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Importance of instrumental and sensory analysis in the assessment of

oxidative deterioration of omega-3 long-chain polyunsaturated fatty acid-
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Article  in  Journal of the Science of Food and Agriculture · January 2007

DOI: 10.1002/jsfa.2733

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Journal of the Science of Food and Agriculture J Sci Food Agric 87:181–191 (2007)

Review
Importance of instrumental and sensory
analysis in the assessment of oxidative
deterioration of omega-3 long-chain
polyunsaturated fatty acid-rich foods
Wojciech Kolanowski,1∗ Danuta Jaworska1 and Jenny Weißbrodt2
1 Department of Analysis and Quality Assessment of Food, Faculty of Human Nutrition and Consumer Sciences, Warsaw Agricultural
University, Warsaw, Poland
2 Institut für Ernährungs und Lebensmittelwissenschaften, Universität Bonn, Bonn, Germany

Abstract: Omega-3 long-chain polyunsaturated fatty acids (LC PUFA) positively influence human health. Their
main dietary source is fish, especially fish oil. Owing to low fish consumption in many Western countries the
average intake of omega-3 LC PUFA is below the recommended level. This raises interest in diet supplementation
and food enrichment with fish oil. However, due to a high degree of unsaturation fish oil is extremely susceptible
to oxidation. Oxidation of fish oil increases when added to food products, which may be enhanced by some
antioxidants, under certain conditions. For quality control of omega-3 LC PUFA-containing foods adequate and
combined methods of oxidation assessment should be used, beginning from the raw material and continuing
during processing, storage and distribution. To achieve this goal correlation of instrumental and sensory methods
with multivariate data analysis may give the best results. In this paper problems of oxidation of fish oil and fish
oil-containing foods, as well as methods for its assessment, are reviewed.
 2006 Society of Chemical Industry

Keywords: omega-3 LC PUFA; fish oil; supplementation; food enrichment; lipid oxidation

INTRODUCTION even cultured fish contain less omega-3 PUFA than

The omega-3 polyunsaturated fatty acid (PUFA)
family consists of α-linolenic acid (LNA C18:3) and
its long-chain metabolites eicosapentaenoic acid (EPA
C20:5) and docosahexaenoic acid (DHA C22:6).
The beneficial health effect of omega-3 fatty acids,
especially long-chain EPA and DHA (LC PUFA), are
well demonstrated in the prevention of cardiovascular
diseases, some types of cancer and rheumatoid
arthritis. Additionally DHA acts as an important factor
ensuring proper development and function of the brain
and visual function.1 – 6
The main natural sources of omega-3 LC PUFA
are fish and other sea animals. Depending on fish
species, age, season and area of living, fish oils may
contain 100–300 g kg−1 of omega-3 LC PUFA EPA
and DHA.7,8 However, there is a large gap between
the actual consumption of fish and the recommended
intake of omega-3 LC PUFA. Due to low acceptance
of oily fish in many societies where so-called Western-
style diet is predominant, average fish intake is
currently far below the recommended minimum of
two fish servings per person per week. Additionally,
modern agriculture with its emphasis on production
has decreased the omega-3 PUFA content in many
foods.5 Animal meats, green leafy vegetables, eggs and
those in the wild.9,10 Currently an average level of
omega-3 LC PUFA intake in the developed countries
of the Western-style diet is approximately 0.15 g per
person per day, which is below the recommended
minimum.11 – 13
The United Kingdom Department of Health (1994)
recommends intake of 1.5 g of EPA plus DHA per
week (i.e., approximately 0.2 g per day), equivalent to
two servings of oily fish, for normal health. Similarly,
the European Academy of Nutritional Sciences
(EANS) recommends intake of an average of 0.2 g of
EPA plus DHA per day.14,15 The recommendations of
the International Society for the Study of Fatty Acids
and Lipids (ISSFAL) suggest an adequate intake of
omega-3 LC PUFA to be 0.65 g of DHA plus EPA
per day (minimum 0.22 g per day each) and the ratio
of omega-6 to omega-3 in the diet should be 4 to
1 instead of the current ca 10 to 1.4,15 As an upper
limit in the year 2000 FDA stated that daily intake
of EPA and DHA should not exceed 3.0 g per person
per day in the form of fish oil from food and dietary
supplement sources.16
Besides fish consumption, alternative ways to
ensure an optimal omega-3 LC PUFA intake are
dietary supplements and food enrichment with

∗
Correspondence to: Wojciech Kolanowski, Department of Analysis and Quality Assessment of Food, Faculty of Human Nutrition and Consumer Sciences,
Warsaw Agricultural University, 02-774 Warsaw, Poland
E-mail: wojciech kolanowski@sggw.pl
(Received 3 September 2005; revised version received 4 November 2005; accepted 8 September 2006)
Published online 17 November 2006; DOI: 10.1002/jsfa.2733

 2006 Society of Chemical Industry. J Sci Food Agric 0022–5142/2006/$30.00

W Kolanowski, D Jaworska, J Weißbrodt
purified fish oil or fish oil powder obtained by
microecapsulation.15,17,18 The production flow sheet
of food-grade fish oil is shown in Fig. 1. By taking
fish oil supplements or enrichment of various food
products with fish oil, the average intake of omega-
3 LC PUFA may be increased, resulting in better
health protection.11,17 – 21 Fish oil can be added to
food either after partial hydrogenation or with the
fatty acid composition unaltered. Hydrogenated oil
is much more stable to oxidation but hydrogenation
causes saturation of double bonds of unsaturated fatty
acids and formation of trans isomers, which damage the
desirable health properties of omega-3 LC PUFA.22,23
Each year the assortment of food products enriched
with fish oil and fish oil-containing supplements
increases.15,24 However, due to very high level of
unsaturation, omega-3 LC PUFA are extremely
susceptible to oxidation. Thus, when incorporating
fish oil into food products, the main concern
is oxidative deterioration, provoking subsequent
development of unacceptable ‘fishy’ off-flavour.25
To avoid this problem producers often use small
amounts of fish oil and extra addition of flavourings or
seasonings into enriched foods. That may effectively
mask unacceptable off-flavour of gradually oxidized
fish oil in the product when processed and/or stored in
the presence of oxygen. Such practice makes fish oil-
enriched food palatable after longer storage periods:
however, masked oxidation significantly impacts safety
of such foods and may be harmful for the consumer’s
health.
Nutritional supplements containing omega-3 LC
PUFA are generally supplied as unsaturated, purified
fish oil embedded in gelatin hard capsules, protecting
them against oxidation. However, the complete
protection of encapsulated fish oil is difficult to
Figure 1. Production flow sheet of food-grade fish oil.24
182
achieve and some level of oxidized compounds is often
detected in these products.26,27 Many more problems
with oxidative stability may be caused by fish oil
microencapsulation, due to greatly increased surface
area and difficulties in sufficiently dense coating
formation, which facilitate oxidation.27 – 31
OXIDATION OF OMEGA-3 LC PUFA
Omega-3 LC PUFA are the most unsaturated fatty
acids occurring in nature (five and six double bounds),
which makes them especially susceptible towards
oxidation. Oxidation of fish oil, as other PUFA-
containing lipids, involves the reaction of unsaturated
fatty acids with oxygen and occurs in three phases: an
initiation or induction phase, a propagation phase and
a termination phase.32 During the initiation phase,
molecular oxygen combines with unsaturated fatty
acids to produce peroxides and free radicals, both
of which are very reactive. For this phase to occur,
besides oxygen, some oxidative initiators must also be
present, such as chemical oxidizers, transition metals
(i.e., iron and copper) or enzymes (i.e., lipoxygenases).
Heat and light (UV) also increase the rate of this and
other phases of lipid oxidation.33,34
The reactive products of the initiation phase
react with additional lipid molecules creating other
reactive chemical products. This chain reaction, i.e.,
propagation of further oxidation by propagation phase
products, gives rise to the term ‘autoxidation’.35
Peroxides are particularly unstable due to the
presence of active methylene groups. These promote
the formation of dihydroperoxides that, in turn,
decompose to secondary oxidation products. In
the final, termination phase, relatively unreactive
secondary oxidation products are formed, including
J Sci Food Agric 87:181–191 (2007)
DOI: 10.1002/jsfa
 Instrumental and sensory assessment of omega-3 LC PUFA-rich foods
hydrocarbons, aldehydes and ketones. Unsaturated
aldehydes and ketones undergo further autoxidation,
producing volatile compounds, which are perceived as
off-flavours and as a warning that food is no longer
edible.34,36,37 Due to the low odour threshold the
presence of volatile secondary oxidation products,
even at low concentrations, significantly decreases
the sensory quality of fish oil and fish oil-containing
foods.25,38 – 40
Oxidation may easily decrease quality of omega-
3 LC PUFA-rich foods, which makes it important
to assess chemical and physical factors promoting
oxidation during processing, storage and distribu-
tion. Access to oxygen and light, developed surface
area, heating and irradiation accelerate lipid oxida-
tion, decreasing stability and shelf-life of products
containing fish oil.34 Additionally contact and possi-
ble contamination with iron and copper ions, which
are catalysts of lipid oxidation, during processing
and storage should be minimised.41 Moreover, high-
and low-molecular-weight compounds formed by the
decomposition of PUFA during refining can impact
oxidative stability and flavour of fish oil and fish oil-
containing foods. Refining of fish oil also decreases the
natural tocopherol level and high temperature of the
deodorization process leads to promotion of oxidation.
Fish oil may be processed by molecular distillation at
lower than the conventional deodorization tempera-
ture (180 ◦ C or below) under high vacuum to decrease
polymer formation.34,42 Although improved deodor-
ization processes have been developed, off-flavours
continue to limit the use of fish oil for food enrichment,
supplements and other nutritional purposes.
Oxygen is the main factor promoting lipid oxida-
tion, which is accepted to be limited by the addition of
antioxidants.43,44 However, addition of antioxidants
cannot completely prevent oxidation of highly unsat-
urated fish oil. The most effective method to stabilize
fish oil or other foods containing omega-3 LC PUFA
against oxidation is to remove all the oxygen and other
oxidation-promoting factors during handling the raw
materials, processing, packaging, storage and distribu-
tion. Modern technology allows vacuum or inert gas
(nitrogen, carbon dioxide) conditions during process-
ing and packaging, but residual oxygen levels of less
than 1% volume in food products are very difficult
to achieve.15,45 – 48 To stabilize omega-3 LC PUFA
of fish oil refining, addition of antioxidants, inert gas
or vacuum conditions during processing and packag-
ing and low temperature of storage and distribution
are needed. Subsequently effective methods of quality
control should be applied for detection of oxidative
deterioration and stability of the product.
Quality of foods has an impact on consumers’
behavior.49 In the view of the consumer, flavour is the
most important among other attributes and parameters
characterizing food quality.50,51 The flavour of various
foods containing omega-3 LC PUFA may undergo
great changes, beginning with handling the raw
material and continuing during processing, storage,
J Sci Food Agric 87:181–191 (2007)
DOI: 10.1002/jsfa
distribution and culinary treatment. Primary flavour
compounds can be lost and secondary ones can be
formed, which impact sensory quality.52 – 54 Fish oils
and fish oil-containing foods are a good example of
products where flavour changes easily, mainly due
to oxidation of omega-3 LC PUFA. The oxidative
deterioration of fish oil and fish oil-containing foods
generates particularly offensive off-flavours even at low
levels of oxidation. Trimethylamine, as well as volatile
compounds formed during oxidation of fish oil, have
been shown to be responsible for ‘fishy’ off-flavour,
which predominantly affect the sensory quality of food
products containing fish oil.25,40,55,56 Additionally,
oxidation reduces the nutritional value of foods by
decreasing the content of LC PUFA in the product and
increasing formation of harmful free radicals, which
promote development of atherosclerosis, cancer and
other degenerative diseases.57 – 59
METHODS FOR THE ASSESSMENT OF
OMEGA-3 LC PUFA OXIDATION
Methods used for the assessment of lipid oxidation
measure either products of the initiation and propa-
gation phases, products of the termination phase, or
depletion of oxygen or substrate. In complex food sys-
tems, the products of each of these phases increase and
decrease with time, making it difficult to quantitatively
measure oxidation. This requires multiple tests for
oxidative quality control of foods containing omega-3
LC PUFA.60 Correlation to sensory testing is the basis
for determining which chemical tests are appropriate
for measuring lipid oxidation.25,34,61 In many food
products the term ‘rancid’ is often used to describe
off-flavour caused by lipid oxidation, but it may also be
used to describe off-flavours that develop through the
hydrolysis of fats containing short-chain fatty acids.
Thus, ‘rancid’ may describe another sensory attribute
in different food products.62 Since fish oil oxidation
causes the formation of volatile secondary oxidation
products, several other sensory attributes should be
used to describe off-flavours of oxidized oil.38,39,63
So far many methods have been used for assessment
of quality of fats and fat-containing foods. The
most commonly used is peroxide value (PV),
which measures total concentration of peroxides and
hydroperoxides formed in the initial phase of oxidation
(ISO 3960: 2001).64 Milliequivalents (meq) of active
oxygen per kilogram of oil are measured by titration
with iodide ion. Due to the unstable and intermediate
nature of hydroperoxides and their sensitivity to
temperature, the PV is an approximate indicator of
the state of oxidation but particularly in the early stage
of oxidation.34 High peroxide values are a definite
indication of a oxidized fat, but moderate values may
be the result of depletion of peroxides after reaching
high concentrations. The main limitations of PV are
low sensitivity, need for large amounts of lipid sample
and difficulties in determining the titration end-point.
To overcome these limitations some novel methods
183
W Kolanowski, D Jaworska, J Weißbrodt
based on the same reaction have been proposed.
Colorimetric (iodometric) determination at 560 nm
and potentiometric end-point determination allow the
detection of hydroperoxides at levels as low as 20
neq.65,66
The TBARS test (thiobarbituric acid reacting sub-
stances) is an often used method based on the mea-
surement of the absorbance of TBA–malonaldehyde
complex at 532–535 nm and other aldehydes.67 Mal-
onaldehyde (MDA) is a three-carbon dialdehyde
formed in the termination phase of lipid oxidation.
The reaction produces a red colour, which can be
measured using a spectrophotometer. The objections
to this method point out that depending on the alde-
hyde type peaks at different absorbance maxima are
observed and TBARS can react with other substances,
which are not formed during lipid oxidation. As with
peroxide value, a low TBARS value is not an abso-
lute indicator of oil quality. Aldehydes may have not
yet formed or volatile aldehydes may have been lost
during processing and storage.
Anisidine value (AV) is used for measurement
of hydroperoxide decomposition products. When
hydroperoxides break down, they produce volatile
aldehydes like hexanal, leaving behind a non-volatile
portion of the fatty acid that remains a part of
the glyceride molecule. This non-volatile reaction
product can be measured by reaction with anisidine.
High anisidine value may be an indication that a
fat has been oxidized even when TBARS and other
aldehyde tests give low results due to volatile aldehydes
being incidentally or intentionally removed during
processing. Anisidine value is defined as 100 times
the absorbance (at 350 nm) of a solution resulting
from reaction of 1 g of fat in 100 mL of solvent.
Its conjunction with peroxide value gives the Totox
value.33,68
Total carbonyl compounds (TCC) are volatiles
related to off-flavours. As an indicator of the oxidation
process TCC can be evaluated by measurement of
orange-coloured hydrazones formed in reaction of
carbonyls with 2,4-dinitrophenylhydrazine.25,34
Ultraviolet detection of conjugated dienes is another
method of lipid oxidation assessment.34,35 During the
formation of hydroperoxides from PUFA conjugated
dienes are typically produced. Conjugated dienes
have a strong absorption at 230–235 nm, due to
rearrangement of the double bonds. Because of the
large molar extinction coefficient of hydroperoxides,
dilute solutions are adequate for spectrophotometric
measurements. The method is limited because the
value obtained greatly depends on the fatty acid
composition of the analysed sample, as well as on
the presence of other conjugated dienes, which are not
hydroperoxides.62
Electron spin resonance (ESR) spectroscopy is
another tool that can be used for detection of
fish oil oxidation products. Lipid oxidation products
in the propagation phase are very reactive species
and their steady state concentration falls below the
184
ESR detection limit, which can reach 10−9 mol
under optimal conditions.69 Consequently, techniques
such as spin trapping or spin scavenging are
required for detection of radical development by
ESR spectroscopy. The spin trapping technique is
based on the reaction of transient radicals with
diamagnetic compounds, known as spin traps, that
are added to the system to form more stable
radicals, known as spin adducts, that accumulate at
detectable concentrations (>10−7 mol). Detection of
spin adducts allows the indirect detection of radicals
involved in lipid oxidation. The ESR spin trapping
technique has provided results that were consistent
with those obtained by TBARS tests, PV, oxygen
depletion, as well as sensory analysis.69
CHROMATOGRAPHIC METHODS AND
ELECTRONIC NOSE
Limitation of most commonly used methods is low
specificity mainly due to interference of minor com-
pounds other than hydroperoxides. This limitation
may be overcome by using both gas and liquid chro-
matographic techniques. Aldehydes, alcohols, ketones,
furanones and lactones are the main volatile com-
pounds generated during decomposition of fish oil
hydroperoxides.53,55,70 The advantage of chromato-
graphic methods is to provide information about
specific oxidation products that have an influence
on the sensory properties of fish oils and fish oil-
containing foods. Aldehydes, especially unsaturated
ones and acids produced during their oxidation, are
the main compounds responsible for characteristic
off-flavours of oxidized fish oil.71 – 73
Gas chromatography (GC) of the headspace
above a sample is used for the measurement of
volatile compounds produced during the termination
phase of fish oil oxidation, e.g. hexanal.63,74,75 The
association between ‘rancid’ off-flavour and contents
of substances such as pentanal, hexanal, 2-heptanal,
octanal and nonanal has been reported.33 Methods
vary, but generally a portion of sample is moderately
heated in a sealed septum bottle. A gas syringe is
used to withdraw a small portion of the headspace
over the sample. The headspace sample is then
injected onto a GC column to separate hexanal from
other volatile components. Hexanal concentrations
can vary widely depending on sample history, fat
content in the product and fat composition. Generally,
data are needed on a variety of samples of the
same product to establish an association between
hexanal concentration and product quality. Once that
correlation is established, hexanal measurement can
be a useful test for fish oil oxidation assessment.
Both static headspace as well as dynamic headspace
methods are used, the latter one especially in cases
where high sensitivity is needed for monitoring volatile
compound levels.56,73,76 – 78
High-performance liquid chromatography (HPLC)
also allows analysis of hydroperoxide decomposition
J Sci Food Agric 87:181–191 (2007)
DOI: 10.1002/jsfa
 Instrumental and sensory assessment of omega-3 LC PUFA-rich foods
products.56,79 Its major advantage compared to GC is
that the method operates at ambient temperature.
This decreases the risk of artefact formation and
permits applications without previous derivatization.
However, quantitative analysis is difficult, since no
universal detector is available. Many different options
are available in HPLC analysis as compared to
GC analysis and a wide range of compounds of
different volatility, molecular size and polarity can
be analysed at high sensitivity of detection, reaching
10−12 mol.62,80
The combination of GC and mass spectrometry
(GC-MS) analysis of secondary oxidation products has
an advantage over the other methods for detection and
identification of particular volatiles responsible for off-
flavours of oxidized fish oil.40,54 – 56,81 In this method,
owing to the instability of hydroperoxides their prior
derivatization to more stable molecules is necessary.
Analysed molecules have to be sufficiently volatile for
GC separation. Hence oxidation products are reduced
to form hydroxy compounds and transesterified to
methyl esters, which can be detected and identified
by mass spectrometry. The use of internal standards
and the prior concentration of the sample facilitate
precise quantification. Sensitivity of GC-MS is very
high, reaching 10−15 g, depending on detection
system.34,55,56
Electronic nose, defined as an array of gas sensors of
diverse specificity, equipped with an identification tool
capable of differentiating simple or complex odours,
is a useful tool, which can be used also in the
quality assessment of fish oil and fish oil-containing
foods.82,83 Electronic nose sensors detect the chemical
species present in the headspace generated by heating
the samples. The most commonly used detector is
composed of a series of sensors, which can be metal
oxides, conducting polymers, electrochemical sensors
or quartz microbalance sensors.84 Recently, mass
selective detectors have been added in place of sensors,
which function similarly to a mass spectrometer to
characterize the aroma of a sample. Electronic nose
combined with GC has been effective in identification
of rancid off-flavour of olive oil or soy and corn oils
and good correlation between sensory profiling and
sensor signals were shown.85,86
SENSORY ANALYSIS OF FISH OIL
OFF-FLAVOUR
Sensory quality of foods is the most important aspect
for the consumer and flavour is a crucial sensory
discriminator for fish oil-containing foods. Association
of specific off-flavours to particular volatile compounds
enables accurate perception and measurement of lipid
oxidation changes, which may significantly improve
control of the oxidative quality of fish oil-containing
foods. Defined sensory languages are necessary to
achieve this goal. Thus, use of methods defining
and describing off-flavour attributes is important
for precise communication in research and quality
J Sci Food Agric 87:181–191 (2007)
DOI: 10.1002/jsfa
control of fish oil-containing food. In this case sensory
analytical tests are the most efficient.51,54,87
Sensory analytical tests involve the use of screened
or trained panellists, whose responses are treated as
instrumental data. Such tests include discriminatory
tests (difference and threshold), and the most
appropriate for off-flavour assessment is descriptive
analysis. The purpose of descriptive analysis is to
train a group of individuals to evaluate specific
sensory properties analytically. Descriptive analysis is
the tool of choice for qualitatively and quantitatively
differentiating foods and for exploring and defining
the relationships between sensory and instrumental
perception. Descriptive analysis of any food requires
a descriptive technique and a lexicon or language
to describe sensory properties. There are several
valid approaches to descriptive analysis, e.g. flavour
profile method, quantitative descriptive analysis, the
spectrum technique and others.88,89 Sensory languages
can be identified for any foods using any of these
approaches. Panellist selection, scales usage and
training are the most important parts of any descriptive
analysis approach. A panel or group of individuals
(generally 8–12) is used for descriptive sensory
analysis. Even highly trained panellists or experts
can vary in sensory function because of age, mood,
foods previously eaten, etc. Thus, use of a group
or panel gives more consistent results than one or
two individuals. Crucial for analytical sensory panel is
panellist training and replication. Although descriptive
analysis does not require expensive instrumentation,
time and training are required. Once trained, a
descriptive panel operates as a precise instrument,
meaning that data must be replicated.50 The panellists
are units of the instrument and each panellist
should replicate each sample. In many studies
quantitative descriptive analysis was successfully used
for description and assessment of flavour profile and
changes of fish oil-containing foods.25,38,40,54,78,87,90
Quality control of fish oil-containing foods needs
an assessment not only of the present oxidation status
of the product but also its stability against oxidation
during time of storage. To determine the induction
period of oxidation several tests are used, in between
sensory methods. Sensory induction period can be
defined as the time required for an oil to become
slightly rancid as determined by a sensory panel.91,92
CORRELATION OF INSTRUMENTAL AND
SENSORY DATA FROM OMEGA-3 LC PUFA
OXIDATION TESTS
Correlation of instrumental and sensory data allows
precise characterization of oxidation quality of fish
oil-containing food products. To achieve this goal,
statistical techniques, especially chemometric, should
be applied. Chemometric methods facilitate inter-
pretation of usually huge amounts of data gener-
ated in analysis of oxidation products, providing
an overview of the obtained data with significantly
185
W Kolanowski, D Jaworska, J Weißbrodt

enhanced interpretability and revealed information Table 1. Volatile compounds associated with odour attributes of
about groupings and correlations in the data.40,93 menhaden oil63
Principal component analysis (PCA), cluster anal- Compound Level (ppm) Odour characteristics
ysis (CA), discrimination function analysis (DFA),
soft independent model class analogy (SIMCA), par- Octane 20 –
tial least squares (PLS) and parallel factor analysis Decane 90 –
(PARAFAC) are used.54,93 Undecane 130 –
Dodecane 280 –
However, in many studies there is a lack of statistical
Tridecane 370 –
correlation between instrumental and sensory data.
Tetradecane 230 –
Moreover, not every instrumental test correlates with Pentadecane 520 –
sensory data. As an example, data obtained from Hexadecane 10 –
the sensory triangle test on fish-oil based mayonnaise Heptadecane 80 –
showed better correlation for TCC and AV data than Pentanal 360 –
for PV and TBA.32 In other experiments with two fish Hexanal 1380 Green (cut grass)
oil-enriched spreads there was no correlation between Heptanal 410 Waxy green, grassy
sensory data and PV or AV.25 The lack of correlation Octanal 240 Citrus, orange, fatty
between sensory data and PV was explained by the fact Nonanal 500 Fatty floral
that lipid peroxides are not responsible for off-flavour Decanal 640 Sweet, green fruity, fatty
with citrus top note
formation and in the case of AV that the sensitivity
t-But-2-enal 130 Painty
of this method is too low to detect the changes in c-Pent-2-enal 70 –
concentration of volatiles resulting in off-flavours. t-Pent-2-enal 930 Greasy green, musty
However, a good correlation between TBARS values t-Hex-2-enal 540 Sharp green, oily
and sensory data was shown.94,95 c-Hept-2-enal 140 Sharp green, greasy
Assessment of lipid oxidation by analysis of volatile t-Oct-2-enal 280 Musty, waxy floral
compounds using GC-MS gives results that have t-Non-2-enal 200 Fatty, waxy, musty
been demonstrated to correlate very well with sensory t-Dec-2-enal 410 –
data.25,95 Analysis based on GC-MS combined t,t-Hexa-2,4-dienal 100 –
with sensory assessment of fish oil allows accurate t,c-Hepta-2,4- 1880 Vegetable green
association of odour and flavour attributes with dienal
t,t-Hepta-2,4- 2180 Vegetable green
particular volatile compounds.32,54,55,96,97 However, it
dienal
was shown that careful interpretation of data obtained Octa-2,4-dienal 10 –
in oxidation studies in which sensory analysis was not t,c-Nona-2,6-dienal 240 –
performed is necessary.25 t,t-Octa-2,4-dienal 40 –
Instrumental analysis of volatiles may deter- Deca-2,4-dienal (2 150 + 250 Oxidized oil
mine particular compounds responsible for sensory isomers)
impression.20,32,54,98 Quantitative descriptive analysis Nonatrienal (2 60 + 130 Oxidized oil
and GC-MS applied simultaneously seem to be the isomers)
best for precise measurement of particular volatile Decatrienal (2 70 + 8 Oxidized fish oil
compounds responsible for off-flavour formation.99 isomers)
Volatile compounds of hydroperoxide decomposition, Heptan-3-one 8530 Sickly sweet, cooling
Nonan-2-one 620 Musty with citrus top-note
even at low concentration, significantly decrease sen-
1-Penten-3-one 240 –
sory acceptability of fish oil and fish oil-containing c,c-Octa-3,5-dien- 130 –
foods.32,39,40,70 Association of fish oil volatile com- 2-one
pounds with sensory odour attributes are shown in t,t-Octa-3,5-dien- 40 –
Table 1. 2-one
Acetic acid 1380 Irritating, vinegar-like
Propanoic acid 2410 Astringent, acidic
STABILIZATION OF OMEGA-3 LC Butanoic acid 8110 Dirty socks
PUFA-CONTAINING FOODS WITH Isobutanoic acid 100 Sweaty, dirty socks
Pentaenoic acid 330 Parmesan cheese, dirty
ANTOXIDANTS socks
Lipid oxidation of fish oil and other PUFA-rich Hexaenoic acid 810 Sweaty, dirty socks
foods is a serious problem that leads to loss of
shelf-life, consumer acceptability, functionality, nutri- t, trans; c, cis.
tional value and safety. Usually, to delay lipid oxi-
dation various antioxidants are added. However, it butylated hydroquinone (TBHQ) was used; how-
is very difficult to stabilize foods containing omega-3 ever, nowadays it is banned in most countries. In
LC PUFA. Synthetic antioxidants like propyl gal- theory oxygen reacts preferentially with these sub-
late or butylated hydroxyanisole (BHA) and butylated stances rather than oxidizing PUFA, thus protecting
hydroxytoluene (BHT) are fat-soluble phenolic com- them against oxidation.100 Nevertheless, some stud-
pounds that are often used for fat stabilization. Also ies indicate that addition of most antioxidants may

186 J Sci Food Agric 87:181–191 (2007)

DOI: 10.1002/jsfa
 Instrumental and sensory assessment of omega-3 LC PUFA-rich foods
negatively influence stability of fish oil-enriched foods
under certain conditions.25,101
Recently interest in natural antioxidants found
in plants has grown due to the increasing trend
towards use of ‘natural’ food additives.102 – 104
Moreover, many natural antioxidants are shown
to have health benefits. Tocopherols and β-
carotene are two important commercial natural
antioxidants that have been intensively investi-
gated for many years. Additionally several plant
polyphenols have also been identified as natural
antioxidants.102,105
Tocopherols are antioxidants widely employed in
the food industry. They act as primary antioxi-
dants by reacting directly with free radicals and
may further intercept the lipid hydroperoxide decom-
position processes.104 It was shown that addition
of α-tocopherol itself to fish oil does not improve
its stability against oxidation.106,107 Better protec-
tion may be achieved when combinations of α-
tocopherol with L-ascorbic acid or ascorbyl palmitate
and lecithin are used; such a combination exhibits
a synergistic antioxidative effect.108 – 110 However, it
was demonstrated that substances with antioxidant
activity may exhibit pro-oxidant behaviour under cer-
tain conditions.111,112 The ability of α-tocopherol to
have an antioxidant, neutral or pro-oxidant effect
in foods depends on temperature, lipid composi-
tion, physical state (bulk phase or emulsion) and its
concentration.101,112 Increasing levels of α-tocopherol
can result in increasing levels of its decomposi-
tion products, which are highly reactive radicals and
can initiate and propagate processes of lipid oxida-
tion by themselves, as well as impact on off-flavour
formation.111,112 Also β-carotene, lutein and lycopene
have been demonstrated to show antioxidant or pro-
oxidant activity under certain conditions in a wide
variety of lipid systems.52,111,113 It is proposed that
when exposed to oxygen carotenoids trap peroxyl
radicals by an addition mechanism. This results in
the formation of a carbon-centred carotenoid radical,
which may be easily oxidized into a mixture of prod-
ucts with epoxy, hydroxy and carbonyl groups. The
autoxidation consumes carotenoids without scaveng-
ing peroxyl radicals, thus attenuating its antioxidant
activity.100,111,112
However, it was shown that some plant-derived
polyphenolic substances (flavonoids) may be used as
potent antioxidants for fish oil stabilization.114 – 117
Moreover, health implications of flavonoids as
nutraceuticals have fostered research on their use in
nutrition, as well as for stabilization of fish oil and
fish oil-containing foods.118 – 120 Antioxidative effects
of catechins, quercetin, myricetin and morin in the
stabilization of refined, bleached and deodorized fish
oils during storage at elevated temperature have been
described.119 – 121 These flavonoids were more effective
in lowering peroxide formation than BHA and BHT.
Myricetin was the most potent and its antioxidative
effect was similar to that of TBHQ. Green tea extracts
J Sci Food Agric 87:181–191 (2007)
DOI: 10.1002/jsfa
provoked oxidation of fish oil. However, after removal
of chlorophyll the purified extract showed a potent
antioxidative activity in fish oil, stronger than BHA
and α-tocopherol.121 – 123 Individual green tea cate-
chins prevented oxidation of fish oil during storage
tests at elevated temperatures.121 Similarly, epicate-
chin gallate was shown to be an effective antioxidant
agent comparable to TBHQ. Rosemary extract con-
taining carnosol and carnosic acid effectively inhibited
oxidation of fish oil stored at elevated temperature.118
A strong synergistic effect was observed between α-
tocopherol and rosemary extract in sardine oil, possibly
due to the regeneration of α-tocopherol.124,125 How-
ever, flavonoids with a phenol-type substitution in their
B ring, such as apigenin and naringenin, have been
reported to result in an increase of reactive oxidant
species formation.112,114,115
FINAL REMARKS
Pro-oxidative effects of various antioxidants occur
more often in complex food systems, where fish oil
is added and emulsified, e.g. margarine, spreads, milk
products, sausages, salad dressing or mayonnaise, than
in bulk oil.38,56,75,101,126 Different emulsifiers are in
direct contact with oil on the developed surface area
of the emulsion droplets and are known to affect
the rate of lipid oxidation.126,127 Additionally, sensory
threshold values of volatile compounds formed during
oxidation of fish oil are much lower when released from
water than from oil.128 Thus, if off-flavour compounds
are present in the aqueous phase of food product
containing fish oil, their volatility is higher than the
volatility of the same compounds in bulk oil. If this
is the case the negative flavour impact of volatile
oxidation products in fish oil-enriched foods may be
much greater than the flavour impact of the same
compounds in bulk oil.25
Many factors affect oxidation of fish oil emulsified
in food products and more studies are required to
fully understand the pro-oxidant mechanisms behind
some of these factors. However, care should be taken
when antioxidants are added to fish oil-containing
foods as functional ingredients due to their ability
to promote omega-3 LC PUFA oxidation under
certain conditions. Special attention should be given to
infant formula enriched with microencapsulated fish
oil powder, which owing to its highly developed surface
area is extremely exposed to oxidation, resulting in
formation of toxic substances.
Problems with oxidative stability of fish oil,
especially when added to food products, raise the
question of whether such a food is safe for the
consumer and whether food should be enriched with
fish oil at all. Nevertheless, for quality control of
omega-3 LC PUFA-containing foods adequate and
combined methods of oxidation assessment should be
used, beginning with the raw material and continuing
during processing, storage and distribution. To
achieve this goal correlation of instrumental and
187
W Kolanowski, D Jaworska, J Weißbrodt
sensory methods and multivariate data analysis may
give the best results.
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