Table Of ContentProduction
and
Fractionation
of
Antioxidant
Peptides
from
Soy
Protein
Isolate
using
Sequential
Membrane
Ultrafiltration
and
Nanofiltration
By
Sahan
Ranamukhaarachchi
A
thesis
presented
to
the
University
of
Waterloo
In
fulfillment
of
the
thesis
requirement
for
the
degree
of
Master
of
Applied
Science
In
Chemical
Engineering
Waterloo,
Ontario,
Canada,
2012
©
Sahan
Ranamukhaarachchi
2012
I
hereby
declare
that
I
am
the
sole
author
of
this
thesis.
This
is
a
true
copy
of
the
thesis,
including
any
required
final
revisions,
as
accepted
by
my
examiners.
I
understand
that
my
thesis
may
be
made
electronically
available
to
the
public.
i
i
Abstract
Antioxidants
are
molecules
capable
of
stabilizing
and
preventing
oxidation.
Certain
peptides,
protein
hydrolysates,
have
shown
antioxidant
capacities,
which
are
obtained
once
liberated
from
the
native
protein
structure.
Soy
protein
isolates
(SPI)
were
enzymatically
hydrolyzed
by
pepsin
and
pancreatin
mixtures.
The
soy
protein
hydrolysates
(SPH)
were
fractionated
with
sequential
ultrafiltration
(UF)
and
nanofiltration
(NF)
membrane
steps.
Heat
pre-‐treatment
of
SPI
at
95
oC
for
5
min
prior
to
enzymatic
hydrolysis
was
investigated
for
its
effect
on
peptide
distribution
and
antioxidant
capacity.
SPH
were
subjected
to
UF
with
a
10
kDa
molecular
weight
cut
off
(MWCO)
polysulfone
membrane.
UF
permeate
fractions
(lower
molecular
weight
than
10
kDa)
were
fractionated
by
NF
with
a
thin
film
composite
membrane
(2.5
kDa
MWCO)
at
pH
4
and
8.
Similar
peptide
content
and
antioxidant
capacity
(α=0.05)
were
obtained
in
control
and
pre-‐heated
SPH
when
comparing
the
respective
UF
and
NF
permeate
and
retentate
fractions
produced.
FCR
antioxidant
capacities
of
the
SPH
fractions
were
significantly
lower
than
their
ORAC
antioxidant
capacities,
and
the
distribution
among
the
UF
and
NF
fractions
was
generally
different.
Most
UF
and
NF
fractions
displayed
higher
antioxidant
capacities
when
compared
to
the
crude
SPI
hydrolysates,
showing
the
importance
of
molecular
weight
on
antioxidant
capacity
of
peptides.
The
permeate
fractions
produced
by
NF
at
pH
8
displayed
the
highest
antioxidant
capacity,
expressed
in
terms
of
trolox
equivalents
(TE)
per
total
solids
(TS):
5562
μmol
TE
g-‐1
TS
for
control
SPH,
and
5187
μmol
TE
g-‐1
TS
for
pre-‐heated
SPH.
Due
to
the
improvement
in
antioxidant
capacity
of
peptides
by
NF
at
pH
8,
the
potential
for
NF
as
a
viable
industrial
fractionation
process
was
demonstrated.
Principal
component
analysis
(PCA)
of
fluorescence
excitation-‐emission
matrix
(EEM)
data
for
UF
and
NF
peptide
fractions,
followed
by
multi-‐linear
regression
analysis,
was
assessed
for
its
potential
to
monitor
and
identify
the
contributions
to
ORAC
and
FCR,
two
in
vitro
antioxidant
capacity
assays,
of
SPH
during
membrane
fractionation.
Two
statistically
significant
principal
components
(PCs)
were
obtained
for
UF
and
NF
peptide
fractions.
Multi-‐linear
regression
models
(MLRM)
were
developed
to
estimate
their
fluorescence
and
PCA-‐captured
ORAC
(ORAC )
and
FCR
FPCA
(FCR )
antioxidant
capacities.
The
ORAC
and
FCR
antioxidant
capacities
for
FPCA FPCA FPCA
NF
samples
displayed
strong,
linear
relationships
at
different
pH
conditions
(R2>0.99).
Such
relationships
are
believed
to
reflect
the
individual
and
relative
combined
ii
i
contributions
of
tryptophan
and
tyrosine
residues
present
in
the
SPH
fractions
to
ORAC
and
FCR
antioxidant
capacities.
Therefore,
the
proposed
method
provides
a
tool
for
the
assessment
of
fundamental
parameters
of
antioxidant
capacities
captured
by
ORAC
and
FCR
assays.
iv
Acknowledgements
First
of
all,
I
would
like
to
express
my
sincere
gratitude
my
advisor,
Christine
Moresoli
for
accepting
me
as
a
graduate
student
and
providing
the
opportunity
to
conduct
exciting
research.
The
attention,
training,
support,
and
advice
provided
by
Christine
have
tremendously
influenced
my
ability
to
conduct
independent
research,
and
have
been
indispensible
to
the
completion
of
this
thesis.
I
would
like
to
acknowledge
the
National
Science
and
Engineering
Research
Council
of
Canada
for
providing
the
financial
support
to
this
research
project.
I
am
also
thankful
to
the
Department
of
Chemical
Engineering,
and
the
University
of
Waterloo
for
providing
financial
assistances
in
many
ways
during
the
course
of
this
Masters
program.
Ramila
Peiris
has
been
a
mentor
and
has
assisted
this
research
in
many
different
avenues.
I
am
grateful
for
his
guidance
and
presence
for
the
duration
of
my
Masters
program.
Special
recognitions
of
appreciation
are
expressed
to
Raymond
Legge
and
the
wonderful
members
of
the
Legge-‐Moresoli
research
group.
Andrew
Yeh,
Jamie
Cousineau,
Katharina
Hassel,
Barbara
Guettler,
Nicholas
Ignagni,
Sarah
Meunier,
Rachel
Campbell,
and
Nikhil
Kumar
have
greatly
influenced
my
life
at
the
University
of
Waterloo,
and
I
am
thankful
for
everything.
I
am
thankful
to
the
reviewers
of
my
thesis,
Xianshe
Feng
and
Marc
Aucoin
for
their
time
and
valuable
feedback;
and
to
Robert
Lencki
(University
of
Guelph)
for
his
assistance
and
influence
in
my
decision
to
attend
the
University
of
Waterloo.
I
would
also
like
to
express
my
immense
gratitude
to
Chitral
Angammana,
Suramya
Mihindukulasuriya,
Nandana
Jayabahu,
Ishari
Jayabahu,
and
Subodha
Gunawardena
for
all
the
care
and
comfort
provided
during
the
past
six
years.
The
constant
presence,
words
of
encouragement
and
incomparable
attention
from
my
family
have
propelled
me
to
excel
at
my
studies
and
to
become
the
person
I
am
today.
I
am
forever
in
debt
to
Senaratne
and
Sandya
Ranamukhaarachchi
(my
beautiful
parents),
and
Himesha
and
Sithumini
Ranamukhaarachchi
(my
two
wonderful
sisters),
without
whom
I
definitely
would
not
be
here
today.
I
am
also
extremely
thankful
to
Lal,
Samanthi,
and
Ayumi
Samarakoon.
Finally,
I
express
my
deepest
appreciation
to
Mayumi
Samarakoon
for
sharing
every
second
of
my
life
in
Waterloo.
v
Table
of
Contents
List
of
Figures
x
List
of
Tables
xii
List
of
Abbreviations
xiii
1.
Introduction
______________________________________________________________________________
1
1.1.
Research
Motivation
________________________________________________________________________
1
1.2.
Project
Objectives
___________________________________________________________________________
2
1.2.1.
Goals
.............................................................................................................................................................
2
1.2.2.
Hypotheses
................................................................................................................................................
2
1.2.3.
Objectives
...................................................................................................................................................
3
1.3.
Thesis
Organization
_________________________________________________________________________
3
2.
Theoretical
Knowledge
and
Principles
______________________________________________
5
2.1.
Proteins
_______________________________________________________________________________________
5
2.1.1.
Soybeans
and
soy
proteins
.................................................................................................................
5
2.1.1.1.
Soy
proteins
in
foods
__________________________________________________________________________
6
2.1.1.2.
Characteristics
of
soy
proteins
_______________________________________________________________
7
2.2.
Peptides
_______________________________________________________________________________________
7
2.2.1.
Production
of
peptides
.........................................................................................................................
8
2.2.1.1.
Enzymatic
hydrolysis
_________________________________________________________________________
9
2.2.1.2.
Microbial
fermentation
______________________________________________________________________
11
2.2.2.
Peptide
and
amino
acid
analysis
techniques
............................................................................
11
2.2.2.1.
Reverse-‐phase
high
performance
liquid
chromatography
________________________________
12
2.2.2.2.
Nuclear
magnetic
resonance
spectroscopy
________________________________________________
13
2.3.
Antioxidants
________________________________________________________________________________
14
2.3.1.
In
vitro
antioxidant
assays
................................................................................................................
15
2.3.2.
Differences
between
in
vitro
versus
in
vivo
antioxidant
assays
.......................................
17
2.3.3.
Antioxidant
soy
peptides
...................................................................................................................
17
2.4.
Membrane
Filtration
______________________________________________________________________
19
2.4.1.
Membrane
fouling
................................................................................................................................
20
2.4.2.
Ultrafiltration
.........................................................................................................................................
22
2.4.3.
Nanofiltration
.........................................................................................................................................
22
2.4.4.
Selection
of
operating
parameters
................................................................................................
25
2.5.
Fluorescence
Spectroscopy
______________________________________________________________
25
2.5.1.
Principal
Component
Analysis
........................................................................................................
26
v
i
3.
Production
and
Fractionation
of
Antioxidant
Peptide
Fractions
from
Soy
Protein
Isolate
using
Ultrafiltration
and
Nanofiltration
_________________________
27
3.2.
Abstract
_____________________________________________________________________________________
28
3.3.
Introduction
________________________________________________________________________________
29
3.4.
Materials
and
Methods
____________________________________________________________________
31
3.4.1.
Preparation
of
soy
protein
hydrolysates
....................................................................................
31
3.4.1.1.
SPI
solution
___________________________________________________________________________________
31
3.4.1.2.
Enzymatic
hydrolysis
of
SPI
solutions
______________________________________________________
31
3.4.1.3.
Ultracentrifugation
___________________________________________________________________________
31
3.4.2.
Filtration
experiments
........................................................................................................................
32
3.4.2.1.
Ultrafiltration
experiments
__________________________________________________________________
32
3.4.2.2.
Nanofiltration
experiments
_________________________________________________________________
33
3.4.3.
Analytical
methods
..............................................................................................................................
34
3.4.3.1.
Total
solids
determination
__________________________________________________________________
34
3.4.3.2.
O’phthaldialdehyde
(OPA)
assay
____________________________________________________________
34
3.4.3.3.
Oxygen
Radical
Absorbance
Capacity
(ORAC)
assay
______________________________________
34
3.4.3.4.
Folin
Ciocalteau
Reagent
(FCR)
assay
______________________________________________________
35
3.4.4.
Statistical
analysis
................................................................................................................................
36
3.5.
Results
and
Discussion
____________________________________________________________________
36
3.5.1.
Effect
of
temperature
on
peptide
yield
during
enzymatic
hydrolysis
...........................
36
3.5.2.
Ultrafiltration
of
hydrolysates
........................................................................................................
37
3.5.2.1.
Effect
of
SPI
heat
pre-‐treatment
on
total
solids
distribution
_____________________________
37
3.5.2.2.
Effect
of
SPI
heat
pre-‐treatment
on
total
peptide
distribution
___________________________
38
3.5.2.3.
Effect
of
ultrafiltration
on
antioxidant
capacity
___________________________________________
39
3.5.3.
Nanofiltration
of
hydrolysates
........................................................................................................
41
3.5.3.1.
Effect
of
SPI
heat
pre-‐treatment
and
pH
on
total
solids
distribution
____________________
41
3.5.3.2.
Effects
of
SPI
heat
pre-‐treatment
and
pH
on
total
peptide
distribution
_________________
42
3.5.3.3.
Effect
of
nanofiltration
on
antioxidant
capacity
___________________________________________
44
3.5.4.
Potential
for
SPI
hydrolysates
as
a
source
of
antioxidants
.................................................
45
3.6.
Conclusion
__________________________________________________________________________________
47
4.
Assessment
of
the
Contribution
of
Biological
Species
to
Antioxidant
Capacity
of
Ultrafiltration
and
Nanofiltration-‐derived
Soy
Protein
Hydrolysate
using
Fluorescence
Spectroscopy
and
Principal
Component
Analysis.
_______________
49
4.2.
Abstract
_____________________________________________________________________________________
50
4.3.
Introduction
________________________________________________________________________________
51
4.4.
Materials
and
Methods
____________________________________________________________________
53
4.4.1.
Preparation
of
soy
protein
hydrolysates
....................................................................................
53
vi
i
4.4.1.1.
Enzymatic
hydrolysis
of
SPI
solutions
______________________________________________________
53
4.4.2.
Filtration
experiments
........................................................................................................................
53
4.4.2.1.
Ultrafiltration
experiments
__________________________________________________________________
53
4.4.2.2.
Nanofiltration
experiments
_________________________________________________________________
54
4.4.3.
Analytical
methods
..............................................................................................................................
54
4.4.3.1.
Total
solids
(TS)
determination
_____________________________________________________________
54
4.4.3.2.
O’phthaldialdehyde
(OPA)
assay
____________________________________________________________
55
4.4.3.3.
Oxygen
Radical
Absorbance
Capacity
(ORAC)
assay
______________________________________
55
4.4.3.4.
Folin
Ciocalteau
Reagent
(FCR)
assay
______________________________________________________
55
4.4.4.
Fluorescence
analysis
.........................................................................................................................
56
4.4.4.1.
Principal
Component
Analysis
______________________________________________________________
56
4.4.5.
Multi-‐linear
regression
analysis
.....................................................................................................
57
4.4.6.
Statistical
analysis
................................................................................................................................
58
4.5.
Results
and
Discussion
____________________________________________________________________
58
4.5.1.
Effects
of
UF
and
NF
on
peptide
distribution
and
antioxidant
capacity
.......................
58
4.5.2.
Fluorescence
EEMs
for
UF
and
NF
peptide
fractions
............................................................
59
4.5.3.
Fluorescence
loading
plots
for
NF
peptide
fractions
............................................................
60
4.5.4.
Fluorescence
and
PCA-‐captured
relative
ORAC
and
FCR
antioxidant
capacities
during
fractionation
of
SPH
by
NF
................................................................................................
61
4.5.5.
Correlation
between
FCR
and
ORAC
measurements
............................................................
63
4.5.6.
Verification
of
results
by
PCA
of
UF
samples
............................................................................
64
4.5.7.
Potential
for
analysis
of
bioactive
compounds
and
future
applications
.......................
65
4.6.
Conclusion
__________________________________________________________________________________
66
5.
Amino
Acid
Analysis
of
Antioxidant
Soy
Protein
Hydrolysate
Fractions
Separated
by
UF
and
NF
_______________________________________________________________
68
5.1.
Introduction
________________________________________________________________________________
68
5.2.
Materials
and
Methods
____________________________________________________________________
69
5.2.1.
Enzymatic
hydrolysis
of
peptide
fractions
................................................................................
69
5.2.2.
Analytical
methods
..............................................................................................................................
69
5.2.2.1.
Total
solids
determination
__________________________________________________________________
69
5.2.2.2.
Reverse-‐phase
HPLC
_________________________________________________________________________
69
5.2.2.3.
1H
NMR
Spectroscopy
________________________________________________________________________
70
5.3.
Results
and
Discussion
____________________________________________________________________
71
5.3.1.
Amino
acid
analysis
by
reverse-‐phase
HPLC
............................................................................
71
5.3.2.
Amino
acid
analysis
by
1H-‐NMR
spectroscopy
........................................................................
74
vi
ii
5.3.3.
Potential
of
reverse-‐phase
HPLC
and
NMR
for
amino
acid
analysis
of
soy
hydrolysate
fractions
.........................................................................................................................
75
5.3.4.
Future
work
............................................................................................................................................
77
5.4.
Conclusion
__________________________________________________________________________________
77
6.
Conclusions
______________________________________________________________________________
78
7.
References
_______________________________________________________________________________
81
8.
Appendix
_________________________________________________________________________________
86
8.1.
Peptide
Concentrations
of
UF
and
NF
Samples
________________________________________
86
8.2.
Total
Material
Balances
for
UF
and
NF
Experiments
_________________________________
86
8.3.
Permeate
Flux
Analysis
___________________________________________________________________
90
8.3.1.
Sample
Calculations:
...........................................................................................................................
94
8.3.1.1.
Normalized
flux
estimation:
_________________________________________________________________
94
8.3.1.2.
Total
resistance
(R )
estimation:
__________________________________________________________
94
tot
8.4.
Fluorescence
analysis
and
PCA
__________________________________________________________
95
8.4.1.
Flow
chart
of
process
..........................................................................................................................
95
8.4.2.
Enhancement
of
antioxidant
capacity
during
peptide
fractionation
.............................
95
8.4.3.
PCA
of
NF
and
UF
data
sets
...............................................................................................................
96
8.4.4.
Linear
regression
models
..................................................................................................................
97
8.4.5.
Residual
Plots
.........................................................................................................................................
99
ix
List
of
Figures
Figure
1:
Mechanism
of
OPA
assay
to
detect
free
amino
acids
and
peptides
_____________
9
Figure
2:
Possible
fluorescein
oxidation
pathway
induced
by
AAPH
_____________________
16
Figure
3:
Mechanism
of
FCR
assay
to
measure
antioxidant
capacity
_____________________
16
Figure
4:
Model
for
mass
transfer
through
a
NF
membrane
______________________________
21
Figure
5:
Components
and
configuration
of
a
lab
scale
UF
system
_______________________
22
Figure
6:
Components
and
configuration
of
a
lab
scale
NF
system
_______________________
23
Figure
7:
Progress
of
enzymatic
hydrolysis
of
control
and
pre-‐heated
soy
protein
hydrolysates
assessed
by
OPA
____________________________________________________
36
Figure
8:
A
comparison
of
peptide
content
estimated
by
OPA
of
UF
fractions
from
control
and
pre-‐heated
soy
protein
hydrolysate
________________________________
38
Figure
9:
A
comparison
of
the
antioxidant
capacity
estimated
by
ORAC
of
UF
fractions
from
control
and
pre-‐heated
soy
protein
hydrolysate
__________________________
39
Figure
10:
A
comparison
of
the
antioxidant
capacity
estimated
by
FCR
of
UF
fractions
from
control
and
pre-‐heated
soy
protein
hydrolysate
__________________________
40
Figure
11:
A
comparison
of
peptide
content
estimated
by
OPA
of
NF
fractions
from
control
and
pre-‐heated
soy
protein
hydrolysate
at
pH
4
and
8
________________
42
Figure
12:
A
comparison
of
the
antioxidant
capacity
estimated
by
ORAC
of
NF
fractions
from
control
and
pre-‐heated
soy
protein
hydrolysate
at
pH
4
and
8
__________
44
Figure
13:
A
comparison
of
the
antioxidant
capacity
estimated
by
FCR
of
NF
fractions
from
control
and
pre-‐heated
soy
protein
hydrolysate
at
pH
4
and
8
__________
45
Figure
14:
Plot
of
observed
ORAC
versus
observed
FCR
antioxidant
capacities
for
96
NF
samples
for
heat
pre-‐treated
soy
protein
hydrolysate
at
pH
4
and
8
__________
59
Figure
15:
Plots
of
observed
ORAC
versus
observed
FCR
antioxidant
capacities
for
UF
and
NF
samples
____________________________________________________________________
59
Figure
16:
Fluorescence
features
observed
in
typical
fluorescence
EEMs
for
UF
permeate
and
NF
permeate
at
pH
8
for
pre-‐heated
soy
protein
hydrolysate
_
60
Figure
17:
3D
illustrations
of
loading
matrices
obtained
by
PCA
of
NF
spectral
data
for
PC
and
PC
_________________________________________________________________________
61
1 2
Figure
18:
Plot
of
ORAC
versus
FCR
antioxidant
capacity
for
96
NF
samples
for
FPCA FPCA
heat
pre-‐treated
soy
protein
hydrolysate
at
pH
4
and
8
________________________
63
x
Description:of Ultrafiltration and Nanofiltration-‐derived Soy Protein Hydrolysate using Effects of UF and NF on peptide distribution and antioxidant capacity .
