Chapter 5

DETERMINATION OF TOTAL FAT AND

FREE FAT CONTENT

Fats play an essential role in food:

. From a nutritional point of view, fats provide energy (37.7 kJ g1),

essential fatty acids (linoleic acid, a-linoleic acid), fat-soluble
vitamins (A, D, E, K), phytosterols and antioxidants.
. From a sensory point of view, fats contribute to food texture and
palatability as well as acting as flavour carriers and precursors of
aromatic molecules. The melting and crystallisation properties of
fats, which depend on chain length and the unsaturation of fatty
acid residues as well as the structure of triacylglycerols, signifi-
cantly affect the sensory perception of foods.

The main problem with lipids is that they oxidise easily. This is one of
the main causes of food spoilage since, in most cases, the production
of compounds with unpleasant flavours or odours (rancidity) is
undesirable. Moreover, the oxidation of lipids can reduce the nutri-
tional quality, change the texture and colour of food and produce
compounds unsuitable for human consumption. In powders, the lipid
substrates mostly affected by oxidation are fractions exposed to air, in
the form of free fat. Three different definitions for free fat can be
found in the literature based on its properties:
1. Structural properties – associated with the presence of a damaged
protective membrane (original intact membrane of fat globules

Analytical Methods for Food and Dairy Powders, First Edition. Pierre Schuck, Anne Dolivet and
Romain Jeantet.
Ó 2012 John Wiley & Sons, Ltd. Published 2012 by John Wiley & Sons, Ltd.

99
100 Analytical Methods for Food and Dairy Powders

or reconstituted coating following technological treatments like

homogenisation). As such, it can also be considered as the non-
emulsified fraction of fat.
2. Biochemical properties – linked to composition. Recent studies
have attempted to highlight the differences in the composition of
saturated and unsaturated fatty acids between free fat and coated
fat, which would influence their respective melting points. How-
ever, these results remain controversial.
3. Analytical properties – generally method-dependent. Free fat can
be defined as the fraction of fat extractable by organic solvents in
standardised conditions, or fat extractable by centrifugation.

From these elements, Vignolles et al. (2007) proposed the following

definition, based on a structural approach: any element of a lipid nature
(i) not completely protected by a protective membrane, be it either the
original membrane (phospholipids and proteins) or reconstituted
following one or more technological treatments (adsorbed proteins)
and/or (ii) partly protected by an ‘amorphous lactose-protein’ matrix
artificially reconstituted during the drying process, can be considered
as a free fat. This can be found on the surface as well as on the inside of a
powder particle, in pores formed during drying.
While free fat may be desired in specific applications, it poses
serious quality problems in other cases: it causes both a decrease in
the stability of powders by oxidation of lipids and a decrease in their
physical flow properties (Chapter 8), wettability, dispersibility and
solubility (Chapter 13).

5.1. Determination of total fat content

5.1.1. Purpose and range of application
This section outlines Gerber’s acid-butyrometric method for mea-
suring the total fat content applicable to whole milk, partly skimmed
milk, buttermilk, reconstituted milk products and milk and food
powders. It is also applicable to milk containing legal preservatives
(potassium dichromate, bronopol). It does not apply to formalde-
hyde-treated milk nor does it apply to milk that has undergone
homogenisation.
 Determination of Total Fat and Free Fat Content 101

5.1.2. Definition
The acid-butyrometric method is a conventional technique, which,
when applied to whole milk with an average fat content and an
average density, at 20 C, yields a fat content that is equivalent to that
obtained by a gravimetric reference method.

5.1.3. Principle
5.1.3.1. Dissolution of proteins by the addition of sulphuric acid.
5.1.3.2. Separation of milk fat by centrifuging the butyrometer,
with the separation being facilitated by the addition of a small
quantity of amyl alcohol.
5.1.3.3. Obtaining the fat content in grams per 100 ml of milk or in
grams per 100 g of milk by direct reading on the butyrometer scale.

5.1.4. Reagents and other products

All reagents should be of recognised analytical quality, in particular:

a, Concentrated sulphuric acid, with density at 20 C (r20) ¼ 1820

 5 kg m3, colourless or slightly amber, free from any impu-
rities that may affect the result.
b, Amyl alcohol, with density at 20 C (r20) ¼ 813  5 kg m3,
distillation range 130  2 C, free from any components that may
affect the result, such as secondary amyl alcohol, tertiary amyl
alcohol, 2-furfuraldehyde, petroleum and benzene derivatives.
c, Distilled water.

5.1.5. Instruments and glassware

Standard laboratory equipment as well as the following:
d, Milk or milk powder butyrometers with suitable stoppers. Use a
butyrometer with a scale that best corresponds to the assumed fat
content of the sample.
e, 11 ml milk pipettes. They allow the result to be directly obtained in
grams of fat per 100 ml or 100 g of milk by direct reading on the
butyrometer scale.
102 Analytical Methods for Food and Dairy Powders

f, 10.0  0.2 ml pipette graduated in milliltres.

g, Automatic dispenser suitable for dispensing 10.0  0.2 ml of
concentrated sulphuric acid.
h, Automatic dispenser suitable for dispensing 1.00  0.05 ml of
amyl alcohol.
i, Butyrometer centrifuge capable of achieving a centrifugal force of
350  50 g at the end of the butyrometer neck. Such a centrifugal
force is obtained by a centrifuge of 52  1 cm in diameter
(distance between the outer ends of the diametrically opposed
butyrometer stoppers) operating at 1100  50 r.p.m. The formula
for calculating the radial acceleration is as follows:
a ¼ 1181  n2  R ð5:1Þ
where a is the radial acceleration in g, i.e. in gravity acceleration, n is
the number of revolutions per minute divided by 1000 and R is the
radius in m.
j, Water bath for the butyrometer with a temperature control set at a
temperature of 65  2 C, and a rack for holding butyrometers
upright. The temperature must be uniform throughout the water
bath. The graduated butyrometer stems should be completely
immersed.
k, Water bath at 38–40 C.
l, Thermometer to check the temperature of the water baths.

5.1.6. Safety
5.1.6.1. Personal protection
Safety goggles and acid resistant gloves are essential.

5.1.6.2. Toxicity of products used

Sulphuric acid (a): MSDS No. 30 – INRS: causes severe burns.
Amyl alcohol (b): MSDS No. 206 – INRS: harmful.

5.1.7. Procedure
5.1.7.1. Preparation of a powder sample
To analyse the fat from a reconstituted liquid, rehydrate the powder to
10% (w/w) dry matter.
 Determination of Total Fat and Free Fat Content 103

5.1.7.2. Preparation of sample for analysis

Using a water bath (k), bring the temperature of the sample to be
analysed to 38–40 C.
Mix the sample thoroughly to obtain a homogenous sample by
repeated inversion of the container, without causing foaming or
churning the fat. Quickly cool to approximately 20 C. For buttermilk
analysis, the samples should not be cooled down as they should be
weighed at 30–40 C.
Note: A correct measurement of the fat content cannot be obtained
from samples having the following characteristics:

. churned milk
. distinct odour of fatty acid
. presence of white particles on the walls of the sample container or
fat globules on the surface of the sample.

5.1.7.3. Samples and reagents

5.1.7.3.1. Powders
Successively add the following to the milk powder butyrometer:

. 10 ml of sulphuric acid (a) using an automatic dispenser (g).

. 8 ml of distilled water using a milk pipette (f ).
. 2.500  0.005 g of powder, on a small square of parchment paper
for example.
. 1 ml of amyl alcohol (b) using an automatic dispenser (h).

Tightly seal the butyrometer without mixing the contents.

5.1.7.3.2. Liquids
When filling the milk butyrometers, take care:

. to avoid wetting the neck of the butyrometer

. to let the liquids flow along the side of the butyrometer
chamber
. not to mix the liquids
. not to let in any air.
104 Analytical Methods for Food and Dairy Powders

Successively add the following to the butyrometer:

. 10 ml of sulphuric acid (a) using an automatic dispenser (g).

. 11 ml of milk using a milk pipette (e).
. 1 ml of amyl alcohol (b) using an automatic dispenser (h).

Tightly seal the butyrometer without mixing the contents.

5.1.7.4. Dissolution of proteins

Mix the contents of the butyrometer by repeated inversion, keeping
the stopper secure and being aware of the potential risk of breakage. It
is also recommended to wrap the butyrometer in absorbent paper
since mixing sulphuric acid and milk produces an exothermic reac-
tion. Continue agitation until the protein material is completely
dissolved or until there are no more white particles.

5.1.7.5. Centrifugation
Place the butyrometer in the centrifuge (i), with the neck downwards
and the cross section of the graduated stem parallel to the lid. It is
important to use an even number of butyrometers, taking care to
balance the load. Centrifuge for 6 min.

5.1.7.6. Reading
Remove the butyrometers from the centrifuge by adjusting the
stopper if necessary, to move the fat layer into the graduated stem.
Beware of acid leaks. Place the butyrometer, with the neck facing
downwards, in a water bath at 65  2 C ( j) for approximately 5 min;
the water level should be above the top of the fat layer. Remove the
butyrometer from the water bath, keeping the neck downwards at all
times, and carefully adjust the stopper in the neck, moving the layer
as little as possible, to bring the lower end of the fat layer to the
nearest gauge mark, preferably a main reference mark. Note the value
at level A corresponding to the lower end of the fat layer, and then,
taking care not to move it, and as quickly as possible (in less than
10 s), note the value at level B corresponding to the upper end of the
fat layer, which coincides with the lowest point of the meniscus. Take
the reading to the closest 0.025 g. Make at least two replicates of the
same sample for analysis.
 Determination of Total Fat and Free Fat Content 105

When taking the readings, the butyrometer should be kept upright

and read at eye level. It should not take more than 10 s between
removing the butyrometer from the water bath and finishing the
reading. If the result is not obtained in less than 10 s or if a verification
of the result is necessary, place the butyrometer back in the water
bath for about 5 min before removing it and taking the reading as
outlined above.
Note: It is recommended to take graduation 0 on the butyrometer
as gauge mark A. If the fat is cloudy or dark, or if there is a black or
white deposit at the bottom of the fat layer, the value obtained for the
fat content will not be accurate. If phase separation is not distinct, a
second centrifugation would yield too high a result: it is preferable to
repeat the analysis.

5.1.7.7. Maintenance of butyrometers

After taking the readings, the butyrometers should be put back into
the rack with the neck upright. After approximately half an hour, the
contents of the butyrometers will still be hot. Turn on the extractor
hood and place the container for collecting waste sulphuric acid and
amyl alcohol under the hood. Carefully unplug the butyrometers and
empty the contents into the container. Rinse the butyrometers and the
stoppers with tap water before placing in the washing-up rack.
If the butyrometers need to be used again straight away, immerse
them and their stoppers in water containing a suitable detergent.
Agitate and empty several times. The same applies to the terminal
bulb. Do not use a bottle brush. Rinse the tube in hot water three times
(successive filling, agitation and emptying). Take care to remove
as much residual water as possible from the butyrometer column
before reusing.

5.1.8. Expression of results

The fat content (F) of milk is:
F ¼ BA ð5:2Þ
where A is the value read at the lower end of the fat layer and B is the
value read at the upper end of the fat layer. It is expressed in grams per
100 ml of milk or in grams per 100 g of milk depending on the
106 Analytical Methods for Food and Dairy Powders

butyrometer used. The result is expressed as the average of two

measurements. Round the result off to the nearest 0.1%.

5.1.9. Remarks
Using a butyrometer graduated in grams per 100 g of milk gives a
direct value for a product having an average density of milk. In other
cases, it is advisable to make a correction by including the specific
density of the product for analysis.

5.1.10. Precision values

5.1.10.1. Repeatability
The difference between two separate results, obtained for the same
product subjected to the same test by the same analyst within a short
space of time should not exceed 0.05 g per 100 ml.

5.1.10.2. Reproducibility
The difference between two separate and independent results, ob-
tained by two analysts working in different laboratories on an
identical product subjected to the same test should not exceed
0.1 g per 100 ml.

5.1.11. Examples
This method was only carried out on powders containing fat (Table 5.1).
For these products, and given their specific composition, the fat
content ranges from 25% (w/w) (milk with 26% fat) to 52.8% (egg
yolk). Standard deviations are in the range of 1 per 1000 in absolute
terms.
Table 5.1. Fat content (wt%) of dairy and food powders
Fat (g 100 g1) Mean  SD (n ¼ 3)
Milk 26% fat 25.0  1.0
Whey 40% fat 38.9  0.9
Whole egg 36.1  1.0
Egg yolk 52.8  1.1
SD, standard deviation; n, number of tests.
 Determination of Total Fat and Free Fat Content 107

5.2. Determination of free fat content

5.2.1. Purpose and range of application
This section outlines the method for the gravimetric determination of
the extractable free fat content of milk and food powder particles.

5.2.2. Definition
Free fat refers to the percentage of substances extracted by following
exactly the outlined method.

5.2.3. Principle
5.2.3.1. Extraction of fat by agitation of a sample in petroleum
ether. The core of the dried milk particles is not accessible in these
conditions.

5.2.3.2. Evaporation of the solvent.

5.2.3.3. Weighing of residue.

5.2.4. Reagents and other products

a, Petroleum ether, distilled between 30 and 60 C.

5.2.5. Instruments and glassware

Standard laboratory equipment as well as the following:

b, Analytical balance.
c, 250 ml bottle with stopper.
d, Paper filter, unfolded, ash-free and fat-free, of approximately
110 mm in diameter.
e, Flask that can be used in an extraction apparatus (e.g. round-
bottom flask of a solvent extractor).
f, Oven set at 102  1 C.
g, Desiccator with an effective desiccant.
h, Solvent extractor.
108 Analytical Methods for Food and Dairy Powders

5.2.6. Safety
As regards personal protection, the wearing of safety goggles and
solvent resistant gloves is essential.

5.2.7. Procedure
5.2.7.1. Preparation of material
Put the flasks in the oven for 1 h, leave them to cool down and then
weigh them. Note the value (w).

5.2.7.2. Preparation of the sample for analysis

Weigh 10  0.05 g of the sample.

5.2.7.3. Samples and reagents

5.2.7.3.1. Put the sample in the bottle (c).

5.2.7.3.2. Add 50 ml of petroleum ether (a).

5.2.7.3.3. Agitate vigorously for 5 min.

5.2.7.3.4. Filter (d ) into a pre-weighed flask (e), taking care not to

remove the sediment.

5.2.7.3.5. Add 40–50 ml of petroleum ether to the bottle.

5.2.7.3.6. Repeat steps 5.2.7.3.3 and 5.2.7.3.4 as often as required

depending on the product (cf. 5.2.9.2).

5.2.7.3.7. Rinse the filter with a few millimetres of solvent.

5.2.7.3.8. Remove the solvent by distillation (h).

5.2.7.3.9. Lay the bottle on its side for leave for 1 h in the oven ( f ).

5.2.7.3.10. Leave to cool in the desiccator ( g) and weigh.

5.2.7.3.11. Repeat the heating process for periods of 30 min until

 Determination of Total Fat and Free Fat Content 109
the minimum weight has been achieved (to nearest 2 mg). Note the
value (W).

5.2.8. Expression of results

The free fat content (FreeF) can be expressed in grams per 100 g of
total fat of the sample by the following formula:
ðW  wÞ
FreeF ¼  1000 ð5:3Þ
F
where W is the weight in grams after the procedure (5.2.7.3.11), w is the
weight in grams of the empty bottle (5.2.7.1), and F is the total fat content
(%) according to the method ‘Determination of the total fat content’. The
result is expressed as the average of two measurements. Round the result
off to the nearest 0.1%.
The free fat content (FreeF) can also be expressed in grams per
100 g of powder by the following formula:
FreeF ¼ ðW  wÞ  10 ð5:4Þ
where W is the weight in grams after the procedure and w is the weight in
grams of the empty bottle.

5.2.9. Remarks
5.2.9.1. Given that the analysis and comprehension of the data
concerning free fat in milk powders was so complex, Vignolles et al.
(2007) recently published a literature review on the different
analytical and physicochemical factors that could influence the
free fat content and consequently the quality of fat-enriched milk
powders.

5.2.9.2. Vignolles et al. (2009b) tested the influence of the number

of successive extractions on the value of measured free fat. Their
results show that within the limit of the powders tested, a single
extraction is not enough. The authors suggest carrying out a number
of extractions, showing that depending on the products and the
quality of free fat, a plateau is reached between two and five
successive extractions.
110 Analytical Methods for Food and Dairy Powders

5.2.10. Precision values

5.2.10.1. Repeatability
The difference between two separate results, obtained for the same
product subjected to the same test by the same analyst within a short
space of time should not exceed 0.05 g per 100 g.

5.2.10.2. Reproducibility
The difference between two separate and independent results, ob-
tained by two analysts working in different laboratories on an
identical product subjected to the same test should not exceed
0.1 g per 100 g.

5.2.11. Analysis report

The analysis report should indicate the method used and the results
obtained. It should also mention all the operating details not specified
in this method, or optional details, as well as any incidents which may
have influenced the results, and in particular any comments indicat-
ing that the result is of questionable accuracy. The analysis report
should give all the information necessary for the complete identifi-
cation of the sample.

5.2.12. Examples
This method was only carried out on powders containing fat
(cf. Table 5.1and Table 5.2). For these products, the free fat content
ranges from 2.1% (grams per 100 g of fat) (milk with 26% fat) to 7.6%
(egg yolk).

Table 5.2. Free fat content (wt%) of dairy and food powders
Free fat g 100 g1 of fat g 100 g1 of powder
(n ¼ 3) Mean  SD Mean  SD
Milk 26% fat 2.1  0.1 0.5  0.0
Whey 40% fat 5.1  0.2 2.0  0.1
Whole egg 7.4  0.2 2.7  0.1
Egg yolk 7.6  0.1 4.0  0.1
SD, standard deviation; n, number of tests.
 Determination of Total Fat and Free Fat Content 111

These values depend on the fat content: an increase in the fat

content generally leads to an increase in the free fat content.
However, recent studies have shown that the influencing factors are
multifactorial (Vignolles et al., 2009a, 2010): the free fat content also
depends on the parameters of the procedure used, the composition of
the non-fat matrix and the type of fat (melting point).
Standard deviations are in the range of two per 1000 in absolute
terms.

5.3. Bibliography
AFNOR 1990. Norme NF V 04-210. Lait – D etermination de la Teneur en
Matiere Grasse – M ethode Acido-Butyrom etrique. Association Française de
Normalisation, Paris.
FIL-IDF, Norme internationale 152A 1997. Lait et Produits Laitiers – D etermi-
nation de la Teneur en Mati ere Grasse – Guide de Directives G enerales
Appliquees aux M ethodes Butyrom etriques. Federation Internationale de
Laiterie, Brussels.
Vignolles, M.L., Jeantet, R., Lopez, C. and Schuck, P. 2007. Free fat, surface fat and
dairy powder: interactions between process and product. A review. Le Lait, 87:
187–236.
Vignolles, M.L., Lopez, C., Madec, M.N., Ehrhardt, J.J., Mejean, S., Schuck, P. and
Jeantet, R. 2009a. Protein-lactose matrix affects fat encapsulation during the
overall spray-drying process of dairy powders. Australian Dairy Journal, 64:
75–79.
Vignolles, M.L., Lopez, C., Ehrhardt, J.J., Lambert, J., Mejean, S., Jeantet, R. and
Schuck, P. 2009b. Methods’ combination is a key tool to elucidate the
suprastructure, composition and properties of fat in fat-filled dairy powders.
Journal of Food Engineering, 94: 154–162.
Vignolles, M.L., Lopez, C., Le Floch, C., Ehrhardt, J.J., Mejean, S., Jeantet, R. and
Schuck, P. 2010. Fat supramolecular structure in fat-filled dairy powders: a tool
to adjust drying kinetics. Dairy Science and Technology, 90: 287–300.