confidence Interval for Whey Sample and

Pea Sample

n mean p-value df 95% CI for difference

alcalase 290.461

promod 29 0.331

Supplementary Table 3: Two-Sample T-Test and Confidence Interval for Alcalase Sample and

Promod Sample

95% CI for difference

0.03 56 [ 0.007, 0.25 ]

mean p-value df n

Treatments Estimate(diff) Std.Error Lower Bound Upper Bound

pea+alcalase- pea+promod 0.123 0.082 -0.09 0.3

pea+alcalase-whey+alcalase -0.15 0.083 -0.4 0.07

pea+alcalase-whey+promod -0.012 0.083 -0.2 0.2

pea+promod-whey+alcalase -0.273 0.08 -0.4 -0.05 *pea+promod-whey+promod -0.135 0.083 -0.3 0.09

whey+alcalase-whey+promod 0.138 0.084 -0.08 0.4

Supplementary Table 4: Tukey multiple comparisons of means 95% family-wise confidence level.

Intervals excluding 0 are flagged by '*' (where there is a significant difference between the

treatments).

- 2 -

Annex 2: Supplementary Figures

(a). Whey protein hydrolysed with promod for 1h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(b). Whey protein hydrolysed with promod for 3h

-30.00%

-10.00%

10.00%

30.00%

50.00%

70.00%

90.00%

110.00%

130.00%

150.00%

170.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(c). Whey protein hydrolysed with promod for 6h

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(d). Whey protein hydrolysed with alcalase for 1h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se exp1

exp2

exp3

exp4

exp5

(e). Whey protein hydrolysed with alcalase for 3h

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(f). Whey protein hydrolysed with alcalase for 6h

-40.00%

0.00%

40.00%

80.00%

120.00%

160.00%

200.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

Supplementary Figure 1: The net response induced by increasing concentrations (0.005 –

3mg/ml) of whey protein hydrolysed with promod/alcalase enzymes for 1, 3 and 6h in five

repeated experiments.

- 3 -

(g). Pea protein hydrolysed with promod for 1h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(h). Pea protein hydrolysed with promod for 3h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(i). Pea protein hydrolysed with promod for 6h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(j). Pea protein hydrolysed with alcalase for 1h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(k). Pea protein hydrolysed with alcalase for 3h

-60.00%

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

(l). Pea protein hydrolysed with alcalase for 6h

-40.00%

-20.00%

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

120.00%

3- 2- 1- 0 1

log (conc) (mg / ml)

% o

f m

ax r

esp

on

se

exp1

exp2

exp3

exp4

exp5

Supplementary Figure 2: The net response induced by increasing concentrations (0.005 – 3

mg/ml) of pea protein hydrolysed with promod/alcalase enzymes for 1, 3 and 6h in five

repeated experiments.

- 4 -

Legend: In both Supplementary Figures 1 and 2 we can see the five different colors represent

results of 5 replicates for each concentration. The maximum response plotted versus the

logarithm of hydrolysate concentration. (a, b, and c) for results from whey hydrolysed with

promod for 1, 3 and 6h, whereas (d, e, and f) for results from whey hydrolysed with alcalase

for 1, 3 and 6h, while (g, h and I) for results from pea hydrolysed with promod for 1, 3 and 6h.

(j, k and l) for pea hydrolysed with alcalase for 1, 3 and 6h.

(1) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 10 20 30 40

Time (s )

fi/f

0

1nM CCK

CHO-CCK1R

CHO-K1

(2) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 10 20 30 40

Time (s)

fi/f

01nM CCK

CHO-CCK1R

CHO-K1

(3) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 10 20 30 40

Time (s)

fi/f

0

1nM CCK

CHO-CCK1R

CHO-K1

(4) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 10 20 30 40

Time (s)

fi/f

0

1nM CCK

CHO-CCK1R

CHO-K1

(5) 3g/l whey + alcalase 3h

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40

Time (s)

fi/f

0

1nM CCK

CHO-CCK1R

CHO-K1

Supplementary Figure 3: The fluorescence kinetics responses of the CHO-CCK1R to 1 nM CCK and those of CHO-CCK1R and CHO-K1 cells to whey hydrolysed with alcalase for 3h of the 3g/l concentration. (1, 2, 3, 4 and 5) are representative of the results of five repeated experiments monitored with a plate reader.

- 5 -