Global Supply Chain

is owned by Heineken Supply Chain B.V., The Netherlands. This document may also contain information, drawings, designs or plans owned by third parties and such information may be proprietary and confidential and protected by intellectual property rights of such third parties. This document
This document including all information, drawings, designs or plans contained therein, is the property of Heineken Supply Chain B.V., The Netherlands and all intellectual property therein, including all proprietary and confidential information, know-how, designs and/or data, remains vested in and

Laboratory Method
Wort
Free Amino Nitrogen by Ninhydrin method
(International method)
may not be used, copied, and/or reproduced in whole or in part for any purpose, unless such usage, copying and/or reproduction has been specifically permitted in writing by Heineken Supply Chain B.V., The Netherlands.

HeiRule
Food Safety and Laboratory Method
Product Quality

CONTENTS

1 REVISION HISTORY ........................................................................................................... 2

2 WARNING AND SAFETY PRECAUTIONS ............................................................................... 2
3 SCOPE ............................................................................................................................. 2
4 FIELD OF APPLICATION ..................................................................................................... 2
5 PRINCIPLE ........................................................................................................................ 2
6 REAGENTS ....................................................................................................................... 3
7 APPARATUS ..................................................................................................................... 3
8 PREPARATION OF SAMPLES ............................................................................................... 3
9 PROCEDURE ..................................................................................................................... 4
10 EXPRESSION OF RESULTS .................................................................................................. 4
11 NOTES ............................................................................................................................. 6
12 RELATED DOCUMENTS ..................................................................................................... 6

Wort - Free Amino Nitrogen by Ninhydrin method (International method)

HMESC: 02.12.01.011 Date: December 2018 Issue: 05 Page 1 of 6
 Global Supply Chain
is owned by Heineken Supply Chain B.V., The Netherlands. This document may also contain information, drawings, designs or plans owned by third parties and such information may be proprietary and confidential and protected by intellectual property rights of such third parties. This document
This document including all information, drawings, designs or plans contained therein, is the property of Heineken Supply Chain B.V., The Netherlands and all intellectual property therein, including all proprietary and confidential information, know-how, designs and/or data, remains vested in and

1 REVISION HISTORY

Issue Change description Author Date

05 Some textual changes (see tracked changes) Marcel de Koning December 2018

Lab Standard became Lab Method, specification

04 Marcel de Koning June 2018
water bath 20°C changed

2 WARNING AND SAFETY PRECAUTIONS

may not be used, copied, and/or reproduced in whole or in part for any purpose, unless such usage, copying and/or reproduction has been specifically permitted in writing by Heineken Supply Chain B.V., The Netherlands.

2.1 For details of safety standards of a physical/chemical laboratory consult the recommendations
included in Safety Standard 02.09.01.001, Safety Standards for Laboratories.

2.2 Ninhydrin is harmful and must be used with care.

3 SCOPE

3.1 The determination of the free amino nitrogen content of wort using colorimetry with ninhydrin.

3.2 The method gives an estimate of amino acids, ammonia, and, to some extent, end group α-amino
nitrogen in peptides and proteins. Proline is partially estimated at the wavelength used.

3.3 The method is not specific for α-amino nitrogen since γ-amino butyric acid which is present in
wort also gives a colour reaction with ninhydrin.

4 FIELD OF APPLICATION

4.1 The method can be applied to all kind of wort samples.

4.2 A correction must be made for dark worts (higher than 100 EBC units) due to the colour of the
sample. This correction is not needed for the measurement by Segmented Flow Analysis (see 12.3).

5 PRINCIPLE

The sample, a standard and a blank solution are heated in the presence of ninhydrin at pH 6.7, and
the absorbances at 570 nm are measured against distilled water. For dark coloured wort samples
(higher than 100 EBC units) a correction for the colour of the sample is applied.

Wort - Free Amino Nitrogen by Ninhydrin method (International method)

HMESC: 02.12.01.011 Date: December 2018 Issue: 05 Page 2 of 6
 Global Supply Chain
is owned by Heineken Supply Chain B.V., The Netherlands. This document may also contain information, drawings, designs or plans owned by third parties and such information may be proprietary and confidential and protected by intellectual property rights of such third parties. This document
This document including all information, drawings, designs or plans contained therein, is the property of Heineken Supply Chain B.V., The Netherlands and all intellectual property therein, including all proprietary and confidential information, know-how, designs and/or data, remains vested in and

6 REAGENTS

6.1 Ninhydrin colour reagent. Dissolve 20 g disodium hydrogen phosphate dodecahydrate (Na2HPO4 ⋅
12H2O), 12 g potassium dihydrogen phosphate (KH2PO4), 1.0 g ninhydrin and 0.6 g fructose in a
beaker. In case chemicals do not dissolve, use magnetic stirrer and gently warm up to maximum 47
°C, cool down after heating. Transfer to beaker and check pH. The pH must be between 6,6 and 6,8,
adjust if necessary with droplets of 85% Phosphoric acid or 50% Sodium hydroxide solutions.
Transfer solution to 200 ml volumetric flask and bring up to 200 ml with distilled water. The
solution will keep for 2 weeks if stored cold (0 to 4 °C) in an amber bottle.
6.2 Diluting solution. Dissolve 2 g potassium iodate (KIO3) in 600 ml of distilled water and add 400 ml
of 96 % (V/V) ethanol. The solution will keep for 1 week if stored at room temperature. Note: use
may not be used, copied, and/or reproduced in whole or in part for any purpose, unless such usage, copying and/or reproduction has been specifically permitted in writing by Heineken Supply Chain B.V., The Netherlands.

only measuring cylinders; do not dilute to 1 litre.

6.3 Glycine stock solution. Accurate weigh 0.1072 g of glycine and dissolve in about 50 ml of distilled
water in a glass-stoppered 100 ml volumetric flask, and make up to the mark with distilled water.
The solution will keep for 2 months if stored at 0 to 4 °C.
6.4 Glycine standard solution. Dilute 1 ml of the glycine stock solution to 100 ml with distilled water in
a glass-stoppered 100 ml volumetric flask so that the glycine standard solution contains 2 mg
amino nitrogen per litre. The standard solution should be prepared freshly.

7 APPARATUS

7.1 Boiling water bath

7.2 Water bath, set at 20.0 °C ± 1 °C
7.3 Spectrophotometer, to determine absorbance at 570 nm
7.4 Cuvettes, polystyrene or glass, optical path length 10 mm
7.5 Analytical balance, accurate to 0.1 mg
7.6 pH meter
7.7 Test tubes, approx. 16 x 150 mm (O.D. x l)
7.8 Pipettes with rubber suction bulbs or with pipette filler pumps, or automatic dispensers
7.9 Glass marbles, 20 – 25 mm diameter
7.10 Volumetric flasks, with a ground glass stopper, 50 ml, 100 ml, 200 ml and 1000 ml
7.11 Laboratory centrifuge, with a current swing-out rotor radius of about 14 cm, giving a relative
centrifugal field of approx. 1400 x g (see Note 11.1).

8 PREPARATION OF SAMPLES

8.1 If necessary clarify the wort sample by centrifuging a 20 ml aliquot of the sample for 10 minutes
at 1400 x g (see Note 11.1).
During handling of the wort take care not to introduce traces of amino acids by contamination
with saliva or skin. Use pipettes with rubber suction bulbs or with pipette filler pumps or use
automatic dispensers whenever possible (see Note 11.2).

Wort - Free Amino Nitrogen by Ninhydrin method (International method)

HMESC: 02.12.01.011 Date: December 2018 Issue: 05 Page 3 of 6
 Global Supply Chain
is owned by Heineken Supply Chain B.V., The Netherlands. This document may also contain information, drawings, designs or plans owned by third parties and such information may be proprietary and confidential and protected by intellectual property rights of such third parties. This document
This document including all information, drawings, designs or plans contained therein, is the property of Heineken Supply Chain B.V., The Netherlands and all intellectual property therein, including all proprietary and confidential information, know-how, designs and/or data, remains vested in and

9 PROCEDURE

9.1 Dilute the sample with distilled water in such a way that a solution is obtained containing 1 to 3 mg
free amino nitrogen per litre. It is recommended that wort is diluted 1 ml to 100 ml.

9.2 Wort: pipette 2 ml of the diluted wort sample into test tubes in triplicate.

9.3 Blank: pipette 2 ml of distilled water into test tubes in triplicate.

9.4 Standard: pipette 2 ml of the glycine standard solution into test tubes in triplicate.
may not be used, copied, and/or reproduced in whole or in part for any purpose, unless such usage, copying and/or reproduction has been specifically permitted in writing by Heineken Supply Chain B.V., The Netherlands.

9.5 Pipette 1 ml of ninhydrin colour reagent to each test tube.

Stopper all tubes with glass marbles to avoid loss by evaporation.
Heat all tubes for exactly 16 minutes in a boiling water bath and then cool in a water bath at 20 °C
for 20 minutes. After this time pipette 5 ml of diluting solution into each tube. Mix contents of each
test tube thoroughly by shaking, and measure absorbance at 570 nm in 10 mm cuvettes against
distilled water within 30 minutes after addition of diluting solution.

9.6 For dark coloured wort samples (higher than 100 EBC units) a correction due to the colour of the
sample must be made (see Note 11.4).
9.6.1 Pipette 2 ml of the diluted wort sample into three test tubes and add 1 ml of distilled water. Stopper
the three tubes with glass marbles, heat for exactly 16 minutes in a boiling water bath and then
cool in a water bath at 20 °C for 20 minutes. After this time pipette 5 ml of diluting solution into
each tube, mix well, and measure absorbance at 570 nm in 10 mm cuvettes against distilled water
within 30 minutes after addition of diluting solution.

10 EXPRESSION OF RESULTS

10.1 Calculation

Average absorbance readings obtained for the triplicate tubes from each sample.
Calculate the free amino nitrogen (FAN) content of the sample using the following formula:

2• d•(At - Ab - A c )
FAN (mg/l) =
A s - Ab

Where:
2 = concentration of glycine standard solution (in mg per l)
d = dilution factor (e.g. 100 if dilution was 1 ml to 100 ml)
At = average absorbance of diluted wort sample
Ab = average absorbance of blanks
Ac = average absorbance for correction of dark coloured samples according 9.6.1 (see
Note 11.4)
As = average absorbance of glycine standard

Wort - Free Amino Nitrogen by Ninhydrin method (International method)

HMESC: 02.12.01.011 Date: December 2018 Issue: 05 Page 4 of 6
 Global Supply Chain
is owned by Heineken Supply Chain B.V., The Netherlands. This document may also contain information, drawings, designs or plans owned by third parties and such information may be proprietary and confidential and protected by intellectual property rights of such third parties. This document
This document including all information, drawings, designs or plans contained therein, is the property of Heineken Supply Chain B.V., The Netherlands and all intellectual property therein, including all proprietary and confidential information, know-how, designs and/or data, remains vested in and

10.1.1 Example

For wort:
Average absorbance of blanks = 0.050.
Average absorbance of glycine standard = 0.490.
Average absorbance of wort diluted 1 to 100 = 0.380.
Net absorbance of glycine standard after blank subtraction = 0.440.
Net absorbance of wort after blank subtraction = 0.330.

0.330
Free Amino Nitrogen (FAN), mg/l = x 2 x 100 = 150.0
0.440
may not be used, copied, and/or reproduced in whole or in part for any purpose, unless such usage, copying and/or reproduction has been specifically permitted in writing by Heineken Supply Chain B.V., The Netherlands.

Report FAN in mg/l rounded-off to whole numbers, i.e., 150 mg/l.

10.2 Reporting

Express the result of Free Amino Nitrogen (FAN) in mg/l to the nearest whole number.

10.3 Precision

The precision values below (mg/l) were determined from the data of a collaborative trial carried out
by the EBC Analysis Committee in 1992. Between 11 to 14 laboratories analysed wort samples at 3
levels in the range 110 to 180 mg/l according to the method described in Analytica-EBC (see
Related Documents 12.1). This EBC method is completely comparable with the above-described
procedure. However, the latter is more detailed.
In this EBC trial malt wort samples were prepared by the participating laboratories.

Range r95 R95

110 to 180 28 - 0.105m 47 - 0.173m

where:
m = the mean value
r95 = the within laboratories repeatability
R95 = the between laboratories reproducibility

Wort - Free Amino Nitrogen by Ninhydrin method (International method)

HMESC: 02.12.01.011 Date: December 2018 Issue: 05 Page 5 of 6
 Global Supply Chain
is owned by Heineken Supply Chain B.V., The Netherlands. This document may also contain information, drawings, designs or plans owned by third parties and such information may be proprietary and confidential and protected by intellectual property rights of such third parties. This document
This document including all information, drawings, designs or plans contained therein, is the property of Heineken Supply Chain B.V., The Netherlands and all intellectual property therein, including all proprietary and confidential information, know-how, designs and/or data, remains vested in and

11 NOTES

11.1 See for the connection between relative centrifugal field (x g) value, centrifuge rotor radius and
run speed (in rpm): note 11.1 in Laboratory Method 02.12.01.013 Wort, Residue: Centrifugal
Method.
11.2 Since the quantities of amino nitrogen reacting in this method are very small, it is necessary to
avoid introducing traces from outside sources by taking the following measures:
- use suction bulbs for pipettes or use pumpettes or automatic dispensers whenever possible
- use always forceps when handling glass marbles
- carefully cleaning of the glassware and only handling on external surfaces.
11.3 Times and temperatures stated are critical and should be adhered to very closely. A standard
may not be used, copied, and/or reproduced in whole or in part for any purpose, unless such usage, copying and/or reproduction has been specifically permitted in writing by Heineken Supply Chain B.V., The Netherlands.

and blank sample must be included in each test to compensate for temperature variations in the
boiling water bath.
11.4 For light coloured samples Ac = 0.

12 RELATED DOCUMENTS

12.1 Analytica – EBC of the European Brewery Convention, Method 8.10: Free Amino Nitrogen in Wort by
Spectrophotometry (IM).
12.2 Methods of Analysis of the ASBC, method Wort-12A: Free Amino Nitrogen (International Method) –
Ninhydrin method
12.3 HEINEKEN Laboratory Method, HMESC 02.12.01.025, Free Amino Nitrogen by Segmented Flow
Analysis
12.4 HEINEKEN Laboratory Method, HMESC 02.12.01.063, Packaged Beer - Free Amino Nitrogen with
CDR Beerlab®

Wort - Free Amino Nitrogen by Ninhydrin method (International method)

HMESC: 02.12.01.011 Date: December 2018 Issue: 05 Page 6 of 6