UNIT IX

Fatty acid composition of fish liver and body oils, auto-oxidation of fatty
acids, rancidity, lipasas and phospholipases, pro- and anti-oxidants, oxidation indices, lipid-
protein interactions, oxidized lipids-protein interactions and their impact on quality.

Fatty acid composition

Introduction
Fish and other sea foods remain an important source of white meat for the human diet due to
its beneficial effect in reducing coronary heart diseases. This effect is especially due to the fat in fish,
which is probably responsible for converting fatty dienoic acid into tetraenoic, pentaenoic and hexaenoic
acid1−5.
Like other oils and fats, fish oils may be divided into their lipid fractions such as
triacylglycerol, diacylglycerol, monoacylglycerol, phospholipids, sterylesters, sterols and free fatty acids.
The quantity of total lipids may differ between various tissues and organs and also between different
species. The type of fatty acids present as free acid or as neutral lipid differs to a great extent from species
to species and water environments6−8.

In a study of both freshwater and ocean fish oil preparations, it was observed that fish oil preparations
from ocean fish contained 35 unsaturated fatty acids with 1-6 double bonds.
In contrast, freshwater fish oil preparations contained 14 unsaturated fatty acids having 1-5
double bonds. Ocean fish oil preparations contained considerable amounts of unsaturated fatty acids of
≥= 20 C, such as C20:5 and C22:6, whereas all freshwater fish fatty acids showed a chain length of < 20
of carbon atoms. Fatty acids except 20:1 and 22:1, which are of exogenous origin, are a basic composition
fish oil from temperate and northern latitudes, with similar totals for saturated (14:0 and 16:0),
monounsaturated (16:1 and 18:1) and polyunsaturated (primarily ω-3) fatty acids.
The fatty acid pattern of triacylglycerol and
phospholipids of various fish oils were also assessed, showing that they contained highly unsaturated fatty
acids. The polyunsaturated fatty acids of triacylglycerol and lecithin were preferentially located in the β-
position. Various scientists have studied the composition of fish liver oil and found that 91-99% of the
fatty acids in the β-position are unsaturated and 36-86% of the fatty acids in the α-position are saturated.

NATURE OF FATTY ACIDS AND CHEMICAL DISTRIBUTIONS

Fish oils and marine-animal oils are
generally characterized by a rather large group of saturated and unsaturated fatty acids, which are
commonly associated with mixed triglycerides. In addition to triglycerides, body oils from fish and
marine animals usually include minor amounts of fatty acids as substituents of phospholipids and other
lipids. In comparison to body oils, on the other hand, liver oils and oils from particular parts of fish and
marine animals can often contain large amounts of fatty acids associated with phospholipids, glyceryl
ethers (alkoxydiglycerides), and wax esters, depending on the source of oils and lipids.

ENVIRONMENTAL INFLUENCE ON FATTY ACID COMPOSITION

It has been observed in the past that

environmental factors such as geographic locations of catch and seasons of the year, which may be related
to water temperatures, are related to the proportions of fatty acids of fish oils. Re evaluations of these
factors have been important in light of modern instrumentation like gas-liquid chromatography (GLC)
(Farquhar et al. 1959). Seasonal variations in fatty acids of commercial cod-liver oils were investigated
by DeWitt ( 1963). He observed a range in iodine values, 146 to 168, for GLC analyzed fatty acids of the
liver oils sampled from May to November.

Auto-oxidation of fatty acid

Products that contain fats and oils turn rancid and deteriorate in other
ways when they arc exposed to air. The action,known as autoxidation, imparts disagreeable flavors and
odors to fats and foods containing them. Specifically, the basic problem was the mechanism of
autoxidation—how oxygen attacks a fat. On that point the investigators made several discoveries in
1943. They have developed moderately effective methods of preventing rancidification, and believe now
that the solution of the problem is in sight. It is important to prevent autoxidation because fat and fat-
containing foods valued at several billion dollars are produced and marketed annually. The oil, meat,
fishery, dairy, and bakery industries suffer serious losses from autoxidation of their products. Nutritional
tests indicate that autoxidation reduces or destroys the food value of fats. For example, the vitamin E
content of vegetable oils and the vitamin A content of fish oils are reduced, if not entirely destroyed,
during autoxidation.

Oxidative rancidity of fats such as lard, shortenings, salad and cooking oils refers to the undesirable
odors and flavors which develop when such products are exposed to the oxygen in the air. Products
containing these fats, including but not limited to food products such as fish, poultry, meat, frozen
vegetables and dry milk can become rancid as the fats in the products react to air. The poly-unsaturated
fatty acid portions of these foods react with oxygen to form peroxides.
The peroxides decompose to yield a
complex of mixtures, including aldehydes, ketones, and other volatile products. These products are
responsible for "rancid" odors and flavors. It is important to note that fish contain highly unsaturated
(poly-unsaturated and mono-unsaturated) fatty acids which make some fish products particularly
susceptible to oxidative deterioration. Highly saturated products, such as butter, are not as prone to
oxidative rancidity due to the absence of poly-unsaturated fatty acid compounds. These products also tend
to be more solid at room temperature.

Hydrolytic rancidity refers to the odor that develops when triglycerides are hydrolyzed and free fatty
acids are released. This reaction of lipid with water sometimes requires a catalyst, but results in the
formation free fatty acids and salts from free fatty acids (soaps). In particular, short chain fatty acids, such
as common butter fats, are odorous.

Factors Influencing Fat Oxidation

Temperature: The rate of fat oxidation is highly dependent on temperature. Considerable improvement
in storage stability can therefore be gained by lowering the storage temperature. As an example, it has
been found that the storage time for frozen raw, lean meat can be extended approximately by 3 times by
lowering the temperature from 5 to -13°F (-15 to -25°C). Temperature fluctuations during storage should
also be minimized.
Oxygen: Oxygen in the air may be displaced by an inert gas such as nitrogen or carbon dioxide to retard
oxidative rancidity, or the products may be packed under vacuum. These methods require the use of
packaging materials with low oxygen permeability.

Type of fat: In general, the softer the fat, the more unsaturated are the fatty acids and the more
susceptible they are to oxidation and oxidative rancidity. However, vegetable fats, although unsaturated,
are usually more stable than animal fats because they contain natural antioxidants. The most common
antioxidant found in vegetable fats is vitamin E.

Light. Packages that exclude light can be used to protect the products against fat oxidation.

Metals. Metals such as copper, iron, manganese, and chromium increase rate of fat oxidation. As a result,
the preferred storage containers are steel drums, tin, or nonmetallic materials such as plastic. Stainless
steel is commonly used in processing plants so as to avoid excessive contact with metals that increase fat
oxidation. It is important to note that water with trace metal is often a cause of rancidity in food products.

Products from fat oxidation. Traces of oxidized fat in ingredients can accelerate oxidative rancidity in
the remainder of the products. Blending oxidized products with un-oxidized products is not
recommended. Steam treatment under vacuum conditions has been effective in removing products of
deterioration (odorous substances) from some oils and fats.

Antioxidants
Food antioxidants are compounds that increase the resistance of fats to oxidation and consequent
deterioration or rancidity. Only specifically approved antioxidants are able to be added to lard, tallow, and
other foods susceptible to rancidity. In the United States, authorization of antioxidants for use in meat
products is a responsibility of the Food Safety Inspection Service (FSIS) of theUnited States Department
of Agriculture (USDA). Antioxidants used in dairy products, salad dressings and oils are regulated by the
Food and Color Additives Division of the U. S. Food and Drug Administration (FDA). Antioxidant
inclusion is restricted to specific limits and must be declared on product labels. Some of the approved
antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), propyl gallate, and
tocopherols.

These primary antioxidants are often used in combination with citric or phosphoric acid.

Natural antioxidants, such as those contained in some spices, such as

rosemary, sage, and marjoram, have met acceptance for the retardation of rancidity in meat products.
These and other natural antioxidants not only retard the warmed over flavor (WOF) in precooked meat
products, but provide an agreeable aromatic aroma and flavor. Some spice extracts, particularly rosemary,
are prepared primarily for their antioxidant activity and do not include strong flavor components.
 Lipases
INTRODUCTION
Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are a class of hydrolase which
catalyze the hydrolysis of triglycerides to glycerol and free fatty acids over an oil–water interface. In
addition, lipases catalyze the hydrolysis and transesterification of other esters as well as the synthesis of
esters and exhibit enantioselective properties. The ability of lipases to perform very specific chemical
transformation (biotransformation) has make them increasingly popular in the food, detergent, cosmetic,
organic synthesis, and pharmaceutical industries. Lipases have emerged as one of the leading biocatalysts
with proven potential for contributing to the multibillion dollar underexploited lipid technology bio-
industry and have been used in in situ lipid metabolism and ex situ multifaceted industrial applications .
Lipases are produced by animals, plants, and microorganisms. Microbial
lipases have gained special industrial attention due to their stability, selectivity, and broad substrate
specificity

General characteristics of lipase

Lipase (triacylglycerol acylhydrolase) is an omnipresent enzyme with important physiological

significance and industrial potential. Lipases catalyze the hydrolysis of triglycerides to glycerol and free
fatty acids. They are soluble in water and hydrolyze insoluble substrates to more polar lipolytic products.
The first lipase was identified by Claude Bernard in 1856 and from then on they have been identified in
microorganisms, plants and animals.

Lipases are serine hydrolases containing G-X1-S-X2-G sequences as the catalytic part of the particle,
where G = glycine, S = serine, X1 = histidine, X2 = glutaminic or aspartic acid. Such structure is
characteristic also for serine proteases. The knowledge of their 3-dimensional structure plays a significant
role in designing and structuring lipases for specified purposes [3]. It has been shown that many lipases
are phylogenetically connected.
The properties of lipases,

1. A definitely desirable feature is the thermal stability of this enzyme

2. The source of thermally resistant lipases are first and foremost bacteria, and the thermal
durability is linked to their structure.
3. The thermal stability is influenced by such factors as pH and the presence of heavy metals.
4. All activities at the level of protein engineering are meant to improve their stability
5. A considerable improvement of the resistance of many lipases may be obtained by their
immobilization on substrates and in many types of carriers.

Application of lipase

Lipases are commonly used in processing fats and oils, as additions to detergents and degreasing
agents, in food processing, chemical and pharmaceutical syntheses, in paper production and the cosmetic
industry. They may also be used in increasing the speed of fatty and polyurethane waste decomposition.
Lipases used as additions to detergents must fulfill the following criteria: low substrate selectivity,
resistance to fairly raw washing conditions (pH 10-11, 30-60°C), resistance to destructive surfactants and
enzymes (e.g. linear AAS and proteases) which are very important components of detergents.

Antioxidant

An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is

a chemical reaction that can produce free radicals, leading to chain reactions that may damage cells.
Antioxidants such as thiols or ascorbic acid (vitamin C) terminate these chain reactions. The term
"antioxidant" is mainly used for two different groups of substances: industrial chemicals which are added
to products to prevent oxidation, and natural chemicals found in foods and body tissue which are said to
have beneficial health effects.
To balance the oxidative state, plants and animals maintain complex systems of overlapping antioxidants,
such as glutathione and enzymes (e.g., catalase and superoxide dismutase) produced internally or the
dietary antioxidants, vitamin A, vitamin C, and vitami E.
Antioxidant dietary supplements do not improve health nor are they effective in preventing
diseases. Randomized clinical trials including supplements of beta-carotene, vitamin A, and vitamin E
singly or in different combinations found no effect on mortality rate and cancer risk, or may even
increase cancer risk. Supplementation with selenium or vitamin E does not reduce the risk
of cardiovascular disease. Oxidative stress can be considered as either a cause or consequence of some
diseases, an area of research stimulating drug development for antioxidant compounds for use as
potential therapies.

Pro-oxidant activities
Antioxidants that are reducing agents can also act as pro-oxidants. For example, vitamin C has
antioxidant activity when it reduces oxidizing substances such as hydrogen peroxide, however, it will also
reduce metal ions that generate free radicals through the Fenton reaction.
 2 Fe3+ + Ascorbate → 2 Fe2+ + Dehydroascorbate
2 Fe2+ + 2 H2O2 → 2 Fe3+ + 2 OH· + 2 OH−
The relative importance of the antioxidant and pro-oxidant activities of antioxidants is an area of
current research, but vitamin C, which exerts its effects as a vitamin by oxidizing polypeptides,
appears to have a mostly antioxidant action in the human body. However, less data is available for
other dietary antioxidants, such as vitamin E, or the polyphenols. Likewise, the pathogenesis of
diseases involving hyperuricemia likely involve uric acid's direct and indirect pro-oxidant properties.