Further decomposition of hydroperoxides

C-C-C=C-CHO

C-C-C=C-CHO O O H

R-CHO + OHC-CH2-CHO (malonaldehyde)

Formation of malonaldehyde is one of the major

products of lipid oxidation.

Malonaldehyde can cross-link with proteins,

enzymes and DNA and cause health problems.

Thiobarbituric acid (TBA) test

Measuring TBARS (Thiobarbituric acid reactive

substances) is a general test used to evaluate the

extent of lipid oxidation.

Oxidation products of unsaturated systems produce

a colour reaction with TBA.

Colour results from condensation of two molecules

of TBA and one molecule of malonaldehyde.

2 mol TBA + malonaldehyde red colour

The product can be measured quantitatively at 530

nm using a spectrophotometer.

Role of metal ions in lipid oxidation

Metal ions can catalyze the oxidation of lipids.

Metals possessing two or more valency states and a

suitable oxidation-reduction potential between them

are effective pro-oxidants. e.g. Fe, Cu, Mn, Co

Even at concentrations as low as 0.1 ppm, they can

decrease the induction period and thereby increase

the rate of oxidation.

Trace amounts of heavy metals are found in edible

oils originating from:

• the soil in which the plant was grown

• the animal

• metallic equipment used in processing or storage

Role of metal ions in lipid oxidation

They are also naturally occurring components of all

foods and are present in both free and bound forms.

Mechanisms for metal catalysis of oxidation are as

follows.

1. Activation of molecular oxygen to give singlet oxygen

and peroxy radical.

-e- 1O2

Mn+ + O2 M(n+1) + 3O2

+H+

HOO0

2. Direct reaction with the unoxidized substrate.

Mn+ + RH M(n-1)+ + H+ + Ro

Role of metal ions in lipid oxidation

3. Acceleration of hydroperoxide decomposition

Mn+ + ROOH M(n+1)+ + OH- + ROo

Mn+ + ROOH M(n-1)+ + H+ + ROOo

Lipoxydase (Lipoxygenase) catalysed oxidation:

This is an enzymatic reaction which needs O2.

1,4 pentadiene structure is also needed.

RH + Lipoxydase + O2 RH + O2

Lipoxydase

ROOH ROOH R + OOH

+ Lipoxydase Lipoxydase

Lipoxydase

Lipoxygenase catalysed oxidation ……

• Lipoxygenase is present in plants and animals.

• Activation energy for the above reaction (3-4 kcal/

mol) is low compared with autooxidation hence it can

take place at low temperature, even at refrigeration

temperature.

• Under frozen conditions aw is low and the mobility of

reactants is low. Hence the rate of reaction reduced.

• Enzymatic cleavage of ROOH yields a variety of

breakdown products which are responsible for the

characteristic flavor of natural products.

• This reaction can be inhibited by phenolic anti-

oxidants (tocopherol, hydroquinones etc.)

Hematin catalysed oxidation ……

• Hematin compounds present in many food tissues

are also important pro-oxidants. E.g. myoglobin,

haemoglobin, cytochrome

• Even in well bled tissues very low amount of hematin

is present. Fe3+ is involved in this reaction.

• This reaction is different to other 2 forms due to the

requirement of pre-formed hydroperoxides.

ROOH + hematin

RH

Ro + carboxyl compounds + hematin

The activation energy for reaction is 3.3 kcal /molecule.

ROOH + hematin

Antioxidants

• Substances that can delay the onset or slow down

the rate of oxidation

• Main lipid soluble antioxidants used in foods are

monohydric or polyhydric phenols

E.g. Tocopherol, BHT, BHA, PG, TBHQ

• For maximum efficiency, they are used in combination

with metal sequestering agents

Mechanism of action:

A substance delays autooxidation reaction, if;

• it inhibits formation of free radicals

• it interrupts propagation of free radicals

Antioxidants

• Antioxidants inhibit free radical formation by:

• Quenching singlet oxygen

• Chelating metal ions

• Decomposing hydroperoxides

• An antioxidant inhibits the chain reaction by acting as

hydrogen donor (free radical acceptor) for Ro and

ROOo radicals.

Ro + AH RH + Ao

ROOo + AH ROOH + Ao

• The resulting antioxidant radical will not initiate new

free radicals and they may undergo a variety of

reactions forming stable products.

Antioxidants…..

Ao + Ao AA

Ao+ ROOo ROOA

Dihydric phenols dismute to yield quinones with the

formation of original antioxidant.

Ao + Ao AH + quinone

Effectiveness of an antioxidant is influenced by its,

• Chemical potency

• Solubility in oil (accessibility to free radical)

• Volatility (stability during heating, storage)

Synergism of Antioxidants

• Synergism occurs when a mixture of antioxidants

produces a greater activity than the sum of the

activity of each antioxidant in the mixture, when

tested individually.

• Two types of synergism are recognized:

1. Action of mixed free radical acceptors:

ROOo + AH ROOH + Ao

Ao + BH AH + Bo

The presence of the second antioxidant (BH) will

have a sparing effect since it regenerates the primary

antioxidant (AH).

E.g. Phenolic antioxidant and ascorbic acid

Synergism of Antioxidants

• Phenolic antioxidant is the primary antioxidant (more

effective one) while ascorbic acid is the synergist.

• It is possible for two phenolic antioxidants to exhibit

synergism in a similar way.

2. Combined action of a free radical acceptor and a

metal chelating agent:

• Metal chelating agents are compounds which can

partly deactivate trace metals present.

• When antioxidant property of a free radical acceptor

is enhanced by the presence of a metal chelating

agent synergism occurs. E.g. citric acid, phosphoric

acid, polyphosphates.

Choice of the Antioxidant

• Antioxidants exhibit substantial differences in their

effectiveness when used with different types of fatty foods

and under different processing and handling conditions.

• Factors to be considered in selecting an anti-oxidant are;

• Chemical potency of the antioxidant

• Ease of incorporation into the food

• Carry-through characteristics

• Sensitivity to pH

• Hydrophilic-lipophilic properties

• Tendency to produce off-flavour or off-colour

• Availability

• Cost

Choice of the Antioxidant

• In bulk oils – TBHQ and PG are more effective.

• In oil-water emulsions, polar lipid membranes,

intracellular micelles of neutral lipids - more lipophilic

antioxidants, such as BHA, BHT and tocopherols are

the most effective.

Thermal Decomposition

• Heating of food produces various chemical changes,

some of which are important to flavor, appearance,

nutritive value and toxicity.

• Different nutrients in food undergo decomposition

reactions and also interact among themselves in

extremely complex ways to form a large number of

new compounds.

• Lipid oxidation at high temperatures is complicated:

thermolytic and oxidative reactions taking place

simultaneously.

• Both saturated and unsaturated fatty acids undergo

decomposition when exposed to heat.

Thermal decomposition

I. Thermal, non-oxidative reactions of SFA:

Heating of saturated triglycerides to >200 oC yields

detectable amounts of hydrocarbons, acids and

ketones due to thermolysis.

II. Thermal, oxidative reactions of SFA:

Even though SFA are more stable to heat than their

unsaturated analogs, above 150 oC they can also

undergo oxidation, giving rise to a complex decomposition

pattern.

Major oxidative products are, series of carboxylic acids

and hydrocarbons: 2-alkanones, n-alkanals, n-alkanes,

1-alkenes and lactones.

Thermal decomposition

III. Thermal non-oxidative reactions of USFA:

Unsatutared fatty acids form dimeric compounds and low

molecular weight compounds during high heat in the

absence of oxygen.

IV. Thermal oxidative reactions of USFA:

At elevated temperatures oxidative decomposition of

USFA takes place very rapidly.

Major compounds formed at high temperatures are

qualitatively the same as that of room temperature

autooxidation. But at elevated temperatures

hydroperoxide decomposition and secondary oxidation

are extremely rapid.

Fatty acids, esters

and triglycerides

Saturated Unsaturated

Thermolytic Oxidative Thermolytic Oxidative

reactions reactions reactions reactions

(α,β,γ attack)

acids alkanes acyclic and volatile and

hydrocarbons alkenes cyclic dimeric

propenediol alkanals dimers products of

acrolein lactones autooxidation

ketones carboxylic acids

Generalized scheme for thermal decomposition of lipids

Chemistry of Frying

• Foods fried in oil, contribute significantly to the

energy in the diet because 5-40% of the oil can be

absorbed to food.

• During deep-fat frying, foods contact oil at high

temperatures (around 180 oC) and is exposed to air

for a variable period of time.

• Thus frying has the greatest potential for causing

chemical changes in food.

Chemistry of Frying

Behaviour of the frying oil:

• The physical and chemical changes that can be

observed in the oil during frying include;

• Increase in viscosity

• Increase in free fatty acid content

• Development of a dark colour

• Decrease in iodine value

• Decrease in surface tension

• Changes in refractive index

• Increased tendency to foam

Above changes are due to following classes of

compounds produced from the oil, during frying.

Chemistry of Frying……

1. Volatiles:

• Oxidative reactions involving the formation and

decomposition of hydroperoxides, lead to the

formation of saturated and unsaturated aldehydes,

ketones, hydrocarbons, lactones, alcohols, acids

and esters.

• Volatiles produced vary widely depending on the type

of oil, type of food and the heat treatment.

• They reach a plateau value with time probably

because a balance is achieved between the formation

and loss of volatiles.

Chemistry of Frying

2. Non-polymeric polar compounds of moderate

volatility (E.g. hydroxyl and epoxy acids):

These compounds are produced due to various oxidative

pathways involving the alkoxy radical.

3. Dimeric- and polymeric acids, and dimeric- and

polymeric glycerides:

These compounds occur from thermal and oxidative

reaction combinations of free radicals. Polymerization

results in an increase of viscosity of the frying oil.

4. Free fatty acids:

These compounds arise from the hydrolysis of

triglycerides in the presence of heat and water.

Chemistry of Frying

Behaviour of the food during frying:

• Water is continuously released from the food into the

hot oil. This produces a steam distillation effect,

sweeping volatile oxidative products from the oil.

• Released moisture also agitates the oil and hastens

the hydrolysis making more FFA available.

• Blanket of steam produced above the surface of oil

tends to reduce the amount of oxygen available for

oxidation.

• Volatiles may develop in the food itself or from the

interaction between food and oil.

Chemistry of Frying

• Food absorbs varying amount of oil during deep fat

frying. E.g. in potato chips the final fat content is

about 35 %.

• Food itself may release some of its endogenous lipids

(e.g. fat from chicken) into the frying medium and

consequently the oxidative stability of the new mixture

may be different from that of the original oil.

• The oil may get darken at an accelerated rate due to

the presence of food.

• Extensive decomposition due to uncontrolled frying

operation can be a potential source of damage to

sensory properties, nutritive value and safety of food.

Browning Reactions in Foods

Browning reactions in foods are of 2 major types:

1. Enzymatic:

2. Non-enzymatic:

• Maillard browning

• Caramelization

• Ascorbic acid oxidation

Enzymatic browning (phenolase / oxidative browning):

• Enzymatic browning is the common form of browning

occurring in fruits and vegetables when they are

damaged or bruised. E.g. apple, banana, pear, guava,

avocado, mango, grapes, potato, brinjal, lettuce etc.

Also found in shrimp and lobsters.

• It is the reaction between oxygen and a phenolic

substrate catalyzed by the enzyme phenolase.

Enzymatic browning

• It is a major contributor to the desirable colour of tea,

apple juice, cocoa and cider.

• Responsible for the normal colour of raisins, prunes,

dates and figs.

• Phenolase activities in fruits and vegetables are not

desirable because the ensuing brown colour is not

pleasing. Enzymatic browning is detrimental to quality,

particularly in post-harvest storage of fresh fruits, juices

and some shellfish.

• In intact plant tissues phenolic substrates are separated

from phenolase and browning does not occur.

• In cut surfaces of light-coloured fruits and vegetables

enzymatic browning is prominent.

Enzymatic browning …..

• Exposure of cut surface (cutting, peeling, bruising) to

air results in rapid browning due to oxidation of

phenols to ortho-quinones, which then rapidly

polymerize into brown colour melanin pigments.

• Enzymes catalyzing oxidation of phenols are known

as “phenolase” and it includes phenolases,

polyphenol oxidases, tyrosinases or catecholases.

• Phenol enzymes have a copper prosthetic group.

• Phenolases catalyse two types of reactions.

Enzymatic browning ……

1. Hydroxylation reaction (known as phenol hydroxylase or

cresolase activity)

2. Oxidation reaction (known as polyphenol oxidase or

catecholase activity)

• First reaction results in ortho-hydroxylation of a phenol and

the second, oxidation of the diphenol to an ortho-quinone.

• Phenolases have a Cu prosthetic group.

Substrates:

1. Monophenols – e.g. tyrosine

2. Ortho-diphenols – e.g.

• catechol

Enzymatic browning ……

• Caffeic acid

• Protocatechuric acid

• Chlorogenic acid

3. Flavanoids in apple – e.g. rutin, quercetin

4. Tannins in peach, tea

5. Catechins in tea

Reaction:

• When tyrosine is the substrate, phenolase first catalyzes its

hydroxylation to DOPA (L-3,4 dihydroxy phenylalanine) and

subsequently catalyzes oxidation of DOPA to DOPA quinone.

Enzymatic browning …..

enzyme + H2O enzyme +O2

Tyrosine DOPA DOPA quinone fast

fast

Hallochrome (red) Leuco-compound

polymerization

Indole 5,6 quinone Melanin

• DOPA quinone formation is enzyme and O2 dependent.

• After the quinone step reactions will proceed without the

involvement of enzymes.

• From hallochrome onwards a gradual change of colour

occurs.

• Melanin, the final product, can interact with protein to form

complexes.

Enzymatic browning …..

• Hydroxylation of monophenol is the rate-limiting step.

• Some phenolase enzymes (e.g. in tobacco, tea and sweet

potato) do not hydroxylate monophenols but some (e.g. in

mushrooms and potato) perform both functions.

• Ortho-diphenols are readily attacked by catecholase

component of the phenolase.

• Meta-diphenols (e.g. rescorcinol) do not participate in the

oxidation reaction. It acts as an inhibitor.

Rescorcinol (benzene-1,3 diol)

• Para-diphenols (e.g. quinol) can act as a substrate but the

reaction rate is slower than ortho-diphenol since monophenol

hydroxylation reaction is required.

Quinol (benzene-1,4 diol)

• Although enzymatic browning is desirable in tea and apple

juice industries, in most fruits and vegetables and frozen

products, it should be prevented.

Methods to control enzymatic browning:

1. Blanching to destroy the enzyme, most commonly used method

2. HTST Pasteurization for fruit puree and not for fruit slices

(textural changes occur).

3. For potato, microwave heating causes more rapid inactivation

of phenolases than hot water treatment.

4. Dipping in water – limits O2 access to cut surfaces. E.g. potato

chips. Textural and flavour changes may occur.

5. Surface treatment of fruit slices (apple, pear, peach) with

excess ascorbic acid (antioxidant).

6. Vacuum packaging to exclude air.

7. Immersing in sucrose solution before freezing of fruits

(reduces dissolved oxygen).

8. Reduction of pH by adding acids. Citric, malic, phosphoric and

ascorbic acids are used to lower pH. They also serve as

chelators for Cu. Addition of lemon juice or vinegar is also

effective to lower pH.

Optimum pH for the reaction is pH 5-7. Below pH 3, the

enzyme is irreversibly inactivated. In apple juice production

malic acid is used to get pale brown colour (rate of reaction is

only reduced by malic acid).

9. Use of 1% NaCl solution: Cl- inhibits enzymic browning.

10. Addition of sulphites: Na2S, KMS, SO2 to inactivate enzyme.

11. Methylation of the substrate using SAM (methyl donor) and

ortho-methyl transferase enzyme. E.g.

Catechol + SAM + enzyme guaicol

Caffeic acid + SAM + enzyme ferulic acid

12. High Pressure Processing (HPP):

HPP is a technique of food processing where food is subjected

to high pressure (500-700 atmosphere) to achieve microbial

and enzyme inactivation.

High pressure processing causes minimal changes in foods.

Compared to thermal processing, HPP results in foods with

fresher taste and better appearance, texture and nutrition.

13. Ultrafiltration:

Ultrafiltration is a membrane separation process, driven by a

pressure gradient. The membrane separates liquid

components according to their size and structure. In the food

industry this technique is applied for white wine and fruit juices.

Ultrafiltration is able to remove larger molecules like enzyme

polyphenol oxidase, but not lower-molecular-weight

compounds like polyphenols.

Maillard Browning

• Maillard browning is a non-enzymic browning reaction

that takes place during the processing or storage of

protein foods containing reducing carbohydrates or

carbonyl compounds (e.g. aldehydes or ketones derived

from lipid oxidation).

• The minimum reactant requirements for Maillard browning

are the presence of an amino-bearing compound, usually

a protein, a reducing sugar and some water.

• The rate of Maillard reaction is markedly enhanced during

cooking, heat processing, evaporation and drying.

Maillard Browning

The Maillard reaction is responsible for many colors

and flavors in foodstuffs such as:

• caramel made from milk and sugar

• the browning of bread into toast

• the color of beer, chocolate, coffee, and maple

syrup

• the flavor of roast meat

• roasted nuts

• the color of dried or condensed milk

Maillard Browning

• Reaction rates are greatest in foods with an intermediate

moisture content such as roasted nuts, toasted breakfast

cereals and roller-dried milk powders.

• Maillard reaction begins with condensation of a non-

ionized amino group (terminal α-NH2 group of amino

acid) and a reducing sugar (aldose or ketose).

• During the first step, aldosylamines or ketosylamines are

formed by aldose and ketose sugars, respectively.

• Next, aldosylamines are transformed by „Amadori

rearrangement‟ into ketosamines while ketosylamines are

transformed by „Heyn‟s rearrangement‟ into aldosamines,

both of which are stable compounds.

Maillard Browning

Aldose sugar carbonyl Aldosyl Ketosamine

+ amino acid amine amine (Amadori

product)

Ketose sugar carbonyl Ketosyl Aldosamine

+ amino acid amine amine (Heyn‟s

product)

• During the second step, ketosamines and aldosamines

evolve into numerous carbonyl and polycarbonyl

unsaturated derivatives, such as reductones.

R – C = C – C - R‟

| | || - Reductone

OH OH O

Maillard Browning

• Some of these derivatives may react with amines and amino

acids leading to formation of ammonia and new carbonyl

compounds (Strecker degradation).

• Decarboxylation of free amino acids may also take place.

• Commercially the Strecker degradation is used to produce

distinctive flavours of chocolate, honey, maple syrup and

bread. Common Strecker aldehydes include ethanal (fruity,

sweet aroma), methylpropanal (malty) and 2-phenylethanal

(flowery/honey like aroma). Thus at times Maillard and

Strecker reactions are favourable.

• In the next step, polycarbonyl unsaturated derivatives undergo

both scission and polymerization reactions leading on one

hand to volatile compounds (aldehydes, pyrazines) and on the

other hand to brown or black pigments known as melanoidins,

with high molecular weight and complex structures.

Maillard Browning

• Such pigments are responsible for the colour of bread

and bakery products.

• It is important to control both Maillard and Strecker

reactions because of their adverse contribution to flavour

and odour and possible toxicity of degradation products.

• `Premelanoidin‟ products may also contribute to

nitrosamine formation, which is mutagenic.

Factors affecting the rate of browning

• Browning reaction occurs at a pH range of 7.8 - 9.2. In

less than pH 6, browning is reduced. In strong acidic

solutions browning does not occur.

• Browning is high at intermediate water levels.

• Copper and iron enhance browning. Fe3+ is more effective

than Fe2+

• Sugar type has an effect on the rate of Maillard reaction.

Pentoses > hexoses > disaccharides

• Among hexoses fructose is less reactive than aldoses.

Nutritional effects of Maillard reaction

1. Loss of biologically available lysine (in bread, milk). Other

basic amino acids such as L-arginine and L-histidine are

also susceptible.

2. Amadori products may inhibit intestinal absorption of

certain essential amino acids.

3. Formation of melanoidins destroys the digestibility of the

protein fraction of the molecules.

4. Melanoidins may be carcinogenic although they are

generally less absorbable (colloidal nature).

Methods to control Maillard browning

1. Decreasing moisture to very low levels or if

the food is a liquid, diluting it.

2. Lowering the pH

3. Lowering temperature

4. Removing one of the substrate, e.g. sugar

5. Addition of sulphur dioxide or sulphites.

Caramelization

• Direct heating of carbohydrates, particularly sugars and

sugar syrups, produces a complex group of reactions

called caramelization. It is used extensively in cooking for

the resulting nutty flavor and brown color. Caramelization

generally occurs at high temperatures (~150 °C), low

water content or high sugar content.

• Reactions are facilitated by small amount of acid and

certain salts.

• Thermolysis causes dehydration with formation of

anhydro rings or introduction of double bonds into sugar

rings.

• The latter produces intermediates to unsaturated rings,

such as furans. Conjugated double bonds absorb light

and produce colour.

Caramelization

• Maltol, isomaltol, hydroxyl methyl furans contribute to

the flavour and can be used to enhance various

flavours and sweeteners.

• In unsaturated ring systems, condensation will occur to

polymerize ring systems, yielding useful colours and

flavours.

• Catalysis speed up the reaction and often is used to

direct the reaction to specific types of caramel colours,

solubilities and acidities.

• Sucrose is commonly used to produce caramel colours

and flavours. It is heated in solution with acid or acidic

ammonium salts to produce a variety of products used

in food, candies and beverages (cola drinks, beer).

Caramelization

• Caramel pigments contain hydroxyl groups of varying

acidity, carbonyl, carboxyl, enolic and phenolic

hydroxyl groups.

• Rapid initial reaction is oxygen dependent and

proceeds until oxygen is completely exhausted.

• Increasing the temperature and increasing pH

increases the reaction rate. At pH 10 the reaction is 10

times faster than at pH 5.9.

• In the absence of buffering salts, humic substances

are formed which produce a bitter taste.

Ascorbic Acid Oxidation

• Decomposition of ascorbic acid is reported to be the major

deteriorative reaction occurring during the storage of orange

juice. Further, there exists a high correlation between the

percentage loss of ascorbic acid and an increase in browning

in grapefruit juices.

• Ascorbic acid breakdown in orange juice results in furfural

production and furfural buildup closely parallels quality loss in

citrus products.

• It has been demonstrated that amino acids accelerate ascorbic

acid breakdown, and in the presence of amine it is the

dehydroascorbic acid (DHA) that is the reactive intermediate in

the pathway to furfural and brown pigment production.

• If DHA has already been formed in juices brown color is

produced more intensely under non-oxidative conditions than

under oxidative conditions.

Ascorbic acid oxidation