ISO 13082:2011

INTERNATIONAL ISO
STANDARD 13082
IDF
First edition
2011-11-15
Milk and milk products — Determination
of the lipase activity of pregastric lipase
preparation
Lait et produits laitiers — Détermination de l'activité de lipase de la
préparation de lipase prégastrique

Reference numbers
IDF 218:2011(E)
©
ISO and IDF 2011
IDF 218:2011(E)
©  ISO and IDF 2011
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ii © ISO and IDF 2011 – All rights reserved

IDF 218:2011(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has
been established has the right to be represented on that committee. International organizations, governmental
and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 13082IDF 218 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5,
Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and
IDF.
IDF 218:2011(E)
Foreword
IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide.
IDF membership comprises National Committees in every member country as well as regional dairy
associations having signed a formal agreement on cooperation with IDF. All members of IDF have the right to
be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in
the development of standard methods of analysis and sampling for milk and milk products.
The main task of Standing Committees is to prepare International Standards. Draft International Standards
adopted by the Standing Committees are circulated to the National Committees for endorsement prior to
publication as an International Standard. Publication as an International Standard requires approval by at least
50 % of IDF National Committees casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. IDF shall not be held responsible for identifying any or all such patent rights.
ISO 13082IDF 218 was prepared by the International Dairy Federation (IDF) and Technical Committee
ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF
and ISO.
All work was carried out by the Joint ISO-IDF Project Group Lipase activity of the Standing Committee on
Analytical methods for processing aids and indicators under the aegis of its project leaders, Mrs M. Harboe
(DK) and Dr J. Jacobsen (DK).
iv © ISO and IDF 2011 – All rights reserved

IDF 218:2011(E)
Introduction
Lipases (EC 3.1.1.3) are the group of esterases that hydrolyse emulsified triacylglycerolesters, which are the
main component of milk fat.
Commercial pregastric lipase and some rennet preparations (paste or liquid) contain lipases from calf, kid-
goat or lamb sources. These lipase preparations are used particularly in the production of Italian type cheeses,
e.g. in Romano, Provolone, and Asiago and in other similar cheese varieties and in enzyme-modified dairy
[6]
products as described in IDF Bulletin 294 . Lipase is not allowed in Feta, but it is often used in Feta-type
cheese.
[7]
The method is based on the principle of the FCCIV method for forestomach lipase activity , but in its current
form the FCCIV method is not sufficiently developed. As such, it does not provide adequate details in several
critical areas, most notably in sample and substrate preparation. However, the FCCIV method served as a
useful model for the development of this International Standard.

INTERNATIONAL STANDARD
IDF 218:2011(E)
Milk and milk products — Determination of the lipase activity of
pregastric lipase preparation
1 Scope
This International Standard specifies a method for the determination of the lipase activity. It is intended for the
preparation of pregastric lipase and rennet paste, both of animal origin.
NOTE No reference method was used to check this method as no stable standard can be found. On the other hand,
a reference method can be omitted as the substrate is reproducible and well defined.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
international lipase unit
ILU
amount of lipase activity that releases butanoic acid, also known as butyric acid, at a rate of 1,25 µmol/min
under specified conditions
NOTE 1 Lipase activity is expressed either in international lipase units (ILU) per gram of product or ILU per millilitre of
product.
NOTE 2 The definition is based on the direct consumption of titrant while not considering that a small molar fraction of
the butyric acid (4 %) is not dissociated and thus cannot be titrated. As such, that creates a small error in the definition.
3 Principle
Triglyceride esters are hydrolysed by lipase. The free fatty acids (as butyric acid) released from the substrate
tributyrin are titrated in a pH-stat with sodium hydroxide. The amount of sodium hydroxide consumed within a
defined period is used to calculate the activity in ILU per millilitre or ILU per gram.
Due to the non-existence of a reference standard, it is recommended that a control (known) sample be
included in the test.
4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled
water or demineralized water or water of equivalent purity.
The brand of chemicals can affect the result. Therefore, before using a brand other than the one mentioned,
verify whether it gives the same result.
IDF 218:2011(E)
1)
4.1 Tributyrin (glycerintributyrate or glyceryl tributyrate), e.g. Merck No. 1.01958.0100 or similar.
1)
4.2 Sodium caseinate, e.g. Sigma C8654 or similar.
1)
4.3 Lecithin, from soya bean, e.g. BDH Prod. 29863 or similar.
4.4 Liquid paraffin. Use paraffin which is highly liquid (or similar light mineral oil), e.g. Merck
1)
No. 7174.1000 , or similar.
1) 1)
4.5 Soda lime granules [Carbosorb ], e.g, BDH no 331104 or similar.
4.6 Sodium hydroxide solution, c(NaOH)  0,025 mol/l, which can either be purchased or be prepared as
follows.
Using a pipette (5.1), add 25,00 ml of 1 mol/l sodium hydroxide with an accurately known titre into a container.
Dilute with water to 1 000 ml.
The 0,025 mol/l NaOH solution can be kept in a closed container, protected against carbon dioxide in the air
by use of a CO trap with soda lime (4.5) at room temperature for at least 1 month. If necessary, seek advice
from the supplier of the equipment or reagent. Change the soda lime at least once a year.
When changing the sodium hydroxide batch, check the actual stability of the titre by comparing the old and
new titrant, e.g. using a control sample.
For samples with low activity and manual titrations, use a 0,010 mol/l NaOH instead of a 0,025 mol/l NaOH
solution. As such, the 0,010 mol/l NaOH solution gives a higher and more useful consumption of titrant.
Prepare the 0,010 mol/l NaOH solution freshly before use (unless the titre has been checked) as it is unstable.
If using the 0,010 mol/l NaOH solution, correct the calculation according to the formulae in 8.1.
4.7 Lecithin solution, with a mass per volume fraction of 10 %. Weigh 10,0 g of lecithin in a suitable bottle.
Use magnetic stirring to dissolve it in approx. 95 ml of liquid paraffin, which may take between 1 day and
2 days of mixing. When the lecithin is completely dissolved, make it up to a total volume of 100 ml with the
liquid paraffin.
When stored in a refrigerator, the lecithin solution is stable for 1 year.
4.8 Control sample. Include a control sample of known activity in each series of test for lipase samples.
Collect the results and use them for the evaluation of the variation of the test.
The control sample can be the last sample analysed or another well-known sample.
When carrying out the method for the very first time, use a control sample obtained from another laboratory or
the first sample analysed being kept as control sample for the next series of analyses. If needed, store the
control sample(s) in a freezer.
NOTE It can be difficult to get a suitable control sample for rennet paste.
5 Apparatus
Usual laboratory equipment and, in particular, the following.
The laboratory equipment can be substituted by other equipment verified as giving similar results.
5.1 Micropipette or any other pipette, of capacities 1 ml and 10 ml with a repeatability of 0,5 % or higher.

1) Example of a suitable product available commercially. This information is given for the benefit of users of this
document and does not constitute an endorsement by ISO of this product.
2 © ISO and IDF 2011 – All rights reserved

IDF 218:2011(E)
[3]
5.2 One-mark volumetric flasks, of required capabilities, ISO 1042 class A.
5.3 Water bath, capable of circulating the water externally and of maintaining a constant temperature in the
reaction vessel of 42 °C  0,5 °C.
1) 1)
5.4 Blender, Warren , Ultraturax or any equivalent apparatus.
5.5 pH stated equipment, including the following components:
a) a thermostated reaction vessel capable of stirring effectively, e.g. mechanical or magnetic stirring;
b) a burette for titration;
c) a recorder, printer or computer.
1)
A Metrohm 718 Stat Titrino is suitable for the purpose. A manual titration set-up may also be used but that
can reduce the precision of the method.
For control purposes, therefore, mention the equipment used in the test report.
5.6 Stomacher and stomacher bags, for dissolving rennet paste, e.g. standard bags BA 6041 from
1)
Seward or equivalent.
6 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this International Standard. A recommended sampling method
[2]
is given in ISO 707IDF 50
.
Test samples may be stored at a temperature of 5 °C or lower for 2 months. In case of a long storage period,
store the test samples frozen, e.g. at 18 °C, as that will significantly improve the stability of the lipase powder.
7 Procedure
7.1 Substrate
Disperse 600 mg of sodium caseinate (4.2) in 95 g water in the blender vessel. Add 0,5 ml lecithin solution
(4.3) and 1,0 ml tributyrin (4.1). Blend for 60 s at low speed. Pour the substrate into a flask or beaker and keep
it at room temperature on a magnetic stirrer using slow speed.
Use the substrate within 4 h.
7.2 Preparation of lipase test solution
7.2.1 Liquid lipase sample
Accurately pipette the required amount of the liquid lipase sample or control into a 100 ml one-mark volumetric
flask (5.2) to obtain a 100 ml lipase solution with a concentration of (4  1) ILU/ml. Make up to the mark with
water.
NOTE Volumetric flasks of different capacities can be used or the sample can be analysed und
...