Pressing oilseeds Solvent extraction Fats and Oils

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Lipids
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Class Presentations Will be Discussed at the End
of Class

• Exams back next Monday
• No class this Wednesday !!!!!

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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

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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

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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

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Lipids

• Structure
• Triglycerides or triacylglycerols
• Glycerol 3 fatty acids
• gt20 different fatty acids

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Lipids 101

• Fatty acids- the building block of fats
• A fat with no double bonds in its 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 (160, 161, 182 etc)

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Lipids

• Oil- liquid triacylglycerides Oleins
• Fat- solid or semi-solid mixtures of crystalline
  and liquid TAGs 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.

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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

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Saturated Fatty Acids
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Saturated Fatty Acids
Octanoic Acid
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Mono-Unsaturated Fatty Acids
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Poly-Unsaturated Fatty Acids
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Fatty Acids Melting Points and Solubility in
Water
Melting Point
Response
Solubility in H2O
z
2
Fatty acid chain length
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Unsaturated Fatty Acids
3 - Octenoic Acid
3, 6 - Octadienoic Acid
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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

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Fatty Acids Melting Points and Solubility in
Water
Melting Point
Response
Solubility in H2O
z
2
Fatty acid chain length
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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
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Fatty Acids
1 Carbon
Acid Group
Polar End - Hydrophilic End
Non-polar End - Hydrophobic End
(Fat-soluble tail)
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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.

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Lipids 101

• For the 18-series (180, 181, 182, 183) the
  double bonds are usually located between carbons
  910 1213 1516.

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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 1516 double bond is a 34 double bond or
  as the medical folks call it.an w-3 fatty acid

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Tuning Fork Analogy-TAGs

• 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

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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 glycerophospholipid
  s, or simply phospholipids
• contain a polar phosphorus moiety and a glycerol
  backbone
• or glycolipids, which contain a polar
  carbohydrate moiety instead of phosphorus.

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Lipids
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Other types of lipids

• Phospholipids
• Structure similar to triacylglycerol
• High in vegetable oil
• Egg yolks
• Act as emulsifiers

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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.

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Fats and Oils Processing
Peanut

• Extraction
• Rendering
• Pressing oilseeds
• Solvent extraction

Rape Seed
Safflower
Sesame
Soybean
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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)

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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.

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Where Do We Get Fats and Oils?

• Mechanical Pressing
• Mechanical pressing is often used to extract oil
  from seeds and nuts with oil gt50. 
• 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.

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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.

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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.

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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

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Hydrogenating Vegetable oils can produce
trans-fats
Cis-
Trans-
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The cis- and trans- forms of a fatty acid
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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 fats 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.

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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)

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Lipid Oxidation
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Effects of Lipid Oxidation

• Flavor and Quality Loss
• Rancid flavor
• Alteration of color and texture
• Decreased consumer acceptance
• Financial loss
• Nutritional Quality Loss
• Oxidation of essential fatty acids
• Loss of fat-soluble vitamins
• Health Risks
• Development of potentially toxic compounds
• Development of coronary heart disease

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LIPID OXIDATION and Antioxidants

• Fats are susceptible to hydrolyis (heat, acid, or
  lipase enzymes) as well as oxidation. In each
  case, the end result can be RANCIDITY.
• For oxidative rancidity to occur, molecular
  oxygen from the environment must interact with
  UNSATURATED fatty acids in a food.
• The product is called a peroxide radical, which
  can combine with H to produce a hydroperoxide
  radical.
• The chemical process of oxidative rancidity
  involves a series of steps, typically referred to
  as
• Initiation
• Propagation
• Termination

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Simplified scheme of lipoxidation
Oxygen
Catalyst
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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

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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.

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Oleic acid
Radical Damage, Hydrogen Abstraction
Formation of a Peroxyl Radical
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Propagation Reactions
Peroxyl radical
Ground state oxygen
Initiation
Hydroperoxide
New Radical
Hydroxyl radical!!
Hydroperoxide decomposition
Start all over again
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Propagation of Lipid Oxidation
Oxygen
Catalyst
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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.

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Antioxidants and Lipid Oxidation

• BHT butylated hydroxytoluene
• BHA butylated hydroxyanisole
• TBHQ tertiary butylhydroquinone
• Propyl gallate
• Tocopherol vitamin E
• NDGA nordihydroguaiaretic acid
• Carotenoids

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Chemical Tests for Lipid Characterizations
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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.

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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 starch indicator
• I2 Starch thiosulfate colorless endpoint
• (Blue colored)

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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

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Iodine value g absorbed I2/ 100 g fat
Highly saturated
High in 181
High in 181 and 182)
181, 182, 183
181, 182, 183 (longer chains)
What can we conclude about the COMPOSITION or
STRUCTURE of each of these oil types?
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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)
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Chemical Tests

• Saponification Value

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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 ---gt 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

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Saponification Value

• The mg KOH required to saponify triacylglycerides
  into glycerol plus fatty acids is related to
• average fatty acid chain length or
• average fatty acid molecular weight
• Divide molecular weight by 3 to get average of
  the fatty acids present

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Chemical Tests for Oxidation

• Lipid Oxidation
• Hydrolysis
• Peroxide Value
• Oxidation Tests

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LIPID OXIDATION
Lipid System Under Oxidizing Conditions
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Free Fatty Acids (FFAs)

• Degree of hydrolysis (hydrolytic rancidity)
• High level of FFA means a poorly refined fat or
  fat breakdown after storage or use.

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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.

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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.

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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

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• 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

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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

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• 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.

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• 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

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• MEASURING OXIDATIVE STABILITY
• Active Oxygen Method - Air is bubbled through oil
  or fat at 97.8C. 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
  (110C). 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.

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Free Radicals
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• What are free radicals?
• Where are free radicals from?
• How damaging are free radicals?
• How do we control free radicals?

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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).
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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.

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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.

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Our Body vs. Our Food

• Biological radicals
• Food-based radicals
• Where do these 2 areas cross?

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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

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Free Radicals

• Early 1950s 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

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Reactive Oxygen Species (ROS)

• Primary target list protein, lipid, DNA, and
  carbohydrates
• End results cancer, CHD, stroke, arterial
  disease, rheumatoid arthritis, Parkinsons/Alzheim
  er disease, cataracts, macular degeneration.many
  more
• Aging by slow oxidation?

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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

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Best DefenseA Good Offense

• Nutrients that cant 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).

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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

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Antioxidants in Food Systems
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Oxidative Stress-the food remedy

• Diet
• Inflammation- tocopherol
• Smoke- ascorbic acid
• Physical stress- carotenoids
• Pollution- carotenoids
• Environment
• Radiation- glutathione
• Carcinogens- antioxidant enzymes, diet
  modification

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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

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Magic Bulletsfor 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.

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Magic Bulletsfor 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)

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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 Alzheimers 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

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Structure-Based AOX

• Polyphenolics

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Structure of flavonoids
3
4
8
2
7
5
3
6
4
5
Flavonols XOH Flavones XH Flavanones No 2-3
db
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B-Ring Substitutions
B
Kaempferol
A
C
Quercetin
Myricetin
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Quercetin 4 OH groups
2-3 db
3-OH
4-oxo function
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Catechin 4 OH groups
3-OH
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Cyanidin4 OH groups
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Structurally Similar Compounds
Catechin AOX 2.4
Quercetin AOX 4.7
Cyanidin AOX 4.4
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Importance of the 3-OH group
Quercetin-3-glucoside AOX 2.5
Quercetin AOX 4.7
Luteolin AOX 2.1
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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.
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Importance of the 2-3 db
Quercetin AOX 4.7
Taxifolin AOX 1.9
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More on the Phenolic Acids
Hydroxyphenylacetic Acid (HPA)
Hydroxybenzoic Acid (HBA)
Cinnamic Acid (CA)
101
p-OH-benzoic p-coumaric
0.08
2.22
Protocatechuic Caffeic
1.19
1.26
Vanillic Ferulic
1.43
1.90
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Antioxidants in Food Systems
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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

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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 gt R- CAR
• Chain breakers
• ROO CAR gt ROO-CAR
• ROO-CAR ROO gt ROO-CAR-ROO
• Singlet oxygen quenchers
• 1O2 1CAR gt 3O2 3CAR

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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.

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Antagonism-Synergism-Metals

• Many antioxidant work for and against each other
• An antioxidant in a biological system my be
  regenerated
• In mixed ROSinefficiency 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.

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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 ?
108
AOX with Quercetin Interactions(ß-Carotene
Bleaching)
450 ppm Caffeic 47 880 ppm Ascorbic 15
109
AOX with Luteolin Interactions(ß-Carotene
Bleaching)
450 ppm Caffeic 47 880 ppm Ascorbic 15
110
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
111
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
112
Antioxidant Activity after Dilution
Inhibition of Carotene Bleaching
113
Antioxidant Activity with Chelator
Inhibition of Bleaching
114
Antioxidant Methods
115
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

116
HAT

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

117
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

118
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.

119
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.

120
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.

121
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

122
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

123
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