Methods of Flavor

Analysis

Submitted by : Monika Mathur

2014FST29D
ALLPPT.com _ Free PowerPoint Templates, Diagrams and Charts
 Flavor
The word is not found in many languages.
Scientifically used to indicate two meanings.
1. Sensation (e.g. Mumm Yummy!!!)
- Flavour, sight, mouth feel- 3 important sensation of food
2. components (e.g. Flavour compounds in strawberry ice cream)
Flavour
- Flavour, appearance, texture- 3 important quality parameters.
Flavour (sensation)
Defination :-

• Complex combination of the olfactory, gustatory and trigeminal sensati

on perceived during tasting (ISO 5492:2008)
• Flavour is the result of the correct balance and concentration of a wide
variety of volatile flavour compounds.
Problem in flavor analysis
The task of identifying volatile flavour components in natural products is formidable for s
everal reasons
1. Laboratory instrumentation is not as sensitive to many odors as is the human olfactor
y system.
2. Food Flavor is distributed throughout a food matrix.
3. Flavor isolation and analysis are made difficult also by the fact that flavors comprises
a large number of chemical classes. The flavor chemist cannot focus only on one func
tional group and thus greatly simplify the procedure
4. Large number of flavor compounds complicates flavor analysis.
5. Once the food has been extracted, concentrated, separated and detected, a major quest
ion arise concerning each chemical importance to flavor, analytical instruments has n
o sense of taste and smell.
Analysis

•Objectives
•Analyte
•Analytical steps
Objectives

1. To obtain aroma isolate to accurately identify and qu

antify every aroma constitute in food.
2. To identify only key components of an aroma profile.
3. To identify and off note in a food product.
4. To monitor aroma changes with time.
5. To predict sensory attributes.

Based on the objective the methodology of analysis will change

The Analyte

• Volatile compounds
• Low boiling point
• Trace amounts
• Highly reactive
• Varying concentration with time
• Varying threshold detection level (odor activity)
• Varying functional moieties
• Varying molecular mass (up to 300)
The Analyte
These characteristics of the volatile flavor compounds makes
it difficult to analyze.

The methods of sample preparation and analysis will vary

vastly the compound or food of consideration.
Analytical Steps

1. Isolation of volatile Flavor compounds (VFC)

2. Extraction
3. Fractionation
4. Identification
Isolation

1.Objective
2.Methods
Objective of Isolation

• To remove interfering compounds as much as possible from analyte (

volatile Flavor compounds)
• To get a representative sample, a near complete flavor profile of the fo
od matrix
• To avoid artifacts formation.

The inactivation of enzyme of fresh plant and animal tissue when the isolation
procedure exceeds only a few minutes is essential. A common method is to
homogenize the food in methanol.
Methods of isolation

1. Headspace Extraction
2. Distillation and extraction
Headspace Extraction
1. Static head space method
1. Headspace condensation method
2. Syringe Method
2. Dynamic Head Space Method
3. Solid Phase Micro Extraction (SPME)
Head space method
Direct injection
Method- this method is simple in which
• Draw 10 ml of headspace in to a syringe and inject it into a gas chromatograph, it is
rapid , reproducible and samples only what the nose receives.
• The primary problem with direct head space analysis is that too little sample is available for instru
mental analysis.
• Direct headspace injection are generally limited to 10 ml or less, one can see that only volatiles
present at concentrations exceeding 10~7 g/1 (headspace) will be detected by gas chromatography,
and only those at concentrations exceeding 10~5 g/1 will be adequate for mass spectrometry.
• Since the concentration of volatiles above a food product generally ranges from about 10~4 to 10"
10 g/1 (or less) (Weurman, 1974), only the most abundant volatiles will be detected by direct heads
pace sampling.
• second disadvantage of headspace methods is that it is difficult to do quantitative studies using
them.
• used in quality control situations where only major components need to be measured.
Headspace Concentration
• Trace analysis of food volatiles may be ac
complished via headspace concentration
techniques.
• The equilibrium headspace vapors above
a food or the food itself may be purged
with an inert gas in order to obtain a la
rge volume of headspace gas for analysis.
• The apparatus used for purging the food
sample is most commonly a simple
flask with a means to deliver inert gas so
that the gas sweeps the headspace into a
trap (Fig. 2-2) or bubbles through the sa
mple and then into a trap.
Cryogenic trapping
Headspace trapping methods are commonly called dynamic headspace or
purge and- trap methods.

The sample is purged with an inert gas, such as nitrogen or helium, which strips aroma con
stituents from the sample. The volatiles in the purge gas must then be trapped (somehow re
moved) from the gas stream. The aroma constituents may be trapped via a cryogenic,
Tenax (or alternative polymer), charcoal, or other suitable trapping system.

• The simplest means of concentrating headspace vapors is by passing the he

adspace or purge gas through a series of cold traps.
• Assuming proper design and operation of the traps , organic volatiles will
be condensed from the purge gas.
• A major problem with cryogenic trapping is that water is the most abundant
volatile in most foods and, therefore, the trap condensate is primarily water.
 A typical headspace cryogenic trap system is shown in Fig. 2-4 (Chang et al. 1977).
The sample chamber of this system has a volume of 18.5 L and will hold 27 .lbs. Of
french fries. Inert gas flows into the bottom of the sample chamber, through the fo
od sample and then out into the series of condensers. The initial condenser is
generally cooled with ice water while the other condensers may be cooled with dry
ice. acetone or liquid N2.

The condensates are typically pooled and the

n extracted with diethyl ether, dichlorometha
ne, or some other organic solvent. The solven
t extract is dried with anhydrous magnesium
sulfate or sodium sulfate and then concentrat
ed for GC analysis .

Adsorbent trapping
Volatile flavors are often trapped on adsorbe
nt materials to avoid co-condensation of
water with the flavor volatiles.
Extraction

1. Steam distillation
2. Solvent extraction
3. Supercritical fluid extraction
4. Simultaneous distillation extraction
5. Pressurized fluid extraction
6. Solvent assisted flavor evaporation
7. Microwave assisted hydro distillation
8. Cryofocussing
Steam Distillation

1. Steam
2. Continuous stirring
3. Cold trap
1. Ice water
2. Dry ice / acetone
3. Liquid nitrogen
4. Low temperature 30°-60°.
5. Vacuum 8psi
Falling film evaporators/climbing film evaporators.

Distillate of very dilute solution of flavor compound

Steam distillation continue

1. Extract the distillate with the orgenic solvent e. g. penatane, diethlye ether,
dichloro methane
2. Dry the isolate with MgSO3 or the other drying agents.
3. Filtered and concentrated for instrumental analysis.
Solvent extraction

1. Pure solvents 99.9% (a blank solvent also should be run to monitor solven
t artifact detection)
2. Diethly ether, hexane, dichloro methane
3. Need further processing ot separate aroma components from lipids- mole
cular distillation, steam distillation, purge and trap or dialysis
4. Simple procedure
5. Fat free foods- wines , bread, fruit and berry juices, some vegetables and
alcoholic beverages.
Solvent selection

Solvent selection
1. Polarity
1. Non polar solvents for alcoholic bevrages
2. Pentane, hexane, tricholoromethane
2. Polar solvents for non alcoholics food matrics
Supercritical Fluid Extraction
1. Same procedure as in SE
2. Supercritical CO2 is used
3. pressurized chamber (80-100)
4. Very low boiling point (35-40 °C)
5. No residue or left overs of the solvent
6. Penetrates food metrics effectively
7. Solvent properties controlled through T and P or chemical modifiers (metha
nol
SFE

Drawbacks
• Expensive
• Instrumentation
• Non-polar nature
• Small Sample size
SDE

1. Both distillation and solvent extraction done simultaneous

ly.
2. First introduced by Lickens and Nickersons in 1964.
3. Efficient Flavour extraction by intimate molecular mixing
of steam and solvent vapour.
4. Two types
1. Atmospheric SDE
2. Reduced pressure SDE
SDE
Furthuer SDE combined with

• Tenax or charcoal trap

1. Flavour molecules adsorbed to the rrap and then desorb
ed with solvent
• Purged with inert gas
2. sample is purged with inert gas and then the flavour
compounds are trapped in water which continuously refl
uxed with the solvent.
SAFE
Solvent assisted fluid extraction

• Vacuum low T distillation

• Nitrogen trapping ;- little changes to extract
• Volatiles and non volatiles
• GCO
Solid Phase Micro Extraction

• Solvent free micro extraction

• Principle- Equilibrium Adsorption and desorption process
• Stationary phase- organics coated on a silica fibre
• Solid sample – Head space equilibrate to 15-30 min.
• Liquid samples- salting out of aroma compounds from foo
d to head space
Identification

• Flame Ionization detector

• Mass Spectrometry
• GC-MS/ Olfectometery
The question is ‘how should flavours be analysed?’ There is no debate as to whe
ther chemical analysis or sensory evaluation should be utilized to determine flav
our products and the flavour quality of foods; both must be used. Sensory evalua
tion looks at the whole sample, is very reproducible and the analysis is usually
done by averaging individual responses of trained judges. Objective chemical an
alysis is dependent upon sampling techniques and sample handling and/or separ
ation techniques before measurement. But, as noted previously, some chemical
s that cause large gas chromatography (GC) peaks have little or no odour.
Thus, instrumental methods can only be utilized to measure flavours when they
are calibrated by analytical sensory methods.
 PSYCHOPHYSICS AND SENSORY
EVALUATION

 Psychophysics is the study of the relationship between the psychological percep

tion of a stimulus and the physical stimulus that causes that perception.
 Sensory evaluation is the utilization of psychophysical techniques in the food in
dustry to answer three types of questions:
(1) Description: What does the product taste like? How is one product different fro
m another in quality? How do changes in the process, formulation, packaging, or
storage conditions affect its perceived sensory characteristics?

(2) Discrimination: Would people notice the difference? How many people would d
etect it? If there is a difference, how great is it?

(3) Afective or hedonics: How much do people like this product? Is it an improve
ment over another product? Which attributes are liked or disliked? Which product
would the consumer select?
Discrimination Tests
Discrimination or difference testing is used to determine whether there is a
perceptible difference between two or more products and, in some cases, the
magnitude of the difference.
These tests involve comparative judgements, they can be very sensitive in dete
rmining small differences between products.
Types of difference tests:
1. paired comparison test
2. triangle test
3. duo-trio test
Paired comparison test

Paired comparison. This test is used to determine whether two samples differ in
a specific character; it is a directional test with a named attribute. For
example, the panellist is presented with two samples and asked which one is
more bitter. Positional bias is an important factor; thus, the sample order of
presentation must be balanced so that there are equal numbers ofAB and BA
presentations. The statistical chance of obtaining the correct answer by
guessing is 1 in 2, or 50%. Therefore, the detection threshold is normally
placed at 75% correct responses, indicating that the difference has been
detected on half the trials.
Multiple comparison. Efficiently used to evaluate four or five samples at a time, t
his test only measures the direction and magnitude of differences in one or
perhaps two characteristics. A known standard is labelled as a reference or
control and presented to the panellist along with several blind coded samples.
The panellists are then asked to compare each coded sample to the known
standard on the basis of an identified characteristic.
Triangle test. This test is used to determine an unspecified sensory difference
between two products. The panellist is provided with three samples and told
that two are the same and one is different. The objective of the test is to
identify the different sample. In addition, the panellist is often asked to give
the reason for his or her decision.
triangle test is popular because: (1) it is rapidly administered, (2) it is
an easily understood task and (3) data analysis is simple.
Duo-trio test. This test is used to measure unspecified differences between
samples. It is another three-sample difference test, where the panellist is first
given a control sample and then asked which, of the next two samples, is the
same as the control. The probability of guessing the correct answer in the
duo-trio test is one in two, or 50%. This test is useful for panellist screening
and training. Using immediate feedback, this practice helps teach panellists
to discriminate, especially if allowed to retaste the samples and attempt to
associate the similarities in the identical combination and then decide what is
different in the odd sample.
Ranking test . This test is used to establish a magnitude of difference between
samples on a specified attribute. The panellists are presented with 3-5 coded
samples and asked to rank them in order according to a single specific attribute,
e.g. sweetness. The taste order should be prescribed and a balanced design
used.
Magnitude estimation. In this test, two or more coded samples are presented i
na specified order, which is balanced across panellists. An arbitrary value for
the attribute in question is assigned to the first sample. When panellists
analyse (via tasting or smelling) the next sample, they assign a higher or lower
value to it according to their estimate of the magnitude of the difference.
Scales used could be
category
Scales
line scales
ratio scales.
The simplest means of evaluating perceived intensity is to have panellists
assign each stimulus to one of several categories, ranging from ‘no flavour’ t
o ‘very strong flavour’.
The categories can be replaced with a line scale, along which the panellist
places a mark corresponding to the intensity of the odorant. This takes more
training, as standard concentrations of the attribute to be determined are
used as anchors to define the ends of the line.
When a highly trained panellist assigns a series of numbers that represent
the ratio of the perceived intensities of the presented flavourants to the first
presented sample, a taste or odour’s psychological magnitude can be related
directly to its physical concentration by a logarithmic function.
Flavour Legislation
Flavourings represent a class of food additives that has had comparatively little le
gislation. This is probably due to the relatively large number of ingredients used i
n flavourings, the very small quantities involved and the difficulty of regulating ad
ded substances (which are also naturally present in foodstuffs) by analytical meth
ods.
• Without detailed knowledge regarding the composition of flavourings, they
were laying themselves open to criticism by food activists.
• flavour legislation is carried out at national and international levels.
METHODS OF LEGISLATION
There are fundamentally two basic methods of legislation:
(1) Positive: Only those materials that are listed are permitted to be used, to t
he exclusion of all others. This type of legislation has the disadvantage that re
search into possible new materials is frustrated by the requirement for publica
tion before use, unless the time required for commercialisation is built into th
e regulation. However, regulatory authorities generally prefer positive listing b
ecause they believe that it offers the consumer maximum protection and is rel
atively easy to police.
(2) Negative: All materials are allowed except those that are listed. In the case
of food legislation, this needs to be supplemented by a statement that none o
f the materials used are injurious to health. This firmly puts the requirement fo
r safety of the materials onto the manufacturer, but is strongly objected to by
most national legislative bodies on the basis that they do not have control ov
er what is used. The system encourages the research and development of nov
el materials to provide more authentic flavourings, to the overall benefit of the
consumer.
• The initial work was undertaken by the USA Food and Drug Administration (FD
A) as part of Title 21 of the Code of Federal Regulations. Some 27 flavouring su
bstances were evaluated and classified as generally recognized as safe (GRAS),
presumably on the basis of long usage without untoward effect.
• Flavor and Extract Manufacturing Association (FEMA) proposed to assist the FD
A by using independent experts to evaluate the other flavouring ingredients tha
t were known to be in use at the time, classifying most of them as GRAS and al
locating the numbers 2001-3124 to them.
• This group was later named FEXPAN and consists of independent international
toxicologists who undertake the safety evaluation of novel flavouring ingredient
s.
• term ‘natural flavor’ or ‘natural flavoring’ is defined in Title 21 of the Code of Fe
deral Regulations,
It has become normal in the USA to designate various categories of natural flavo
urings as follows:
FTNF (from the named fruit): As the name suggests, these consist solely of extr
acts or distillates derived from the named fruit. For instance, strawberry FTNF c
ould consist of concentrated strawberry juice with added strawberry distillate. It
may not contain material from any other natural source. Flavourings of this cate
gory tend to be very expensive in use (they are usually very weak) and not very
stable.
WONF (with other natural flavourings): These must contain more than 51% der
ived from the named source but may contain other natural flavouring ingredients
. For instance, strawberry WONF could consist of 51% concentrated strawberry
juice fortified with other fruit juices or natural chemicals. These flavourings are
still expensive in use because of the price of the named ingredient.
Natural flavour: These must contain only natural ingredients, but the type or so
urce is not defined. For instance, natural strawberry flavour may contain ingredi
ents from any source so long as they are classified as natural.
INTERNATIONAL SITUATION: JECFA

• JECFA is the Joint Food and Agriculture Organisation of the United Nations (F
AO)/World Health Organisation (WHO) Expert Committee on Food Additives.
• Set up in 1956 to evaluate the safety of food additives, residues of veterinary
drugs in food, and naturally occurring toxicants and contaminants in food.
• JECFA serves as a scientific advisory body to FAO, WHO, their member states
and the Codex Alimentarius Commission, primarily through the Codex Commi
ttee on Food Additives and Contaminants (CCFAC), regarding the safety of fo
od additives including flavouring substances.
• The committee establishes acceptable intakes on the basis of toxicological da
ta and related information on the substance being evaluated.
• It also develops general principles for assessing the safety of chemicals in foo
d. The requirement to keep up-to-date with scientific developments in toxicol
ogy and related disciplines necessitates a constant review of evaluation proce
dures (Munro et al., 1999).
• JECFA has to date evaluated some 600 flavouring substances and, more impor
tantly, indicated that many flavouring agents are used in such small quantity t
hat a full evaluation may not be appropriate.
• JECFA evaluations and procedures are of international significance since they a
re not influenced by any particular group. JECFA evaluations are taken into ac
count by both FEXPAN and the European Food Safety Authority (EFSA) in their
evaluation of the safety of flavourings for the USA and Europe, respectively.
COUNCIL OF EUROPE
The Council of Europe ad hoc Working Party on Natural and Artificial Flavouring S
ubstances was set up in 1965 as a subsidiary body to the Sub-Committee on the
Health Control of Foodstuffs. The
The initial aims of the Council of Europe were
• To draw up a list of natural and artificial flavourings that could be used in fo
odstuffs without hazard to public health;
• To draw attention to those flavourings that presented hazard to public health
.
It is important in the context of the Council of Europe lists to understand the definitions they
have used in classifying flavourings in their initial publication (Council of Europe, 1974).
(i) A flavouring is a substance that has predominantly odour-producing properties and that possi
bly affects the taste.
(ii) A natural flavouring is a substance obtained from vegetable and sometimes animal
sources, exclusively through the appropriate physical processes. Those biological processes
that occur spontaneously, and roasting, are assimilated to physical processes.
(iii) An artificial flavouring is a substance that has flavouring properties and that has been
obtained by a chemical process. This term includes
(a) substances that exist in natural products;
(b) substances not present, or as yet undiscovered in natural products.
(i) Flavouring properties are those that are predominantly odour-producing and th
at possibly affect the taste.

(ii) A flavouring substance is a chemically defined compound that has flavouring p

roperties. It is obtained either by isolation from a natural source or by synthesis
.

(iii) Natural sources of flavourings are products of plant or animal origin from whic
h flavourings may be obtained exclusively through appropriate physical process
es or by biological processes that occur spontaneously (e.g. fermentation).

(iv) Natural flavourings may be defined as complex mixtures derived from natural
sources that have flavouring properties.
Thank You