Natural Antioxidants: Maximizing Effectiveness for Shelf

Natural Antioxidants:
Maximizing Effectiveness for
Shelf-life Extension
Fereidoon Shahidi
University Research Professor
Department of Biochemistry
Memorial University of Newfoundland
St. John’s, NL, A1B 3X9, Canada
[email protected]
October 29, 2013
www.GlobalFoodForums.com/CleanLabel
ANTIOXIDANTS
Antioxidants are compounds that when present
in food or in the body at very low
concentrations, they delay , control or prevent
oxidative processes leading to food quality
deterioration or initiation and propagation of
degenerative diseases in the body.
Antioxidants in Food and nonFood Applications






These are generally phenolic and polyphenolic in
nature
They are synthetic or natural
They are effective at low concentrations
They are non-toxic
They generally have good carry-through
properties
They are reasonable in cost
Antioxidants
Primary antioxidants: They are good free radical
scavengers and reducing agents
*Synthetic: BHA, BHT, TBHQ, PG
*Synthetic/Natural: ascorbic acid,
tocopherol
* Natural: mixed tocopherols, rosemary,
sage, green tea
Secondary antioxidants: EDTA, citric acid, etc
Antioxidants…
Synthetic antioxidants
OH
OH
C(CH3)3
C(CH3)3
CH2OH
OCH3
OCH3
3-BHA
2-BHA
H C OH
O
OH
(H3C )3C
OH
OH
C(CH3)3
HO
CH2COO(CH2)14CH3
H C OH
O
OH
Ascorbic acid
BHT
COOR
Gallate
O
C( CH3)3
OH
HO
CH3
O
OH
TBHQ
HO
OH
Ascorbyl palmitate
ANTIOXIDANTS IN FOOD



Consumers now demand clean labels with no
artificial ingredients
Retailers expect longer shelf life and more
stability
Food processors can meet the needs of both
groups by using plant-derived natural extracts
Antioxidants…

Natural antioxidants
OH
OH
HO
COOH
HO
CH CH COOH
p-coumaric acid
p-hydroxybenzoic acid
OH
HO
OH
O
HO
O
OH
OH
OH
C H3O
HO
COOH
Vanillic acid
HO
HO
C O OH
HO
Syringic acid
Epigallocatechine
OH
OH
Caffeic acid
OH
OH
HO
HO
O
O
C H C H C OO H
OH
C H 3O
OH
Epicatechine
CH CH COOH
C H 3O
C H3O
HO
OH
O
OH
O
OH
C
O
Ferulic acid
OH
OH
C
O
OH
OH
OH
Epicatechine gallate
OH
Epigallocatechine
gallate
Antioxidants…

Natural antioxidants
OH
HO
HOOC
OH
HO
R1
O C
HO
O
R2
Carnosic Acid
HO
O C
C OO H
O
Tocopherols
R1
HO
R2
O
CH3
OH
Rosmanol
OH
OH
O
O
CH3
Carnosol
OH
O
Rosmarinic acid
Tocotrienols
Over 5000 polyphenolics have been identified in
different plants
Number of publications
7000
6000
Total
5000
4000
No of Publications
24, 021
3000
2000
1000
Number of publications related to polyphenols/antioxidants (1989-2011)
(SciFinder)
2011
2010
2009
2008
2007
2006
2005
2004
2003
2002
2001
2000
1999
1998
1997
1996
1995
1994
1993
1992
1991
1990
0
Plant Phenolics





They are present in plants to protect them
against herbivours, attack by micro-organisms,
and other preditors
To protect plants from stress under sunlight
To participate in wound-healing process
To attract polinators
Others
Biosynthesis of Phenolics
OH
HO
Calvin cycle
CH2CHCOOH
COOH
NH2
Phenylalanine
Shikimic
acid
pathway
OH
Phenols
Shikimic acid
-CO2
Hydroxybenzoic acid
Quinones
HO
-C2
CH
p- Hydroxycinnamic acid
+
-CO2
o-Oxidation
Reduction
3 Malonate
HO
Reduction
Polymerization
Condensed tannins
Hydroxyacetophenones
Hydroxycoumarins
-CO2
Flavonoids
-NH3
CHCOOH
Xanthones
Hydroxystilbenes
CH
CHCH2OH
p - Hydroxycinnamyl alcohol
-O
Phenylpropenes
Dimerization
Lignans
Polymerization
Lignins
Antioxidants are important components
of Functional Foods and Occur as:








Phenolic acids (benzoic acid derivatives)
Phenylpropanoids (cinnamic acid derivatives)
Tocols (tocopherols and tocotrienols)
Flavonoids
Isoflavones
Coumarins
Tannins (condensed and hydrolysable)
Others (eg. carotenoids, phospholipids, amino
acids and protein hydrolysates, vitamin C, etc)
Mechanisms of Action of Phenolics









Antioxidant effect & direct removal of ROS/RNS or
potentiation of cellular antioxidant defense capacity
Effect on cell differentiation
Increased activity and modulation of enzymes
involved in detoxification, oxidation and reduction
processes
Blocking the formation of N-nitrosamines
Alter estrogen metabolism & colonic milieu
Increase apoptosis of cancerous cells and maintain
normal DNA repair
Affect DNA methylation
Decrease cell proliferation
Preserve integrity of intracellular matrices and
strengthening immune system by regulating gene
expression, cell signaling and hormone metabolism
Lipid Oxidation
• Autoxidation
• Photooxidation
• Thermal Oxidation
• Enzymatic Oxidation
Oxidation Triangle
O2
Lipid
Initiator
Lipid Autoxidation
RH
Dimers, polymers
. HydroperoxiCyclic Peroxides
I
n
i
t
i
a
t
i
o
n
ROO
Propagation
R
.
RH
Acyclic & cylic compounds
ROOR and ROR Dimers
Aldeydes
Aldehydes
Ketones,furans,
acids
ROOH
RO
.
OH
Alkyl radicals
Semialdehydes
bis-allylic
allylic
 88 kcal/mol
R
 75 kcal/mol
H
H
H
R’
H
H
H
H
Alkyl
 101 kcal/mol
H H
H
Olefinic
 105 kcal/mol
All Foods Have Metals
•
Fe > Cu
-- Cu is 50 fold more reactive than iron
•
Iron
-- Ferrous (Fe 2+) is 1013 to 1017 more soluble than
ferric (Fe 3+)
-- Ferrous is over 100 times more reactive than ferric
Major aldehydes formed from decomposition of
hydroperoxides
Fatty Acid
Oleic
CH2 Groups
Aldehydes
11
Octanal, 2-decenal
8
nonanal, 2-undecenal
Linoleic
11
Hexanal, 2-octenal, 2,4-decadienal
Linolenic
14
Propanal, 2-pentenal, 2,4-heptadienal
11
3-hexenal, 2,4-decadienal, 2,4,7decatrienal
13
Hexanal, 2-octenal, 2,4- decadienal
10
3-nonenal, 2,4-undecadienal, 2,4,7tridecatrienal
7
3,6-dodecadienal, 2,5,8-tetradecatrienal,
2,4,7,10-hexadecatetraenal
Arachidonic
PHOTOOXIDATION: Photooxidation or photooxygenation
requires singlet oxygen, produced
from triplet oxygen, by interaction
of light and a sensitizer.
Sensitizers may be:
Chlorophyll
Erythrosine
Rose bengal
Methylene blue
Sen* + 3o2
Sen + hν
R2
Sen + 1o2
R2
H
H
O
O
R1
O
R1
O
This reaction is unaffected by antioxidants, but is inhibited by
quenchers of singlet oxygen such as β-carotene
Autoxidation
8-OOH Δ9 (26%)
10-OOH Δ8 (23%)
9-OOH Δ10 (24%)
11-OOH Δ9 (26%)
Photooxidation
9-OOH Δ10 (50%)
10-OOH Δ8 (50%)
AUTOXIDATION VERSUS PHOTOOXIDATION OF OLEATE
RELATIVE RATES OF OXIDATION
Fatty acid
Autoxidation
Photooxidation
1
30 x 103
C18:2
50
40 x 103
C18:3
100
70 x 103
C18:1
Threshold Values of Compounds Formed from
Oxidized Oils
Compounds
Threshold (ppm)
Hydrocarbons
90-2150
Substituted furans
2-27
Vinyl alcohols
0.5-3
1-Alkenes
0.02-9
2-Alkenals
0.04-2.5
Alkanals
0.04-1.0
trans,trans-2,4-Alkadienals
0.04-0.3
Isolated alkadienals
0.002-0.3
Isolated cis-alkenals
0.0003-0.1
trans,cis-Alkadienals
0.002-0.006
Vinyl ketones
0.00002-0.007
Natural Antioxidant Extracts



Rosemary – Has FDA GRAS status (21 CFR
182.10) ; it contains carnosic acid and carnosol;
it is used in meat, poultry and seafood products,
edible oils, snack foods, sauces, dairy products
Green tea – It contains catechins; it can be used
in the same products listed for rosemary
Mixed tocopherols
Rosemary
(Rosmarinus officinalis L.)

Rosemary extract
Antioxidants
Carnosic acid
Carnosol
Rosmarinic acid
 Aromatic “essential oils”
Cineol or Eucalyptol
Pinene
Camphor

Green tea
(Camellia sinensis L.)

Extracts of immature tea leaves are rich in
flavanols and their gallic acid derivatives


(+)-catechin, (–)-epicatechin, (+)-gallocatechin, (–)epicatechin gallate, (–)-epigallocatechin, and (–)epigallocatechin gallate
Natural green-tea flavor components,

terpenes, oxygenated terpenes, sesquiterpenes, and
organic acids
 Catechins (flavan-3-ols) predominate in green tea
• (-)-epicatechin (EC, 6.4%)
• (-)-epicatechin gallate (ECG, 13.6%)
• (-)-epigallocatechin (EGC, 19%)
• (-)-epigallocatechin gallate (EGCG, 59%)
Natural Tocopherols



These are usually a mixture from deodorizer
distillate
The most abundant and commonly used one is
that from soybean oil processing
It contains mainly gamma-, delta- and alphatocopherol
Method of Incorporation
Addition of oil-soluble/dispersible liquid
antioxidants into freshly deodorized oil
Method of Incorporation
Method of incorporating water-soluble/dispersible
antioxidants into a product
Method of Incorporation
Method of incorporating powdered antioxidants into
a product
Application Opportunities with
Rosemary and Green Tea
Extracts
Effect of antioxidants on the
inhibition of TBARS in chilled (4C)
turkey burgers
Effect of Different Antioxidants on the TBARS
Values of Cooked White Muscle of Mackerel
Stored at 4C
Effect of green-tea extract on the
inhibition of TBARS in chilled
(4C) roasted turkey burgers
Effect of green-tea extract and rosemary
extract on the inhibition of
hexanal in chilled (4C) roasted beef
burgers
Effect of green-tea extract and rosemary
extract on the inhibition of
hexanal in chilled (4C) roasted beef
burgers
Effect of Different Antioxidants on the TBARS
Values of Cooked White Muscle of Mackerel
Stored at 4C
Peroxide values (meq/kg) of a treated fish
oil with tea extracts and stored at 60 C
Additive
Control
Polyphenon G(P-25;500)
Polyphenon G(P-60;500)
Nikken polyphenon 60
(NPP-60; 500)
Storage time at 60 C, days
1
7
11.0
10.5
8.1
8.2
31.6
61.8
43.0
40.9
Benefits of Green tea extracts








Natural alternative to synthetic antioxidants
Promotes good health
Extended food product shelf-life
Retards development of off-flavors and off-odors
Consistent product quality
Easy to handle and apply
Low cost-in-use
Availability of optimized tailor-made solutions
 Epigallocatechin gallate (EGCG)
1. The most predominant catechin in tea: 90 mg/cup
2. Prepared by solvent extraction of crude catechin mixture
followed by purification processes: CPC, HSCCC, HPLC,
HPCE, etc.
3. Highly hydrophilic
4. Low bioavailability
5. Powerful antioxidant: free radical scavenger, reducing
agent, metal chelator, Redox enzyme inhibitor, etc
6. Potential food preservative and pharmaceutical agent
Oxidative stress
Oxidative stress
(UV, smoke, metabolic diseases)
Mitochondrial dysfunction
Mitochondria
Oxygen radical species
(ROS) production
DNA mutations &
deletions
Lipid
peroxidation
Apoptosis/
Necrosis
Cancer, neurodegenerative diseases, aging
Interest in conceiving antioxidants that would target
mitochondria
Strategies to target mitochondria
Phenolic Antioxidants are generally too polar for optimized
membrane crossing
VEHICLES
- Micelles, liposomes
ROS
ROS
COVALENT MODIFICATIONS
ROS
-Covalent bonding of lipid moieties
-(fatty alcohols, fatty acids, acylglycerols)
- to antioxidant molecules (e.g. phenolics)
PHENOLIPIDS
Phenolipids



As the name implies, phenolipids include a
phenolic compound and a lipid molecule joined
together via covalent bonding
The phenolic compound could be a simple
phenolic or a polyphenolic in nature
The lipid could be a simple lipid fatty acid, a
triacylglycerol or a phospholipid; these are
perferably of those with health benefits
H3C
COOH
Eicosapentaenoic acid (EPA, 20:5n-3)
H3C
COOH
Docosahexaenoic acid (DHA, 22:6n-3)
Omega-3’s EPA/DHA Benefits
O
EGCG
+ RCOOH
OH
O
R
O
O
R
O
O
O
HO
OH
CO
OC
O
O
OH
O
O
R
(EGCG ester)
R
OH
DPPH radical scavenging capacity
µmol TE/g
EGCG
217.77 ± 0.43
EGCG tetra-esters
217.90 ± 0.04
•EGCG esters
•TE: Trolox equivalents
Inhibition against LPS-induced NO production in RAW 264.7
macrophage
• LPS-stimulated NO production monitored
• % inhibition by EGCG and its derivatives calculated
• IC50 (concentration to achieve 50% inhibition) obtained for
anti-inflammatory activity evaluation
IC50:
EGCG > EGCG derivatives
Anti-inflammatory activity:
EGCG derivatives > EGCG
Blood channel
Thrombus
Atherosclerotic Artery
Cross
section
Plaque
progression
Plaque
rupture
Table 2. Inhibition (%) of EGCG and its derivatives against copperinduced LDL-cholesterol oxidation.
% inhibition
EGCG
EGCG-SA
EGCG-EPA
EGCG-DHA
6.53 ± 0.92 c
16.33 ± 1.07 a
11.80 ± 2.26 ab
8.18 ± 2.90 bc
Results
Effect of EGCG-ester and EGCG-DHA on colon
tumors multiplicity in AOM-treated ICR mice
Adenoma
Adenocarcinoma
1. Hepatitis C virus (HCV)
• 170 million people worldwide are chronically infected with HCV
• associated with liver cirrhosis, hepatic failure and hepatocellular
carcinoma
• current approved therapies show limited virologic response rates
and side effects
F1
F5
F2
F6
F3
F7
F4
Figure 4 - Schematic diagram showing pearling action
Trolox equivalent antioxidant capacity (TEAC)
60
Falcon
Metcalfe
50
40
30
20
10
0
F1
F2
F3
F4
F5
F6
F7
Fractions
Figure 6 - Trolox equivalent antioxidant capacity of Falcon and
Metcalfe barley fractions
ORAC (μmol TE/g defatted material)
Oxygen Radical Scavenging Capacity (ORAC)
200
Falcon
Metcalfe
180
160
140
120
100
80
60
40
20
0
F1
F2
F3
F4
F5
F6
F7
Fraction
Figure 10 – Oxygen radical scavenging capacity of Falcon and
Metcalfe barley fraction (defatted material)
Effects of mustard flour (MF) on TBA
cooked comminuted meat
Additive
Fresh Stored (3 weeks)
No additive
MF (1%)
MF (2%)
NaNO2(150ppm)
6.16
1.14
0.31
0.32
10.28
2.54
0.37
0.43
Protein Hydrolyzates
Multiple bioactivities




Antioxidant activity
Angiotensin inhibitory activity
Immune-enhancing properties
Etc.
Antioxidative peptides from gelatin hydrolyzate of Alaska Pollack
skin in comparison with that of soy 7S protein.
Peptide
P1
P2
P1
P2
P3
P4
P5
P6
Amino acid sequence
ALASKA POLLACK SKIN
Gly-Glu-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-ProHyp-Gly-Pro-Hyp-Gly
Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-Pro-Hyp-Gly-ProHyp-Gly
Soy 7S PROTEIN
Val-Asn-Pro-His-Asp-His-Glu-Asn
Leu-Val-Asn-Pro-His-Asp-His-Glu-Asn
Leu-Leu-Pro-His-His
Leu-leu-Pro-His-His-Ala-Asp-Ala-Asp-Tyr
Val-Ile-Pro-Ala-Gly-Tyr-Pro
Leu-Glu-Ser-Gly-Asp-Ala-Leu-Arg-Val-Pro-Ser-GlyThr-Tyr-Tyr
Nutrient Content Claims Using the Term
“Antioxidant”
•
According to 21 CFR 101.54(g), the term
“antioxidant” can be used in a nutrient
content claim when:
-An RDI has been established for each of the
nutrients;
-The nutrients claimed have recognized
antioxidant activity;
-The level of each nutrient claimed is
sufficient to qualify for the nutrient content
claims
-The names of the nutrients claimed are
included as part of the claim (e.g., High in
antioxidant vitamins C and E)
AOs with DRIs or DRVs
Nutrient
RDI/DRV
Vitamin A (alpha-carotene, betacarotene, beta-cryptoxanthin)
500 IU
Vitamin C
60 mg
Vitamin E
30 IU
Riboflavin (Vitamin B2)
1.7 mg
Selenium
70 µg
Copper
2 mg
Manganese
2 mg
Zinc
15 mg
DRV = Daily Reference Value; RDI = Reference Daily Intake
Implications of 21 CFR 101.54(g)
•
•
Nutrients without RDIs/DRVs are not
recognized as AOs, even if they have AO
activity
Many FDA Warning Letters have been issued
due to the misuse of the term “antioxidant”
FDA Warning Letters Related to the
Improper Use of the Term “Antioxidant”
Company
Date
Product
Claim
Issue
Sunsweet
Growers Inc.
Feb. 22, 2010
Sunsweet®
“High antioxidant“,
Antioxidant
“Full of nutritious
Blend™ dried antioxidants"
fruit mix
Claim is in violation of 21
CFR 101.54(g)
Dr. Pepper
Snapple
Group
Aug. 30, 2010
Canada Dry
Sparkling
Green Tea
Ginger Ale
“Enhanced with 200
mg of antioxidants
from green tea and
vitamin C”
Product contained only 60
mg vitamin C; the
remaining 140 mg were
presumably from green tea
flavonoids, which do not
have recognized AO
activity according to 21
CFR 101.54(g)
Hardy
Peanuts, Inc.
July 15, 2011
Boiled
Peanuts and
Hot n’ Spicy
Boiled
Peanuts
“Four times more
antioxidants than raw
or roasted peanuts”
Claim is in violation of 21
CFR 101.54(g)
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