1. Characterization and identification of hypotensive,
immunomodulatory and metabolic disorder benefiting peptides from
Atlantic mackerel (Scomber scombrus) hydrolysate separated based
on molecular weight, charge and hydrophobicity
Dissertation Defense by
Soheila Abachi
June 2021
Supervisor: Prof. Lucie Beaulieu
Co-supervisor: Prof. Laurent Bazinet
2. Outline of presentation
Introduction
Hypothesis and objective
Design and analysis
Methodology, results and conclusion
(objectives 1, 2 & 3)
General conclusion
Future directions
Acknowledgment & references
2
5. Non communicable diseases: >50% of deaths
CVD: >30% of deaths
Diabetes: 592 million by 2035
Overweight: >1.9 billion adults
Obesity: 13% of population
T2D costs France:
Indirect €8.5 billion/y
Direct €19.5 billion (2003)
Inflammation cause or effect of metabolic diseases
Introduction: global issues
5
7. Introduction: treatment
Synthetic treatments
Side effects & non-compliance issue
Usually mono-functional
Natural treatments
Commonly pose no adverse effects
Food-derived biomolecules
Non-cytotoxic, generally dual- & multi-functional
Could be incorporated into diet or treatment regimen
In demand by health-conscious customers
7
8. Introduction: fish biopeptides
Byproducts, discards &
Underutilized species (e.g., Atlantic mackerel)
Different compositional features & properties
Some marketed as therapeutics
Beneficial on immunity, MetS & risk factors
Derived, separated & purified by various techniques
Mainly enzymatic hydrolysis, filtration & chromatography
Newly designed EDUF technique
8
10. Hypothesis
Atlantic mackerel (Scomber scombrus) bioactive-peptide fraction(s)
isolated and separated by different techniques based on various
molecular characteristics possess beneficial biological activity on
hypertension, inflammation and other metabolic syndrome (MetS)
factors.
10
11. Main objective
Fractionation and identification of hypotensive,
immunomodulating and anti-MetS peptides with different
molecular weight, charge and hydrophobicity characteristics using
chromatographic, membrane filtration & electro-separation
techniques
11
13. Design and analysis
Experimental design
Completely randomized design
Conducted in triplicates, 2-7 independent studies
Statistical analysis
Anderson-Darling Normality test (P > 0.05)
One way-ANOVA, Tukey’s multiple comparison (P < 0.05)
Student t-test for determination of significant differences between
control and treatment (P < 0.05)
13
14. OBJECTIVE 1
Isolation of immuno-modulatory biopeptides from Atlantic mackerel
(Scomber scombrus) protein hydrolysate based on molecular
weight, charge, and hydrophobicity
would be submitted for publication to the Journal Of Food Bioactives
14
15. Objective 1
Separation of anti- and pro-inflammatory peptides from hydrolysate using:
I- EDUF: MWCO 20 kDa (pH 3, 6 and 9)
II- Pressure driven UF: MWCO 1 kDa
III- SPE: hydrophobic peptides
15
16. Obj.1- Methodology: hydrolysis
Mackerel lysate provided by Merinov, QC, Canada
Enzymatic hydrolysis: protamex
E: S ratio 1: 1000
Process: 120 min.
Heat enzyme inactivation
200 – 10,000 Da
16
24. Obj.1- Results: migration rate
Isolation efficiency of EDUF
Effect of pH on migration pattern
Measured by BCA
Cationic
pH3 > pH6 > pH9
a
a
a
a
a
a
a
ab
ab
b
a a
b
b
b
0
250
500
0 30 60 120 180
Concentration
(µg
mL
-1
)
Time (min)
Cationic peptides
pH3 pH6 pH9
24
25. Obj.1- Results: migration rate
Isolation efficiency of EDUF
Effect of different pH on migration pattern
Measured by BCA
Cationic
pH3 > pH6 > pH9
Anionic
pH6 & pH9 > pH3
Total migration
pH3 > pH9 > pH6
a
a
a
b
b
a
a
a
a
ab
a
a
a
a
a
0
250
500
0 30 60 120 180
Concentration
(µg
mL
-1
)
Time (min)
Anionic peptides
pH3 pH6 pH9
25
26. Obj.1- Methodology: immunomodulation
activity
NO reduction and or stimulation effect
As index of iNOS activity
Based on Griess reaction
Read at 540nm
Positive control
Metformin and phenformin
% inflammation reported
J774A.1 macrophages
ATCC® TIB-67™
Overnight treatment of
LPS stimulated cells
Cytotoxicity of compounds
BCA assay
26
27. Obj.1- Results: anti- & pro-inflammatory
activity
Nitrite accumulation quantified as measure of NOS activity
pH3: pro-NO
*
*
*
0
100
LPS
2.5
ng
mL-1
P+3
1
ng
mL-1
P+3
1
ug
mL-1
P+3
10
ug
mL-1
P-3
1
ng
mL-1
P-3
1
ug
mL-1
P-3
10
ug
mL-1
F3
1
ng
mL-3
F3
1
ug
mL-1
F3
10
ug
mL-1
Phen
100
uM
%
inflammation
pH3
Cationic
peptides
Feed
recovery
Anionic
peptides
27
35. Obj.1- Results: peptide identification
EDUF: protein precursors mainly
Enzymes such as:
NADH dehydrogenase,
ATP synthase,
cytochrome oxidase & …
Tissue proteins such as:
α-actin, cardiac muscle & …
50 different precursor proteins
35
36. Obj.1- Conclusion
Hydrophobicity more vital for anti-inflammatory effect
Charge more vital for pro-inflammatory effect
pH3 more efficient in separating pro-inflammatory biopeptides
Negative charge more effectual on immuno-stimulating
attribute
Small size not important for immunoregulatory properties of
biopeptides
36
37. OBJECTIVE 2
Identification of gastrointestinal digestion stable antihypertensive
fish peptides from Atlantic mackerel (Scomber scombrus)
would be submitted for publication to the Journal Of Food Science And Human
Wellness
37
38. Objective 2
Separation of hypotensive peptides from hydrolysate using:
I- EDUF: MWCO 20 kDa (pH 3, 6 and 9)
II- Pressure driven UF: MWCO 1 kDa
III- SPE: hydrophilic and hydrophobic peptides
38
40. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
a
a
b
c
a
b
bc
c
a
a
a
a
a
b
b
b
40 20 10 5
a
a
b
c
a
ab
bc
c
a
b
c
d
a
b
c c
0
50
100
40 20 10 5
a
a
b
c
a
b
bc
c
a
a
b
b
a
ab
b
c
40 20 10 5
Total hydrolysate Feed recovery Positive peptides Negative peptides
pH3 pH9
pH6
Peptide concentration (µg)
%
Inhibition
40
41. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
UF could not improve anti-ACE effect of hydrolysate
UF
Peptide concentration (µg)
%
Inhibition
a
a
b
c
a
ab ab
b
a
a
b
b
0
50
100
40 20 10 5
Total hydrolysate
Retentate
Permeate
41
42. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
UF could not improve anti-ACE effect of hydrolysate
SPE enhanced bioactivity of hydrolysate
a
ab
bc
c
a
a
ab
b
a
ab
bc
c
a
ab
bc
c
0
50
100
40 20 10 5
Total hydrolysate
70% ACN
55% ACN
10% ACN
SPE
Fraction concentration (µg)
%
Inhibition
42
51. Obj.2- Conclusion
Most hydrophobic fraction having highest anti-ACE activity
Charge and size not as important for anti-hypertensive effects
Most potent anti-ACE fraction being GI digestion stable
Leucine rich motifs presumably vital for hypotensive effects
51
52. OBJECTIVE 3
Bioactivity of Atlantic mackerel peptides on obesity and insulin
resistance, an in-vivo study
would be submitted for publication to the Journal Of Food And Function
52
53. Separation of anti-obesity and anti-insulin resistance peptides from
hydrolysate using:
I- EDUF: MWCO 20 kDa (pH 3)
setting different from
objectives 1 & 2
Objective 3
53
54. Obj.3- Methodology: anti-MetS activity
In-vivo effects in diet-induced obese and diabetic rats
Effects compared against hypercaloric & or chow diet
Samples:
Hydrolysate & EDUF pH3 cationic peptide fraction
8-weeks experimental diet, 208 mg/kg BW
Modulation of obesity, dyslipidemia & diabetes markers
Food intake, total & organ weight gain, TG & TC, adiposity,
insulin & glucose levels
54
56. Obj.3- Results: in-vivo analysis
Insignificant effect on
Weight gain: total, organ & tissues
Body composition
Food intake
56
57. Obj.3- Results: in-vivo analysis
Insignificant effect on
Weight gain: total & organ
Body composition
Food intake
Glycemia & hyperinsulinemia
Dyslipidemia &
hepatic steatosis
57
58. Obj.3- Conclusion
Fish type dependent obesity and insulin resistance benefiting
effects
Immunomodulatory and hypotensive attributes not correlated
with anti-obesity and anti-IR activities of biopeptides
Possibly higher dosage required for tested bioactivities
58
59. General conclusion
Peptide migration rate rather pH dependent
Importance of separation technique in isolation efficiency of
biopeptides
In-vitro experiments:
59
60. General conclusion
Peptide migration rate rather pH dependent
Importance of separation technique in isolation efficiency of
biopeptides
In-vitro experiments:
Hydrolysate: pro-inflammatory & hypotensive
EDUF fractions: pro-inflammatory
SPE fractions: anti-inflammatory & hypotensive
UF fractions: insignificant effects
Non-cytotoxic biological activities
60
61. General conclusion
Peptide migration rate rather pH dependent
Importance of separation technique in isolation efficiency of
biopeptides
In-vitro experiments:
In-vivo experiments:
Insignificant on diet-induced obesity &
Metabolic syndrome associated risk factors
61
62. Future directions
Study of SAR of selected fractions
Further examination of hypotensive peptides in well controlled
animal & or cell studies on MetS risk factors
Purification of active peptide & in-depth characterization
Process optimization for efficient production of biomolecules
Incorporation of therapeutic biopeptide into commercial
products for general & clinical use
62
63. Acknowledgment
Supervisors:
Dr. Lucie Beaulieu
Dr. Laurent Bazinet
Supervisory committee members:
Dr. André Marette
Dr. Ismail Fliss
Funded by Natural Sciences & Engineering Council of Canada
(NSERC)
63