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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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
i
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
iii
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
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
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)
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
- 39 -
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
- 40 -
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.
- 41 -
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.
- 42 -
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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
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.