Download Enzymatic methods in omega-3 concentration - Lectures For UG-5

Polyunsaturated Fatty Acids and
Their Therapeutic Functions
MTB Lecture # 9
PUFA
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Many of the fatty acids can be synthesized by humans, but there is a
group of PUFA, the essential fatty acids, that the human body cannot
produce: omega-3 (n−3) and omega-6 (n−6) fatty acids.
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The parent omega-6 fatty acid is linoleic acid (C18:2n−6, LA) and the
parent omega-3 fatty acid is α-linolenic acid (C18:3n−3, ALA).
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Omega-6 fatty acids as arachidonic acid (C20:4n−6; AA) can be
synthesized by humans from LA, and omega-3 fatty acids, as
eicosapentaenoic acid (C20:5n−3; EPA), docosapentaenoic acid
(C22:5n−3, DPA) and docosahexaenoic acid (C22:6n−3, DHA), from αLA.
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The conversion of α-LA in EPA, DPA and DHA is low and these omega3 fatty acids are considered essential fatty acids too. Therefore, both n−3
and n−6 PUFA are entirely derived from the diet and necessary for
human health.
Omega-3 fatty acids are a class of polyunsaturated fatty acids. They all
have a double carbon-to-carbon bond in the third position from the
omega (or methyl, or n) end of the fatty acid chain.
Omega-3 family include
Eicosapentaenoic acid (EPA)
Docosahexaenoic acid (DHA)
Alpha-linolenic acid (ALA).
.
PUFA

Both n−3 and n−6 PUFA are entirely derived from the diet and
necessary for human health.

An n−6:n−3 fatty acid ratio of 5:1 or less is desired, as suggested by
nutrition experts (WHO/FAO, 1994).
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Modren trends in food a high consumption of meat, seed oils, fast food
(pizzas, hamburgers…) and snack food (cakes, biscuits…), that contain a
large amount of saturated fatty acids and a low proportion of PUFA
(Fernández-SanJuan, 2000).
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The researches concluded that the n−6:n−3 ratio in blood, in people who
consume Japanese or Mediterranean food, is close to 2:1, while in people
who consume fast food, it can reach values up to 25:1, much higher than
desired
EPA and DHA
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EPA and DHA are found primarily in fatty fish; α-LA is abundant in
flaxseed, walnuts, and soybeans.

The human body can convert small amounts of α-LA into EPA and
DHA: only about 5% of α-LA is converted to EPA and less than 0.5%
is converted to DHA.
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It is not known whether α-LA is active itself or only via these
metabolites
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Healthy people should consume fish (preferably oily fish) at least
twice a week, according to the American Heart Association

Cod and Catfish contain 0.2 g of EPA/DHA per 100-g serving;
others, such as Atlantic salmon, contain about 10 times as much
Omega-3 fatty acids and human health
The omega- 3 index (percentage of EPA+DHA of total fatty acids in red
blood cells) as a risk factor for sudden cardiac death should be
higher than 8%. There are also several studies that propose the
mechanisms by which the omega-3 PUFA act in humans
Fish oil supplements lower triglyceride levels and may have other
benefits such as preventing arrhythmias, reducing inflammation
(although they have minimal impact on C-reactive protein (CRP)),
inhibiting platelet aggregation, and lowering blood pressure, all of
which should reduce cardiovascular risk.
CRP binds to the phosphocholine expressed on the surface of dead or
dying cells and some bacteria. This activates the complement system,
promoting phagocytosis by macrophages, which clears necrotic and
apoptotic cells and bacteria.
Omega-3 fatty acids and human health

Hypertriglyceridemia is thought to increase the risk of coronary
heart disease by two mechanisms.

Firstly triglyceride-rich lipoproteins such as very low-density
lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) are
thought to be atherogenic promote ASVD.
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Secondly, triglyceride-lipoprotein metabolism involves competition
with high-density lipoprotein (HDL), leading to a decrease in HDL
production and to denser LDL particles (bad cholesterol,
attract macrophages, and thus drive atherosclerosis. ).
How omega-3 fatty acids lower triglyceride
levels

One mechanism, seen in animal studies, is by decreasing hepatic
synthesis and secretion of VLDL particles by inhibiting various enzyme
transcription factors.

Another proposed mechanism is that EPA and DHA increase the
activity of lipoprotein lipase (pancreatic lipase, hepatic lipase, and
endothelial lipase), leading to an increase in chylomicron (Transport
dietary lipids) clearance.

It is a water soluble enzyme that hydrolyzes triglycerides in lipoproteins, such as
those found in chylomicrons and very low-density lipoproteins (VLDL), into two
free fatty acids and one monoacylglycerol molecule. It is also involved in promoting
the cellular uptake of chylomicron remnants, cholesterol-rich lipoproteins, and free
fatty acids

This was validated by Khan et al (2011)who showed that lipoprotein
lipase activity increased in patients who received omega-3 fatty acids 3
g/day for 6 weeks.
How much do they lower triglycerides
Data from the makers of Lovaza3 indicate that in a patient population
with a mean baseline triglyceride level of 816 mg/dL, 4 g/day of
omega-3 fatty acids lowered triglyceride levels to 488 mg/dL, a 45%
reduction (P < .0001).
In addition, HDL cholesterol (HDL-C) levels increased by 9%.
Higher the base line more efective is this conversion.
People with known coronary artery disease should take in 1 g of EPA/DHA
per day according to the American Heart Association
Procedures to obtain long chain omega-3 PUFA

The procedures to obtain long chain omega-3 PUFA concentrates,
usually from the natural sources that contain them, have been
investigated and some new procedures are being proposed.
Production of omega-3 fatty acids from fish:

The most important natural sources of omega-3 PUFA are marine
organisms (fish, seafood, algae), that are fed, directly or indirectly, from
marine phytoplankton, the primary producer of omega-3 in the trophic
chain
In general, traditional processes to obtain fish oil involve two stages:

oil extraction from raw material and refining.

Some other new processes to obtain omega-3 concentrates for
pharmaceutical or nutritional purposes include enzymatic methods and
methods that use supercritical fluids.

Traditional Fish oil extraction processes

Fish oil is produced from the antiquity by Nordic towns that used it
as fuel in lamps. At the beginning of the 19th century, USA began to
produce fish oil from menhaden, using a process with two steps:

Fish cooking and rock-weighted pressing later this press was
replaced by mechanical screw presses and later by hydraulic presses.

To improve the yield of the extraction, the quality of the fish oil
extracted and the profitability of the process for industrial purposes,
especially using fish by-products as raw material

The traditional methods that use organic solvents for oil extraction
are widely applied for analytical purposes but not for production
due to the several drawbacks of using solvents with restrictions in
the food
wet pressing method

Nowadays, the most common process to obtain crude fish oil from
fresh fish at industrial scale is the wet pressing method, as
describedby the Food and Agriculture Organization of the United
Nations (FAO,1986).

This process involves the production of fish oil and fish meal
through several steps, i.e. cooking of the raw material, pressing of
the cooked material and final filtration or centrifugation to recover
the oil from the miscella. The use of a 3-phase centrifuge can greatly
simplify the separation stages after cooking.

Different fish by-products have also been proposed as sources of fish
oil such as separation of oil from tuna heads.
Flow diagram of the conventional method to
obtain crude fish oil
Supercritical fluid extraction (SFE)

The fluid possesses intermediate properties between a gas and a liquid, i.e.:
liquid-like density and gas-like viscosity and diffusivity. That is, supercritical
fluids (SCF) have at the same time a good solvent power and good transport
properties

The most widely used SCF is carbon dioxide that is considered a green solvent;
CO2 is non toxic, cheap and non flammable. As is gaseous under ambient
conditions and therefore easy to separate from the processed products after
processing.

Fish oil solubility increased significantly with pressure, whereas, at constant
pressure, oil solubility decreased with temperature up to a pressure value from
which oil solubility began to increase as temperature increased.

Solubility of fish oil from sardine in SC-CO2 in a pressure range of 20 to 35 MPa
at four different temperatures (313, 333, 343 and 353 K)
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The extraction of sardine oil with SC-CO2, concluded that it is possible to find the
conditions to recover most of the oil (95%) without degradation of the omega-3
PUFA
Oil extraction by enzymatic methods

This technology requires neither organic solvents nor high temperatures

The use of enzymatic technology to release lipids from fish avoiding the
use of solvents and high temperatures: Liaset, Julshamn,&Espe (2003)
studied the enzymatic hydrolysis of salmon frames with proteases and
the composition of the different fractions obtained after separation by
centrifugation.

They reported that this process enables to obtain omega- 3 enriched oil
with a good recovery (about 77%) as well as several interesting products
such as peptides or essential amino acids.

Linder, Fanni, and Parmentier (2005) developed another enzymatic
method to extract oil from ground salmon heads at middle temperature
(55 °C) different commercial enzymes: a protease (Alcalase), an
exopeptidasen(Neutrase) and an endo-peptidase (Flavourzyme).
Fish oil refining

The oil extracted from a natural material is a mixture of several compounds as
free fatty acids, glycerides, phospholipids, sterols, pigments or tocopherols, and,
sometimes, toxics such as heavy metals, dioxins or PCBs (Cheryan, 1998).

Oil refining needs to be performed to remove non-triglyceride, colorants, smelly
and toxic compounds in the production of edible oils.

The conventional oil refining in industry is usually made by chemical methods,
which include several steps
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degumming, to separate phospholipids
neutralization or de-acidification, to clear free fatty acids and
decrease oil acidity
bleaching to absorb pigments or contaminants and de-odorization
to remove smelly compounds.
This process presents several drawbacks since it involves the use of chemical
products (alkalis) that contaminate the environment and some neutral oil is lost,
mainly in oils with high free fatty acid content
Physical Refining Processes,

based on the application of superheated steam under low pressure have been
proposed as alternative to remove free fatty acids and volatile compounds.

However, these methods require a preliminary step of chemical refining and,
due to the use of high temperatures, they are not suitable for thermolabile oils
such fish oil.

Physical adsorption on activated carbon has been proposed recently to remove
contaminants, such as dioxins and PCBs, from fish.

Traditional oil deodorization is based on the application of high temperatures is
not good for fish oil above 180ºC the an important PUFA degradation, involving
the formation of many undesirable compounds such as polymers, PUFA isomers,
mono and di-trans and cyclic fatty acid monomers.

Vacuum steam distillation at low temperatures followed by a treatment in a silica
gel column, adsorption with a resin or treatment with diatomaceous earth have
been proposed for removing smelly compounds from fish oil.
Supercritical fluid technology, together with membrane and enzymatic
processes, is one of the most recent technologies proposedas
alternative to oil refining with chemical products or high
temperatures.
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Omega-3 concentration from fish oil
Enzymatic methods in omega-3 concentration
Stabilization of omega-3 PUFA
Alternative sources of omega-3 fatty acids
Medical applications
Omega-3 concentration


Omega-3 fatty acids are better absorbed by human organism when they
are as acylglycerides than as esters and their stability against oxidation
is also higher when omega-3 are as acylglycerides than as esters
Enzymatic methods in omega-3 concentration
Normally lipases, which are able to catalyse reactions such hydrolysis,
ethanolysis or transesterification of triglycerides.

Due to the fatty acid distribution in the glycerol backbone found in
several marine triglycerides (Ando et al., 1992) and the stereo-specifical
activity of certain lipases (Wong, 2003), these methods are very useful
both in omega-3 concentration from fish oil and in the production of
structured lipids.

In the last years, a great amount of enzymatic methods followed by a
separation process such membrane filtration, urea complexation or
molecular distillation have been proposed in the literature to obtain
omega-3 concentrates, especially EPA and DHA, as different forms such
free fatty acids, ethyl esters or 2-acylglycerides
Alternative sources of omega-3 fatty acids

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The benefits of fish as source of omega-3 are sometimes questioned
(Domingo, 2007) and several alternative sources for omega-3 PUFA
have been explored.

Marinemicroalgaes, algaes or transgenic plants. A study comparing
microalgae oil and fish oil concluded that both oils present a similar
amount of omega-3, although microalgae oil has the advantage of
presenting neither an unpleasant odour nor a high amount of
cholesterol, and contains squalene and phytosterols, which offer
additional benefits to human health.

In addition, microalgaes are easily cultivated, which avoid
differences in seasonal production and enables increasing the
productivity of PUFA from an industrial point of view.

two main techniques used at the time for obtaining highly pure
PUFA: urea fractionation and HPLC, and detailed what were
“potentially useful techniques” such as supercritical fluid extraction
and lipase-catalyzed processing.

From then to now, several authors have proposed new PUFA
extraction methods from different microorganisms, many of them
using supercritical fluid technology, similar to the fish oil extraction
processes.

Transgenic plants have been also proposed as an alternative source
of omega-3 fatty acids. Napier (2006) has recently published a review
with the last advances in the production of enriched vegetables
species through genetic modifications. Robert (2006) has also
compiled the most recent studies about production of transgenic
seed oils and their use in human and aquaculture nutrition.