CHARACTERIZATION AND PROPERTIES
OF FLAXSEED PROTEIN FRACTIONS
BY
Anwer Ali Ayad
Department of Food Science and Agricultural Chemistry
McGill University, Montreal
A thesis submitted to McGill University in partial fulfillment of
the requirements of the degree of Doctor of Philosophy
May, 2010
©Anwer Ali Ayad, 2010
Suggested Short Title
PROPERTIES OF FLAXSEED PROTEINS
ii
This thesis is dedicated to my wife Zaineb, my son Mohamed
and my parents Ali and Sheika
iii
ABSTRACT
The albumin, globulin and glutelin fractions were fractionated from defatted flaxseed
meal by sequential solvent extraction. Albumin and glutelin fractions were the
predominant protein fractions, accounting for 38.1 and 33.9% respectively while the
globulin accounted for 27.9%. The protein content of albumin, glutelin and globulin
fractions were 64.9, 22.4 and 18.1% of the total protein. Native-PAGE patterns of
albumin, globulin and glutelin, exhibited that each fraction has two bands. SDS-PAGE
profile of the protein fractions showed two intense bands corresponding to 22 and 24
kDa and two bands corresponding to 9 and 33 kDa were observed from albumin.
Globulin showed one intense band at 23 kDa and three minor bands, with a molecular
weight (MW) of 10, 24 and 33 kDa. Glutelin showed the presence of two intense bands
with a MW 22 and 35 kDa and three minor bands with MW of 35, 45 and 55 kDa. ESIMS results were as follows; two protein subunits with MW of 9 and 27 kDa were
observed with albumin, globulin indicated the presence of two subunits with MW of 17
and 28 kDa and glutelin showed the presence of one subunit with MW of 14 kDa. FTIR
analysis of protein fractions suggested that -helix is a common peak in all protein
fractions. DSC showed that the albumin had the highest peak temperature (Td) of
122.3C, globulin with Td of 118.3C and glutelin with a peak temperature (Td) value of
106.8C. RP-HPLC analysis showed more than one major component for the globulin,
glutelin and albumin fractions. The albumin fraction showed two peaks; a dominant
peak at a retention time of 26.2 min and one minor peak at 25.1 min. The globulin
fraction demonstrated one dominant peak at retention time of 24.9 min and one minor
iv
peak at 30.4 min. Glutelin exhibited a major peak at 25.0 min and two minor peaks at
18.0 and 26.2 min.
In-vitro tryptic digestion of albumin, globulin and glutelin protein fractions indicated
that the degree of hydrolysis (DH) was for glutelin with 24.5% DH, albumin with 16.0%
DH, and globulin with 9.4% DH: by comparison the DH of a commercial soybean
protein isolate was 14.1%. Tandem mass spectrometric (LC-ESI-MS/MS) analysis of
tryptic hydrolysates resulted in the identification of some peptides from the albumin,
globulin and glutelin fractions which were found to be homologous with conlinin and
chitinase IV. Investigation of selected biological properties of tryptic digests of flaxseed
proteins showed that the highest antioxidant (90%) and effective fungal inhibition
activity was obtained with glutelin hydrolysates, while the highest ACE inhibition
(60%) activity was observed with glutelin protein.
v
RESUMÉ
Une extraction séquentielle par solvant de graines de lin a permis d’obtenir des
fractions protéiques d’albumine, globuline and glutéline. Les principales fraction
protéiques étaient celles d’albumine et de glutéline, représentant 38.1 et 33.9%
respectivement, tandis que la globuline ne représenta que 27.9%. La teneur en protéine de
la fraction d’albumine fut de 64.9%, tandis que celles des fractions de glutéline and
globuline furent, respectivement, de 22.4 et 18.1% de la protéine totale. Une PAGE
d'échantillons à l'état natif indiqua que chacune des fractions protéiques (albumine,
globuline et glutéline) consistait de deux bandes. La PAGE des fractions d’albumine en
présence de SDS montra deux bandes foncées (p.m. 22 et 24 kDa), ainsi que deux bandes
plus faibles (p.m. 9 and 33 kDa); la fraction des globulines montra une bande foncée (p.m.
23 kDa) and trois bandes plus faibles (p.m. 10, 24 et 33 kDa); tandis que la fraction des
glutélines montra deux bandes foncées (p.m. 22 and 35 kDa) et trois bandes plus faibles
(p.m. 35, 45 and 55 kDa.). L’analyse ESI-MS indiqua que l’albumine comportait deux sousunités protéiques (p.m. 9 et 27 kDa), la globuline deux sous-unités (p.m. 17 et 28 kDa); et la
glutéline une sous-unité (p.m. 14 kDa). Une analyse des différentes fractions protéiques
par spectromètre infrarouge à transformée de Fourier (IRTF) indiqua qu’au moins une
l’hélice  était présente dans chacune des fractions. Des thermogrammes de DSC
indiquèrent que l’albumine avait la température de pointe (Td) la plus élevée à 122.3C,
tandis que celles de la globuline et de la glutéline étaient de 118.3C et 106.8C,
respectivement. L’analyse par RP-HPLC des fractions de globuline, gluténine et albumine
indiquèrent qu’il existait plus d’un composant dans chaque fraction. La fraction d’albumine
donna deux pics, un important pic à un temps de rétention de 26.2 min et un moindre à
25.1 min; la fraction de globuline donna un important pic à 24.9 min et un moindre à 30.4
vi
min; la fraction de glutéline donna un important pic à 25.0 min et deux moindres à 18.0
and 26.2 min. Une digestion in-vitro par trypsine des fractions protéiques de glutéline,
albumine et globuline afficha des degrés d’hydrolyse de 24.5, 16.0 et 9.4%, respectivement,
comparativement à 14.1% pour un concentré commercial de protéines de fève soja. Une
analyse des hydrolysats par trypsine par chromatographie en phase liquide suivi de LCESI-MS/MS a permis l’identification de peptides provenant des fractions d’albumine,
globuline et glutéline, ainsi que des fragments de protéine dont l’origine serait la conlinine
et chitinase IV. Lors d’une étude des propriétés biologiques des hydrolysats tryptiques,
l’activité antioxidante la plus élevée (90%) appariée à une inhibition adéquate de l’activité
fongique fut obtenue avec les hydrolysats de glutéline, tandis que l’inhibition la plus élevé
de l’activité de l’ACE (60%) fut obtenu avec la glutéline intacte.
vii
ACKNOWLEDGMENTS
“In the name of Allah, the Beneficent, the Merciful”
“Read: In the name of thy Lord Who createth, (1) Createth man from a clot (2) Read:
And thy Lord is the Most Bounteous, (3) Who teacheth by the pen, (4) Teacheth man that
which he knew not” (5) Al-Alaq (Holy Quran).
I would like to sincerely thank my supervisor Dr. Inteaz Alli for his kind support,
academic advice, as well as his understanding, expertise and help in the preparation of
this work. I would like to thank his family, his wife Farida and their sons Alam and Asif
for their friendship. I would like to thank Dr. Selim Kermasha for his valuable help and
giving me access to his facilities during my research work. Sincere thanks and
appreciation is extended to Dr. Jasim Ahmed (Polymer Source Inc.) for his scientific and
technical help during my work and his help of DSC and FTIR analyses. Mrs. Beata
Usakiewicz help in MS and LC-ESI-MS/MS analyses is highly appreciated
(Biotechnology Research Institute, Montreal, Canada). My lab mate Mr Yu-Wei Chang
help is highly valued. Lastly, sincere thank you to all of my friends in Libya, Canada
and other countries especially former student; Dr. Muhamed Aludatt for his brotherly
friendship and help. Staff of Food Science department also deserve a big greeting for
their help especially Ms Leslie Ann LaDuke and Ms Diane Chan-Hum.
I would like to express my deepest appreciation and thanks from the bottom of my
heart to my lovely home country Libya for full financial support and care of me and my
family and giving me the chance to accomplish this Ph. D degree through the Libyan
cultural section staff who offered me all kinds of help during my entire Ph. D program.
viii
Finally, my deepest gratitude goes to my family members and a special thank
you goes to my wife Zaineb Gebril IL-Mgerbi and to my loving son Mohamed Anwer
Ayad, for their help, encouragement, and patience during my program. Their strong
belief in me, support and encouragement helped me to pursue higher education as well
as kept me energetic for all those years in my studies. A very special thank you to my
Father Ali Ahmed Ayad and my Mother Sheika Salem Ayad, who initiated me in
learning how to read and write that helped me build up my knowledge toward the rest of
my entire life.
ix
CLAIMS OF ORIGINAL RESEARCH
1.
This is the first study to investigate the isolation, identification and
characterization of individual protein fractions albumin, globulin and glutelin from
commercially defatted flaxseed meal and the investigation of their structural,
molecular and thermal properties using gel electrophoresis, mass spectrometry (ESIMS), Fourier transform infrared spectroscopy (FTIR), reversed-phase high
performance liquid chromatography (RP-HPLC) and differential scanning calorimetry
(DSC).
2.
This is the first study to investigate the use of tryptic hydrolysis of individual
flaxseed protein fractions albumin, globulin and glutelin and to characterize the
hydrolysis products using gel-electrophoresis, RP-HPLC and LC-ESI-MS/MS.
3.
This is the first study on the proteomic investigation of the peptides obtained
from in-vitro and in-gel tryptic digestion of flaxseed protein fractions albumin,
globulin and glutelin.
4.
This is the first study to investigate selected biological properties, antioxidant,
antifungal and angiotensin converting enzyme inhibitory activities using tryptic
hydrolysates from the flaxseed protein fractions albumin, globulin and glutelin.
x
TABLE OF CONTENTS
ABSTRACT
iv
RESUME
vi
viii
ACKNOWLEDGMENTS
x
CLAIMS OF ORIGINAL RESEARCH
LIST OF TABLES
xvi
LIST OF FIGURES
xviii
AMINO ACIDS ABBREVIATIONS
xxv
LIST OF ABBREVIATIONS
xxvi
CHAPTER I
INTRODUCTION
2.1 General Introduction
1
2.1 Rationale and Objective of Study
3
CHAPTER II
LITERATURE REVIEW
2.1 Importance of Flaxseed
5
2.2 Flaxseed Protein Applications
6
2.3 Flaxseed Proteins
8
2.3.1 Flaxseed Protein Fractions
11
2.3.1.1 Flaxseed Globulins (Linins)
11
2.3.1.2 Flaxseed Albumins (Conlinins)
13
2.3.2 Minor Flaxseed Proteins
15
xi
2.3.2.1 Oil-Binding Proteins (Oleosins)
15
2.3.2.2 Cadmium-Binding Proteins
15
2.3.2.3 Antifungal Proteins (Linusitins)
15
2.4 Other Flaxseed Components
16
2.4.1 Dietary Fiber (Mucilage or Gum)
16
2.4.2 Cyanogenic Glycosides
17
2.4.3 Lignans and Phenolic Acids
18
2.4.4 Phytic Acid
20
2.4.5 Lintaine
20
2.5 Isolation of Flaxseed Proteins
20
2.6 Micellisation and Isoelectric Precipitation
24
2.7 Biological Properties of Flaxseed Proteins
26
2.7.1 Digestibility of Flaxseed Proteins
28
2.7.2 Bioactive Peptides from Food Proteins
29
2.7.3 Antihypertensive Properties of Flaxseed Proteins
30
2.7.4 Antioxidant Properties of Flaxseed Proteins
31
2.7.5 Antimicrobial Properties of Flaxseed Proteins
32
2.8 Functional and Thermal Properties of Flaxseed Proteins
33
CHAPTER III
Isolation, Fractionation and Characterization of Proteins from Defatted Flaxseed Meal
3.1 Justification
36
xii
3.2 Materials and Methods
36
3.2.1 Materials
36
3.2.2 Protein Content and Protein Yield
37
3.2.3 Preparation of Flaxseed Protein Fractions from Defatted Meal
37
3.2.4 Characterization of Protein Fractions
40
3.2.4.1 Native Polyacrylamide Gel Electrophoresis (Native-PAGE) of Protein
40
Fractions
3.2.4.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-
40
PAGE) of Protein Fractions
3.2.4.3 Two Dimensional Gel Electrophoresis
41
3.2.4.3.1 IPG Strip Rehydration
42
3.2.4.3.2 Isoelectric Focusing (IEF) Run
42
3.2.4.3.3 IPG Strip Equilibration and Running Gels
42
3.2.4.4 Electrospray Ionization Mass Spectrometric (ESI-MS) Analysis of
43
Protein Fractions
3.2.4.5 Reversed Phase High Performance Liquid Chromatography (RP-HPLC)
43
Separation of Protein Fractions
3.2.4.6 Fourier Transform Infrared Spectroscopic (FTIR) Analysis of Protein
44
Fractions
3.2.4.7 Differential Scanning Calorimetric (DSC) Analysis of Protein Fractions
3.3 Results and Discussion
45
45
3.3.1 Fractionation of Protein Fractions from Defatted Flaxseed Meal
xiii
45
3.3.2 Characterization of Flaxseed Protein Fractions
47
3.3.2.1 Gel Electrophoresis
47
3.3.2.2 Two-Dimensional Gel Electrophoresis
51
3.3.2.3 ESI-MS Analysis of Protein Fractions
54
3.3.2.4 Reversed-Phase HPLC
58
3.3.2.5 Characterization of HPLC Fractions by SDS-PAGE
61
3.3.2.6 FTIR Spectroscopy
63
3.3.5 Differential Scanning Calorimetry
67
3.4.1 Conclusion
70
CHAPTER IV
Study of Tryptic Hydrolysis Effects
on Flaxseed Protein Fractions and
Characterization of In-vitro and In-gel Tryptic Digests from Flaxseed Protein Fractions
4.1 Justification
71
4.2 Materials and Methods
72
4.2.1 Materials
72
4.2.2 Preparation of Flaxseed Protein Hydrolysates from Protein Fractions
72
4.2.2.1 Enzymatic Hydrolysis of Flaxseed Protein Isolates
73
4.2.2.2 Determination of Degree of Hydrolysis
73
4.2.3 Characterization of Protein Hydrolysates
73
4.2.3.1 Polyacrylamide Gel Electrophoresis of Protein Hydrolysates
73
4.2.3.2 RP-HPLC Separation of Peptides
74
4.2.3.3 In-Gel Tryptic Digestion of Proteins
75
xiv
4.2.3.3.1 Processing of Silver Stained Proteins from Gels
75
4.2.3.3.2 Reduction and Alkylation of Selected Proteins
75
4.2.3.3.3 In-Gel Digestion of Proteins
76
4.2.3.4 Liquid Chromatography Electrospray Ionization Mass Spectrometry of
76
Protein Hydrolysates
4.2.3.5 Mass Spectra Interpretation and Database Search
4.3 Results and Discussion
77
78
4.3.1 Enzymatic Hydrolysis
78
4.3.2 Gel Electrophoresis of Flaxseed Protein Hydrolysates and In-Gel Digests
82
4.3.3 RP-HPC Separation of Protein Hydrolysates
86
4.3.4 Identification of Peptides from Protein Hydrolysates Using LC-ESI-
93
MS/MS
4.3.5 Identification of Peptides With Potential Bioactivity from In-Gel Tryptic
113
Digests Using LC-ESI-MS/MS
4.4.1 Conclusion
115
CHAPTER V
Biological Properties of Flaxseed Protein Fractions
5.1 Justification
116
5.2 Materials and Methods
116
5.2.1 Materials
116
5.2.2 Preparation of Flaxseed Protein Isolates and Tryptic Digests
117
xv
5.2.3 Evaluation of Biological Activities
117
5.2.3.1 Assay of Antioxidant Activity
117
5.2.3.1.1 Statistical Analysis
118
5.2.3.2 Determination of Total Phenolic Compounds Content
118
5.2.3.3 Assay of Antifungal Activity
118
5.2.3.4 Assay of Angiotensin Converting Enzyme (ACE) Inhibition
119
5.3 Results and Discussion
120
5.3.1 Antioxidant Activity and Phenolic Compounds Content
120
5.3.2 Antifungal Activity
126
5.3.3 Angiotensin Converting Enzyme (ACE) Inhibitory Activity
127
5.4.1 Conclusion
129
Chapter VI
General Conclusions
6.1 Conclusions
130
References
134
LIST OF TABLES
CAHPTER II
Table 2.1
Composition of Flaxseed and its Processed Products
7
Table 2.2
Components of Extractable Flaxseed Protein Fractions
8
Table 2.3
Amino acid Composition of Proteins from Flaxseed, Soybean and
10
Canola
Table 2.4
Physicochemical Properties of Flaxseed Proteins (Albumins and
xvi
14
Globulins)
Table 2.5
Composition of Relative Neutral Sugars in Flaxseed and Commercial
17
Gums (%)
Table 2.6
Cyanogenic Glycoside Content of Flaxseed at Various Stages of
18
Processing
Table 2.7
Phenolic Compounds Contents of Flaxseed Products
19
Table 2.8
Protein Content of Flaxseed Protein Isolates Using Micellisation and
22
Isoelectric Precipitation
Table 2.9
Comparison of Biological and Nutritional Properties of Flaxseed and
27
Soybean Meals
Table 2.10
Degree of Hydrolysis, Angiotensin I-Converting Enzyme Inhibitory
28
Activity, Antioxidant Activity from Flaxseed Protein Hydrolysates
Table 2.11
Inhibition of Candida albicans by Seed Extracts
33
Sequential Solvent Extraction, Protein Content and Yield of the Different
47
CHAPTER III
Table 3.1
Solubility Classes of Flaxseed Proteins
Table 3.2
Molecular Weight of Subunits Identified by SDS-PAGE of the Protein
51
Fractions
Table 3.3
Molecular Weight (Da) of Subunits of Protein Fractions from Flaxseed;
58
Albumin, Globulin and Glutelin Determined by ESI-MS
Table 3.4
SDS-PAGE Estimated Molecular Weight (MW) of RP-HPLC Protein
Fractions from Defatted Flaxseed
xvii
62
Table 3.5
Major Bands Assignments of the Deconvoluted Amide I Spectral Region
67
of Protein Fractions from Flaxseed
CHAPTER IV
Table 4.1
Proteins and Peptides Detected in Protein Hydrolysates from Flaxseed by
100
LC-ESI-MS/MS Following Trypsin Digestion of Isolated Protein
Fractions
CHAPTER V
Table 5.1
Total Phenolic Compounds Content (mg/ml Gallic- Acid) in Flaxseed
123
and Soybean Protein Fractions and Hydrolysates
Table 5.2
Growth Inhibition of P. camemberti Treated with Different Flaxseed
126
Protein Fractions and Soybean Protein Hydrolysates
LIST OF FIGURES
CHAPTER II
Figure 2.1
Nitrogen Solubility of Flaxseed Products as Function of pH
24
Procedure for Sequential Solvent Extraction of Flaxseed Protein
39
CHAPTER III
Figure 3.1
Fractions Albumin, Globulin and Glutelin
Figure 3.2
Native-PAGE of Flaxseed Protein fractions (M) Standard Protein
49
Markers; (A) Albumin Fraction; (B) Globulin Fraction and (C) Glutelin
Fraction
Figure 3.3
SDS-PAGE of Flaxseed Protein Fractions. (M) Standard Protein
Markers; (A) Albumin ; (B) Globulin and (C) Glutelin Fractions
xviii
50
Figure 3.4
Standard Curve Generated by Plotting the Log of the Molecular Weight
50
of Protein Standards vs. the Relative Mobility
Figure 3.5
Two Dimensional Gel Electrophoresis Patterns of the Separation of
53
Flaxseed Protein Fractions; Albumin (A), Globulin (B) and Glutelin (C)
Figure 3.6
(A) ESI-MS Spectra of Albumin Fraction Indicating the Net Charge of
55
the Multiprotonated Ions and (B) Deconvoluted ESI-MS Spectra of
Albumin
Figure 3.7
(C) ESI-MS Spectra of Globulin Fraction Indicating the Net Charge of
56
the Multiprotonated Ions and (D) Deconvoluted ESI-MS Spectra of
Globulin
Figure 3.8
(E) ESI-MS Spectra of Glutelin Fraction Indicating the Net Charge of the
57
Multiprotonated Ions and (F) Deconvoluted ESI-MS Spectra of Glutelin
Figure 3.9
RP-HPLC Chromatograms of Protein Fractions; Albumin (A), Globulin
60
(B) and Glutelin (C) from Defatted Flaxseed
Figure 3.10 SDS-PAGE of RP-HPLC Fractions Isolated from Flax Proteins. (M)
62
Molecular Weight Markers; (AF2) Albumin; (BF1) Globulin and (CF2)
Glutelin
Figure 3.11 Full Range FTIR Spectra of Albumin, Globulin and Glutelin Flaxseed
65
Figure 3.12 Deconvoluted FTIR Spectra of Protein Fractions from Flaxseed;
66
Albumin (A); Globulin (B) and Glutelin (C)
Figure 3.13 DSC Thermograms of Albumin (A), Globulin (B) and Glutelin (C)
Fractions Isolated from Flaxseed
xix
69
CHAPTER IV
Figure 4.1
Degree of Tryptic Hydrolysis of Protein Fractions from Flaxseed (A)
Albumin Hydrolysate,
Hydrolysate
Figure 4.2
and (B) Globulin
, Control Globulin
Degree of Tryptic Hydrolysis of Protein Fractions from Flaxseed (A)
Glutelin Hydrolysate
Protein Hydrolysate
Figure 4.3
, Control Albumin
80
, Control Glutelin
82
; and (B) Soybean
, Control Soybean Protein
Native-PAGE of Isolated Albumin Fraction (1), Control Albumin (2),
84
Enzymatic Hydrolysate of Albumin (3), Isolated Globulin Fraction (4)
Control Globulin (5), Enzymatic Hydrolysate of Globulin (6), Isolated
Glutelin Fraction (7) and Control Glutelin (8), Enzymatic hydrolysate of
Glutelin (9), and Standard Protein Markers (M)
Figure 4.4
SDS-PAGE of Isolated Albumin Fraction (1), Control Albumin (2),
85
Enzymatic Hydrolysate of Albumin (3), Isolated Globulin Fraction (4),
Control Globulin (5), Enzymatic Hydrolysate of Globulin (6), Isolated
Glutelin Fraction (7), Control Glutelin (8), Enzymatic Hydrolysate of
Glutelin (9) and Standard Protein Markers (M)
Figure 4.5
Electrophoreogram of Selected Spots of Flaxseed Protein Fractions Used
86
for In-gel Digestion; Albumin (1), Globulin (2), Glutelin (3), Soybean (4)
and Standard Protein Marker (M)
Figure 4.6
RP-HPLC Chromatograms (A) Albumin Isolate ; (B) Control Albumin;
xx
89
and (C) Hydrolyzed Albumin Fraction
Figure 4.7
RP-HPLC Chromatograms (A) Globulin Isolate; (B) Control Globulin;
90
and (C) Hydrolyzed Globulin Fraction
Figure 4.8
RP-HPLC Chromatograms (A) Glutelin Isolate; (B) Control Glutelin;
91
and (C) Hydrolyzed Glutelin Fraction
Figure 4.9
RP-HPLC Chromatograms (A) Soybean Protein Isolate ; (B) Control
92
Soybean Protein; and (C) Hydrolyzed Soybean Protein Fraction
Figure 4.10 LC-ESI-MS/MS Spectra of Enzymatic Hydrolyzed Albumin Fraction
Isolated
from
Defatted
Flaxseed
Meal.
Inset
Represents
95
the
Fragmentation Spectra of the Peptide RDPVLAWRT from Chitinase IV
(m/z 429, MW 856 Da)
Figure 4.11 LC-ESI-MS/MS Spectra of Enzymatic Hydrolyzed Globulin Fraction
Isolated
from
Defatted
Flaxseed
Meal.
Inset
Represents
96
the
Fragmentation Spectra of the Peptide RQDIQQQGQQQEVERW from
Conlinin (m/z 857.4, MW 1712.8 Da)
Figure 4.12 LC-ESI-MS/MS Spectra of Enzymatic Hydrolyzed Glutelin Fraction
Isolated
from
Defatted
Flaxseed
Meal.
Inset
Represents
97
the
Fragmentation Spectra of the Peptide QIQEQDYLRS from Conlinin
(m/z 597, MW 1191 Da)
Figure 4.13 (A) Sequences of Peptides Derived from Albumin Hydrolysate Showing
the Possible Cleavage Location and Homology According to the Tryptic
Peptides Identified from Conlinin Protein from Flaxseed
xxi
98
Figure 4.13 (B) Sequences of Peptides Derived from Albumin Hydrolysate Showing
98
the Possible Cleavage Location and Homology According to the Tryptic
Peptides Identified from Chitinase IV from the Flaxseed
Figure 4.14 Sequences of Peptides Derived from Globulin Hydrolysate Showing the
99
Possible Cleavage Location and Homology According to the Tryptic
Peptides Identified from Conlinin Protein from Flaxseed
Figure 4.15 Sequences of Peptides Derived from Glutelin Hydrolysate Showing the
99
Possible Cleavage Location and Homology According to the Tryptic
Peptides Identified from Conlinin Protein from Flaxseed
Figure 4.16 (A): LC-ESI-MS/MS Spectra of In-Gel Digested Albumin Fraction from
101
Flaxseed (FA1). Inset Represents the Fragmentation Spectra of the
Peptide FFLAGNPQRQ (m/z 525, MW 1048.5 Da)
Figure 4.16 (B): LC-ESI-MS/MS Spectra of In-Gel Digested Albumin Fraction from
102
Flaxseed (FA2). Inset Represents the Fragmentation Spectra of the
Peptide AMPQLAASAPVPATALATPGKPPR (m/z 772.4, MW 2313.1
Da)
Figure 4.16 (C): LC-ESI-MS/MS Spectra of In-Gel Digested Albumin Fraction from
103
Flaxseed (FA3). Inset Represents the Fragmentation Spectra of the
Peptide IQSMPKLALK (m/z 578.3, MW 1155.6 Da)
Figure 4.17 (A): LC-ESI-MS Spectra of In-Gel Digested Globulin Fraction from
Flaxseed (FB1). Inset Represents the Fragmentation Spectra of the
Peptide VTRWSAASLSPR (m/z 673.9, MW 1345.8 Da)
xxii
104
Figure 4.17 (B): LC-ESI-MS Spectra of In-Gel Digested Globulin Fraction from
105
Flaxseed (FB2). Inset Represents the Fragmentation Spectra of the
Peptide LPSTISKIELGGMLSK (m/z 558.6, MW 1672.8 Da)
Figure 4.17 (C): LC-ESI-MS Spectra of In-Gel Digested Globulin Fraction from
106
Flaxseed (FB3). Inset Represents the Fragmentation Spectra of the
Peptide QKLLSAQK (m/z 458.3, MW 914.5 Da)
Figure 4.18 (A): LC-ESI-MS Spectra of In-Gel Digested Glutelin Fraction from
107
Flaxseed (FC1). Inset Represents the Fragmentation Spectra of the
Peptide LIEGGLTPLAPKPVPPRFR (m/z 724.4, MW 2170.1 Da)
Figure 4.18 (B): LC-ESI-MS Spectra of In-Gel Digested Glutelin Fraction from
108
Flaxseed (FC2). Inset Represents the Fragmentation Spectra of the
Peptide ELAVQIHSMIQNLAQFTDIK (m/z 772.4, MW 2313.1 Da)
Figure 4.18 (C): LC-ESI-MS Spectra of In-Gel Digested Glutelin Fraction from
109
Flaxseed (FC3). Inset Represents the Fragmentation Spectra of the
Peptide EDGAPAAIVDR (m/z 557.3, MW 1112.5 Da)
Figure 4.19 (A): LC-ESI-MS Spectra of In-Gel Digested Soybean Protein Isolate
110
(FS1). Inset Represents the Fragmentation Spectra of the Peptide
LLANASGAMSFAVIVP (m/z 780.9, MW 1559.8 Da)
Figure 4.19 (B): LC-ESI-MS Spectra of In-Gel Digested Soybean Protein Isolate
111
(FS2). Inset Represents the Fragmentation Spectra of the Peptide
RSQSDNFEYVSFKT (m/z 725.8, MW 1449.6 Da)
Figure 4.19 (C): LC-ESI-MS Spectra of In-Gel Digested Soybean Protein Isolate
(FS3). Inset Represents the Fragmentation Spectra of the Peptide
xxiii
112
RLSAEFGSLRKN (m/z 554.3, MW 1106.6 Da)
CHAPTER V
Figure 5.1
Antioxidant Activity of (A): (10 mg/mL) Non-Hydrolyzed Albumin
124
(ALB1), Hydrolyzed Albumin (ALBH1); (5 mg/mL) Non-Hydrolyzed
Albumin (ALB2), Hydrolyzed Albumin (ALBH2). B: (10 mg/mL) NonHydrolyzed Globulin (GLB1), Hydrolyzed Globulin (GLBH1); (5
mg/mL) Non-Hydrolyzed Globulin (GLB2), Hydrolyzed Globulin
(GLBH2)
Figure 5.2
Antioxidant Activity of (A): (10 mg/mL) Non-Hydrolyzed Glutelin
125
(GLT1), Hydrolyzed Glutelin (GLTH1); (5 mg/mL) Non-Hydrolyzed
Glutelin (GLT2), Hydrolyzed Glutelin (GLTH2). B: (10 mg/mL) NonHydrolyzed Soybean (SOY1), Hydrolyzed Soybean (SOYH1); (5
mg/mL) Non-Hydrolyzed Soybean (SOY2), Hydrolyzed Soybean
(SOYH2)
Figure 5.3
Angiotensin Converting Enzyme (ACE) Inhibitory Activity of Flaxseed
Protein
Fractions;
(ALB)
Non-Hydrolyzed
Albumin;
(HALB)
Hydrolyzed Albumin; (GLB) Non-Hydrolyzed Globulin; (GGLB)
Hydrolyzed Globulin; (GLT) Non-Hydrolyzed Glutelin; (HGLT)
Hydrolyzed Glutelin and (SOY) Non-Hydrolyzed Soybean; (HSOY)
Hydrolyzed Soybean
xxiv
128
AMINO ACIDS ABBREVIATIONSA
Amino Acid
Three Letters
Single Letter
MW (Da)B
Alanine
Ala
A
89
Arginine
Arg
R
174
Asparagine
Asn
N
132
Aspartic Acid
Asp
D
133
Cysteine
Cys
C
121
Glutamic Acid
Glu
E
147
Glutamine
Gln
Q
146
Glycine
Gly
G
75
Histidine
His
H
155
Isoleucine
Ile
I
131
Leucine
Leu
L
131
Lysine
Lys
K
146
Methionine
Met
M
149
Phenylalanine
Phe
F
165
Proline
Pro
P
115
Serine
Ser
S
105
Threonine
Thr
T
119
Tryptophan
Trp
W
204
Tyrosine
Tyr
Y
181
Valine
Val
V
117
A
Tezcucano-Molina, 2006; Bwww.imgt.org
xxv
LIST OF ABBREVIATIONS
AA
Amino Acids
AA
Antioxidant Activity
ABS
Absorbance
ACE
Angiotensin I- Converting Enzyme
AFP
Antifungal Peptide
ANOVA
Analysis of Variance
APS
Ammonium Persulfate
ºC
Celsius
1D
One Dimension
2D
Two Dimension
D2O
Deuterium Oxide
Da
Dalton
DH
Degree of Hydrolysis
DPPH
1,1-diphenyl-2-picrylhydrazyl
DSC
Differential Scanning Calorimetry
xxvi
ESI-MS
Electrospray Ionization-Mass Spectrometry
FAO/WHO
Food and Agriculture Orginization of the United
Nations/World Health Organization
FTIR
Fourier Transform Infrared Spectroscopy
HCl
Hydrocholoric Acid
HHL
Hippuryl-L-Histidyl-L-Leucine
pI
Isoelectric Point
IEF
Isoelectric Focusing
IPG
Immobilized pH Gradient
kDa
Kilo Dalton
LC-ESI-MS
Liquid
Chromatography-Electrospray
Ionization-
Mass Spectrometry
LC-ESI-MS/MS
Liquid Chromatography- Electrospray IonizationTandem-Mass Spectrometry
M
Mole
mg
Milligram
min
Minute
xxvii
ml
Milliliter
MS/MS
Tandem Mass Spectrometry
m/z
Mass-to-Charge Ratio
MW
Molecular Weight
NaCl
Sodium Chloride
NaOH
Sodium Hydroxide
OPA
o-phathadialdehyde
PAGE
Polyacrylamide Gel Electrophoresis
PDA
Potato Dextrose Agar
Res
Residue
RP-HPLC
Reverse
Phase
High
Performance
Liquid
Chromatography
RT
Retention Time
SDS-PAGE
Sodium
Dodecyl
Electrophoresis
T.o.F
Time of Flight
TCA
Tricholoroacetic Acid
xxviii
Sulfate-Polyacrylamide
Gel
Td
Peak Temperture of Transition
TEMED
Tetramethylethylene-Diamine
TFA
Trifluoroacetic Acid
UV
Uultra Violet
xxix
CHAPTER 1
INTRODUCTION
1.1 General Introduction
Flaxseed (Linum usitatissimum L.) has been used as human food and as animal feed
within different areas of Europe and Middle East since thousands of years ago (Oomah
and Mazza, 2000; Daun et al., 2003). It has been cultivated also for many industrial uses
such as the use of its fiber as linen, oil in paints, stains and in the production of oleo
chemicals based on -linolenic acid (Daun et al., 2003). Flaxseed contains high amounts
of α-linolenic acid (52% of the total fatty acids), an essential fatty acid making flaxseed a
unique oilseed crop for oil production as well as for incorporation in foods and as edible
flaxseed (Chung et al., 2005). Flaxseed also contains relatively high content of lignans
making it a potential source of health food (Oomah, 2001). The use of flaxseed
components such as oil, fiber polysaccharides or the whole flaxseed meal in food is
becoming more attractive due to their specific health advantages and disease preventive
properties (Oomah et al., 1994; Oomah, 2001).
Studies performed on flaxseed indicated that consumption of flaxseed either raw or
defatted can be beneficial for human health and can enhance the performance of kidney
function in some patients, providing evidence of the healthy use of flaxseed components
in cardiovascular protection and renal function ( Oomah and Mazza, 2000; Oomah,
2001). More recently, interest has increased for the incorporation of flaxseed in a number
of products developed for the health food market and bioactive ingredients for the human
health including lignans, secoisolariciresinol diglucoside (SDG), α-linolenic acid (ALA),
and non starch polysaccharides (gum or fiber) (Hall et al., 2006). For example, flaxseed
1
meal has been introduced as feed for many farm animals (for milk and eggs) and fish to
enrich their products with omega-3 fatty acids (Jiang et al., 1991; Kennelly and
Khorasani, 1992; Dick and Yang, 1995).
The relatively high protein content of flaxseed is of interest for reduction of
hypertension and heart diseases, because plant proteins with other components tend to
reduce serum cholesterol (Oomah, 2001; Oomah et al., 2006). Beyond the primary
importance of flaxseed proteins as a source of nutrients and their status as functional
foods, the need has surfaced for the study of biological properties of flaxseed proteins due
to their desirable amino acid profile (Oomah, 2001). The modification of flaxseed
proteins for specific applications is needed as a potential source of bioactive peptides.
These peptides may already be present in flaxseed proteins as natural components or may
be released after enzymatic hydrolysis. Flaxseed has been reported to contain albumin,
globulin, glutelin and prolamin, the globulin being the major seed protein fraction
(Oomah and Mazza, 1993). However, there is still limited information on identification,
characterization and molecular and biological properties of the various flaxseed protein
fractions. Therefore this study was undertaken to fractionate flaxseed proteins by
solubility and to characterize individual flaxseed protein fractions by their
electrophoertic, molecular and structural properties, reversed phase high-performance
liquid chromatography (RP-HPLC), Fourier transform infrared spectroscopic (FTIR)
analysis, differential scanning calorimetry and enzymatic hydrolysates profiles for better
understanding of their structural, molecular and biological properties.
2
1.2 Rationale and Objective of Study
Use of proteins as physiological active components is related to numerous nutritional,
functional and biological properties of the proteins. The occurrence of the physiological
action of protein, or peptides or amino acids could happen naturally in raw food material
or via a biochemical metabolism. These types of foods area also known as „„functional
foods‟‟ and defined to prevent and treat certain disorders and diseases a long with their
nutrition value (Jimenez-Colmenero et al., 2001).
The biological activity among different dietary proteins has been observed to be
associated with the formation of bioactive peptides through their release from their parent
proteins during the food digestion in the gastrointestinal tract. These peptides are able to
cross the digestive epithelial barrier and reach the blood vessels, which allow them to
reach peripheral organs and have beneficial effects for the organism (Yust et al., 2003).
Flaxseed has been valued for its laxative effects and its ability to relieve gastric disorders
since the time of ancient Greeks and Romans. Recently, flaxseed has drawn the attention
of the consumers for their health benefits such as cholesterol lowering, protection against
heart disease and possibly certain types of cancer; and mediation of the immune response
(Oomah, 2001). The rationale of this study is to investigate selected biological properties
of flaxseed proteins and their use as nutraceuticals/functional products for foods
applications. Since the hydrolysis of many food proteins such as soybean has been
reported to yield bioactive peptides with different biological activities such as angiotensin
converting enzyme (ACE) inhibition, antimicrobial, antifungal activity (Pihlanto-Leppala,
2001), the physiologically active components of flaxseed proteins will be investigated
using in-vitro digested flaxseed protein isolates. The overall objective of this research is
3
to investigate the preparation of individual protein fractions from flaxseed and to study
selected biological properties of the protein hydrolysates produced from the protein
fractions. The specific objectives are:
1. Sequential solvent extraction, fractionation and isolation of homogenous proteins;
albumins, globulins and glutelins from defatted flaxseed.
2. Molecular, structural and thermal characterization of protein fractions using gel
electrophoresis, reversed phase high performance liquid chromatography (RP-HPLC),
Fourier transform infrared spectroscopic analysis of protein isolates (FTIR), differential
scanning calorimetric analysis of protein isolates (DSC) and electrospray ionization
mass spectrometry (ESI-MS).
3. Study of in-vitro and in-gel tryptic hydrolysis effects on isolated flaxseed proteins and
to study the extent of hydrolysis.
4. Identify, characterize and investigate the molecular and structural characteristics of
tryptic hydrolysis products using gel electrophoresis, reversed phase high performance
liquid chromatography (RP-HPLC) and liquid chromatography electrospray ionization
mass spectrometry and tandem mass spectrometry.
5. Proteomic database search of bioactive peptides and peptide sequencing results of
both in-vitro and in-gel tryptic flaxseed protein digests using liquid chromatography
electrospray ionization mass spectrometry and tandem mass spectrometry (LC-ESIMS/MS).
6. Investigation of selected biological properties of flaxseed protein hydrolysates
including their angiotensin-I-converting enzyme inhibitory, antioxidant and antifungal
activities.
4
CHAPTER 2
LITERATURE REVIEW
2.1 Importance of Flaxseed
The importance of flaxseed lies in its relatively high contents of fat (30-40%), protein
(20-30%), ash (4%) and dietary fiber (20%) (Daun et al., 2003; Madhusudhan and Singh,
1983; Oomah and Mazza, 1998). Flaxseed contains an average of 38-45% oil depending
on the variety, location and environment (Oomah et al., 1997; Daun et al., 2003).
Flaxseed oil contains 50-62% of α-linolenic acid (ALA) as major fatty acid as well as
small amounts of other fatty acids such as linoleic, oleic, palmitic and stearic (Oomah et
al., 1997; Daun et al., 2003). The high content of α-linolenic acid in flaxseed makes
flaxseed a unique oilseed crop for oil production as well as for incorporation in foods, and
as edible flaxseed (Chung et al., 2005).
Flaxseed also contains relatively high content of lignans making it a potential source of
health food (Oomah, 2001). Flaxseed contains some compounds that are considered to be
undesirable or might have some antinutritional effects (Vassel and Nesbitt, 1945). These
compounds include cyanogenic glycosides, linatine, antipyridoxine factor, trypsin
inhibitors, phytic acid, allergens, and goitrogens (Klosterman et al., 1967; Perlman, 1969;
Cunnane and Thompson, 1995). Flaxseed contains minor components such as sulfur (2.52.5 mg/g), potassium (5.5-10.6 mg/g), magnesium (3.2-4.1 mg/g), calcium (2.0-4.4
mg/g), phosphorus (4.4-7.6 mg/g), minerals such as iron (36.7-164.4 mg/kg), zinc (38.293.6 mg/kg), manganese (13.0-42.8 mg/kg) and water-soluble vitamins such as folate
(278 mg/100g), vitamin B6 (0.4-10 mg/100g), vitamin B5 (1.5-7.0 mg/100g) (Daun et al.,
2003). Flaxseed is a rich source of phytochemicals or phenolics, having anticarcinogenic
5
and chemoprotective action (Thompson and Cunnane, 2003). Pretova and Vojtekova
(1985) reported the presence of lutein, β-carotene, and violaxanthin in flaxseed. These
carotenoids may be used as secondary antioxidants and chain-breaking antioxidants
(Belitz et al., 2004).
Flaxseed contains tocopherols (vitamin E) which are fat soluble antioxidants with the
ability to protect fats and oils from rancidity (Anttolainen et al., 1995). Tocopherols in
flaxseed range from 0.88 to 12.74 mg/100 g seed (Oomah et al., 1997; Thompson and
Cunnane, 2003); they can be in four homologous isomers: α (5,7,8-trimethyltocol), β (5,8dimethyltocol), γ (7,8-dimethyltocol), and δ (8-methyltocol) (Oomah et al., 1997). Budin
et al. (1995) reported that flaxseed contain 0.88, 2.42, 9.20, 0.24, and 12.74 mg/100 g of
seed of α, β, γ, and δ tocopherols and total tocopherols, respectively.
Recent studies on the biological evaluation of flaxseed protein showed that it contains
small amounts of trypsin inhibitor activity (20-30 units/g of seed) which is relatively
lower than that found in soybean and canola and no amylase inhibitor activity or
hemaglutinating activity. Flaxseed proteins have shown also lower allergenic properties
compared to other oilseed proteins (Thompson and Cunnane, 2003).
2.2 Flaxseed Protein Applications
The utilization of whole flaxseed or its components as diet for human and animal feed
has been established for a long time. Introduction of whole flaxseed or defatted flax meal
in foods due to their potential nutritional and health benefits that have been reported;
flaxseed consumption for its medical properties has also reported (Oomah, 2001). Use of
flaxseed protein in combination with other components such as dietary fiber, phenolic
6
compounds (isoflavones, lignans), glucosinolates and phytic acid might be useful in
prevention and treatment of many diseases (Oomah, 2001). The contents of some
components of flaxseed and its products are protein, ash and soluble carbohydrate, and
decreased the oil content and total phenols are shown in Table 2.1.
Table 2.1: Composition of flaxseed and its processed products (Oomah and
Mazza, 1998)
Product
Protein
Fat
Ash Carbohydrate
Phenolic
(Nx5.41)
(g/kg)
acids
Seed
203
438
48
99
13
Flake
213
394
40
99
15
Cake
304
173
59
150
12
Meal
345
57
73
152
9
Defatted seed
283
99
53
132
16
Defatted flake
306
56
60
152
17
Defatted cake
366
16
68
156
11
Defatted meal
65
6
74
178
9
Flaxseed flour is used in a wide variety of cereals, snacks and other health related
products (Oomah et al., 1994). Recently flaxseed protein has become attractive for use in
bakery products and health bar-products (Oomah, 2001). Addition of flaxseed protein
products containing high polysaccharide had been reported to enhance the emulsion
stability of the canned fish sauce (Dev and Quensel, 1989).
Flaxseed proteins were found to reduce the fat losses during cooking when added to
meat emulsions and can improve the cooking emulsion and meaty flavor (Dev and
7
Quensel, 1989). A flaxseed protein product with a high content of mucilage was added to
dough, and was found to improve dough hardness and bread shelf life (Dev and Quensel,
1989).
Table 2.2: Components of extractable flaxseed protein fractions
Protein component
Total proteins Sedimentation MW (proteins
coefficient
subunits)
(% )
(S20, w)
(kDa)
a,c
Globulin (Linin)
40-80
11-12
14400
or
24.6 (basic)
30.0 (acidic)
35.2 (acidic)
50.9
Albumin (Conlinin)a,b,c
20-40
1.6-2.0
16-18
Glutelinc
13.5
-
-
Prolaminc
6.5
-
-
Oleosind
7.2
-
16-24
Cd-binding proteine
7.0
-
1.5
Linusitinf
<1.0
-
25
a
(Madhusudhan and Singh,1983, 1985b, 1985c); b(Youle and Huang, 1981);
c
(Sammour, 1999); d(Tzen et al., 1993); e(Lei et al., 2003); f(Anzlovar et al., 1998).
2.3 Flaxseed Proteins
The total proteins in flaxseed represent about 20-30% of the seed meal which makes it
as a good source of proteins (Sammour, 1999). Flaxseed proteins have similar nitrogen
extractability at varying pH and ionic strength with other oilseed sources of proteins
(Oomah and Mazza, 1993). Previous research on flaxseed protein molecular structure has
8
indicated that flaxseed contains mixed or heterogeneous proteins comprising different
protein fractions (Sammour et al., 1994). Flaxseed proteins are similar to other oilseed
proteins and have been classified based on their solubility in a series of aqueous and nonaqueous solvents into different protein classes based on the Osborne classification of
proteins (Osborne, 1924). The flaxseed proteins classes include globulins or linins,
albumins or conlinins, glutelins and prolamins (Table 2.2) (Anonymous, 1962; Sammour
et al., 1994).
Flaxseed proteins were reported to be 20% albumins of low molecular weight proteins
(1.6S and 2S) and 80% globulins as high molecular weight proteins (11S and 12S) and
were found to be structurally more lipophilic than soybean proteins due to the influence
of their polysaccharide composition (Sammour, 1999; Oomah and Mazza, 1993).
Flaxseed proteins have comparable amino acid patterns to soybean proteins (Oomah,
2001). They contain relatively higher levels of aspartic acid, glutamic acid and arginine
indicating the high content of amides. Flaxseed proteins (Table 2.3) have less lipidimic
and atherogenic effects when consumed compared to soybean proteins due to their low
lysine/arginine ratio compared with soybean (Oomah, 2001).
Studies on the molecular and structural properties of flaxseed proteins compared with
other oilseed proteins such as soybean have been reported to show a close similarity in
terms of hydrophobicity, secondary structure and surface properties (Prakash and
Narasinga Rao, 1986).
9
Table 2.3: Amino acid composition of proteins from flaxseed, soybean and
canola
Amino acid
Flaxseed
3Soybean
glycinin
(g/100g)
1Total
4Canola
Globulin
(g/100g)
2Globulins
2Albumins
Aspartic acid
(g/100g)
8.3
(g/16gN)
11.3
(g/16gN)
5.5
12.7
8.5
Glutamic acid
22.8
19.8
35
15.5
19
Serine
4.1
5.1
3.9
5.3
5
Glycine
4.9
4.8
8.3
7.7
6
Histidine
2.7
2.5
1.6
1.8
3.3
Arginine
10.4
11.5
13.1
5.5
8.3
Threonine
3.4
3.9
2.1
3.7
3.4
Alanine
4.3
7.9
1.9
5.6
4.2
Proline
3.6
4.5
3
6.2
8.3
Tyrosine
2.2
2.3
1.4
2.8
2.9
Valine
5.7
5.6
2.6
5.7
3.9
Methionine
1.5
1.7
0.8
1.6
1.8
Cysteine
3.3
1.4
3.5
0.7
1.8
Isoleucine
4.8
4.6
2.8
4.6
3.4
Leucine
6.7
5.8
5.4
7
8.2
Phenylalanine
5.1
5.9
2.4
4.3
5.2
Lysine
4.4
3.1
4.9
4.2
4.7
proteins
1
Wanasundara and Shahidi (1997); 2Madhusudhan and Singh (1985a);3Wolf and Nelsen
(1996); 4Gruener and Ismond (1997).
10
2.3.1 Flaxseed Protein Fractions
2.3.1.1 Flaxseed Globulins (Linins)
Flaxseed globulins showed similar properties to globulins from canola (Thompson and
Cunnane, 2003). Globulin was found to be the major flaxseed protein varying between
252-298 kDa (11-12 S) and contain 3% α helical and 17% β structures (Youle and Huang,
1981; Madhusudhan and Singh, 1985b; Dev and Sienkiewicz, 1987). The intrinsic
viscosity of the globulins in phosphate buffer is 0.031 dl/g, average hydrophobicity value
of 880 has been reported by Bigelow (1967) (Oomah and Mazza, 1993). A frinctional
ratio (f/f0) of 1.322 was observed for flaxseed globulins indicating the globular
conformation of the protein when compared with standard proteins (Cantor and
Schimmel, 1980; Madhusudhan and Singh, 1985b). Globulins from flaxseed are similar to
legume globulins of Vicia faba and Pisum sativum legumin-type which were reported to
be heterogeneous disulphide linked subunits do not contain covalently-bound
carbohydrate residues (Sammour et al., 1994; Madhusudhan and Singh, 1985b). Flaxseed
globulins have lower number of disulfide bonds and less S-containing amino acids
compared to albumins from flaxseed (Thompson and Cunnane, 2003). The amino acid
composition of the globulins showed a high amide content (glutamic acid-glutamine,
aspartic acid-asparagine and arginine) that plays a role in storage proteins (Sammour et
al., 1994; Wanasundara and Shahidi, 1997). Madhusudhan and Singh (1985b) isolated
and characterized globulins from defatted flaxseed meal to be 12S with five subunits with
molecular weight (MW) of 11, 18, 29, 42 and 61 kDa with more intensity observed for 18
and 42 kDa. Sammour et al. (1994) reported that flaxseed globulins contain six subunits
with MW of 55, 54, 50, 45, 43, and 41 kDa were disulphide-linked subunits. The same
11
authors reported that the highest molecular weight bands (55, 54 and 50 kDa) were
cleaved with 2-mercaptoethanol into acidic subunits with MW of 40 kDa and basic
subunits with MW about 20 kDa. The other disulphide bonded bands formed two groups;
acidic subunits with MW of 25 kDa and basic subunits with molecular weight of 20 kDa.
Marcone et al. (1998) reported five components from flaxseed globulins with MW of
14.4, 24.6, 30.0, 35.2 and 50.9 kDa. The 24.6 kDa component was reported to correspond
to basic subunits while the 30.0 and 35.2 kDa components were identified as the acidic
subunits. Subunits of the linins are attached together with weak secondary attractive
forces, such as hydrogen, hydrophobic and electrostatic linkages. Oomah and Mazza
(1998) observed the presence of 4 predominant polypeptides in flaxseed meal products
with MW of 14, 24, 25 and 34 kDa, as well as a number of other minor bands.
Krause et al. (2002) reported that four groups of 11S subunits (55, 50, 46 and 36 kDa)
were detected in globulins from flaxseed, each composed of a pair of disulphide-linked αand β-chains. The molecular masses of the α-chains are 38-34 and 25 kDa; those of the βchains are 19-21 kDa. In addition, minor bands were also detected in flaxseed globulins
showing traces of 7S subunits (54, 36 and 21 kDa) and a small amount of low MW
components (7-10 kDa). Chung et al. (2005) isolated a major flaxseed protein from
NorMan flaxseed defatted meal and showed the presence of three bands, the MW of the
identified bands were 20, 23 and 31 kDa with other minor bands of MW ranging between
9 and 17kDa. Native-PAGE profile of the same flaxseed protein showed a major
component with a relative intensity (93.4%) and two minor components (1.0% and 3.9%)
(Chung et al., 2005).
12
2.3.1.2 Flaxseed Albumins (Conlinins)
Flaxseed albumins or conlinins have shown a similarity with the properties of
albumins from other types of plant proteins such as sunflower and rapeseed (Vessel and
Nesbitt, 1945; Madhusudhan and Singh, 1983; Oomah and Mazza, 1995). The albumins
are considered as enzymic or metabolic proteins used by the oilseed plant (Dieckert and
Dieckert, 1985). Albumins from flaxseed are composed of a single polypeptide chain with
a low molecular weight of 16-18 kDa and sedimentation coefficients of 1.6-2S
(Madhusudhan and Singh, 1985c) (Table 2.4). They showed similar basic characteristics
to other low molecular weight proteins from castor bean, sunflower and rapeseed.
Flaxseed albumins consist of 26% α helical and 32% β structures (Bhatty, 1995); these
conlinins showed a more ordered structure (α helix and β structure) compared to high
aperiodic structure of linins (Madhusudhan and Singh, 1985c), contain more disulfide
linkages than flaxseed linins (Thompson and Cunnane, 2003). The N-terminal amino acid
of albumins was found to be alanine and the C-terminal amino acid was lysine
(Madhusudhan and Singh, 1985c).
The albumins from flaxseed were reported to be homogenous proteins and contain no
phosphorus with less than 0.5 % carbohydrate, with a high content of glutamic acid,
lysine, arginine, glycine and cystine suggesting that the albumin represent a group of
sulphur storage proteins that can be used by the plant during the early stages of
germination. (Youle and Huang, 1981; Madhusudhan and Singh, 1985c; Sammour et al.,
1994).
13
Table 2.4: Physicochemical properties of flaxseed proteins (albumins and
globulins) (Oomah and Mazza, 1993)
Property
Albumin
Globulin
Total proteins (%)
20
66
Sedimentation coefficient (S20,w)
1.6
12
Diffusion coefficient (D20,w x107,cm2/S)
10.7
3.7
Archibald method
17
294
Sedimentation diffusion
16
298
From viscosity
-
252
-Helix
26
3
β-Structure
32
17
Aperiodic
42
80
SDS-PAGE
1
5
Urea-PAGE
-
6
Molecular weight (kDa)
Secondary structure
Subunit composition
Madhusudhan and Singh, 1985c isolated and characterized a low molecular weight
proteins of 1.6S from flaxseed meal. The identified protein showed a single polypeptide
chain with 18 kDa using SDS-PAGE. Sammour (1988) reported that the SDS-PAGE
analysis of native albumins from flaxseed indicated the presence of 16 bands with sharp
bands at MW of 42 and 38 kDa (Madhusudhan and Singh, 1985c). Sammour et al. (1994)
14
identified an albumin like protein that has subunit composition of 25 and 11 kDa and no
subunits contained covalent bound carbohydrate.
2.3.2 Minor Flaxseed Proteins
2.3.2.1 Oil Binding Proteins (Oleosins)
Oleosins represent a group of specific flaxseed proteins that are associated with oil
bodies of the seed (Tzen et al., 1993). Oleosins are found embedded with phospholipids
forming the outer surface of the oil bodies. Oleosins are high lipophilic proteins and have
molecular weight of 16-24 kDa and form 7.2% of the total flaxseed proteins (Thompson
and Cunnane, 2003).
2.3.2.2 Cadmium-Binding Proteins
Cadmium-binding proteins are formed as a result of the accumulation of the cadmium
from the soil by flaxseed plant through cadmium-binding components that are rich in SH
group and comprise about 7% of flaxseed proteins with a molecular weight of 1.5 kDa
and cadmium content ranging between 0.08-2.78 ppm (Lei et al., 2003; Thompson and
Cunnane, 2003).
2.3.2.3 Antifungal Proteins (Linusitins)
Linusitins are proteins derived from flaxseed; their molecular weight is ranging
between 22-25 kDa and are considered to have antifungal activity (Anzlovar et al., 1998).
This type activity is used by the flaxseed plant as one of its defense mechanism against
fungal invasion (Thompson and Cunnane, 2003).
15
2.4 Other Flaxseed Components
2.4.1 Dietary Fiber (Mucilage or Gum)
Flaxseed is different from other oilseeds by having a relatively high content of a
mucilaginous material composed of acidic and neutral polysaccharides (Table 2.5)
(BeMiller, 1973; Mazza and Biliaderis, 1989). Flaxseed contains both soluble and nonsoluble fiber, which accounts for about 28% of the weight of full-fat flaxseeds. The major
insoluble fiber fraction in flaxseed consists of cellulose and lignin, and the major soluble
fiber fractions are the mucilage gums. Mucilage gums are polysaccharides that become
viscous when mixed with water. Fiber in flax can be classified as either dietary fiber or
functional fiber (Cui and Mazza, 1996; Payne, 2000; Oomah, 2001). Fiber in flaxseed is
used as a stabilizer additive in cosmetics.
Flaxseed polysaccharides have been suggested to play a role in reduction of diabetes
and coronary heart disease and prevention of some cancer types such as colon and rectal
cancer (Jenkins et al., 1999; Oomah, 2001). As abundant components in flaxseed, both
proteins and soluble polysaccharides interaction has been reported can be useful for
reducing colon luminal ammonia (Clinton, 1992).
16
Table 2.5: Composition of relative neutral sugars in flaxseed and commercial gums
(Cui and Mazza, 1996)
_________________________________________________________________
Flaxseed gums
Commercial gums
________________________________________________________________________
Norman
Omega Foster
Arabic
Guar
Xanthan
(%)
_______________________________________________________________________
Rhamonse
21.2
27.2
25.6
34.0
0.0
0.0
Fucose
5.0
7.1
5.8
Arabinose
13.5
9.2
11.0
Xylose
37.4
28.2
Galactose
20.0
Glucose
Mannose
0.0
0.0
0.0
24.0
24.0
0.0
21.1
0.0
0.0
0.0
24.4
28.4
45.0
33.0
0.0
2.1
3.6
8.2
0.0
0.0
50.7
0.0
0.0
0.0
0.0
67.0
49.3
________________________________________________________________________
2.4.2 Cyanogenic Glycosides
Cyanogenic glycosides such as linamarin, linustatin, lotsutralin and neolinustatin
glycosides have the ability to release hydrogen cyanide upon acidic or enzymatic
hydrolysis (Thompson and Cunnane, 2003). The content of the cyanogenic glycosides
varies from plant to another depending on the location in the plant and stage of
development (Niedzwiedz-Siegien, 1998).
Linustatin and neolinustatin are the most abundant cyanogenic glycosides in flaxseed
as shown in Table 2.6 (Oomah et al., 1992). Linamarin was reported to be at low levels of
32 mg/100 g seed in many flaxseed cultivars. Although the level of cyanide that can be
released from flaxseed would be less than the toxic level, heating of the seeds or an
17
aqueous treatment at high temperature of the flaxseed meal were suggested for removal of
these compounds (Madhusudhan and Singh, 1985d).
Table 2.6: Cyanogenic glycoside content of flaxseed at various stages of processing
(Oomah and Mazza, 1998)
________________________________________________________________________
Linamarin
Linustatin
Neolinustatin Total Total (fat-free basis)
(mg/100g)
________________________________________________________________________
Seed
15
149
144
309
550
Flake
10
148
127
282
465
Cake
14
217
204
435
526
Meal
11
247
218
476
502
Defatted seed
2
207
190
397
440
Defatted flake
-
212
213
425
450
Defatted cake
7
183
167
354
360
Defatted meal
5
236
223
464
466
________________________________________________________________________
2.4.3 Lignans and Phenolic Acids
Flaxseed has been reported to be a natural source of major plant food phytochemicals
such as flavonoids, coumarins, lignans, and phenolic acids (Thompson et al., 1991).
Dabrowski and Sosulski (1984) reported that the major phenolic acids in flaxseed are:
trans-ferulic (46%), trans-sinpaic (36%), p-coumaric (7.5%), and trans-caffeic (6.5%).
The role of phenolic compounds as antioxidants has been suggested to be associated with
some ethanolic extracts of phenolic compounds in flaxseed (Amarowicz et al., 1994). The
total levels of esterified phenolic acids were reported to be between 74 and 81 mg/100 g,
respectively, for dehulled defatted flaxseed meal (Dabrowski and Sosulski, 1984).
18
Table 2.7: Phenolic compounds contents of flaxseed products (Hall and Shultz, 2001)
_________________________________________________________________
Phenolic compounds
NDFEa
DFEb
(mg/100g)
________________________________________________________________________
Ferulic acid
161
313
Coumaric acid
87
130
Caffeic acid
4
15
Chlorogenic acid
720
1435
Gallic acid
29
17
Protocatechuic acid
7
7
p-Hydroxybenzoic acid
1719
6454
Sinapic acid
18
27
Vanillin
22
42
Total
2767
8440
SDGc
2653
4793
a
b
(NDFE, non-defatted flaxseed extract); (DFE) defatted flaxseed extract; c(SDG)
Secoisolariciresinol diglucoside
The contents of phenolic compounds in different flaxseed preparations are shown in
Table 2.7. Varga and Diosady (1994) reported that flaxseed extract contains a total of
phenolic acids of 442 and 355 mg/100 g for hexane-extracted and methanol- ammonia
extracted, respectively. In comparison to other oilseeds, flaxseed has very low levels of
bound phenolic acids at 7.2 mg/100 g and is also a rich source of ferulic acid (Dabrowski
and Sosulski, 1984).
19
242.4.4 Phytic Acid
Phytic acid is known to be a strong chelator of mineral cations such as potassium,
magnesium, iron and it binds to proteins and starch (Thompson and Cunnane, 2003).
Flaxseed contains 23-33 g/kg of its dry seed weight as phytic acid (Oomah et al., 1996).
This amount of phytic acid is comparable to the amount of phytic acid in peanut and
soybean and represent only a minor component of flaxseed (Reddy et al., 1982). Several
studies have suggested that phytic acid has a positive role in eliminating the incidence of
colon cancer in rats by complexing with iron which can reduce the amount of hydroxyl
radicals in the colon (Daun et al., 2003).
2.4.5 Linatine
Klosterman et al. (1967) identified the antipyridoxine factor linatine, as a polar
compound and probably had an amine moiety. Linatine is antivitamin B6 compound is
formed from I-amino-D-proline, a secondary hydrazine, bound to glutamic acid by
peptide bond (Thompson and Cunnane, 2003). Consumption of flaxseed containing
linatine has been shown to be a problem in chicks but has not shown any vitamin B6
deficiency effect in humans (Daun et al., 2003).
2.5 Isolation of Flaxseed Proteins
Many fractionation techniques have been used over the years to explore protein
content, size and charge heterogeneity among the different flaxseed protein fractions
(Oomah and Mazza, 1993; Sammour et al., 1994; Sammour 1999; El-Ramahi, 2003). The
separation of flaxseed proteins from the flaxseed meal is not achieved easily due to the
20
presence of gum or polysaccharides which cause the swelling of these gums in the
aqueous medium (Smith et al., 1946; Sosulski and Bakai, 1969). Removal of the
mucilage by demucilaging or degumming from the whole seed has been reported to
improve the separation process of the proteins from flaxseed (Table 2.8) (Oomah and
Mazza, 1998). The mucilage removal may be done chemically by soaking the extract in
dilute acid (HCl) or alkali (NaOH) for 16-24 h or using enzymatic pretreatment or heat
treatments (Madhusudhan and Singh, 1983; Dev and Quensel, 1988; Oomah and Mazza,
1998; Wanasundara and Shahidi, 1997). Mandokhot and Singh (1979) obtained relatively
higher total flaxseed protein content (55.6%) after degumming the flaxseed by soaking
the seeds in 1% HCl for 16 h.
Solubility of flaxseed proteins show a wide range of nitrogen extractability at different
pH which is similar to proteins from other oilseeds, depends also on other factors such as
pH, solvent-to-meal ratio, ionic strength, solvent composition, salt concentration, heat
treatment and protein concentration (Madhusudhan and Singh, 1983; Dev and Quensel,
1986).
A solubility of 82% of flaxseed protein was observed when 1.28 M NaCl was used as
solvent (Oomah et al., 1994). Panford (1989) reported a nitrogen solubility of 42% using
70% ethanol and 0. 1 N NaOH, 29.4% with NaCl and 25% with water. The minimum
extractability of flaxseed protein from defatted meal was observed at pH 3.0-3.5 (Smith et
al., 1946) and pH 3.5-4.0 and lower for hexane-extracted meals than those for methanol
ammonia-water/hexane-extracted meals (Madhusudhan and Singh, 1983; Oomah and
Mazza, 1993). The highest solubility of the nitrogen of flaxseed proteins was observed at
pH 8 and above with a 75% extraction was obtained at pH 7 (Figure 2.1), (Oomah et al.,
21
1994). The solubility curve generally passes through a minimum in the pH range 4 to 6
for most oilseed proteins such as soybean, groundnut and sunflower seed proteins
(Oomah et al., 1994).
Table 2.8: Protein content of flaxseed protein isolates using micellisation and
isoelectric precipitation
________________________________________________________________________
Protein preparation
Protein (%)
Reference
________________________________________________________________________
Micelle protein isolate
93.0
Krause et al. (2002)
Isoelectric-precipitated protein isolate
89.0
Low mucilage flour
56.4
Low-mucilage protein concentrate
Dev and Quensel (1988)
59.7
High mucilage protein concentrate from seed
63.4
High mucilage protein concentrate from
expeller cake
65.5
Low-mucilage protein isolate
86.6
High-mucilage protein isolate
66.3
Low-mucilage protein isolate
86.5
Zhang (1994)
Osborne (1892) reported the first attempt to isolate flaxseed proteins using salt
extraction and precipitation of the protein by removal of the salt by dialysis. The presence
of globulins and albumins was reported with 18.6% nitrogen and albumin like protein
with 17.7% nitrogen (Chung et al., 2005). 25% of the proteins were found to be watersoluble, 34 to 47% soluble in 5% (w/v) NaCI, 2% soluble in 70% (v/v) ethanol and 3.5%
soluble 36% in 0.2% (w/v) NaOH (Sosulski and Bakai, 1969). Flaxseed meals exhibited
22
higher protein solubility values by alkali extraction as compared with the acidic extraction
(Oomah and Mazza, 1993). Youle and Huang (1981) reported that 93% of conlinins were
soluble in water and 99% was soluble in 0.05 M NaCl.
Linins were also extracted from salt-soluble protein fractions (1 M NaCl) by salting
out with ammonium sulfate precipitation or cryoprecipitation and purified with
chromatographic methods such as size exclusion and ion exchange chromatography
(Oomah and Maza, 1993; Marcone et al., 1998; Thompson and Cunnane, 2003).
Madhusudhan and Singh (1983) found that 1 M NaCl extracted 85% of the total nitrogen
from defatted flaxseed. The prepared protein was separated into three fractions, by gel
chromatography on Sepharose 6B, accounting for 3, 67 and 30% of the total content
(Oomah and Mazza, 1993). Dev and Sienkiewicz (1987) extracted a high molecular
protein in 0.07 M phosphate buffer, pH 7.6, in 1 M NaCl; the supernatant was
cryoprecipitated at 4°C and purified on a Sephadex G-200 column by eluting with
phosphate buffer. Dev and Quensel (1988) used isoelectric precipitation of to extract
flaxseed protein which was resulted in a protein content of 56-86%.
Panford (1989) reported a nitrogen solubility of 42 % using 70 % ethanol and 0.1 N
NaOH, 29.4% with NaCl and 25% with water. An extraction of 97% of flaxseed meal
protein was obtained using 0.8 M NaCl at pH 8.0. Extraction with methanol or methanolwater resulted in lowering the extractability of nitrogen from the meals (Oomah et al.,
1994). Highest solubility of 69-70% was also obtained by Wanasundara and Shahidi
(1994) using alkali medium as compared with the acidic extraction. Extraction with
ethanol and isopropanol-ammonia-water slightly lowered the extractability, but methanol
23
in combination with ammonia was found to improve the extractability of the meals
(Oomah et al., 1994).
100
Nitrogen solubility (%)
80
LMF
60
LMPC
40
HMPC-S
20
HMPC-EC
0
0
1
2
3
4
5
6 7 8 9 10 11 12
pH
Figure 2.1: Nitrogen solubility of flaxseed products as
function of pH (Dev and Quesel, 1998)
LMF, low mucilage flour; LMPC, low mucilage protein concentrate; HMPC-S, high
mucilage protein concentrate from seed; HMPC-EC, high mucilage protein concentrate
from expeller cake.
El-Ramahi (2003) determined the highest protein solubility of 25% to be obtained with
NaOH extraction at pH 11. Chung et al. (2005) reported that proteins from defatted
flaxseed meal were fractionated by anion exchange chromatography yielded a major
fraction with three predominant bands with MW of 20, 23 and 31 kDa.
2.6 Micellisation and Isoelectric Precipitation
Micellisation as an isolation procedure was reported to preserve the native state of the
protein and can remove the non protein components, whereas the isoelectric precipitation
24
may create a partial denaturation and irreversible aggregation of proteins (Krause et al.,
2002). Flaxseed proteins prepared by micellisation and isolelectric point precipitation
were found to consist mainly of 11S globulin and suggested to influence both the amount
of non protein component and the conformation of the isolate (Krause et al., 2002). The
proteins prepared by micellisation have low contents of phytic acid and pentosans
(Krause et al., 2002). Dev et al. (1986) reported the precipitation of flaxseed of 77% of
the flaxseed protein meal at pH 4.1 after alkaline extraction.
Sammour et al. (1994) reported that isoelectric point between 4.5-6.5 for acidic
subunits from major flaxseed proteins and basic subunits with isolelectric points between
7.0 and 8.0. Sammour (1999) reported that isoelectric point of flaxseed proteins was
found to be distributed over a range of pH 3-10.
Krause et al. (2002) reported that using the isoelectric precipitation at acidic pH may
create a higher solubility than at alkaline pH, while the micellisation showed higher
protein solubility at alkaline pH than at acidic pH; the same authors reported that a higher
protein solubility of micellisation isolates (90%) was observed compared to the isoelectric
precipitation isolates (45-50%). El-Ramahi (2003) found that the isoelectric point of 8-11
for major flaxseed proteins with the highest protein yield observed at pH 11 using NaOH;
the obtained results showed an increase in solubility to 43% for NaOH and 37% for the
isoelectric precipitation. Co-precipitation of flaxseed protein with soybean and whey
proteins gave yields of 27.0% and 57.8%, respectively, while, co-precipitation of an acid
soluble extracts of flaxseed proteins with soy and whey protein was found to be 4.6 and
9.8%, respectively (El-Ramahi, 2003). Chung et al. (2005) reported that the isoelectric
25
point of flaxseed proteins were found to be between 5.9-7.2 for acidic subunits and 8.79.2 for basic subunits.
2.7 Biological Properties of Flaxseed Proteins
Most recent biological evaluations have focused on either whole flaxseed, flaxseed
flour or defatted flaxseed meal or a partial flaxseed components such α-linolenic acid
(ALA), lignans, soluble polysaccharides; relatively little work has been done on flaxseed
proteins to investigate their biological properties (Oomah, 2001). Preliminary
investigation on biological properties of flaxseed has shown that it contains small
amounts of trypsin inhibitors activity which is relatively lower than that found in soybean
and canola and no amylase inhibitor activity or hemaglutinating activity (Madhusudhan
and Singh, 1983).
Omoni and Aluko (2006) reported that in-vitro calmodulin-binding activity and
calmodulin dependent neuronal nitric oxide synthase inhibitory activity of Alcalasehydrolyaste of flaxseed. Wu et al. (2004) reported that proteolytic hydrolysis of flaxseed
meal without purifying its protein generates a hydrolysate with angiotensin converting
enzyme inhibitory (ACEI) activity. Bhathena et al. (2002) reported that flaxseed proteins
were effective in lowering plasma cholesterol and triacylglycerol levels compared to
soybean and casein proteins in obese rats. Flaxseed proteins have shown 72.9-91.6%
digestibility and 61.6-77.4% biological value compared to 90.5% and 72.8%,
respectively, for soybean (Oomah and Mazza, 1995).
Flaxseed proteins exhibited a favorable ratio of amino acids, with lysine, threonine and
tyrosine as the limiting amino acids and as a good source of sulfur amino acids;
26
methionine and cystine (Oomah and Mazza, 1995). Table 2.9 shows some nutritional and
biological values of flaxseed and soybean meals.
Flaxseed proteins have shown lower allergenic properties compared to other oilseed
protein (Madhusudhan and Singh, 1983) and can be considered as an important source in
the production of physiological functional foods for specific needs such as in patients
with malnutrition and certain diseases or allergies (Oomah, 2001). Flaxseed proteins
contain 36% as percentage of the essential to the total amino acids, which is considered as
an ideal protein that has been recommended by FAO/WHO in 1973.
The limiting amino acids in flaxseed proteins are lysine, threonine and tyrosine
(Thompson and Cunnane, 2003). Health effects have been associated with flaxseed
consumption including decreased risk of cardiovascular disease, antiviral activity,
antibacterial activity, antifungal activity, laxative effect, anti-inflammatory etc (Oomah,
2001).
Table 2.9: Comparison of biological and nutritional properties of flaxseed and
soybean proteins ( Oomah and Mazza, 1995)
Property
Flaxseed
Soybean
Biological value (%)
61.6-77.4
72.8
Net protein utilization (%)
57.8
61.4
Digestibility (%)
72.9-91.6
90.5
Protein efficiency ratio (g/g)
0.79-1.76
2.32
Protein score (mg/g)
56.5-82.0
47.0
27
2.7.1 Digestibility of Flaxseed Proteins
In recent years plant proteins have become important ingredient in many food
preparations including proteins and their modified products. Plant proteins have indicated
a lower digestibility compared to animal proteins. Protein digestibility depends on
different factors that can be inherent or extrinsic factors (Kakade, 1974). The inherent
factors such as proteins structure and composition, while the extrinsic involve the external
agents that might inhibit the enzymatic hydrolysis such as trypsin inhibitor (Kakade,
1974). Protein hydrolysis of some food proteins can be resulted in the formation of a
specific protein fragments called bioactive peptides that have the ability to exhibit
different biological activities that can have the positive impact on body functions or
conditions such as the reduction to rate of autoxidation of fats in foods; those peptides
may show their biological activity upon release from their parent proteins during the
digestion or food processing (Kitts and Weiler, 2003).
Table 2.10: Degree of hydrolysis, angiotensin-I-converting enzyme inhibitory
activity, antioxidant activity from flaxseed protein hydrolysates (Marambe et al.,
2008)
________________________________________________________________________
Type of activity
Activity
IC50
(%)
(mg solid/ml)
________________________________________________________________________
Degree of hydrolysis (DH)
11.9-70.6
Angiotensin I-converting
enzyme inhibitory activity (ACEI)
71.6-88.3
0.07
Antioxidant activity
12.5-22.1
1.56
_______________________________________________________________________
Losso et al. (1996) reported with the hydrolysis of flaxseed 11S protein, a high
concentration of free glutamine and glutamic acid were released compared to the total
28
amino acid before and after the hydrolysis. The degree of hydrolysis (DH) was
determined to be 11.9-70.6% using isolated flaxseed proteins which were treated with
Flavourzyme (leucine amino peptidase) using different levels of enzyme to substrate
ratios and hydrolysis time (Marambe et al., 2008). Table 2.10 shows the degree of
hydrolysis, angiotensin I-converting inhibitory activity and antioxidant activity of
flaxseed protein hydrolysates.
2.7.2 Bioactive Peptides from Food Proteins
The modification of food proteins for many food applications is needed as demand for
newly designed food products through the incorporation of the modified proteins or their
precursors (Oomah, 2001). Recently the role of dietary proteins has been explored as a
rich source of biologically active peptide; these peptides are inactive within the sequence
of the parent protein and can be released in three ways: (a) through hydrolysis by
digestive enzymes, (b) through hydrolysis by proteolytic microorganisms and (c) through
the action of proteolytic enzymes derived from microorganisms or plants (Korhonen and
Pihlanto, 2006). The research on bioactive peptides has increased since 1979 towards the
separation of other bioactive peptides that are opiate, antithrombotic or antihypertensive
(Pihlanto-Leppala, 2001). In addition, to the importance of flaxseed proteins of being as a
source of nutrients and its status as a functional food after centuries of use as natural
medicine, need has surfaced for nutraceuticals, including the production of flaxseed
bioactive peptides based on the potential health benefits. More recently, a large number of
biologically active peptides have been isolated and characterized from different sources
including bacterial, fungal, plant, food and animals. The composition of these bioactive
29
peptides range between dipeptides to complex linear and cyclic structures (Gill et al.,
1996).
Due to the potential health importance, isolation of bioactive peptides from food
proteins as physiologically active compounds is increasingly become more interesting.
The role of proteins in the formation of bioactive peptides from food was first observed
by Mellander (1950), when he suggested that casein-derived phosphorylated peptides
enhanced vitamin D-independent bone calcification in infants (Korhonen and Pihlanto,
2006).
2. 7.3 Antihypertensive Properties of Flaxseed Proteins
Action of biologically active peptides depends on amino acid composition and
sequence. Milk is currently the main source of a range of bioactive peptides but plant and
animal proteins are also known to contain these bioactive peptides (Pihlanto-Leppala,
2001). ACE is a Zn–metallopeptidase and plays an important role in regulating blood
pressure. The naturally occurring peptides with ACE inhibitory activity were first
obtained from snake venom Ondetti et al. (1971). These ACE inhibitors contained 5–13
amino acid residues per molecule, and most contained a C-terminal sequence of Ala-Pro
or Pro-Pro. Angiotensin I-converting enzyme (ACE) inhibitory peptides as bioactive
peptides were first reported by Oshima and Nagasawa (1979) in the digest of gelatin with
bacterial collagenase. Later, many other sources of ACE inhibitory peptides were
observed in enzymatic hydrolysates of bovine caseins (Maruyama et al. 1985), whey
proteins and fish proteins (Ahn, 2001). However, development of various physiological
active peptides from flaxseed proteins such as angiotensin-converting enzyme inhibitors,
30
antioxidants and antifungal that can be released upon in vitro digestion of flaxseed meal is
a new area that need to be explored and with further investigation.
The study of bioactivity of flaxseed proteins and their protein hydrolysates may
provide a good understanding of the components of flaxseed proteins for designing new
nutraceutical/functional food applications (Liu, 2000). Oshima and Nagasawa (1979)
reported ACE inhibitory peptides produced from food proteins by digestive protease.
Many other ACE inhibitory peptides have been discovered from foods or from enzymatic
digestion of food proteins such as fish, chickpea, and mushroom (Ma et al., 2006).
Marambe et al. (2008) reported the formation of angiotensin I-converting enzyme
inhibiting activity (71.6-88.3%) and hydroxyl radical scavenging activity (12.5-23.0 %)
from protein hydrolysates of isolated flaxseed proteins. Udenigwe et al. (2009) reported
that low molecular weight peptides resulted from thermolysin hydrolysis of flaxseed
proteins displayed the most potent activity with the inhibition of 20% and 61% ACE
inhibitory activity.
2.7.4 Antioxidant Properties of Flaxseed Proteins
Recent work on flaxseed extracts has indicated the role of the bioactive constituents of
its oil for maintaining the oxidative stability; this role was referred to the fatty acid
composition of flaxseed oil and their total content of tocopherols as well as proteins
present that can add to their stability (Shahidi et al., 1995; Abuzaytoun and Shahidi,
2006; Smet et al., 2008).
Flaxseed protein reported to contain higher ratios of cysteine and methionine that
suggested to improve the antioxidant ability of the human body and potentially increase
31
the stability of DNA during cell division and reduce the risk of certain colon cancer
(Oomah, 2001). Carnosine (β-Ala-His) a natural dipeptide found in large amounts in
animal muscles, can inhibit the oxidation of lipids catalyzed by iron, hemoglobin and
lipoxidase. This peptide was reported to have the ability to inhibit oxidative rancidity in
cooked meats during refrigeration (Chan et al., 1993).
Recent studies have reported that the peptide fractions from Alcalase chickpea protein
hydrolysate showed 38% to 81% of antioxidant activity (Li et al., 2008) whereas the
hydrolysis of egg yolk protein yielded hydrolysates that scavenged 74% (Sakanaka and
Tachibana, 2006). Peng et al. (2009) reported that a whey protein hydrolysate and its
peptide fractions showed moderate antioxidant properties ranging between 14.7- 58.7%.
Udenigwe et al. (2009) reported that flaxseed protein hydrolysates showed 82.3-89.9%
antioxidant inhibition activity.
2.7.5 Antimicrobial Properties of Flaxseed Proteins
Flaxseed protein and polyphenolic fractions in flaxseed were reported to possess
antimicrobial (Xu et al., 2006) and fungistatic activities against the growth of some plant
pathogens such as Alternaria solani, Alternaria alternata, and human pathogen Candida
albicans (Vigers et al., 1991; Borgmeyer et al., 1992) (Table 2.11). Plants secrete
different substances through the germination stages some of which posses an inhibitory
action against plant pathogens (Agrios, 1997). In addition, several preformed plant
proteins have been shown to inhibit proteolytic enzymes that are involved in host cell
wall degradation by pathogens, and to inhibit proteases (Shewry and Lucas, 1997).
32
Table 2.11: Inhibition of Candida albicans by seed extracts (Vigers et al., 1991)
________________________________________________________________________
Seed
Name
Minimum inhibitory dose
(mg/disk)
________________________________________________________________________
Barley
Hordeum vulgare
3
Corn
Zea mays
3
Flax
Linum usitatissimum
2
Millet
Panicum miliaceum
>30
Oats
Avena sativa
3
Rice
Oryza sativa
> 30
Rye
Secale cereale
≥ 30
Wheat
Triticum aestivum
7
2.8 Functional and Thermal Properties of Flaxseed Proteins
Functional properties of food proteins describe the characteristics of any protein
quality and determine their food utilisation. The investigation of functional properties of
flaxseed proteins has indicated a comparable profile to soybeans proteins comparing the
hydrophobicity and surface properties such as surface tension and emulsion (Prakash and
Narasinga Rao, 1986). The functional properties of flaxseed proteins were investigated in
order to understand solubility characteristics, rheological behavior, emulsifying
properties, foaming and whipping ability and to address the suitability of flaxseed protein
for the formulation of new food products (Oomah and Mazza, 1993). The solubility
behavior of flaxseed proteins is typical of type II protein such as α-chymotrypsin and
bovine serum albumin (Mattil, 1971). Flaxseed protein solubility is independent of pH in
33
the range of 6-10 and at a solvent-to-meal ratio of 25 (Oomah et al., 1994). Dev and
Quensel (1986, 1988) reported that flaxseed products exhibit a favorable water
absorption, oil absorption, emulsifying activity, and emulsion stability compared with the
corresponding soybean products; however, the alkali extracted- acid-precipitated
flaxseed-protein isolates have higher water-absorption properties and bound four times as
much oil as the soybean isolate.
Differential scanning calorimetry (DSC) or thermal denaturation of proteins is used to
gain more information concerning the denaturation of proteins in proteins as a function of
temperature (Li-Chan and Ma, 2002). DSC involves conformational changes from the
native structure due to the disruption of chemical forces that maintain the structural
integrity of the protein molecules, e.g. hydrogen bonds, hydrophobic bonds, ionic
interactions, and covalent disulfide bonds. Heat treatment of flaxseed meal in hot water
reduced the nitrogen solubility of flaxseed meal in water, NaCl and sodium
hexametaphosphate, and reduced the foam capacity and stability, emulsification capacity
and fat absorption capacity, but it increases water absorption capacity of the flaxseed
meal (Madhusudhan and Singh, 1985d).
A major endothermic transition at 91.3°C was observed for purified flaxseed globulins
by micro-differential scanning calorimetry (Marcone et al., 1998). A protein fraction from
flaxseed eluted with 0.25 M NaCl showed a single peak at 114.7°C with a high enthalpy
of 16.6 J/g (Li-Chan and Ma, 2002). A single endotherm at 106.4°C with an enthalpy of
14.16 J/g was observed for flaxseed protein isolate (90% protein by weight) with 78, 12
and 9% 7S, 12S and 2S, respectively (Green et al., 2005). Protein isolates obtained by
isoelectric precipitation had an elevated endotherm at 121.5°C with low enthalpy of 1.2
34
J/g (Green et al., 2005). DSC performed on four, 0.28, 0.35, 0.45 and 0.50 M NaCl
lyophilized flaxseed protein fractions exhibited two thermal events at 83 and 115°C
(Oomah et al., 2006).
35
CHAPTER 3
ISOLATION, FRACTIONATION AND CHARACTERIZATION OF PROTEINS
FROM DEFATTED FLAXSEED MEAL
3.1 Justification
The potential for flaxseed proteins use as a protein source in the food industry has
increased considerably in recent years. Studies on flaxseed proteins have largely
concentrated on the predominant protein fraction, namely the globulins and albumins
(Oomah and Mazza, 1993; Sammour et al., 1994). The isolation of all the various
flaxseed protein fractions is vital for the characterization of their function, structure and
interactions. This chapter describes the isolation of individual protein fractions from
defatted flaxseed meal on the basis of the Osborne classification of proteins, and
identification and characterization using gel electrophoresis, reversed phase high
performance liquid chromatography (RP-HPLC), Fourier transform infrared spectroscopy
(FTIR), differential scanning calorimetry (DSC) and electrospray ionization mass
spectrometry (ESI-MS).
3.2 Materials and Methods
3.2.1 Materials
Commercial defatted flaxseed meal was obtained as a gift by Bunge Canada
(Winnipeg, Manitoba, Canada). Samples were ground using a coffee grinder, were passed
through #20 mesh screen (1 mm, ASTME II) and kept in air-tight plastic containers at
room temperature prior to use. Native, SDS–PAGE chemicals and molecular weight
36
markers were obtained from Bio-Rad Laboratories, Richmond, CA). Two dimensional gel
electrophoresis chemicals were obtained from GE Healthcare (Uppsala, Sweden). All
other chemicals used in this study were of analytical grade and HPLC grade.
3.2.2 Protein Content and Protein Yield
Protein fractions were analyzed for protein content according to the Kjeldahl method
(AOAC, 1990). Nitrogen content was converted to protein using the factor of 6.25.
Protein yield was calculated on the basis of the obtained isolate weight and the protein
content of defatted meal.
3.2.3 Preparation of Flaxseed Protein Fractions from Defatted Meal
Preparation of flaxseed proteins was carried out according to a modified procedure of
Kwon et al. (1996). Protein isolate fractions were obtained according to Osborne
fractionation scheme (Osborne, 1924). The protocol of protein extraction is outlined in
Figure 3.1. Three solvents; water, sodium chloride and sodium hydroxide were used in
sequence to extract the protein fractions from defatted flaxseed meal. A slurry was
prepared by adding 1 L distilled water to the defatted meal, using a meal to solvent ratio
of 1:10 (w/v) and stirred for 1h using a magnetic stirrer; the proteins were then
precipitated at pH 4.2 using 1 M HCl. The supernatant was centrifuged (10,000xg, 30
min) and the supernatant was filtered through glass wool then dialyzed against distilled
water and lyophilized; the resulting protein powder was designated as albumin and stored
at -20ºC for further use. The residue remaining from water extraction was resuspended in
1 L 0.5 M NaCl solution which was adjusted to pH 8.0 using 1 M NaOH and stirred for
37
1h. The suspension was centrifuged (10,000xg, 30 min), the supernatant was filtered
through glass wool, dialyzed against distilled water, freeze dried, designated as globulin
and stored at -20C for further use. NaOH solution (1 L, 0.1 M) was added to the residue
and adjusted to pH 11.0, stirred for 1h and centrifuged (10,000xg, 30 min). The
supernatant was dialyzed against distilled water, freeze-dried and designated as glutelin
and stored for further analysis.
38
H2O extract (1:10, 1h)
Isoelectric point precipitation (pH 4.2)
Centrifugation
Filtration
Supernatant
Lyophilization
Residue
NaCl extract (10:1, 0.5 M, pH 8.0, 1h)
Centrifugation
Albumin
Filtration
Supernatant
Residue
Lyophilization
NaOH extract (10:1, 0.1 M, pH 11.0, 1h)
Globulin
Centrifugation
Filtration
Supernatant
Residue
Lyophilization
Discard
Glutelin
Figure 3.1: Procedure for sequential solvent extraction of flaxseed protein fractions
albumin, globulin and glutelin.
39
3.2.4 Characterization of Protein Fractions
3.2.4.1 Native Polyacrylamide Gel Electrophoresis (Native-PAGE) of Protein Fractions
Native-PAGE was carried out according to the method of Davis (1964) with 12% and
4% (w/v) acrylamide gels as separating and stacking gels, respectively, using a MiniProtein III electrophoresis cell unit (Bio-Rad, Hercules, CA). Samples of freeze dried
protein fractions (15-25 µL) at a concentration of 5-10 mg protein /mL sample buffer
were loaded into the sample wells. A sample of protein standard (Amersham Pharmacia
Biotech, Montreal, Quebec) was also injected in the gel as high molecular weight marker.
The standard protein mixture consisted of thyroglobulin (669 kDa), ferritin (440 kDa),
catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (66
kDa). A tris-base glycine buffer (pH 8.3) was used as running buffer. The electrophoresis
was performed at a constant voltage of 50-60 V /gel at room temperature, for 2-3h and
terminated when the tracking dye front reached the bottom of the gel. Gels were then
removed from the electrophoresis unit and stained overnight in Coomassie brilliant blue
R-250. The gels were destained using a 20% (v/v) methanol and 10% acetic acid (v/v) in
water. The destaining step was repeated several times until the background color
disappeared.
3.2.4.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) of
Protein Fractions
SDS-PAGE was performed under reducing conditions (2-mercaptoethanol) according
to the discontinuous system that has been described by Laemmli (1970). Gels of 12% and
40
4% (w/v) acrylamide as separation and stacking gels were used. An amount of protein
powder was solubilized to a final concentration of 5-10 mg/mL in sample buffer
containing 2% SDS, 10% glycerol, and 0.05% bromophenol blue and 5% of 2mercaptoethanol in tris-HCl, pH 6.8. Samples were heated at 95°C for 5 min before
loading (10-15 L) into each well. The gels were run for 1.5-2h at a constant voltage of
50-60 V/gel using a mini Protein III cell unit (Bio-Rad, Hercules, CA). At the end of
electrophoresis, the gels were stained overnight (14-16 h) in a solution of Coomassie Blue
R-250 and destained in 20% methanol and 10% acetic acid solution. The destaining
solution was replaced as necessary. Protein bands were compared in relation to the
mobilities of the following marker proteins and a standard curve of log MW verses RF
(relative mobility) values was established for determination of molecular weights of
unknown proteins: myosin (200 kDa), β-galactosidase (116 kDa), phosphorylase b (97
kDa), serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa),
trypsin inhibitor (22 kDa), lysozyme (14 kDa) and aprotinin (7 kDa).
3.2.4.3 Two Dimensional Gel Electrophoresis
The first dimension isoelectric focusing (IEF) separation was performed as described
by Chassaigne et al. (2007) using immobilized pH gradients (IPG). The IPG gel strips
dimensions were as follow: 70 × 3.0 ×0.5 mm and 240 × 3.0 × 0.5 mm, pH range: 3–10,
on a polyacrylamide gel matrix) using an Ettan IPGphor unit (GE Healthcare, Uppsala,
Sweden). Separation on the SDS-PAGE was carried out with Bio-Rad electrophoresis
unit (Bio-Rad Laboratories, Richmond, CA) using separating gel (12.5%) and SDS-buffer
as described by Laemmli, 1970.
41
3.2.4.3.1 IPG Strip Rehydration
Protein samples were solubilised in 150 μL of 2% rehydration buffer (20 mM
dithiothreitol (DTT), 8 M urea, 2% CHAPS; cholamido-propyl-dimethylammoniopropane sulfonate, 0.5% (v/v) IPG buffer and 0.002% bromophenol blue, pH 3-10).
Subsequently IPG gel strips with a linear pH range (3–10) were rehydrated for at least 10
h at 20°C into the strip holder. An amount of 50-100 μg of protein was loaded on each
individual gel and a total volume of 125 μL was loaded per IPG strip.
3.2.4.3.2 Isoelectric Focusing (IEF) Run
Strip holder was positioned on the IPGphor and isoelectric focusing was run with the
initial voltage limited to 500 V for 30 min, and then stepped up to 1000 V for 30 min and
finished at 5000 V for 1h and 45 min.
3.2.4.3.3 IPG Strip Equilibration and Running Gels
After isoelectric focusing, the IPG gel strip was prepared for transfer to the second
dimension by soaking with gentle agitation for 15 min in an equilibration solution 50 mM
tris–HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue and
dithiothreitol (DTT). The equilibrated IPG gel strip was embedded at the top of the SDSPAGE gel in molten 2% (w/v) agarose in electrode buffer using tris–glycine pH 8.3 as
buffer for the second dimension. SDS-PAGE gels (12.5%) were run at 50-60 V per gel
for 90-120 min.
42
3.2.4.4 Electrospray Ionization Mass Spectrometric (ESI-MS) Analysis of Protein
Fractions
Samples of flaxseed protein fractions; albumin, globulin and glutelin were dissolved
in 0.2% formic acid, vortexed, centrifuged (5,000xg, 5 min) and filtered through a
membrane filter (0.45µm, ChromSpec, Brockville, ON, Canada). A sample of 1 μL was
injected into ESI-MS (Waters Micromass QTOF Ultima Global, Micromass, Manchester,
UK); hybrid mass spectrometer equipped with a nano flow electrospray source was used.
It was operated in positive ionization mode (+ESI) at 3.80 KV; with source temperature
of 80°C and desolvation temperature of 150°C. The time of flight (TOF) was operated at
an acceleration voltage of 9.1 kV, a cone voltage of 100 V and collision energy of 10 eV
(for MS survey). For the MS survey mass range, m/z was 300-1990, scanned
continuously over the chromatographic run. The calibration and tuning of the mass
spectrometer was performed with [Glu]-Fibrinopeptide B (Sigma Chemicals Co; St.
Louis, MO). Data analysis was carried out by MassLynx V4.0 software (Waters
Corporation, 2005).
3.2.4.5 Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC)
Separation of Protein Fractions
Reversed phase high performance liquid chromatography (RP-HPLC) analysis of
protein fractions was carried out as described by El-Ramahi (2003) using analytical
HPLC. The analysis was performed using a liquid chromatograph (Beckman; CA, USA)
equipped with a Programmable Solvent Module (model 126) for high pressure solvent
delivery, a Programmable Detector Module (model 166) and protein and peptide C18
43
reversed phase column (Octadecyl, 4.6 x 250 mm column, Mallinckrodt Baker, Inc., New
Jersey, USA) was used. The analysis was performed by injecting 100 µL previously
prepared sample into a manual injector (200 µL loop) and the elution was done at 1 mL
/min using two-solvent gradient system at room temperature: solvent A, 0.1%
trifluoroacetic acid (TFA) in water; solvent B, 0.1% TFA in 70/30 acetonitrile/water, 10%
solvent B for 10 min, and increasing to 90% over 30 min. The linear condition was then
re-established over 10 min. Detection was carried out at 210 nm. Eluted fractions were
collected, concentrated, lyophilized and stored for further work. The spectral and
chromatographic data were analyzed by the Gold System (version V810), translated into
PRN format for Microsoft Excel processing.
3.2.4.6 Fourier Transform Infrared Spectroscopic (FTIR) Analysis of Protein
Fractions
Fourier transform infrared (FTIR) spectrophotometric analysis of protein fractions was
performed for determination of proteins secondary structure of flaxseed protein fractions.
Previously prepared protein fractions albumin, globulin and glutelin were suspended in
deuterium oxide (D2O). The samples (5-6 mg) were mixed with potassium bromide (KBr,
100 mg) and made as pellets of lyophilized albumin, globulin and glutelin samples.
Measurements were performed in the mid-infrared region, with a resolution of 4 cm-1,
using a Nicolet Avatar 330 Fourier transform infrared spectrophotometer (FTIR) (Nicolet,
WI, USA). The measurements were carried out at 25°C and the spectra were recorded in
the range of 400- 4000 cm-1.
44
3.2.4.7 Differential Scanning Calorimetric (DSC) Analysis of Protein Fractions
Differential scanning calorimetry (DSC) was performed to study the thermal properties
of the protein fractions. Flaxseed protein fractions albumin, globulin and glutelin, were
investigated using a TA Q100 differential scanning calorimeter (TA Instruments,
Newcastle, DE, USA) calibrated with indium for heat flow and temperature as described
by Ahmed et al. (2006). The DSC was equipped with a refrigerated cooling system that
monitors the temperature. Sealed aluminum pans were used to avoid any moisture loss
during analysis and an empty aluminum pan was used as a reference. Protein samples
were placed in pre-weighed DSC medium pressure pans, which were hermetically sealed
and reweighed. Samples and reference pans were cooled to -90C, held for 10 min for
equilibration, and then loaded to the DSC chambers using a four axis robotic device.
Nitrogen was used as purge gas at a flow rate of 50 mL/min and the heating rate was set
to 5C /min over a range of -90 to 180C. DSC data were analyzed with the Universal
Analysis Software (version 3.6 C) for thermal analysis.
3.3 Results and Discussion
3.3.1 Fractionation of Protein Fractions from Defatted Flaxseed Meal
Table 3.1 shows the results of flaxseed protein fraction isolation using sequential solvent
extraction based on solubility in different solvents. For the defatted flaxseed meal the
components were 34.1% protein (dry weight basis), % fat is 4.2%, 5% H2O and 4% ash.
In accordance with the Osborne (1924) classification of protein, the albumin and glutelin
fractions were the predominant protein fractions, accounting for 38.1 and 33.9%
45
respectively while the globulin accounted for 27.9%. The protein content of albumins
fraction was 64.9% and for glutelin and globulin fractions were 22.4 and 18.1% of the
total proteins, respectively. The results showed higher protein content of albumin fraction
and lower protein content for globulin and glutelin fractions which can be explained as
due to the presence of polysaccharide gum as major contaminant of flaxseed protein
isolates and its interference with settling and extraction of flaxseed proteins (Oomah and
Mazza, 1993; Agboola et al., 2005). Sosulski and Bakai (1969) reported the solubility of
flaxseed protein using water was between 42 and 52%; soluble in NaCl, 1-2% soluble in
70% ethanol and 3-3.5% soluble in NaOH. Mandokhot and Singh (1979) obtained a
protein content of 55.6% after degumming the flaxseed by soaking the seeds in 1% HCl
for 16 h with water while the highest solubility of the nitrogen in the isolate was observed
at pH 8 and 75% extraction at pH 7.0. Youle and Huang (1981) extracted 93% of watersoluble proteins from flaxseed and 99% soluble in 0.5 M NaCl in 0.035 M sodium
phosphate buffer. Madhusudhan and Singh (1983) extracted 85% of the total nitrogen
protein from defatted flaxseed meal with 1 M NaCl at pH 7.0. Recently, Marambe et al.
(2008) reported that flaxseed meal defatting and subsequent removal of flaxseed mucilage
using salt solution at 50C resulted in an isolated flaxseed protein with 80% protein
content.
46
Table 3.1: Sequential solvent extraction, protein content and yield of the different
solubility classes of flaxseed proteins
________________________________________________________________________
Protein Solvent
pH
Protein content
Protein yield
fractions
(%, N x 6.25)
(%)
________________________________________________________________________
Albumin H2O
4.2
64.9 (±3.1)*
38.1 (±1.4)
Globulin NaCl (0.5 M)
8.0
18.1 (±2.1)
27.9 (±1.9)
Glutelin NaOH (0.1 M) 11.0
22.4 (±1.3)
33.9 (±2.2)
_______________________________________________________________________
*
Numbers in parenthesis represent standard deviation of the means
Sammour et al. (1994) reported that extraction of protein fractions from defatted
flaxseed meal yielded albumin (40.2%) using water, globulin (40.0%) using NaCl,
glutelin (13.3%) using NaOH and prolamin (6.5%) using ethanol. Oomah et al. (1994)
extracted 97% of the flaxseed meal protein using 0.8 M NaCl at pH 8.0 and 82% of
flaxseed protein at neutral pH of 6.8, ionic strength of solvent of 1.28 M NaCl and a
solvent-to-meal ratio of 16.
3.3.2 Characterization of Flaxseed Protein Fractions
3.3.2.1 Gel Electrophoresis
Figure 3.2 shows Native-PAGE patterns of the flaxseed protein fractions. The albumin
fraction, the globulin fraction and the glutelin fraction each showed two bands. Albumin
and glutelin contain one band which is likely to be a linin (≈ 300 kDa), while the second
band that appears in albumin is possibly a conlinin (less than 66 kDa). Figure 3.4 shows
the standard curve used for the estimation of MW of protein fractions. Table 3.2
summarizes the MW of subunits of the flaxseed protein fractions. Figure 3.3 shows the
47
SDS-PAGE profile of the protein fractions. Two intense bands corresponding to 22 and
24 kDa and two minor bands corresponding to 9 and 33 kDa were observed from
albumin; globulin showed one intense band at 23 kDa and three minor bands with MW of
10, 24 and 33 kDa; glutelin showed the presence of two intense bands with a MW 22 and
35 kDa and three minor bands with MW of 35, 45 and 55 kDa. Oomah and Mazza (1993)
reported that the molecular weight of linins ranges from 252-298 kDa by Archibald
method. Sammour et al. (1994) estimated the MW of flaxseed globulins to be 320 kDa
using Native-PAGE; the SDS-PAGE of flaxseed globulin showed six major bands with
MW of 55, 54.5, 50, 45, 43 and 41 kDa; the albumin contained two subunits with MW of
11 and 25 kDa. El-Ramahi (2003) reported the identification of two major protein
components from flaxseed protein using Native-PAGE estimated to be at MW of 320 and
514 kDa when the flaxseed proteins exposed to both NaCl and NaOH solvents.
Madhusudhan and Singh (1985b,c) reported that SDS-PAGE of flaxseed globulins
showed five non identical subunits with molecular weights of 11, 18, 29, 42, and 61 kDa,
and albumins contained three subunits of 17, 16 and 15 kDa.
48
(M)
(A)
(B)
669
440
(C)
Glutelin
Linin
66
Conlinin
ns
Figure 3.2: Native-PAGE of flaxseed protein fractions (M); standard protein
markers; (A) albumin fraction; (B) globulin fraction and (C) glutelin fraction.
49
kDa
(M)
(A)
(B)
(C)
200
116
97
66
45-66 kDa
45
31-35 kDa
31
21-31 kDa
21-25 kDa
21
14
7-10 kDa
7
Figure 3.3: SDS-PAGE of flaxseed protein fractions. (M) standard protein markers;
(A) albumin; (B) globulin and (C) glutelin fractions.
6
LogMW
5
4
3
2
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
Relative Mobility
Figure 3.4: Standard curve generated by plotting the log of the molecular weight of
protein standards vs. the relative mobility.
50
Table 3.2: Molecular weight of subunits identified by SDS-PAGE of the
protein fractions
Protein
Subunits MW obtained Subunit MW reported by
Fraction
in present work
previous researchers
(kDa)
(kDa)
33
36.3b
Albumin
Globulin
Glutelin
24
24b
22
20a
9
11 e
33
34.7b
24
26.2b
23
22.5b
10
10.3d
55
55e
45
45e
35
34.7c
22
20a
a
Marcone et al. (1998); bMadhusudhan and Singh (1985b, 1985c); cAludatt, 2007;dElRamahi, 2003; eSammour (1988, 1994, 1999)
3.3.2.2 Two-Dimensional Gel Electrophoresis
Figure 3.5 shows the 2D electrophoreograms of the two dimensional gel patterns of
protein separation of fractions from flaxseed proteins; albumin (A), globulin (B), glutelin
(C). A total of 21 resolved albumin proteins gave values ranging from 10 kDa to 116 kDa
51
and pI values ranging from 4 to 9. A total of 20 globulin proteins gave values ranging
from 18 kDa to 140 kDa and pI values ranging from 3.5 to 9.5. A total of 20 glutelin
proteins gave values ranging from 25 kDa to 140 kDa and pI values ranging from 3 to 9.
Previous work on two-dimensional gel electrophoresis of flaxseed proteins showed the
different polypeptide profile when they were extracted with water and buffer;
polypeptides with 40 kDa were focused in the acidic range ( subunits) while the 20 kDa
polypeptides were focused in the basic range (β subunits) (Sammour, 1999).
52
kDa
pH 3
10
200
116
A
97
66
45
31
21
14
7
200
B
116
97
66
45
31
21
14
7
C
200
116
97
66
45
31
21
14
7
Figure 3.5: Two dimensional gel electrophoresis patterns of the separation of
flaxseed protein fractions; albumin (A), globulin (B) and glutelin (C).
53
3.3.2.3 ESI-MS Analysis of Protein Fractions
Figures 3.6 (A,B), 3.7 (C,D) and 3.8 (E,F) show the mass spectra of the albumin,
globulin and glutelin fractions. ESI-MS results are summarized in Table 3.3. Two protein
subunits with MW of 9 and 27 kDa were observed with the albumin fraction. The
globulin fraction showed two subunits with MW of 17 and 28 kDa and glutelin fraction
showed the presence of one subunit with MW of 14 kDa. El-Ramahi (2003) reported that
the ESI-MS spectra of one protein fraction from flaxseed showed the protein subunits
with molecular weights of 13.73, 13.79, 14.05, 14.17 and 14.26 kDa, while another
flaxseed protein fraction gave seven subunits with MW ranging from 5.9 to 42.0 kDa.
54
+10
Albumin fraction
+11
○ 9016 Da
27048 Da
+9
A
+24
+25
4
+23
B
Figure 3.6: (A) ESI-MS spectra of albumin fraction indicating the net charge of
the multiprotonated ions and (B) deconvoluted ESI-MS spectra of albumin
fraction.
55
Globulin fraction
C
17005 Da
+12
○28342 Da
+22
+23
+13
+21
+11
D
f
Figure 3.7: (C) ESI-MS spectra of globulin fraction indicating the net charge of
the multiprotonated ions and (D) deconvoluted ESI-MS spectra of globulin
fraction.
56
Glutelin fraction
X 14171Da
E
+11
X
+10
X
+9
X
F
Figure 3.8: (E) ESI-MS spectra of glutelin fraction indicating the net
charge of the multiprotonated ions and (F) deconvoluted ESI-MS spectra
of glutelin fraction.
57
Table 3.3: Molecular weight (Da) of subunits of protein fractions from
flaxseed; albumin, globulin and glutelin determined by ESI-MS
Protein
Fraction
Subunits
by
ESI-MS
(kDa)
Subunits
by
SDS-PAGE
(kDa)
Reported results
SDS-PAGE
(kDa)
ESI-MS
(kDa)
33
Albumin
27
24
25c
9
22
10.3a
9a
42a
9
Globulin
17
33
29b,c
28
24
25a,c
23
22.5d,e
10
11-12.5c,d
55
55c
45
45c
35
36a
20
25a
Glutelin
14
9a
42.5a
14.1a
a
El-Ramahi (2003); Madhusudhan and Singhb (1985b & 1983); cSammour et al. (1988,
1994, 1999); dAludatt (2007).
3.3.2.4 Reversed-Phase HPLC
Figure 3.9 (A, B, C) shows the profile of flaxseed protein fractions chromatogram by
RP-HPLC. The albumin fraction showed two peaks; a dominant peak at a retention time
of 26.2 min and one minor peak at 25.1 min; the globulin fraction gave one dominant
peak at retention time of 24.9 min and one minor peak at 30.4 min; glutelin exhibited a
58
major peak at 25.0 min and two minor peaks at 18.0 and 26.2 min. It can be inferred from
the above mentioned results that the type of solvent and pH has shown an effect on the
proportion of hydrophobic to hydrophilic proteins. The albumin fraction extracted with
water can be expected to be more hydrophobic than globulin fraction that extracted with
NaCl and glutelin fraction extracted with NaOH. The difference in retention times and the
intensity of the peaks can also be explained due to alteration in protein properties during
the extraction and pH adjustments. In addition, exposure of the proteins to alkaline pH
can result in unfolding of the protein structure (Lee et al., 2007). The denaturation of
proteins and their interaction with other components also responsible for the variation of
hydrophobicities among fractionated proteins (Lee et al., 2007). Madhusudhan and Singh
(1983) reported that flaxseed protein extracted using 0.1 M NaCl and subjected to ion
exchange chromatography gave at least three fractions. Sammour et al. (1994) reported
that albumin extracted from flaxseed purified by chromatography on Sephadex G-200
gave two components. Marcone et al. (1998) observed four peaks on Sephacryl S-300
chromatography of crude globulin from flaxseed. Krause et al. (2002) reported that
proteins from flaxseed extracted with 0.5 M NaCl and subjected to size exclusion
chromatography gave one major peak and a NaOH extract also gave one component
within a similar retention time to that of NaCl extract. El-Ramahi (2003) reported that
NaOH isolation gave three major components by RP-HPLC. Chung et al. (2005) reported
that proteins extracted from defatted flaxseed were fractionated by anion exchange
chromatography yielded a major fraction with molecular weight of 365,000 Da, as
determined by Sephacryl S-300 gel permeation chromatography.
59
1.4
A
AF2 (26.2)
1.1
0.8
AF1 (25.1)
0.5
0.2
-0.1
BF1 (24.9)
Absorbance (210nm)
1.4
B
1.1
0.8
BF2 (30.4)
0.5
0.2
-0.1
1.4
C
CF2 (25.0)
1.1
0.8
CF3 (26.2)
CF1 (18.0)
0.5
0.2
-0.1
0
5
10
15
20
25
30
35
40
Retention Time (min)
Figure 3.9: RP-HPLC chromatograms of protein fractions; albumin (A), globulin
(B) and glutelin (C) from defatted flaxseed.
60
3.3.2.5 Characterization of HPLC Fractions by SDS-PAGE
The collected HPLC protein fractions (AF1), globulin (BF1) and glutelin (CF2) were
investigated with SDS-PAGE. Figure 3.10 showed the results of SDS-PAGE done on the
collected fractions from HPLC elutions of albumin, globulin and glutelin and the results
are summarized in Table 3.4. A HPLC collected albumin fraction (AF2) showed the
presence of three bands at 35, 14 and 7 kDa; globulin fraction (BF1) indicated the
presence of two bands at 16 and 10 kDa and glutelin fraction (CF2) exhibited the
presence of four bands at 42, 40, 16 and 7 kDa. Madhusudhan and Singh (1983) noted the
presence of a single symmetrical peak with albumin isolated from flaxseed with subunit
composition of 15, 16, 17 kDa and the formation of four peaks using ion exchange
chromatography and gave five subunits with MW of 11, 18, 29, 42 and 61 kDa. Dev et al.
(1986) also reported that the gel filtration pattern of globulin from flaxseed protein has a
mixture of two high molecular weight components.
Krause et al. (2002) reported that flaxseed protein separated by size exclusion
chromatography with 0.5 M NaCl and observed that the purified proteins contain four
groups of 11S subunits of 36, 46, 50 and 55 kDa. The same authors noticed also low
molecular weight proteins from flaxseed with MW of 7-10 kDa.
El-Ramahi (2003) reported that the major fractions of flaxseed proteins isolated by
sodium hydroxide and analyzed by RP-HPLC gave molecular weights ranging from 78 to
320 kDa by Native-PAGE, 6.5 to 40.1 kDa by SDS-PAGE. The same author also
observed that the flaxseed protein fraction F11 that has been isolated by NaOH and
further purified by RP-HPLC, gave five subunits when it was analyzed by ESI-MS with
61
MW of 13.73, 13.79, 14.05 and 14.26 kDa, whereas a fraction F12 from the same isolate
indicated the presence of 7subnits with a MW ranging between 5.9-42.5 kDa.
kDa
( M)
(A)
(B)
(C)
200
116
97
66
45
21
42
40
kDa
kDa
35
kDa
16
kDa
10
kDa
14
7
Figure 3.10: SDS-PAGE of RP-HPLC fractions isolated from flaxseed proteins. (M)
molecular weight markers; (AF2) albumin; (BF1) globulin and (CF2) glutelin.
Table 3.4: SDS-PAGE estimated molecular weight (MW) of RP-HPLC protein
fractions from defatted flaxseed
________________________________________________________________________
Protein fractions
Albumin
Fraction
No
AF2
Globulin
Glutelin
No of Subunits
3
MW
(kDa)
7, 14 , 35
BF1
2
10, 16
CF2
4
7, 16, 40, 42
62
3.3.2.6 FTIR Spectroscopy
The secondary structure of flaxseed protein fractions was investigated by studying
their FTIR absorption spectrum. The original amide I region spectra of the three protein
fractions albumin, globulin and glutelin is shown in Figure 3.11. Deconvolution of the
infrared spectrum in Figure 3.12 of the protein fractions; albumin, globulin and glutelin in
the amide I absorption region (1700 –1600 cm-1) revealed the presence of seven bands at
1685, 1668, 1655, 1650, 1645 and 1637 cm-1. Table 3.5 presents the assignments of the
amide I bands of the flaxseed protein fractions according to Ellepola et al. (2005). The helix assigned band (1650-1652 cm-1) is observed in all protein fractions.
Albumin showed the presence of 4 bands assigned as antiparallel β-sheet (1685 cm-1),
β-turns (1668 cm-1), -helix (1650 cm-1) and β-sheet (1637). Globulin showed one band
at 1650 cm-1 assigned as -helix. Glutelin showed the presence of two bands at 1655 cm-1
assigned as antiparallel β-sheet and 1645 cm-1 assigned as -helix. The results showed
that the intensity of the major -helix band between 1650-1652 cm-1 as lower in albumin
and higher in globulin and the highest was observed in glutelin. The intensity of β-sheet
band at 1685 cm−1 was obvious with the appearance of a shoulder in albumin than
globulin and glutelin. The results indicate a change in conformation among the three
protein fractions. The relative intensity of a spectral component associated with a given
structural conformation is related to the relative content of the conformation itself and the
peak frequencies of the α, β and turn components slightly vary in different proteins
(Murayama and Tomida, 2004; Carbonaro et al., 2008). Our findings showed some
similarity with earlier reports of the albumin low content of α-helix compared to globulin
63
and glutelin from flaxseed. Madhusudhan and Singh (1985c) reported that albumins from
flaxseed contain 51% of β-structure and 26-32% of α-helix while flax globulins contain
17% β-pleated sheet and 3-4% α-helix. Marcone et al. (1998) reported that a
measurement of diluted solution of flaxseed showed 4.0% α-helix, 62.8% β-sheet, 16.2 %
β-turn and 17.0% random structure.Yu et al. (2005) reported that
golden flaxseed
contained relatively higher percentage of α-helix (47.1%) and lower percentage of β-sheet
(36.9%) than brown flaxseed (37.2%), (46.3%) respectively. Chung et al. (2005)
determined that flaxseed proteins fractionated by anion exchange chromatography contain
an amide I band that is centered near 1245cm-1 with a shoulder at 1684 cm-1 with a low αhelix and a high content of β-sheet and disordered structure.
64
Figure 3.11: Full range FTIR spectra of albumin, globulin and glutelin fractions from flaxseed.
65
Figure 3.12: Deconvoluted FTIR spectra of protein fractions from flaxseed; albumin ; globulin and glutelin.
66
Table 3.5: Major bands assignments* of the deconvoluted amide I spectral region of
protein fractions from flaxseed
__________________________________________________________________
Protein isolate
Frequency (cm-1)
Band assignment
__________________________________________________________________
Albumin
1685
anti-parallel β-sheets
1668
β-turns
1650
α-helix
1637
β-sheet
Globulin
1650
α-helix
Glutelin
1655
α-helix
1645
Unordered (random coil)
________________________________________________________________________
*(Ellepola et al., 2005)
3.3.2.6 Differential Scanning Calorimetry
The DSC thermograms of the protein fractions are presented in Figure 3.13. Albumin
fraction showed endothermic peak at maximum peak temperature (Td) of 122.3C,
globulin fraction gave a peak with a Td value of 118.3C and glutelin fraction a peak with
Td value of 106.8C. The results indicate high thermal stability of flaxseed proteins. The
highest thermal stability was observed with albumin fraction followed by globulin
fraction and the lowest was obtained with glutelin fraction. The maximum thermal
stability of albumins may be attributed partially to the effect of different factors such as
67
pH and salts used in the extraction. Although, the faster rate of heating has been
mentioned as possible effect on the measurement of Td of the proteins. Differences in
purification steps could also lead to protein preparations with different DSC
characteristics (Wright, 1984). It has also been reported that the maximum stability of
food proteins was observed generally between pH 5 and 8 while the extremes of pH, in
either acid or alkaline region resulted in a decreased denaturation temperatures and small
endotherms (Privalov and Khechinashviti, 1974; Harwalkar and Ma, 1987). Li-Chan and
Ma (2002) reported that 11-12S globulin from flaxseed has an exceptional high Td
compared to other globulins. By comparing DSC results with Native-PAGE and RPHPLC results, it can be noticed that the proteins become more stable and aggregate as
could be due to the protein environment and also due to the effect of different solvents
used since the fractionation of plant proteins on the basis of solubility is only an
approximation of the actual protein composition, and formation of more covalent bonding
among proteins and protein interactions (Kwon et al., 1996; Lee et al. 2007). However,
our findings are in partial agreement with previously reported results of high Td value of
114.7 C for the flaxseed globulin that has been reported by Li-Chan and Ma (2002).
Oomah et al. (2006) reported that ion exchange protein fraction from flaxseed exhibited a
Td at 83 and 115C. Aludatt (2007) reported the Td of flaxseed protein fractions at 121.6,
128 and 140.8ºC.
68
mW
Endothermic Heat
Flow
122.3C
20
40
60
80
100
120
140
160
180
160
180
160
180
mW
Endothermic Heat
0
Flow
118.3C
20
40
60
80
100
120
140
mW
Endothermic Heat
0
Flow
106.8C
0
20
40
60
80
100
120
140
Temperature C
Figure 3.13: DSC thermograms of albumin (A), globulin (B) and glutelin (C) fractions
isolated from flaxseed.
69
3.4.1 Conclusion
This chapter investigated the sequential solvent extraction and characterization of
individual protein fractions albumin, globulin and glutelin from defatted flaxseed meal.
The extraction solvents were water, sodium chloride and sodium hydroxide to extract
albumin, globulin and glutelin, respectively. The conditions, which resulted in the highest
yield were a combination of sequential extraction and isoelectric point precipitation. The
highest yield was obtained by water extraction followed by extraction with sodium
hydroxide and sodium chloride. Native-PAGE showed the presence of two major
components in each protein fractions. SDS-PAGE results showed the presence of two
intense bands and one minor band for albumin; globulin showed one intense band and
four minor bands, and glutelin showed the presence of two intense bands and three minor
bands. The FTIR deconvoluted spectra indicated that -helix assigned band (1650-1652
cm-1) was observed in all protein fractions. DSC results showed that the albumin has an
endothermic peak at 122.3C, globulin at 118.3C and glutelin fraction at106.8C.
70
CHAPTER 4
STUDY OF TRYTPIC HYDROLYSIS EFFECTS ON FLAXSEED PROTEIN
FRACTIONS AND CHARACTERIZATION OF IN-VITRO AND IN-GEL
TRYPTIC DIGESTS FROM FLAXSEED PROTEIN FRACTIONS
4.1 Justification
Enzymatic hydrolysis of proteins can result in formulation of functional foods and
nutraceutical products with potential to improve the functional and nutritional properties
of proteins (Pena-Ramos and Xiong, 2002). Protein hydrolysates from different sources,
such as whey, soy protein, and tuna have been reported to have a variety of physiological
activities such as improving cardiovascular system function, antioxidant activity and
antihypertensive activity (Gill et al., 1996; Klompong et al., 2007). The levels and
compositions of free amino acids and peptides were reported to determine the biological
importance of any protein or protein hydrolysates (Wu et al., 2003). However, there is
little information regarding protein hydrolysates from flaxseed proteins and their
hydrolysis products (Marambe et al., 2008). This part of the research is aimed at
investigating protein hydrolysates from flaxseed protein fractions, degree of hydrolysis of
the proteins isolated from defatted flaxseed fractions; on the RP-HPLC and
electrophoretic separation of the hydrolysates and their potential peptide profile content
using liquid chromatography electrospray ionization tandem-mass spectrometric (LCESI-MS/MS) analysis and interpretation of resulted spectra using Mascot database. In
addition, in-gel tryptic digests of flaxseed protein fractions will be investigated.
71
4.2 Materials and Methods
4.2.1 Materials
Flaxseed protein fractions; albumin, globulin and glutelin were prepared as described
previously in Section 3.2.3. A commercial soybean isolate was provided by ADM Protein
technologies Inc. (Decatur, Illinois; Trypsin type IX-S from porcine pancreas, E.C.
3.4.21.4; activity 13100 units/mg protein), OPA (o-phthaldialdehyde) were purchased
from Sigma-Aldrich Co (Oakville, ON). All other chemicals used in this study were of
analytical grade and HPLC grade.
4.2.2 Preparation of Flaxseed Protein Hydrolysates from Protein Fractions
4.2.2.1 Enzymatic Hydrolysis of Flaxseed Protein Isolates
The in-vitro hydrolysis assay was performed according to the method described by
Adebiyi et al. (2008). Dispersions of protein fractions (10 %, w/v) were brought to pH 8.0
with 2N NaOH under mixing and were incubated with trypsin at 1:20 enzyme to protein
ratio, in 50 mM sodium phosphate buffer, pH 8.0 at 37ºC. At time intervals between 0 to
120 min, tubes of the digest were removed from the water bath at 0, 15, 30, 45, 60 and
120 min. The enzyme action was stopped by heating at 95ºC for 10 min. The protein
hydrolysates were then centrifuged at 10,000xg for 30 min, and the supernatant was
lyophilized.
72
4.2.2.2 Determination of Degree of Hydrolysis
The degree of hydrolysis (DH) was determined spectrophotometrically according to
the method of Adebiyi et al. (2008) by measuring the increase in number of free amino
groups released during the tryptic hydrolysis and following the reaction with ophthadialdehyde (OPA). Samples of 50-100 uL of protein hydrolysates were added
directly to 2 mL OPA reagent, vortexed and incubated for 2 min at ambient temperature.
The absorbance of solutions was measured at 340 nm. The amount of free amino groups
in the hydrolysate was calculated as percent of DH using the equation Adebiyi et al.
(2008):
DH (%) = (MW∆340nm )/(d.ε.P) x 100
Where MW = Average molecular weight of amino acids (120)
∆340nm = Absorbance at 340 nm
d = Dilution factor
ε = Average molar absorption of amino acids (6000 M-1 cm-1)
P= Protein concentration
4.2.3 Characterization of Protein Hydrolysates
4.2.3.1 Polyacrylamide Gel Electrophoresis of Protein Hydrolysates
The protein hydrolysates were examined during the course of hydrolysis by NativePAGE and SDS-PAGE. Samples of lyophilized protein hydrolysates prepared in Section
73
4.2.2 were subjected to Native-PAGE and SDS-PAGE following a procedure described in
Section 3.2.4.2.
4.2.3.2 RP-HPLC Separation of Peptides
Separation of peptides in the protein hydrolysates was performed by reversed-phase
high performance liquid chromatography (RP-HPLC) according to a modified procedure
described by Tezcucano-Molina (2006). The HPLC was equipped with a programmable
solvent module (model 126, Beckman, CA) and a dual pump system for high pressure
solvent delivery and a programmable detector module (model 166). The lyophilized
protein hydrolysates at concentration of 5-10 mg/mL (w/v) of filtered samples (0.45 µm
MilliporeTM membrane filters) were resuspended in 0.1% TFA and subjected to RPHPLC. An Octadecyl (C18) reverse phase column; 5µm, 250 x 4.6 mm column
(Mallinckrodt Baker, Inc., New Jersey, USA) was used for the separation of the peptide.
The analysis was performed by injecting 100 µL sample into a manual injector with a
200 µL loop and the elution was done at a flow rate of 1 mL/min using two-solvent
gradient system. Solvent A, 0.1% trifluoracetic acid (TFA) in water; solvent B, 0.1% TFA
in 70/30 acetonitrile/water. Elution was done within 60 min, and a linear gradient started
at 0% of solvent B and increased up to 80% over 50 min. The linear condition was then
re-established over 10 min. Detection of peptides was carried out at 215 nm. Data were
analyzed by the Gold System (V810) and transferred to a disc into print (PRN) format for
Microsoft Excel software applications.
74
4.2.3.3 In-Gel Tryptic Digestion of Proteins
4.2.3.3.1 Processing of Silver Stained Proteins from Gels
Albumin, globulin and glutelin were first separated by electrophoresis on SDS-PAGE
as previously described in Section 3.2.4.2. Gels were stained with silver stain according
to the Bio-Rad protocol Bio-Rad manual for silver stain plus (Bio-Rad Laboratories,
Richmond, CA). After staining, the gels were destained and stored in 5% (v/v) acetic acid
in water.
4.2.3.3.2 Reduction and Alkylation of Selected Proteins
Protein bands were cut into small fragments (as shown in Figure 4.5) from the gel
using a tip of pipette and collected in prewashed Eppendorf tubes. Reduction of the
selected spots was performed by adding 50 µL of 10mM dithiothreitol (DTT) in 50 mM
NH4HCO3 and incubating the samples for 30 min at 56ºC, alkylation step of the proteins
was done by adding 100 µL of iodoacetamide in 50 mM NH4HCO3 at room temperature
for 10 min. The samples were then subjected to centrifugation (5000xg, 1 min), the
supernatant was discarded and the gel fragments were dehydrated with 100% acetonitrile
with agitation at room temperature. When the gel fragments turned white, the acetonitrile
was removed and the samples were rehydrated with 50 µL of 50 mM NH4HCO3. The
dehydration step was repeated with 100% acetonitrile and at last the samples were let to
dry (air-dry) at room temperature.
75
4.2.3.3.3 In-Gel Digestion of Proteins
Digestion of the selected protein bands was performed for 4 h at 37ºC using trypsin
solution prepared by dissolving 20 µg of trypsin (modified porcine trypsin, sequencing
grade, Promega, Madison, WI) in 3 mL of 50 mM NH4HCO3. An amount of 30 µL of
trypsin solution was added to each sample and incubated for 4 hrs at 37ºC. At the end of
digestion, samples were centrifuged and 30 µL of the extraction solvent (90 %
acetonitrile/0.5 M urea) was added to the remaining gel fragments and incubated for 15
min. The resulting gel fragments were then centrifuged and the resulted supernatant was
combined with originally respective supernatant. However, the extraction step was
repeated twice. Finally, the combined supernatants were dried under vacuum and stored at
-20ºC.
4.2.3.4 Liquid Chromatography Electrospray Ionization Mass Spectrometry of Protein
Hydrolysates
Protein hydrolysates and in gel protein digests from flaxseed protein fractions;
albumin, globulin, glutelin and a commercial soybean isolate were dissolved in 0.2%
formic acid, vortexed, centrifuged (5,000xg, 5 min) and filtered through a membrane
filter (0.45 µm, ChromSpec, Brockville, ON, Canada). The liquid chromatography
electrospray ionization mass spectrometric (LC- ESI-MS/MS) analysis was done using
the mass spectrometer coupled to a Waters Cap LC system, operated at a flow rate of 6
µL/min. A splitter placed before the column delivered a final flow rate of 0.3 µL/min.
The guard column consisted of Water Symmetry 300 Nano Ease C18, 5 µm, and
separation was achieved by Waters Atlantis DC18 (3 µm, 75 µm x 50 mm) column. A
76
binary gradient of solvent B (acteonitrile: 0.1% formic acid) and solvent A (water: 0.1%
formic acid) was increased from 5 to 50% in 25 min and the injection volume was 5 µL.
The mass range, m/z for MS/MS survey was 50-1990, scanned continuously over the
chromatographic run (Waters Corporation manual, 2005). The calibration and tuning of
the mass spectrometer was performed with [Glu]-Fibrinopeptide B (Sigma Chemicals Co;
St. Louis, MO). Data analysis and control of the instrument was carried out by
ProteinLynx Global Server v2.1 and MassLynx v4.0 (Waters Corporation, 2005).
4.2.3.5 Mass Spectra Interpretation and Database Search
The mass spectra interpretation, molecular weight estimation and possible sequence of
peptides from the hydrolysates were carried out using ProteinLynx Global server v2.1
(Waters Corporation, 2005), MassLynx and the Mascot software (Perkins et al., 1999).
The acquired peaklists of LC-ESI-MS/MS data were created by ProteinLynx software and
transformed into PKL format and submitted to Mascot (matrix Science, Boston, USA) for
identification of proteins and peptides against the green plants (Viridiplantae). The PKL
data format contained the MS/MS data that have to be searched against a FASTAformatted protein bank (SwissProt protein database, Swiss Institute of Bioinformatics,
2009). Mascot search parameters used were carobxamidomethyl cysteine as fixed
modification, oxidation as variable modification, ±1.2 Da peptide mass tolerance and
±0.6 Da fragment mass tolerance and the enzyme entry was set as trypsin. All peptide
masses were obtained as monoisotopic masses. The Mascot scoring is based on the
77
probability that a peptide identified from the experimental fragment matched a peptide in
a protein database.
4.3 Results and Discussion
4.3.1 Enzymatic Hydrolysis
Figures 4.1 (A,B) and 4.2 (A,B) show the results of the degree of hydrolysis (DH) for
the flaxseed protein fractions hydrolysates and soybean protein hydrolysate during the
120 min period. The rate of hydrolysis increased during an initial period then reached a
steady state after 60 min. The DH of the hydrolysis of the protein fractions ranged from
9.4-24.5%. Glutelin hydrolysate showed the highest rate of hydrolysis of 24.5% DH,
followed by albumin hydrolysate of 16.0% DH and globulin hydrolysate of 9.4% DH; the
DH of the commercial soybean protein hydrolysate was 14.1%. The DH was also
measured for control samples (samples of protein isolates run without enzyme), albumin
fraction showed almost unchanged level of 3.8-4.2% DH, globulin 1.6-1.8% DH, glutelin
showed 3.6-5.4% DH and soybean showed 4.6-5.4% DH. Our findings showed partial
agreement with the results of enzymatic hydrolysis of flaxseed proteins reported by other
workers and this can be explained as due to (a) the difference in the purity of the proteins
used and (b) the effect of removing mucilage from flaxseed during the hydrolysis, (c) the
availability and solubility of protein molecules that can be attacked by the proteolytic
enzyme and (d) other factors such as the time of hydrolysis, enzyme to substrate ratios
and the differences in sensitivity of the methods used for determination of DH. Marame et
al. (2008) reported that a range of 12 to 70% DH was observed with the use of
78
Flavourzyme (EC 232-752-2; leucine amino peptidase) to hydrolyze proteins from
flaxseed. The maximum DH (70%) was achieved when the protein was hydrolysed at an
enzyme to substrate ration 80 (LAPU/g of protein; leucine amino peptidase units) for 20
h. A DH of 42.2% was observed with the use of sunflower protein as substrate for
Flavourzyme within 3 h of hydrolysis (Villanueva et al., 1999). The DH increased with
increase in enzyme/substrate ratio and time of hydrolysis. Degree of hydrolysis of
proteins from other oilseeds such as soybean was found to be 39.5% upon 8 h of
hydrolysis with the use of Flavourzyme (Hrckova et al., 2002). Wu et al. (2004) reported
that 54% DH was obtained using Alcalase enzyme from the hydrolysis of flaxseed meal
without purifying the proteins. Peng et al. (2009) reported that whey protein isolate was
hydrolysed for 0.5–8 h using Alcalase, reached DH of 35–36% after 5 h of hydrolysis.
79
Degree of hydrolysis (%)
20
A
16
12
8
4
0
0
30
60
90
Degree of hydrolysis (%)
12
120
B
8
4
0
0
30
60
Time (min)
90
120
Figure 4.1: Degree of tryptic hydrolysis of protein fractions from flaxseed (A)
albumin hydrolysate,
, control globulin
, control albumin
.
80
and (B) globulin hydrolysate
28
A
Degree of hydrolysis (%)
24
20
16
12
8
4
0
0
30
60
90
120
24
B
Degree of hydrolysis (%)
20
16
12
8
4
0
0
30
60
Time (min)
90
120
Figure 4.2: Degree of tryptic hydrolysis of protein fractions from flaxseed (A) glutelin
hydrolysate
, control glutelin
control soybean protein
.
; and (B) soybean protein hydrolysate
81
,
4.3.2 Gel Electrophoresis of Flaxseed Protein Hydrolysates and In-Gel Digests
Figure 4.3 shows Native-PAGE of isolated protein fraction, controls (samples run
without enzyme) and enzyme hydrolyzed protein fractions from flaxseed; albumin,
globulin and glutelin. The isolated protein fractions; albumin (1), globulin (4) and glutelin
(7) gave two bands of different electrophoretic mobility. The electrophoretic pattern of
control protein samples of albumin (2), globulin (5) and glutelin (8) differ from the
isolated protein samples showing a change in mobility of the bands with more intensity
compared to isolated protein fractions. The electrophorgram of the enzymatic
hydrolysates of albumin (3), globulin (6) and glutelin (9), showed only one band in
albumin hydrolysate, and no bands in globulin hydrolysate and one less intense band in
glutelin hydrolysate. The results showed that both the heat treatment and enzyme have
effect on the protein composition and structure.
Figure 4.4 shows the SDS-PAGE patterns of isolated protein fractions, controls
(samples run without enzyme) and enzyme hydrolyzed protein fractions from flaxseed;
albumin, globulin and glutelin. The isolated albumin fraction (1) gave two intense bands
corresponding to 31 and 35 kDa and one band at 50 kDa, isolated albumin globulin (4)
gave two intense bands corresponding to 25 and 33 kDa, and isolated albumin glutelin (7)
gave two intense bands at 50 and 31 kDa and three minor bands with MW of 60, 90 and
100 kDa. The control protein sample of albumin (2) showed the same patterns except
with the appearance of an additional band at 11 kDa, control protein samples of globulin
(5) gave two intense bands corresponding to 25 and 33 kDa and control protein samples
of glutelin (8) contained a similar profile to the control with a formation of faint band at
10 kDa. The enzymatic hydrolysate of albumin (3) showed one band of 35 kDa and two
82
bands of 16 and less than 10 kDa; enzymatic hydrolysate of globulin (6) showed only two
faint bands between 14 and 7 kDa, the enzymatic hydrolysate of glutelin (9) showed the
disappearance of all bands except one band between 25-45 kDa. The protein bands of 33
and 10 kDa were visible in all the fractions indicating their resistance to tryptic
hydrolysis. It was also observed the formation of new low molecular weight species of
less than 14 kDa in all protein isolates indicating the breakdown of the high molecular
weight proteins into smaller polypeptides. The enzyme treatment has caused high effect
on protein fractions due to proteolytic degradation. The SDS-PAGE results indicate that
the three protein fractions were substantially hydrolyzed although the DH data indicated
hydrolysis ranging from 9.4% to 24.5%. Marame et al. (2008) observed that the high
molecular weight protein band between 34 and 43 kDa from flaxseed was found to be
resistant to enzymatic hydrolysis by Flavourzyme. It was also reported by the same
authors that the high molecular weight protein band disappeared after DH value of
11.94%, while the low molecular weight band disappeared after 25.13% DH. However,
new polypeptide bands were observed between 43 and 11 kDa due to the hydrolytic
activity of the enzyme.
83
kDa
M
1
2
3
4
5
6
7
8
9
669
440
232
140
66
`
Albumin (1,2,3)
Globulin (4,5,6)
Glutelin (7,8,9)
Figure 4.3: Native-PAGE of isolated protein fractions; isolated albumin fraction (1),
control albumin (2), enzymatic hydrolysate of albumin (3), isolated globulin fraction
(4) control globulin (5), enzymatic hydrolysate of globulin (6), isolated glutelin
fraction (7) and control glutelin (8), enzymatic hydrolysate of glutelin (9), and
standard protein markers (M).
84
kDa
M
1
2
3
4
5
6
7
8
9
200
116
97
66
45
31
21
14
Albumin (1,2,3)
Albumin (4,5,6)
Albumin (7,8,9)
Figure 4.4: SDS-PAGE of isolated albumin fraction (1), control albumin (2),
enzymatic hydrolysate of albumin (3), isolated globulin fraction (4) control globulin
(5), enzymatic hydrolysate of globulin (6), isolated glutelin fraction (7) and control
glutelin (8), enzymatic hydrolysate of glutelin (9), and standard protein markers (M).
85
kDa
M
1
2
3
4
200
116
97
66
FC1
45
31
FA1
FA2
21
FA3
FB1
FC2
FB2
FB3
FC3
FS1
FS2
FS3
14
7
Figure 4.5: Electrophoreogram of selected spots of flaxseed protein fractions used
for in-gel digestion; albumin (1), globulin (2), glutelin (3), soybean (4) and
standard protein marker (M).
4.3.3 RP-HPLC Separation of Protein Hydrolysates
Figures 4.6 (A,B,C), 4.7 (A,B,C), 4.8 (A,B,C) and 4.9 (A,B,C) show chromatograms
obtained from RP-HPLC patterns of protein isolates, control samples (protein samples run
without enzyme) and enzymatic hydrolysates of flaxseed proteins; albumin, globulin and
glutelin
and a commercial soybean protein. The chromatograms demonstrated the
disappearance of the protein composition which was observed in the non-hydrolyzed and
control protein profiles with the formation of many hydrolysis products. There were
86
differences in the peptide profiles of the hydrolysates. The albumin isolate showed two
peaks of 26.2 min and 25.1 min, control albumin fraction showed the presence of two
peaks at 17.5 and 20.2 while the enzymatic hydrolysate of albumin gave 12 peaks but
with no dominant peak with majority peptides appeared in the range of 20 to 25 min; the
globulin isolate gave one dominant peak at retention time of 24.9 min and one minor peak
at 30.4 min, globulin control sample gave two peaks at 17.0 and 19.0 min while the
hydrolysis has resulted in the formation of 13 fractions with two dominant components at
retention time of 25 min and 26 min; the glutelin isolate exhibited a major peak at 25.0
min and two minor peaks at 18.0 and 26.2 min, the control glutelin fraction showed three
peaks at 15.3, 17.2 and 20.1 min and the enzymatic hydrolysate of glutelin gave 12 peaks
with several dominant peaks at retention time ranged from 25 to 30 min. Soybean protein
isolate showed three peaks at 17.0, 20.3 and 22.3 min, control soybean protein showed
three peaks at 15.3, 17.2 and 20.1 min, and the soybean hydrolysate gave 13 peaks with
two dominant peaks at retention time of 25 and 27 min. Compared to globulin and
glutelin, albumin contains less hydrophobic, more polar and less strongly adsorbed
peptides; it is likely that these peptides from albumin are short peptides with basic
character and have less adsorption and interaction with the column support (Janssen et al.,
1984). The variation in retention time of the hydrolysates can be attributed to the
difference in the amino acids released and their interaction, ionization state with
separating column as well as the localization of charges and the hydrophobicity of
composing amino acids (Lemieux and Amiot, 1989). Yeboah et al. (1999) reported that
the hydrolysis of proteins isolates from large lima bean and navy bean resulted in the
formation of several peptides within the first five minutes of hydrolysis. Ahmarani (2006)
87
reported that RP-HPLC analysis of fermented soybean flour resulted in the formation of
7-8 peaks with retention time of 3.4-14.8 min. Adebiyi et al. (2008) reported that a
selected fraction rice bran hydrolysates subjected to RP-HPLC resolved into 52 fractions.
88
2
A
Absorbance (215nm)
1
1
2
B
6
8
45 7
2
3
1
C
9
10
11
12
RT (min)
Figure 4.6: RP-HPLC chromatograms (A) albumin isolate; (B) control albumin; and (C) hydrolyzed albumin fraction.
89
1
A
2
Absorbance (215 nm)
1
B
2
10
9 11
678
1
2 3
45
12 13
C
RT (min)
Figure 4.7: RP-HPLC chromatograms of (A) globulin isolate; (B) control globulin and (C) hydrolyzed globulin
fraction.
90
2
1
Absorbance (215 nm)
A
3
1
B
3
2
4
7
5 6
1
23
4
8
9
10
C
11
12
RT (min)
Figure 4.8: RP-HPLC chromatograms of (A) glutelin isolate; (B) control glutelin and (C) hydrolyzed glutelin
fraction.
91
2
Absorbance (215 nm)
1
A
3
2
1
3
B
7
1
2 3
4 5 6 89
10
11
12
13
C
RT (min)
Figure 4.9: RP-HPLC chromatograms of (A) soybean protein isolate; (B) control soybean protein and (C) hydrolyzed
soybean protein.
92
4.3.4 Identification of Peptides from Protein Hydrolysates Using LC-ESI-MS/MS
LC-ESI-MS mass spectral analysis was conducted on samples of hydrolysis products
from the albumin, globulin and glutelin fractions. The LC-ESI/MS spectral data were
processed with ProteinLynx, MassLynx and Mascot database search engine
(http://www.matrixscience.com).
Figure 4.10 shows LC-ESI-MS/MS spectra of tryptic hydrolyzed albumin fraction. By
comparing the sequence of the identified tryptic peptides from albumin fraction with
fragments of flaxseed proteins published through Mascot database, the origin of some
identified peptides was traced to conlinin and chitinase IV proteins from flaxseed. Figures
4.13 (A) and (B) show peptide sequence from albumin hydrolysate fraction that has
indicated homology of KQIQEQDYLRS (AA 53-61, MW 1191.6 Da) with peptide
sequence from conlinin protein extracted from flaxseed (Accession # AJ414733.1) and
peptide sequence RDPVLAWRT (AA 54-75, MW 856 Da) from chitinase IV extracted
from flaxseed (Accession # AJ414733.1).
Figure 4.11 shows LC-ESI-MS/MS spectra of tryptic hydrolyzed globulin fraction. By
comparing the sequence of the identified tryptic peptides from globulin fraction with
fragments of flaxseed proteins published through Mascot database, the origin of some
identified peptides was traced to conlinin protein from flaxseed. Figure 4.14 shows
peptide sequence homology of RQDIQQQGQQQEVERW (AA 121-134, MW 1712.8
Da) with peptide sequence from conlinin protein extracted from flaxseed (Accession #
CAC94011).
93
Figure 4.12 shows LC-ESI-MS/MS spectra of tryptic hydrolyzed glutelin fraction. By
comparing the sequence of the identified tryptic peptides from glutelin fraction with
fragments of flaxseed proteins published through Mascot database, the origin of some
identified peptides was traced to conlinin protein from flaxseed. Figure 4.15 shows
peptide sequence homology with KQIQEQDYLRS (AA 51-61, MW 1191.6 Da) and
RQDIQQQGQQQEVERW (AA 121-134, MW 1713.8 Da) with peptide sequences from
conlinin protein extracted from flaxseed (Accession # CAC94011). All other identified
peptides from albumin, globulin and glutelin tryptic hydrolyzed fractions and the origins
of these peptides are listed in Table 4.1.
94
Figure 4.10: LC-ESI-MS/MS spectra of enzymatic hydrolyzed albumin fraction isolated from defatted flaxseed meal. inset
represents the fragmentation spectra of the peptide RDPVLAWRT from chitinase IV (m/z 429, MW 855 Da).
95
Figure 4.11: LC-ESI-MS/MS spectra of enzymatic hydrolyzed globulin fraction isolated from defatted flaxseed meal. inset
represents the fragmentation spectra of the peptide RQDIQQQGQQQEVERW from conlinin (m/z 857.4, MW 1712.8 Da).
96
Figure 4.12: LC-ESI-MS/MS spectra of enzymatic hydrolyzed glutelin fraction isolated from defatted flaxseed meal. inset
represents the fragmentation spectra of the peptide KQIQEQDYLRS from conlinin (m/z 597, MW1191 Da).
97
Figure 4.13: (A) Sequences of peptides derived from albumin hydrolysate showing the possible cleavage location and homology
according to the tryptic peptides identified from conlinin protein from flaxseed (Truksa et al. 2003, accession number:
ABA39179). The peptide sequence is underlined.
1MAKLMSLAAVATAFLFLIVVDASVRTTVIIDEDTNQGRGGQGGQGQQQQC
51EKQIQEQDYLRSCQQFLWEKVQKGGRSYYY NQGRGGGQQS QHFDSCCDDL
101KQRSECTCRGLERAIGQMRQDIQQQGQQQ EVERWVQQAK QVARDLPGQC
151GTQPSRCQLQ GQQQSAWF
Figure 4.13: (B) Sequences of peptides derived from albumin hydrolysate showing the possible cleavage location and homology
according to the protein isolates from chitinase IV from the flaxseed (Petrovska et al. 2005, accession number: CAC94011).
The peptide sequences are underlined.
Peptide I:
1HVTHETGSMC YIEEINKAEY CDRSRYPCAQ GKRYYGRGPL QLTWNYNYQE AGKANGFDGV
61 ANPDIVARDP VLAWRTALWF WMTNVRAVLP QGFGATIRAI
Peptide II:
1 HVTHETGSMC YIEEINKAEY CDRSRYPCAQ GKRYYGRGPL QLTWNYNYQE AGKANGFDGV
61 ANPDIVARDP VLAWRTALWF WMTNVRAVLP QGFGATIRAI
98
Figure 4.14: Sequences of peptides derived from globulin hydrolysate showing the possible cleavage location and homology
according to the tryptic peptides identified from conlinin protein from flaxseed (Truksa et al. 2003, accession number:
CAC94011). The peptide sequence is underlined.
1 MAKLMSLAAV ATAFLFLIVV DASVRTTVII DEDTNQGRGG QGGQGQQQQC
51 EKQIQEQDYL RSCQQFLWEK VQKGGRSYYY NQGRGGGQQS QHFDSCCDDL
101 KQLRSECTCR GLERAIGQMR QDIQQQGQQQ EVERWVQQAK QVARDLPGQC
151 GTQPSRCQLQ GQQQSAWF
Figure 4.15: Sequences of peptides derived from glutelin hydrolysate showing the possible cleavage location and homology
according to the protein isolates from conlinin protein from flaxseed (Truksa et al. 2003, accession number: CAC94011). The
peptide sequences are underlined.
Peptide I:
1 MAKLMSLAAV ATAFLFLIVV DASVRTTVII DEDTNQGRGG QGGQGQQQQC
51 EKQIQEQDYL RSCQQFLWEK VQKGGRSYYY NQGRGGGQQS QHFDSCCDDL
101 KQLRSECTCR GLERAIGQMR QDIQQQGQQQ EVERWVQQAK QVARDLPGQC
151 GTQPSRCQLQ GQQQSAWF
Peptide II:
1 MAKLMSLAAV ATAFLFLIVV DASVRTTVII DEDTNQGRGG QGGQGQQQQC
51 EKQIQEQDYL RSCQQFLWEK VQKGGRSYYY NQGRGGGQQS QHFDSCCDDL
101 KQLRSECTCR GLERAIGQMR QDIQQQGQQQ EVERWVQQAK QVARDLPGQC
151 GTQPSRCQLQ GQQQSAWF
99
Table 4.1: Proteins and peptides detected in protein hydrolysates from flaxseed by
LC-ESI-MS/MS following trypsin digestion of isolated protein fractions
Origin
Albumin
Conlinin
(Linum usitatissimum)
Globulin (Cucurbita maxima)
Glutelin (Elaeis guineensis)
Chitinase
IV
(Linum
usitatissimum)
Globulin
Conlinin (Linum usitatissimum
Predicted protein (Populous
trichocarpa)
Japonica cultivar-group (Oryza
sativa
Putative (Ricinus communis)
Glutelin
Conlinin
(Linum
usitatissimum)
Hypothetical
vinifera)
protein
AA residue
MW
(Da)
53 – 61
1191.6
317 – 323
412 – 424
54 – 68
817.4
1375.7
855.5
RADVFNPRG
KTIDNAMVNTIVGKA
RDPVLAWRT
69 – 75
1514.2
KANGFDGVANPD IVARD
121 – 134
93 – 104
1712.8
1242.7
RQDIQQQGQQQEVERW
RGLLLPAYSNAPKL
869 – 875
873.4
RNWSILDKY
162 – 168
873.4
RLWSILDKS
51-61
1191.6
KQIQEQDYLRS
1713.8
1252.6
RQDIQQQGQQQEVERW
KAIDEQFVTSDKV
1640.8
KFLKAIDEQFVTSDKV
121 – 134
(Vitis 256- 269
259-269
100
Peptide sequence
KQIQEQDYLRS
Figure 4.16 (A): LC-ESI-MS/MS spectra of in-gel digested albumin fraction from flaxseed (FA1). Inset represents the
fragmentation spectra of the peptide FFLAGNPQRQ (m/z 525, MW 1048.5 Da).
101
Figure 4.16 (B): LC-ESI-MS/MS spectra of in-gel digested albumin fraction from flaxseed (FA2). Inset represents the
fragmentation spectra of the peptide AMPQLAASAPVPATALATPGKPPR (m/z 1772.4, MW 2313.1 Da).
102
Figure 4.16 (C): LC-ESI-MS/MS spectra of in-gel digested albumin fraction from flaxseed (FA3). Inset represents the
fragmentation spectra of the peptide IQSMPKLALK (m/z 578.3, MW 1155.6 Da).
103
Figure 4.17 (A): LC-ESI-MS/MS spectra of in-gel digested globulin fraction from flaxseed (FB1). Inset represents the
fragmentation spectra of the peptide VTRWSAASLSPR (m/z 673.9, MW 1345.8 Da).
104
Figure 4.17 (B): LC-ESI-MS/MS spectra of in-gel digested globulin fraction from flaxseed (FB2). Inset represents the
fragmentation spectra of the peptide LPSTISKIELGGMLSK (m/z 558.6, MW 1672.8 Da).
105
Figure 4.17 (C): LC-ESI-MS/MS spectra of in-gel digested globulin fraction from flaxseed (FB3). Inset represents the
fragmentation spectra of the peptide QKLLSAQK (m/z 458.3, MW 914.5 Da).
106
Figure 4.18 (A): LC-ESI-MS spectra of in-gel digested glutelin fraction from flaxseed (FC1). Inset represents the
fragmentation spectra of the peptide LIEGGLTPLAPKPVPPRFR (m/z 724.4, MW 2170.1 Da).
107
Figure 4.18 (B): LC-ESI-MS spectra of in-gel digested glutelin fraction from flaxseed (FC2). Inset represents the
fragmentation spectra of the peptide ELAVQIHSMIQNLAQFTDIK (m/z 772.4, MW 2313.1 Da).
108
Figure 4.18 (C): LC-ESI-MS spectra of in-gel digested glutelin fraction from flaxseed (FC3). Inset represents the
fragmentation spectra of the peptide EDGAPAAIVDR (m/z 557.3, MW 1112.5 Da).
109
Figure 4.19 (A): LC-ESI-MS spectra of in-gel digested soybean protein isolate (FS1). Inset represents the fragmentation
spectra of the peptide LLANASGAMSFAVIVP (m/z 780.9, MW 1559.8 Da).
110
Figure 4.19 (B): LC-ESI-MS spectra of in-gel digested soybean protein isolate (FS2). Inset represents the fragmentation
spectra of the peptide RSQSDNFEYVSFKT (m/z 725.8, MW 1449.6 Da).
111
Figure 4.19 (C): LC-ESI-MS spectra of in-gel digested soybean protein isolate (FS3). Inset represents the fragmentation
spectra of the peptide RLSAEFGSLRKN (m/z 554.3, MW 1106.6 Da).
112
4.3.5 Identification of Peptides With Potential Bioactivity from In-Gel Tryptic Digests
Using LC-ESI-MS/MS
Figure 4.16 (A,B,C) shows the LC-ESI-MS/MS spectra of in-gel digests of albumin
fraction (FA1, FA2, FA3). Example of possible bioactive peptide from FA1 is
FFLAGNPQRQ MW of 1048.5 Da (Res 1-9) with a predicted biological activity of ACE
probably due to the presence of LA dipeptide (Dziuba and Darewicz, 2007). An example
of possible bioactive peptide from FA2 subunit digest is a peptide sequence of
AMPQLAASAPVPATALATPGKPPR with a MW of 2313.1 Da (Res 3240-3263) with a
possible bioactivity of ACE and antioxidant. This is likely due to the presence of
dipeptide sequence of LA and VP (Nakamura et al., 1995; Dziuba and Darewicz, 2007).
An example of possible bioactive peptide from FA3 is VAEETTGYTGK with a MW of
1154.5 Da (Res 492-502) with a possible bioactivity of ACE and antioxidant activities
that might be expected due to the presence of dipeptide of VA (Dziuba and Darewicz,
2007).
Figure 4.17 (A,B,C) shows the LC-ESI-MS/MS spectra of in-gel digests of globulin
fraction (FB1, FB2, FB3). An example of possible bioactive peptide from FB1 is
VTRWSAASLSPR with a MW of 1345.8 Da (Res 18-29) with a predicted biological
activity of ACE. This is likely due to the presence of the dipeptide sequence LA and PR
(Nakamura et al., 1995; Dziuba and Darewicz, 2007). FB2 globulin fraction contains
many peptide sequences that may have the potential to be bioactive. An example of one
of these peptides is LPSTISKIELGGMLSK with a MW of 1672.8 Da (Res 559-574) with
a possible bioactivity of ACE-inhibitory activity. This is likely due to the presence of
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amino acids sequence LP (Wang et al. 2009). FB3 globulin fraction may contain some
peptide sequences that may have the potential to be bioactive. An example of possible
bioactive peptide from FB3 is QKLLSAQK with a MW of 914.5 Da (Res 853-860). It has
the possibility of antioxidant activity. This is likely due to the presence of amino acid
sequence LL (Chen et al., 1998).
Figure 4.18 (A,B,C) shows the LC-ESI-MS/MS spectra of in-gel digests from glutelin
fraction (FC1, FC2, FC3). FC1 from glutelin contain a peptide sequence of
LIEGGLLTPLAPKPVPPRFR with a MW of 2170.1 Da (Res 493-512) with a predicted
biological activity of ACE. This is likely due to the presence of amino acid sequence LL,
LA, PR (Nakamura et al., 1995; Dziuba and Darewicz, 2007). FC2 contain a peptide
sequence of ELAVQIHSMIQNLAQFTDIK with a MW of 2313.1 Da (Res 187-206) with
a possible bioactivity of ACE. This is likely due to the presence of dipeptide LA (Dziuba
and Darewicz, 2007). FC3 contains a peptide sequence of IGIIPSWGLSVK with a MW
of 1112.5 Da (Res 58-68) with a possible bioactivity of antimicrobial activity. This is
likely due to the presence of dipeptide sequence II (Wang et al., 2009).
Figure 4.19 (A,B,C) shows the LC-ESI-MS/MS spectra of in-gel digests of soybean
protein (FS1, FS2, FS3). FS1 from Soybean contain a peptide sequence of
LLANASGAMSFAVIVP with a MW of 1559.8 Da with a possible bioactivity of
antimicrobial activity. This is likely due to the presence of dipeptide sequence LL (Wang
et al., 2009). FS2 contains a peptide sequence of RSQSDNFEYVSFKT with a MW of
1449.6 Da. FS3 contains a peptide sequence of RLSAEFGSLRKN with a MW of 1106.6
Da.
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4. 4.1 Conclusion
Flaxseed protein fractions showed different degrees of hydrolysis for glutelin, globulin
and albumin. Glutelin showed the highest degree of hydrolysis, followed by albumin and
globulin. SDS-PAGE of protein hydrolysates showed different peptides profile as
compared to the profile before hydrolysis. RP-HPLC chromatograms of protein
hydrolysates suggest differences in the peptide profile of the albumin, globulin and
glutelin fractions with difference in terms of number of peaks and peak intensities.
Analysis of LC-ESI-MS/MS spectra of the in-vitro and in-gel hydrolysis products showed
that flaxseed proteins contain peptide sequences that are of physiological importance. The
liberated peptides were investigated using proteomic database search and matched with
the online published sequencing information which has resulted in detection of possibility
of biological active peptides of different activities such as antioxidant, antihypertensive
and antifungal effects.
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CHAPTER 5
BIOLOGICAL PROPERTIES OF FLAXSEED PROTEIN FRACTIONS
5.1 Justification
Chapter three addressed the molecular, thermal and structural properties of albumin,
globulin and glutelin fractions from defatted flaxseed meal, chapter four described invitro tryptic hydrolysis in-gel digested of protein fractions. Many health effects have been
reported to be associated with flaxseed consumption including decreased risk of heart
disease, antiviral activity, antibacterial activity, antifungal activity, laxative effect, antiinflammatory activity (Oomah, 2001). Preliminary investigation on flaxseed proteins has
shown the formation of angiotensin converting enzyme inhibitory (ACEI) activity and
hydroxyl radical scavenging activity by protein hydrolysates (Wu et al., 2004; Marambe
et al., 2008). This chapter will address the potential application of hydrolysates of the
albumin, globulin and glutelin fractions for antihypertensive, antioxidant and antifungal
activities.
5.2 Materials and Methods
5.2.1 Materials
Angiotensin converting enzyme (ACE) from rabbit, Hippuryl-L-Histidyl-L-Leucine
(HHL),
HEPES
(4-(2-hydroxyethyl)
piperazine-1-ethanesulfonic
acid,
N-(2-
hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl
(DPPH) were obtained from Sigma-Aldrich (Oakville, Ontario). Cultures of Pencillium
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camemberti produced on potato dextrose agar (PDA). Their spores were collected and
inoculated as described by Perraud (2000). Potato dextrose agar was obtained from BDH
(Darmstadt, Germany).
5.2.2 Preparation of Flaxseed Protein Isolates and Tryptic Digests
Flaxseed protein isolates and protein hydrolysates of albumin, globulin and glutelin,
and commercial soybean protein were prepared as described in Section 3.2.3 and Section
4.2.2. The extracts were centrifuged (10,000xg, 30 min, 4-8C). The supernatants were
freeze-dried and resuspended in distilled water prior to use.
5.2.3 Evaluation of Selected Biological Activities
5.2.3.1 Assay of Antioxidant Activity
The free radical scavenging activity was measured using 2,2-diphenyl-1picrylhydrazyl (DPPH) as a stable radical compound according to the method of Silva et
al. (2000). DPPH reacts with the antioxidant and it is reduced as result of hydrogen
donation by antioxidant and the color changes from violet to yellow. Samples of protein
isolates and protein hydrolysates were prepared as mentioned in Section 4.2.2.1,
centrifuged and filtered in a membrane filter (0.45 µm, ChromSpec, Brockville, ON,
Canada) and mixed with an ethanolic DPPH solution. The reduction of DPPH was
followed spectrophotometrically by the decrease of absorbance at 517 nm over a period
of 15 min against a blank without DPPH, control samples contained only ethanol. The
antioxidant activity was expressed as percentage of inhibition which was calculated as
117
(absorbance of the control minus the absorbance of the sample)/ absorbance of the control
x100. All assays were conducted in triplicates.
5.2.3.1.1 Statistical Analysis
Independent sample Students t-test and multiple comparison tests using Tukey's post
were used to compare differences among the proteins and hydrolysates. A p value < 0.05
was considered statistically significant. Data is presented as means ±SD. The
experimental design was completely randomized design. Data were analyzed using GLM
SAS (2003).
5.2.3.2 Determination of Total Phenolic Compounds Content
The total phenolic content of the protein fractions and enzymatic hydrolysates was
determined by the Folin-Ciocatleu spectrohotmetric method as described by Alu'datt
(2006). A standard curve was prepared using a stock solution of gallic acid (50 mg/50
mL). 1 mL of the extract containing phenolic compounds was added to 7.5 mL of distilled
water and followed by 0.5 mL Folin-Ciocatleu reagent. After 4 min, 1 mL of 5% sodium
carbonate (Na2CO3) was added. The contents were mixed, and after 1 h the absorbance
was measured at 725nm.
5.2.3.3 Assay of Antifungal Activity
Assay of antifungal activity against P. camemberti was carried out using a hyphal
extension-inhibition assay as described by Borgmeyer et al. (1992). The assay of protein
fractions and the protein hydrolysates for antifungal activity was executed using 100 mm
x 15 mm Petri dishes containing 10 mL of potato dextrose agar. After the mycelial colony
118
had been developed, sterile blank paper disks (0.625 cm in diameter) were placed around
and at a distance of 1 cm away from the rim of the mycelial colony. An aliquots of 50100 μL containing 50 μg of the protein extract or hydrolysate in sterile water was loaded
on a disk. The plates were incubated at 23ºC for 5-7 days until mycelial growth had
enveloped peripheral disks and had produced crescents of inhibition around disks
containing samples with antifungal activity.
5.2.3.4 Assay of Angiotensin Converting Enzyme (ACE) Inhibition
Determination of ACE inhibitory activity was performed according to the method of
Cushman and Cheung (1970) and as modified by Ahmarani (2006). A 100 µL sample of
freeze dried protein fractions and protein hydrolysates (5%, w/v) were added to 200 µL of
Hip-His-Leu solution (0.3% w/v), mixed and centrifuged. The reaction was started by
adding 50 µL of enzyme solution to the substrate and protein and hydrolysates extracts.
The reaction mixture was mixed by swirling and incubated for 15 minutes at 37°C.The
reaction was terminated by adding 250 µL of 1 M HCl. An amount (2 mL) of ethyl
acetate was added to the reaction mixture, shake vigorously for 60 seconds and then
centrifuged (3000 x g, 2 min) to extract librated hippuric acid. After centrifugation, 1 mL
of the upper ethyl acetate layer was transferred into a clean tube and evaporated by
heating in boiling water for 15 min, 3 mL distilled water was added and mixed with the
remaining residue after the removal of ethyl acetate. The absorbance of the samples and
blanks was determined at 228 nm. The amount of hippuric acid released from the reaction
in the absence of an inhibitor was defined as 100% ACE activity (Ahmarani, 2006).
119
Calculations:
1. Enzyme activity can be calculated from the following equation, and expressed as
U/mL.
(ABS test sample - ABS blank)(2)(3)
(9.8)(15)(0.91)(0.05)
Where,
2 = Conversion factor for the detected hippuric acid (1/2 of the total amount produced)
3 = Total volume of hippuric acid solution
9.8 = Millimolar extinction coefficient of hippuric acid at 228 nm
15 = Time (min) of the assay as per the unit definition
0.91 = Extraction efficiency of ethyl acetate
0.05 = Volume (mL) of enzyme used
The rate of inhibition was calculated as follows:
ACE inhibitory activity (%) =100-ACE activity (%)
5.3 Results and Discussion
5.3.1 Antioxidant Activity and Phenolic Compounds Content
Figures 5.1 (A,B) and 5.2 (A,B) show the results of the antioxidant activity assay of 5
and 10 mg /ml of digested and non-hydrolyzed flaxseed protein fractions and nonhydrolyzed commercial soybean protein isolate. The hydrolyzed protein fractions from
120
flaxseed and soybean exhibited a range of 45-90% DPPH radical scavenging ability in
comparison to the 31-81% of DPPH radical scavenging ability observed with nonhydrolyzed protein fractions of 5 mg/ml. Hydrolyzed glutelin indicated the highest
inhibition of 90% followed by hydrolyzed soybean (85.3%) ; hydrolyzed globulin
(83.4%) and hydrolyzed albumin (69.3%). There was a significant (P < 0.05) decrease in
DPPH scavenging activities at all subsequent incubation times. Non-hydrolyzed globulin
showed the highest inhibition of 81.0% at the initial time of the reaction, followed by
non-hydrolyzed albumin of 78.0%; non-hydrolyzed soybean of 67.0% and nonhydrolyzed glutelin of 63.6%. The rate of DPPH scavenging activity was fluctuated for
all tested proteins during time of the incubation (0-15 min) and it decreased to a level of
31%.
At 10 mg/ml, the antioxidant level exhibited a range of 40-89% of DPPH radical
scavenging ability for hydrolyzed proteins in comparison to the 17-93% of DPPH radical
scavenging ability observed with non-hydrolyzed proteins. Hydrolyzed albumin gave the
highest inhibition of 89%, followed by hydrolyzed globulin of 86%; hydrolyzed globulin
and hydrolyzed glutelin of 58%. The scavenging activity decreased gradually with the
increasing time of the incubation (0-15 min). Non-hydrolyzed globulin gave the highest
inhibition of 83% at the initial time of the reaction, followed by non-hydrolyzed soybean
of 73%; non-hydrolyzed albumin of 58% and non-hydrolyzed glutelin of 45%. Soybean
protein extract demonstrated antiradical activity of 62% against DPPH (Lee et al., 2002).
Chang (2007) reported that hydrolyzed soybean protein showed a range of 47-72% of
antioxidant activity. Marambe et al. (2008) reported that an antioxidant activity of 12.5–
24.6% for flaxseed proteins (0.5 mg/ml) hydrolyzed with Flavourzyme, while no
121
antioxidant activity has been detected in the non hydrolyzed proteins, these authors
suggest that the precursor protein was responsible for generating peptides with highest
antioxidant activity could be from the flaxseed protein around 43–55 kDa and the protein
around 6.5–11.4 kDa. Table 5.1 shows the total phenolic compounds content of flaxseed
protein fractions and the tryptic hydrolysates. The results indicate that the hydrolyzed
glutelin fraction contain the highest amount of phenols 3.73%, followed by hydrolyzed
albumin of 3.09%, hydrolyzed soybean of 2.64 and finally hydrolyzed globulin of 1.70%,
while the non-hydrolyzed glutelin contains the highest percentage of phenols of 1.87%,
non-hydrolyzed soybean protein isolate contains 1.70%, non-hydrolyzed globulin
contains 1.11% and non-hydrolyzed albumin protein isolate contains 1.10%. The results
suggest that flaxseed protein fractions and their hydrolysates possibly contained phenolic
substances that can act as hydrogen donors and could react with free radicals to covert
them to more stable products and terminate the radical chain reaction. Chen et al. (1998)
reported that one or more of the soybean proteins components including isoflavones and
peptides could be responsible for its antioxidant activity. Hu et al. (2007) reported that the
lignans in flaxseed exhibited strong antioxidant and protective effects in quenching the
DPPH. Aludatt (2007) reported antioxidant activity of 15-35% from bound phenolic
compounds extracted from protein isolates of defatted flaxseed meal, 30-35% full fat
flaxseed meal, and 16-32% for full fat soybean meal. Peng et al. (2009) reported that a
whey protein hydrolysate and its peptide fractions showed antioxidant activity ranging
between 14.7- 58.7%. Udenigwe et al. (2009) reported that flaxseed protein hydrolysates
showed 82.3-89.9 % of antioxidant activity.
122
Table 5.1: Total phenolic compounds content (mg/ml gallic acid) in flaxseed and
soybean protein fractions and hydrolysates
Phenolic content
( %)
Protein fraction
Non-hydrolyzed albumin
1.10 (±0.05)a
Hydrolyzed albumin
3.09 (±0.07)
Non-hydrolyzed globulin
1.11(±0.05)
Hydrolyzed globulin
1.81(±0.03)
Non-hydrolyzed glutelin
1.87(±0.06)
Hydrolyzed glutelin
3.73 (±0.09)
Non-hydrolyzed soybean protein isolate
1.70 (±0.04)
Hydrolyzed soybean protein
2.64 (±0.08)
a
Results are means ± standard deviations of triplicates
123
Antioxidant Activity (%)
A
ALB2
ALH1
ALB1
ALH2
100
B
Antioxidant Activity (%)
80
GLB2
GLH2
GLB1
GLBH1
60
40
20
0
0
3
6
9
12
15
Time (min)
Figure 5.1: Antioxidant activity of (A): (10 mg/mL) non-hydrolyzed albumin (ALB1)
,hydrolyzed albumin (ALBH1); (5 mg/mL) non-hydrolyzed albumin (ALB2),
hydrolyzed albumin (ALBH2). B: (10 mg/mL) non-hydrolyzed globulin (GLB1),
hydrolyzed globulin (GLBH1); (5 mg/mL) non-hydrolyzed globulin (GLB2),
hydrolyzed globulin (GLBH2).
124
Antioxidant Activity (%)
A
GLTH2
GLTH1
GLT2
GLT1
B
Antioxidant Activity (%)
SOYH2
SOY1
SOY2
SOYH1
Time (min)
Figure 5.2: Antioxidant activity of (A): (10 mg/mL) non-hydrolyzed glutelin (GLT1)
, hydrolyzed glutelin (GLTH1); (5 mg/mL) non-hydrolyzed glutelin (GLT2),
hydrolyzed glutelin (GLTH2). B: (10 mg/mL) non-hydrolyzed soybean (SOY1),
hydrolyzed soybean (SOYH1); (5 mg/mL) non-hydrolyzed soybean (SOY2),
hydrolyzed soybean (SOYH2).
125
5.3.2 Antifungal Activity
Table 5.2 shows the results of antifungal activity of the protein fractions. The highest
antifungal activity was observed with hydrolyzed glutelin with protein concentration of
62 µg/disk and the lowest effect was observed with non hydrolyzed albumin with protein
concentration of 30 µg/disk suggesting antifungal activity of the protein fractions and
their hydrolysates. However, these antifungal peptides may have differences concerning
their specificity and inhibitory spectrum towards target fungi species. Vigers et al. (1991)
and Borgmeyer et al. (1992) reported on the isolation of antifungal protein linusitin from
flaxseed; the MW of this protein was estimated to be 25 kDa and was reported to strongly
inhibit the growth of Alternaria solani, Alternaria alternate, Candida albicans,
Penicillium chrysogenum, Fusarium graminearum, and Aspergillus flavus (Xu et al.,
2006).
Table 5.2: Growth inhibition of P. camemberti treated with different flaxseed protein
fractions and soybean protein hydrolysates
Protein fraction
Antifungal activitya
MICb
Non-hydrolyzed albumin
+
30
Hydrolyzed albumin
+
35
Non-hydrolyzed globulin
+
40
Hydrolyzed globulin
++
35
+
50
+++
62
Non-hydrolyzed soy protein
+
45
Hydrolyzed soy
++
66
Non-hydrolyzed glutelin
Hydrolyzed glutelin
a
Denotes (+++) high; (++) moderate; (+) low activity, bMinimum inhibition
concentration (MIC) is expressed as micrograms of protein content used per disk.
126
5.3.3 Angiotensin Converting Enzyme (ACE) Inhibitory Activity
Figure 5.3 shows the difference in ACE inhibitory activity among the flaxseed protein
fractions and soybean protein. All hydrolysates showed ACE inhibitory activity between
49-62%. The most potent ACE inhibitory activity was shown by hydrolyzed glutelin at
62.5%, followed by hydrolyzed globulin at 60.2%, and hydrolyzed albumin at 47.5% and
hydrolyzed soybean at 54.3%. ACE has affinity towards aromatic amino acids (Trp, Tyr
and Phe) or with the amino group (Pro). It has been suggested that difference in the
inhibitory activity of protein hydrolysates is related to difference in peptide size and
amino acid composition and sequence of these fractions (Ahn, 2001). It is possible that
glutelin hydrolysate is richer in proline content in its C-terminal compared to other
hydrolysates or a peptide sequence containing a pro-pro or Ala-Pro sequence at its Cterminal so they can competitively bind the active site on the enzyme (Gibbs, 1999). Food
proteins from some sources shown some ACE inhibitory activity even before exposed to
enzymatic hydrolysis, these proteins include bovine casein, gelatin, maize protein and
muscle all of which are reported to have ACE inhibitory activity (Meisel et al., 1997). Wu
et al. (2004) reported that proteolytic hydrolysis of flaxseed meal (without purifying its
protein) generates a hydrolysate with (ACEI) activity. Marambe et al. (2008) reported
that ACE inhibitory peptides with activity ranging between 71.59–88.29% was obtained
from flaxseed protein hydrolysates. Ahmarani (2006) reported that an ACE inhibition of
66-85% was observed from the fermentations of soybean flour.
127
80
70
ACE Inhibition (%)
60
50
40
30
20
10
Albumin
Globulin
Glutelin
Y
YH
SO
SO
LT
G
H
G
LT
LB
G
H
LB
G
A
LB
H
A
LB
0
Soybean
Protein Fractions
Figure 5.3: Angiotensin converting enzyme (ACEI) inhibitory activity of flaxseed
protein fractions; (ALB) non-hydrolyzed albumin; (HALB) hydrolyzed albumin;
(GLB) non-hydrolyzed globulin; (HGLB) hydrolyzed globulin; (GLT) nonhydrolyzed glutelin; (HGLT) hydrolyzed glutelin and (SOY) non-hydrolyzed
soybean; (HSOY) hydrolyzed soybean.
128
5.4.1 Conclusion
The enzymatic digests of the protein fractions from flaxseed demonstrated different
biological effects among protein fractions. The highest antioxidant activity was observed
using hydrolyzed glutelin fraction and the lowest with non-hydrolyzed glutelin fraction;
higher antifungal activity against Penicillium camemberti was detected with hydrolyzed
glutelin fraction and a lower activity with albumin fraction. Inhibition of ACE was
determined to be higher with the hydrolyzed glutelin fraction and lower with nonhydrolyzed glutelin.
129
CHAPTER 6
GENERAL CONCLUSIONS
6.1 CONCLUSIONS
The importance of flaxseed (Linum usitatissimum L) and flaxseed proteins as a source
of nutrients and their status as functional foods has become recognized; this has resulted
in the need for the study of physicochemical, structural and biological properties of
flaxseed proteins. In this research, the properties of the individual protein fractions from
defatted flaxseed and the effects of tryptic hydrolysis of the protein fractions prepared
from defatted flaxseed meal were investigated. This research study has provided more
information about the importance of flaxseed and its proteins as potential source of
bioactive ingredients for functional foods. It has established that the hydrolysis of
flaxseed protein fractions can be resulted in the production of protein digests with
different biological properties.
This study investigated the extraction, isolation and characterization of protein
fractions albumin, globulin and glutelin from defatted flaxseed meal. The highest protein
yield was obtained by water extraction followed by extraction with sodium hydroxide and
sodium chloride. Native-PAGE profile showed the presence of two major components in
each protein fractions. Albumin and glutelin contain a band which is likely to be a linin,
while the second band that appears in albumin to be a conlinin. SDS-PAGE results
showed the presence of two intense bands and one minor band for albumin; globulin
showed one intense band and four minor bands, and glutelin showed the presence of two
intense bands and three minor bands. Two intense bands corresponding to 22 and 24 kDa
and two bands corresponding to 9 and 33 kDa were observed from albumin; globulin
130
showed one intense band at 23 kDa and three minor bands with MW of 10, 24 and 33
kDa; glutelin showed the presence of two intense bands with a MW 22 and 35 kDa and
three minor bands with MW of 35, 45 and 55 kDa. ESI-MS analysis of flaxseed protein
fractions revealed that both albumin and glutelin contained two protein subunits and
glutelins showed the presence of one subunit.
The albumin, globulin and glutelin fractions were separated by RP-HPLC and
their MWs identified by SDS-PAGE, and ESI-MS. RP-HPLC results confirmed that the
globulin, glutelin and albumin fractions are heterogeneous proteins; the albumin fraction
showed two peaks, a dominant peak at retention time of 26.2 min and one minor peak at
25.1 min; the globulin fraction gave one dominant peak at retention time of 24.9 min and
one minor peak at 30.4 min; glutelin exhibited a major peak at 25.0 min and two minor
peaks at 18.0 and 26.2 min. SDS-PAGE of the major fractions from RP-HPLC ( albumin
AF2, globulin BF1 and glutelin CF2) showed the presence of three bands from albumin;
globulin fraction indicated the presence of two bands, and glutelin fraction exhibited the
presence of four bands. The major fractions from albumin (AF2) showed two bands at 35,
14 and 7 kDa; globulin fraction (BF1) indicated the presence of two bands at 10 and 16
kDa and glutelin fraction (CF2) exhibited the presence of two bands at 42, 40, 16 and 7
kDa.
The secondary structure of the flaxseed protein fractions was investigated by studying
their FTIR absorption spectrum. FTIR deconvoluted spectra indicated that -helix
assigned band (1650-1652 cm-1) was observed in all protein fractions. Albumin showed
the presence of 4 bands assigned as β-sheet (1691 cm-1), antiparallel β-sheet (1682 cm-1),
β-turns (1662 cm-1) and -helix (1652 cm-1). Globulin showed three bands of 1693, 1682
131
and 1650 cm-1 assigned as β-sheet, antiparallel β-sheet and -helix, respectively. Glutelin
showed the presence of three bands at 1682 cm-1 assigned as antiparallel β-sheet, 1662
cm-1assigned as β-turns and 1652 cm-1 assigned as -helix.
DSC results showed the high thermal stability of the flaxseed protein fractions.
Albumin showed an endothermic peak at maximum peak temperature (Td) of 122.3C,
globulin gave a peak with a Td value of 118.3C and glutelin fraction a peak with Td value
of 106.8C.
Investigation of degree of hydrolysis of the flaxseed proteins suggested different
degrees of hydrolysis for glutelin, globulin and albumin. Glutelins showed the highest
degree of hydrolysis, followed by albumin and globulin. SDS-PAGE of protein
hydrolysates showed different peptides profile as compared to profile before hydrolysis.
RP-HPLC chromatograms of protein hydrolysates suggest differences in the peptide
profile of the albumin, globulin and glutelin fractions with difference in terms of number
of peaks and peak intensities. Analysis of LC-ESI-MS/MS spectra of the in-vitro and ingel hydrolysis products and liberated peptides using proteomic database search and
matching the results
with the online published sequencing information showed the
possibility of biological active peptides of different activities such as antioxidant,
antihypertensive and antifungal effects.
Evaluation of selected biological activities of flaxseed protein fractions and their
hydrolysates demonstrated that there was a significant difference among the protein
fractions. The highest antioxidant activity was observed using hydrolyzed glutelin
fraction and the lowest was with non-hydrolyzed glutelin fraction. A higher antifungal
activity against P. camemberti was detected with hydrolyzed glutelin fraction and a lower
132
activity with albumin fraction. Inhibition of ACE was determined to be higher with the
glutelin fraction and lower with hydrolyzed globulin.
The overall results of this study demonstrated the potential role of the individual
flaxseed protein fractions and their tryptic hydrolysis in the process of forming biological
active ingredients that have the value to be used in nutraceutical/functional food
applications.
133
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