Lipids
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Lipids. Class Presentations Will be Discussed at the End of Class. Exams back next Monday No class this Wednesday !!!!!. Lipids. Main functions of lipids in foods Energy and maintain human health Influence on food flavor Fatty acids impart flavor Lipids carry flavors/nutrients

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Lipids

Lipids


Class presentations will be discussed at the end of class

Class Presentations Will be Discussed at the End of Class

Exams back next Monday

No class this Wednesday !!!!!


Lipids1

Lipids

Main functions of lipids in foods

  • Energy and maintain human health

  • Influence on food flavor

    • Fatty acids impart flavor

    • Lipids carry flavors/nutrients

  • Influence on food texture

    • Solids or liquids at room temperature

    • Change with changing temperature

    • Participation in emulsions


Lipids2

Lipids

  • Lipids are soluble in many organic solvents

    • Ethers (n-alkanes)

    • Alcohols

    • Benzene

    • DMSO (dimethyl sulfoxide)

  • They are generally NOT soluble in water

  • C, H, O and sometimes P, N, S


Lipids3

Lipids

  • Neutral Lipids

    • Triacylglycerols

  • Waxes

    • Long-chain alcohols (20+ carbons in length)

    • Cholesterol esters

    • Vitamin A esters

    • Vitamin D esters

  • Conjugated Lipids

    • Phospholipids, glycolipids, sulfolipids

  • “Derived” Lipids

    • Fatty acids, fatty alcohols/aldehydes, hydrocarbons

    • Fat-soluble vitamins


Lipids4

Lipids

Structure

  • Triglycerides or triacylglycerols

  • Glycerol + 3 fatty acids

  • >20 different fatty acids


Lipids 101

Lipids 101

  • Fatty acids- the building block of fats

  • A fat with no double bonds in it’s structure is said to be “saturated” (with hydrogen)

  • Fats with double bonds are referred to as mono-, di-, or tri- Unsaturated, referring to the number of double bonds. Some fish oils may have 4 or 5 double bonds (polyunsat).

  • Fats are named based on carbon number and number of double bonds (16:0, 16:1, 18:2 etc)


Lipids5

Lipids

  • Oil- liquid triacylglycerides “Oleins”

  • Fat- solid or semi-solid mixtures of crystalline and liquid TAG’s “Stearins”

  • Lipid content, physical properties, and preservation are all highly important areas for food research, analysis, and product development.

  • Many preservation and packaging schemes are aimed at prevention of lipid oxidation.


Nomenclature

Nomenclature

  • The first letter C represents Carbon

  • The number after C and before the colon indicates the Number of Carbons

  • The letter after the colon shows the Number of Double Bonds

  • ·The letter n (or w) and the last number indicate the Position of the Double Bonds


Saturated fatty acids

Saturated Fatty Acids


Lipids

Saturated Fatty Acids

Octanoic Acid


Mono unsaturated fatty acids

Mono-Unsaturated Fatty Acids


Poly unsaturated fatty acids

Poly-Unsaturated Fatty Acids


Lipids

Fatty Acids

Melting Points and Solubility in Water

Melting Point

Response

Solubility in H2O

z

2

Fatty acid chain length


Lipids

Unsaturated Fatty Acids

3 - Octenoic Acid

3, 6 - Octadienoic Acid


Lipids6

Lipids

Properties depend on structure

  • Length of fatty acids (# of carbons)

  • Position of fatty acids (1st, 2nd, 3rd)

  • Degree of unsaturation:

    • Double bonds tend to make them a liquid oil

      • Significantly lowers the melting point

    • Hydrogenation: tends to make a solid fat

      • Significantly increases the melting point

  • Unsaturated fats oxidize faster

  • Preventing lipid oxidation is a constant battle in the food industry


Lipids

Fatty Acids

Melting Points and Solubility in Water

Melting Point

Response

Solubility in H2O

z

2

Fatty acid chain length


Lipids

Fatty Acids

M.P.(C)

mg/100 ml

in H2O

C18

70

0.04

Characteristics of Fatty Acids

C4

- 8

C6

- 4

970

C8

16

75

C10

31

6

C12

44

0.55

C14

54

0.18

C16

63

0.08


Lipids

O

R C OH

O

R C OH

Fatty Acids

#1 Carbon

Acid Group

Polar End - Hydrophilic End

Non-polar End - Hydrophobic End

(Fat-soluble tail)


Lipids 1011

Lipids 101

  • Fatty acid profile- quantitative determination of the amount and type of fatty acids present following hydrolysis.

  • To help orient ourselves, we start counting the number of carbons starting with “1” at the carboxylic acid end.


Lipids 1012

Lipids 101

  • For the “18-series” (18:0, 18:1, 18:2, 18:3) the double bonds are usually located between carbons 9=10 12=13 15=16.


Lipids 1013

Lipids 101

  • The biomedical field started using the OMEGA (w) system (or “n” fatty acids).

  • With this system, you count just the opposite.

  • Begin counting with the methyl end

  • Now the 15=16 double bond is a 3=4 double bond or as the medical folks call it….an w-3 fatty acid


Tuning fork analogy tag s

Tuning Fork Analogy-TAG’s

  • Envision a Triacylglyceride as a loosely-jointed E

  • Now, pick up the compound by the middle chain, allowing the bottom chain to hang downward in a straight line.

  • The top chain will then curve forward and form an h

  • Thus the “tuning fork” shape

  • Fats will tilt and twist to the lowest free energy level


Lipids7

Lipids

  • Lipids are categorized into two broad classes.

  • The first, simple lipids, upon hydrolysis, yield up to two types of primary products, i.e., a glycerol molecule and fatty acid(s).

  • The other, complex lipids, yields three or more primary hydrolysis products.

  • Most complex lipids are either glycerophospholipids, or simply phospholipids

    • contain a polar phosphorus moiety and a glycerol backbone

  • or glycolipids, which contain a polar carbohydrate moiety instead of phosphorus.


Lipids8

Lipids


Other types of lipids

Other types of lipids

Phospholipids

  • Structure similar to triacylglycerol

  • High in vegetable oil

  • Egg yolks

  • Act as emulsifiers


Where do we get fats and oils

Where Do We Get Fats and Oils?

  • “Crude” fats and oils are derived from plant and animal sources

  • Several commercial processes exist to extract food grade oils

  • Most can not be used without first “refining” before they reach consumers

  • During oil refining, water, carbohydrates, proteins, pigments, phospholipids, and free fatty acids are removed. 

  • Crude fats and oils can therefore be converted into high quality edible oils

  • In general, fat and oil undergo four processing steps:

    • Extraction

    • Neutralization

    • Bleaching

    • Deodorization

  • Oilseeds, nuts, olives, beef tallow, fish skins, etc.

    • Rendering, mechanical pressing, and solvent extraction.


Fats and oils processing

Fats and Oils: Processing

Peanut

Extraction

  • Rendering

  • Pressing oilseeds

  • Solvent extraction

Rape Seed

Safflower

Sesame

Soybean


Fats and oils further processing

Fats and OilsFurther Processing

  • Degumming

    • Remove phospholipids with water

  • Refining

    • Remove free fatty acids (alkali + water)

  • Bleaching

    • Remove pigments (charcoal filters)

  • Deodorization

    • Remove off-odors (steam, vacuum)


Where do we get fats and oils1

Where Do We Get Fats and Oils?

  • Rendering

  • Primarily for extracting oils from animal tissues. 

  • Oil-bearing tissues are chopped into small pieces and boiled in water. 

  • The oil floats to the surface of the water and skimmed. 

  • Water, carbohydrates, proteins, and phospholipids remain in the aqueous phase and are removed from the oil. 

  • Degumming may be performed to remove excess phospholipids.

  • Remaining proteins are often used as animal feeds or fertilizers.


Where do we get fats and oils2

Where Do We Get Fats and Oils?

  • Mechanical Pressing

  • Mechanical pressing is often used to extract oil from seeds and nuts with oil >50%. 

  • Prior to pressing, seed kernels or meats are ground into small sized to rupture cellular structures. 

  • The coarse meal is then heated (optional) and pressed in hydraulic or screw presses to extract the oil.

  • Olive oils is commonly cold pressed to get extra virgin or virgin olive oil. It contains the least amount of impurities and is often edible without further processing.

  • Some oilseeds are first pressed or placed into a screw-press to remove a large proportion of the oil before solvent extraction.


Where do we get fats and oils3

Where Do We Get Fats and Oils?

Solvent Extraction

  • Organic solvents such as petroleum ether, hexane, and 2-propanol can be added to ground or flaked oilseeds to recover oil. 

  • The solvent is separated from the meal, and evaporated from the oil.

    Neutralization

  • Free fatty acids, phospholipids, pigments, and waxes exist in the crude oil

  • These promote lipid oxidation and off-flavors (in due time)

  • Removed by heating fats and adding caustic soda (sodium hydroxide) or soda ash (sodium carbonate). 

  • Impurities settle to the bottom and are drawn off. 

  • The refined oils are lighter in color, less viscous, and more susceptible to oxidation (without protection).

    Bleaching

  • The removal of colored materials in the oil.

  • Heated oil can be treated with diatomaceous earth, activated carbon, or activated clays.

  • Colored impurities include chlorophyll and carotenoids

  • Bleaching can promote lipid oxidation since some natural antioxidants are removed.


Where do we get fats and oils4

Where Do We Get Fats and Oils?

Deodorization

  • The final step in the refining of oils.

  • Steam distillation under reduced pressure (vacuum).

  • Conducted at high temperatures of 235 - 250ºC.

  • Volatile compounds with undesirable odors and tastes can be removed.

  • The resultant oil is referred to as "refined" and is ready to be consumed.

  • About 0.01% citric acid may be added to inactivate pro-oxidant metals.


Fats and oils further processing1

Fats and OilsFurther Processing

Hydrogenation

  • Add hydrogen to an oil to “saturate” the fatty acid double bonds

    • Conducted with heated oil

    • Often under pressure

    • In the presence of a catalyst (usually nickel)

  • Converts liquid oils to solid fats

  • Raises melting point


Hydrogenating vegetable oils can produce trans fats

Hydrogenating Vegetable oils can produce trans-fats

Cis-

Trans-


The cis and trans forms of a fatty acid

The cis- and trans- forms of a fatty acid


Fats and oils in foods

Fats and Oils in Foods

  • SOLID FATS are made up of microscopic fat crystals. Many fats are considered semi-solid, or “plastic”.

  • PLASTICITY is a term to describe a fat’s softness or the temperature range over which it remains a solid.

    Even a fat that appears liquid at room temperature contains a small number of microscopic solid fat crystals suspended in the oil…..and vice versa

  • PLASTIC FATS are a 2 phase system:

    • Solid phase (the fat crystals)

    • Liquid phase (the oil surrounding the crystals).

  • Plasticity is a result of the ratio of solid to liquid components.

    • Plasticity ratio = volume of crystals / volume of oil

    • Measured by a ‘solid fat index’ or amount of solid fat or liquid oil in a lipid

  • As the temperature of a plastic fat increases the fat crystals melt and the fat will soften and eventually turn to a liquid.


Fat and oil further processing

Fat and Oil: Further Processing

  • Winterizing (oil)

    • Cooling a lipid to precipitate solid fat crystals

    • DIFFERENT from hydrogenation

  • Plasticizing (fat)

    • Modifying fats by melting (heating) and solidifying (cooling)

  • Tempering (fat)

    • Holding the fat at a low temperature for several hours to several days to alter fat crystal properties

      (Fat will hold more air, emulsify better, and have a more consistent melting point)


Lipid oxidation

Lipid Oxidation


Lipids

Oleic acid

Radical Damage,

Hydrogen

Abstraction

Formation of a

Peroxyl Radical


Simplified scheme of lipoxidation

Simplified scheme of lipoxidation

+ Catalyst

+ Oxygen


Primary drivers

Primary Drivers

  • Temperature-basic rxn kinetics

  • Water Activity

    • Both high and low Aw

    • At low Aw, peroxides decompose faster and metal ions are better catalysts in a dry environment

  • MetalIons-catalysts

  • Light-energy source

  • SingletOxygen- ROS, highly electrophilic

    • Reacts 1,500 times faster at C=C than ground state O2

  • Enzymes ie. Lipoxygenase (LOX)


Initiation of lipid oxidation

Initiation of Lipid Oxidation

  • There must be a catalytic event that causes the initiation of the oxidative process

    • Enzyme catalyzed

    • “Auto-oxidation”

      • Excited oxygen states (i.e singlet oxygen): 1O2

        • Triplet oxygen (ground state) has 2 unpaired electrons in the same spin in different orbitals.

        • Singlet oxygen (excited state) has 2 unpaired electrons of opposite spin in the same orbital.

      • Metal ion induced (iron, copper, etc)

      • Light

      • Heat

      • Free radicals

      • Pro-oxidants

      • Chlorophyll

      • Water activity


Considerations for lipid oxidation

Considerations for Lipid Oxidation

  • Which hydrogen will be lost from an unsaturated fatty acid?

  • The longer the chain and the more double bonds….the lower the energy needed.


Bond strength

Bond Strength

103 kCal

65 kCal

65 kCal

85 kCal

Fatty acid

.OH or other

Free radicals

.

.

.


Propagation reactions

Propagation Reactions

Peroxyl radical

Ground state oxygen

Initiation

Hydroperoxide

New

Radical

Alkoxyl radical

Hydroxyl radical!!

Hydroperoxide

decomposition

Start all over again…


Mechanism of photooxidation

Mechanism of Photooxidation

Chlorophyll

Chlorophyll

3O2

1O2

or


Singlet oxygen oxidation

Singlet Oxygen Oxidation


Autoxidation

Autoxidation

+

+


Singlet oxygen oxidation1

Singlet Oxygen Oxidation

Nonanal


Secondary products aldehydes

Secondary Products: Aldehydes


Volatile aldehydes from oxidation

Volatile Aldehydes from Oxidation


Lipid oxidation1

Lipid Oxidation

  • Peroxide decomposition can generate aldehydes, ketones, alcohols, various hydrocarbons, and epoxides. Many are volatile, and many are unappealing.


Relative oxidation rates

Relative Oxidation Rates

  • How fast is lipid oxidation?

    Fatty AcidRelative Rate

  • Steric acid (18:0)1

  • Oleic acid (18:1 n9)100

  • Linoleic acid (18:2 n6)1,200

  • Linolenic acid (18:3 n3)2,500


Termination of lipid oxidation

Termination of Lipid Oxidation

  • Although radicals can “meet” and terminate propagation by sharing electrons….

  • The presence or addition of antioxidants is the best way in a food system.

  • Antioxidants can donate an electron without becoming a free radical itself.


Antioxidants and lipid oxidation

Antioxidants and Lipid Oxidation

  • BHT – butylated hydroxytoluene

  • BHA – butylated hydroxyanisole

  • TBHQ – tertiary butylhydroquinone

  • Propyl gallate

  • Tocopherol – vitamin E

  • NDGA – nordihydroguaiaretic acid

  • Carotenoids


Polar antioxidants

Polar Antioxidants

  • Most effective in nonpolar or less polar environment

    • Bulk oils

  • Located at the oil-air interface or in reverse micelles

    • High amount of oxidants present here

Yellow Oil

Blue water

Phospholipids


Non polar antioxidants

Non-polar Antioxidants

  • Most effective in polar environment

    • Oil-in-water emulsions

  • Located at the water-oil interface

    • Dissolved in oil droplets of the emulsion

    • Allows access to oxidizing agents located in the water phase

      • Peroxides

      • Oxidizing metals


Chemical tests for lipid characterizations

Chemical Tests for Lipid Characterizations


Lipids

Iodine Value

  • Measure of the degree of unsaturation in an oil or the number of double bonds in relation to the amount of lipid present

  • Defined as the grams of iodine absorbed per 100-g of sample.

  • The higher the amount of unsaturation, the more iodine is absorbed.

  • Therefore the higher the iodine value, the greater the degree of unsaturation.


Lipids

Iodine Value

  • A known solution of KI is used to reduce excess ICl (or IBr) to free iodine

    R-C-C = C-C-R + ICl R-C-CI-CCl-C-R + ICl [Excess] (remaining)

  • Reaction scheme: ICl + 2KI KCl + KI + I2

  • The liberated iodine is then titrated with a standardized solution of sodium thiosulfate using a starchindicator

  • I2 + Starch + thiosulfate = colorless endpoint

    (Blue colored)


Iodine value

Iodine Value

Used to characterize oils:

  • Following hydrogenation

  • During oil refining (edible oils)

  • Degree of oxidation (unsaturation decreases during oxidation)

  • Comparison of oils

  • Quality control


Lipids

Iodine value: g absorbed I2/ 100 g fat

Highly saturated

High in 18:1

High in 18:1 and 18:2)

18:1, 18:2, 18:3

18:1, 18:2, 18:3

(longer chains)

What can we conclude about the COMPOSITION

or STRUCTURE of each of these oil types?


Lipids

Automated

Iodine Value

Determination

Standard Iodine Value

A = 23

B = 44

C = 67

D = 89

E = 111

Consumption over

time

Measures IBr or ICl

Consumption (neg. peak)


Chemical tests

Chemical Tests

Saponification Value


Lipids

Saponification Value

Saponification is the process of breaking down or degrading a neutral fat into glycerol and fatty acids by treating the sample with alkali.

Heat

Triacylglyceride ---> Fatty acids + Glycerol

KOH

Definition: mg KOH required to titrate 1g fat

(amount of alkali needed to saponify a given amount of fat)

Typical values: Peanut = 190, Butterfat = 220


Lipids

Saponification Value

  • The mg KOH required to saponify triacylglycerides into glycerol plus fatty acids is related to:

  • average fatty acidchain length or

  • average fatty acidmolecular weight

  • Divide molecular weight by 3 to get average of the fatty acids present


Chemical tests for oxidation

Chemical Tests for Oxidation

Lipid Oxidation

Hydrolysis

Peroxide Value

Oxidation Tests


Lipid oxidation2

LIPID OXIDATION

Lipid System Under

Oxidizing Conditions


Lipids

Free Fatty Acids (FFA’s)

  • Degree of hydrolysis (hydrolytic rancidity)

  • High level of FFA means a poorly refined fat or fat breakdown after storage or use.


Lipids

Measures of Oxidation

  • Oxidation is a very complex reaction - no one test will measure all of the reactants or products.

  • Some assays measure intermediates while others measure end products.


Lipids

Peroxide Value

  • Measures peroxides and hydroperoxides in an oil which are the primary oxidation products (usually the first things formed).

  • The peroxide value measures the “present status of the oil”. Since peroxides are destroyed by heat and other oxidative reactions, a seriously degraded oil could have a low PV.


Lipids

Peroxide Value

KI + peroxyl radical yields free Iodine (I2)

  • The iodine released from the reaction is measured in the same way as an iodine value.

  • I2 in the presence of amylose is blue.

  • I2 is reduced to KI and the endpoint determined by loss of blue color.

    4I + O2 + 4H 2I2 + 2H2O


Lipids

Thiobarbituric acid (reactive substances) TBA OR TBARS

Tests for end products of oxidation – aldehydes, Malonaldehyde (primary compound), alkenals, and 2,4-dienals

  • A pink pigment is formed and measured at ~530 nm.

    TBARS is firmly entrenched in meat oxidation research and is a method of choice.

    TBARS measure compounds that are volatile and may react further with proteins or related compounds.

    High TBA = High Oxidative Rancidity


Lipids

HEXANAL Determination

  • Good indictor of the end products of oxidation (if there are any).

  • Standard method in many industries.

  • Aldehyde formation from lipid oxidation.

  • Nonenal is also a common end-product


Lipids

Conjugated Fatty Acids

During oxidation, double bond migration occurs and conjugated fatty acids are formed.

They absorb light efficiently and can be monitored in a spectrophotometer.


Lipids

TECHNIQUES OF MEASURING OXIDATIVE STABILITY

Induction Period: is defined as the length of time before detectable rancidity or time before rapid acceleration of lipid oxidation


Lipids

MEASURING OXIDATIVE STABILITY

  • Active Oxygen Method - Air is bubbled through oil or fat at 97.8°C. Time required to reach peroxide value of 100 meq/kg fat determined. (method replaced by OSI)

  • Oil Stability Index – automated Rancimat (instrumental method). Air bubbled through sample (110°C). Oil degrades to many acidic volatiles (e.g. formic acid) which are carried by the air into a water trap. Conductivity of the water can then be assessed.


Free radicals

Free Radicals


Lipids

  • What are free radicals?

  • Where are free radicals from?

  • How damaging are free radicals?

  • How do we control free radicals?


What are free radicals

What are free radicals?

  • Any molecular species capable of independent existence, which contains one or more unpaired valence electrons not contributing to intramolecular bonding….is a free radical.

The most frequent radicals are oxygen-derived free radicals, also known as reactive oxygen species (ROS):

Superoxide (O2·-)

Peroxyl (ROO˙)

Alkoxyl (RO˙)

Hydroxyl (HO˙)

Nitric oxide (NO˙)

Other ROS are non-radicals such as singlet oxygen (O2), hydrogen peroxide (H2O2), and hypochlorous acid (HClO).


Where do they come from

Where do they come from?

  • Free radicals are produced by oxidation/reduction reactions in which there is a transfer of only one electron at a time, or when a covalent bond is broken and one electron from each pair remains with each atom.

  • Normal ongoing metabolism, especially from the electron transport system in the mitochondria and from a number of normally functioning enzymes

  • 2) Environmental factors such as pollution, radiation, cigarette smoke and toxins can also spawn biologically-derived free radicals.


How damaging are free radicals

How damaging are free radicals?

  • ROS may be very damaging, since they can attack:

    • Lipids in cell membranes

    • Proteins in tissues or enzymes

    • Carbohydrates

    • DNA

  • These cause cell membrane damage, protein modification, and DNA damage.

  • Thought to play a role in aging and several degenerative diseases (heart disease, cataracts, cognitive dysfunction, and cancer).

  • Oxidative damage can accumulate with age.


Our body vs our food

Our Body vs. Our Food

  • Biological radicals

  • Food-based radicals

  • Where do these 2 areas cross?


Functional foods concept

Functional Foods Concept

  • Certain food ingredients have health benefits beyond basic nutrition

  • Recent development only: since ~1975

  • The concept that ‘non-nutrients’ were beneficial has taken off since then

  • First idea in scientific community: antioxidant compounds may protect against chronic diseases


Free radicals1

Free Radicals

  • Early 1950’s: cell damage is due to reactive oxygen species called “free radicals”

  • Unstable, ‘damaged’ molecule that is missing an electron

  • Highly reactive; reacting to some measurable extent with any molecule they come in contact with

  • In living systems, cell injury or disease

  • In foods, quality-degrading impact


Reactive oxygen species ros

Reactive Oxygen Species (ROS)

  • Primary target list: protein, lipid, DNA, and carbohydrates

  • End results: cancer, CHD, stroke, arterial disease, rheumatoid arthritis, Parkinson’s/Alzheimer disease, cataracts, macular degeneration….many more

  • Aging by slow oxidation?


The defense

The Defense

  • Minimize contact between free radicals and important systems (like cellular components)

  • Cell membranes are one of our best barriers

  • Metal chelation system in-place

  • Protease enzymes are in place to remove damaged proteins for replacement by new

  • “Repair enzymes” help to restore DNA

  • “Antioxidant enzymes”-superoxide dismutase, catalase, glutathione peroxidase


Best defense a good offense

Best Defense…A Good Offense

  • “Nutrients” that can’t be synthesized in vivo: vitamin C, vitamin E, (pro)vitamin A

  • “Non-nutrients”: polyphenolics/carotenoids

    • Diet is only source….are they “essential”?

  • What about conditions of “oxidative stress”?

    • This is a condition when pro-oxidants outnumber antioxidants (I.e. decreased immune response, environmental factors, hypertension, poor diet).


Foods and the antioxidant link

Foods and the Antioxidant Link

  • Soy- isoflavones, polyphenolics

  • Tea- polyphenolics, flavans

  • Coffee- polyphenolics

  • Wine- polyphenolics

  • Rosemary- carnosic acid, rosmaric acid

  • Citrus- flavonoids

  • Onions- sulfur cpds, flavonoids

  • Berries- flavonoids, polyphenolics

  • Vegetables- carotenoids, polyphenolics


Antioxidants in food systems

Antioxidants in Food Systems


Oxidative stress the food remedy

Oxidative Stress-the food remedy

  • Diet:

  • Inflammation- tocopherol

  • Smoke- ascorbic acid

  • Physical stress- carotenoids

  • Pollution- carotenoids

  • Environment:

  • Radiation- glutathione

  • Carcinogens- antioxidant enzymes, diet modification


Oxidative stress and foods

Oxidative Stress and Foods

  • Tocopherol- vegetable oil, whole grains, vegetables, fish/poultry

  • Ascorbic acid- citrus, berries, tomato, leafy veggies, brassicas (broccoli, cauliflower)

  • Carotenoids- yellow/orange fruits and veggies, tomatoes, green leafy veggies.

  • Polyphenolics- coffee/tea, grains, all fruits and vegetables


Magic bullets for our body

Magic Bullets…for our body?

  • Most likely not…

  • Will increasing the intake of antioxidants modulate disease prevention? Will we live longer with no health problems?

  • Lung cancer and ß-carotene: Whoa…

  • Antioxidant compounds have demonstrated benefits (both acute and long-term) of preventing or postponing the onset of many degenerative diseases, but clinical trials are full of holes, and conclusive evidence of “the bullet” is still not with us.


Magic bullets for our foods

Magic Bullets…for our foods?

  • Will increasing the use of antioxidants in foods modulate all oxidative damage?

  • Will food products “live” longer with no quality problems?

  • Pro-oxidant nature of ascorbic acid: Whoa…

  • Ascorbic acid does not always act linearly in food systems

  • In the presence of metal ions (ie. Fe/Cu) it can generate reactive oxygen species (peroxides) or free radicals (hydroxyl radicals)


Causes and effects

Causes and Effects

  • ß-carotene and lung cancer: small, but significant increase with smokers

  • Tocopherols and CHD: protect lipoproteins or inhibit blood clotting (which initiates heart attacks)

  • Tocopherols and Alzheimer’s: reducing oxidative stress by supplementation

  • Cataracts and vitamins (A,C,E): inverse association

  • Macular degeneration and carotenoids: inverse

  • Vitamin C and the Common Cold: shorter, milder colds


Structure based aox

Structure-Based AOX

Polyphenolics


Structure of flavonoids

Structure of flavonoids

3’

4’

8

2

7

5’

3

6

5

4

Flavonols: X=OH

Flavones: X=H

Flavanones: No 2-3 db


B ring substitutions

B-Ring Substitutions

B

Kaempferol

A

C

Quercetin

Myricetin


Quercetin 4 oh groups

Quercetin 4 –OH groups

2-3 db

3-OH

4-oxo function


Catechin 4 oh groups

Catechin 4 –OH groups

3-OH


Cyanidin 4 oh groups

Cyanidin4 –OH groups


Structurally similar compounds

Structurally Similar Compounds

Catechin

AOX = 2.4

Quercetin

AOX = 4.7

Cyanidin

AOX = 4.4


Importance of the 3 oh group

Importance of the 3-OH group

Quercetin-3-glucoside

AOX ~ 2.5

Quercetin

AOX = 4.7

Luteolin

AOX = 2.1


Importance of the 4 oxo function

Importance of the 4-Oxo Function

  • Works with the 2-3 double bond in the C-ring and is responsible for electron delocalization from the B-ring.

  • 3-OH and 5-OH substitutions with the 4-oxo function are best for maximum AOX properties

Structurally, quercetin has

all the right components to

make for the “perfect” antioxidant.


Importance of the 2 3 db

Importance of the 2-3 db

Quercetin

AOX = 4.7

Taxifolin

AOX = 1.9


More on the phenolic acids

More on the Phenolic Acids

Hydroxyphenylacetic

Acid

(HPA)

Hydroxybenzoic

Acid

(HBA)

Cinnamic

Acid

(CA)


Lipids

p-OH-benzoic

p-coumaric

0.08

2.22

Protocatechuic

Caffeic

1.19

1.26

Vanillic

Ferulic

1.43

1.90


Antioxidants in food systems1

Antioxidants in Food Systems


What makes a good antioxidant

What Makes a Good Antioxidant?

  • Polyphenolics- Radical scavengers

    • Number of hydroxy groups (-OH)

    • Location of hydroxy groups (on benzene ring)

    • Presence of a 2-3 double bond (flavylium ring)

    • 4-oxo function (flavylium ring)

  • Synergistic/antagonistic reactions with other antioxidant compounds


What makes a good antioxidant1

What Makes a Good Antioxidant?

  • Carotenoids

  • The number of conjugated double bonds (9+ is best)

  • Substitutions on ß-ionone group (on the end)

    • Radical scavengers

      R* + CAR => R- + CAR*+

    • Chain breakers

      ROO*+ CAR => ROO-CAR*

      ROO-CAR* + ROO* => ROO-CAR-ROO

    • Singlet oxygen quenchers

      • 1O2* + 1CAR => 3O2 + 3CAR*


Tocopherol

Tocopherol

  • Alpha-tocopherol = Vitamin E

    • beta and gamma forms also

  • Synergist with carotenoids and selenium and is regenerated by vitamin C

  • Efficiency determined by the bond dissociation energy of the phenolic -OH bond

  • The heterocyclic chromanol ring is optimized for resonance stabilization of an unpaired electron.


Antagonism synergism metals

Antagonism-Synergism-Metals

  • Many antioxidant work for and against each other

  • An antioxidant in a biological system my be regenerated

  • In mixed ROS…inefficiency of one antioxidant to quench all the different radicals.

  • No way of knowing if the “better” antioxidant for a particular radical is doing all the work or not.

  • Will a better antioxidant for a given food system “beat out” a lesser antioxidant (antagonistic response) in order to quench the radicals.


Example factors affecting aox of bell peppers

Example: Factors Affecting AOX of Bell Peppers

Chemical interactions

  • In vitro models

  • Find synergistic/antagonistic effects

    Free metal ions

  • Diluted isolates

  • Add metal chelator

Flavonoid

Ascorbic

AOX ?


Aox with quercetin interactions carotene bleaching

AOX with Quercetin Interactions(ß-Carotene Bleaching)

450 ppm Caffeic = 47 %

880 ppm Ascorbic = 15%


Aox with luteolin interactions carotene bleaching

AOX with Luteolin Interactions(ß-Carotene Bleaching)

450 ppm Caffeic = 47 %

880 ppm Ascorbic = 15%


Theoretical quercetin regeneration scheme

Theoretical Quercetin “Regeneration” Scheme

Delocalization

of C-ring

Quercetin

Reduced resonation in A and B rings

Minor regeneration by ascorbic acid

Minor regeneration by caffeic acid


Theoretical luteolin regeneration scheme

Theoretical Luteolin “Regeneration” Scheme

Electron donation

by C-ring

Excellent resonance stability in A-ring

Highly regenerated by ascorbic acid

No regeneration by caffeic acid

Luteolin


Antioxidant activity after dilution

Antioxidant Activity after Dilution

% Inhibition of Carotene Bleaching


Antioxidant activity with chelator

Antioxidant Activity with Chelator

% Inhibition of Bleaching


Antioxidant methods

Antioxidant Methods


Hat and set reactions

HAT and SET Reactions

  • Hydrogen Atom Transfer (HAT) vs. Single Electron Transfer (SET)

  • Antioxidants can work in one of two ways (HAT or SET).

  • End result is the same for both, differing in kinetics and side rxns.

  • HAT and SET rxns may occur in parallel

    • Determined by antioxidant structure and properties

    • Solubility and partition coefficient

    • System solvent, system pH


Lipids

HAT

  • HAT-based methods measure the classical ability of an antioxidant to quench free radicals by hydrogen donation (AH = any H donor)


Lipids

SET

  • SET-based methods detect the ability of a potential antioxidant to transfer one electron to reduce any compound, including metals, carbonyls, and radicals.

  • Also based on deprotonation, so pH dependent


Hat vs set

HAT vs SET

HAT

  • Selectivity in HAT rxs are determined by the bond dissociation energy of the H-donating group in the antioxidant

  • Antioxidant reactivity or capacity measurements are therefore based on competition kinetics.

  • Reactions are solvent and pH independent and are very fast

  • Common reducing agents (Vitamin C) are an interference

    SET

  • Usually slow and can require long times to reach completion

  • Antioxidant reactivity is based on a percent decrease, rather than kinetics

  • Very sensitive to ascorbic acid and other reducing agents.

  • Trace amounts of metal ions will interfere, and cause over-estimation and inconsistent results.


Antioxidants and radicals

Antioxidants and Radicals

  • Four sources of antioxidants:

    • Enzymes

      • Superoxide dismutase, glutathione peroxidase, and catalase

    • Large molecules

      • albumin, ferritin, other proteins

    • Small molecules

      • ascorbic acid, glutathione, uric acid, tocopherol, carotenoids, phenols

    • Hormones

      • estrogen, angiotensin, melatonin

  • Multiple free radical and oxidant sources

    • O2, O2·-, HO˙, NO˙, ONOO-, HOCl, RO(O)˙, LO(O)

  • Oxidants and antioxidants have different chemical and physical characteristics.


Complex systems singlet oxygen

Complex Systems: Singlet Oxygen

  • Carotenoids are not good peroxyl radical quenchers compared to polyphenolics

  • Carotenoids are exceptional singlet oxygen quenchers compared to polyphenolics

  • However, singlet oxygen is not a radical and does not react via radical mechanisms

  • Singlet oxygen reacts by its addition to fatty acid double bonds, forming endoperoxides, that can be reduced to alkoxyl radicals, that initiate radical chain reactions.

  • Now we have multiple reaction characteristics and multiple mechanisms

  • No single assay will accurately reflect all of the radical sources or test all the antioxidants in such a complex system.


Method selections for antioxidants

Method Selections for Antioxidants

  • Controversy exists over standard methods for antioxidant determination

  • Historical use and peer-review acceptance is critical

  • Use my multiple labs to highlight strength, weakness, and effectivness

  • New methods take time to adopt and accept

  • An “ideal” method:

    • Measures chemistry actually occurring in potential application

    • Utilizes a biologically relevant radical source

    • Simple to run

    • Uses a defined endpoint and chemical mechanism

    • Instrumentation is readily available

    • Good within-run and between-day reproducibility

    • Adaptable for both hydrophilic and lipophilic antioxidants

    • Adaptable for multiple radical sources

    • Adaptable for high-through-put analysis

    • Understanding of the range of use and recognition of interfering agents


Hat assays

HAT assays

  • ORAC

    • Oxygen Radical Absorbance Capacity

    • Measures inhibition of peroxyl radical induced oxidations in chain breaking activity by H atom transfer

  • TRAP

    • Total Radical-Trapping Antioxidant Parameter

    • Measures the ability to interfere with peroxyl radicals or stable free radicals


Set assays

SET assays

  • FRAP

    • Ferric Reducing Antioxidant Power

    • The reaction measures the reduction capacity of a ferric compound to a color end-product

  • CUPRAC

    • Copper Reduction Assay

    • Variant of FRAP assay using Cu instead of Fe

  • Folin-Ciocalteu assay

    • Reduction of oxidized iron and molybdenum


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