Production and Fractionation of Antioxidant Peptides from Soy Protein Isolate using

114 Pages·2012·5.41 MB·English

by  Sahan Ranamukhaarachchi

Production
 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