ISO/PRF 4134

Meat and meat products -- Determination of L-(+)-glutamic acid content -- Reference method

Viande et produits à base de viande -- Détermination de la teneur en acide L-(+)-glutamique -- Méthode de référence

General Information

• Status: Published
• ICS: 67.120.10 - Meat and meat products
• Technical Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Drafting Committee: ISO/TC 34/SC 6/WG 25 - Determination of glutamic acid content

RELATIONS

• Revised: ISO 4134:1999 - Meat and meat products -- Determination of L-(+)- glutamic acid content -- Reference method
• Effective Date: 23-Apr-2020

Standards Content (sample)

INTERNATIONAL ISO
STANDARD 4134
Third edition
Meat and meat products —
Determination of L-(+)-glutamic acid
content — Reference method
Viande et produits à base de viande — Détermination de la teneur en
acide L-(+)-glutamique — Méthode de référence
PROOF/ÉPREUVE
Reference number
ISO 4134:2021(E)
ISO 2021
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ISO 4134:2021(E)
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© ISO 2021

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Published in Switzerland
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ISO 4134:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 1

5 Sampling ........................................................................................................................................................................................................................ 2

6 Preparation of test sample ......................................................................................................................................................................... 2

7 Test method of spectrophotometer .................................................................................................................................................. 2

7.1 Reagents........................................................................................................................................................................................................ 2

7.2 Apparatus .................................................................................................................................................................................................... 4

7.3 Procedure .................................................................................................................................................................................................... 5

7.3.1 General...................................................................................................................................................................................... 5

7.3.2 Test portion .......................................................................................................................................................................... 5

7.3.3 Preparation of extract ........................................................................................................................................... ....... 5

7.3.4 Determination .................................................................................................................................................................... 5

7.4 Calculation and results ..................................................................................................................................................................... 6

7.4.1 Absorbance difference of L-(+)-glutamic acid standard solution .......................................... 6

7.4.2 Absorbance difference of L-(+)-glutamic acid test solution ....................................................... 7

7.4.3 Formula of linear regression of L-(+)-glutamic acid standard curve ................................. 7

7.4.4 L-(+)-glutamic acid concentration of the test solution ................................................................... 7

7.4.5 L-(+)-glutamic acid content of test sample ............................................................................................... 8

7.5 Precision ....................................................................................................................................................................................................... 8

7.5.1 Interlaboratory test ....................................................................................................................................................... 8

7.5.2 Repeatability ....................................................................................................................................................................... 8

7.5.3 Reproducibility .................................................................................................................................................................. 8

7.6 Detection limit ......................................................................................................................................................................................... 8

8 Test method of light absorption microplate reader ........................................................................................................ 9

8.1 Reagents........................................................................................................................................................................................................ 9

8.2 Apparatus .................................................................................................................................................................................................... 9

8.3 Procedure .................................................................................................................................................................................................... 9

8.3.1 General...................................................................................................................................................................................... 9

8.3.2 Extraction of L-(+)-glutamic acid in the test portion ....................................................................... 9

8.3.3 Determination .................................................................................................................................................................... 9

8.4 Calculation and results ..................................................................................................................................................................10

8.4.1 Absorbance difference for L-(+)-glutamic acid standard solution ....................................10

8.4.2 Absorbance difference for L-(+)-glutamic acid test solution..................................................10

8.4.3 Formula of linear regression for L-(+)-glutamic acid standard curve ............................11

8.4.4 L-(+)-glutamic acid concentration of the test solution ................................................................11

8.4.5 L-(+)-glutamic acid content of test sample ............................................................................................11

8.5 Precision ....................................................................................................................................................................................................12

8.5.1 Interlaboratory test ....................................................................................................................................................12

8.5.2 Repeatability ....................................................................................................................................................................12

8.5.3 Reproducibility ...............................................................................................................................................................12

8.6 Detection limit ......................................................................................................................................................................................12

9 Test report ................................................................................................................................................................................................................12

Annex A (informative) Safety practices ..........................................................................................................................................................14

Bibliography .............................................................................................................................................................................................................................15

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ISO 4134:2021(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 6,

Meat, poultry, fish, eggs and their products.

This third edition cancels and replaces the second edition (ISO 4134:1999), which has been technically

revised. The main changes compared with the previous edition are as follows:

— a new test method, the light absorption microplate reader method, has been added;

— the order of the clauses has been rearranged;

— the Scope (Clause 1) has been revised to specify free L-(+)-glutamic acid in meat and meat products;

— the Normative references (Clause 2) have been updated;

— the Terms and definitions (Clause 3) have been modified by adding the term “free L-(+)-glutamic

acid”;

— in Clause 4, the description of “extraction of L-(+)-glutamic acid of test portion” has been modified

and the detection wavelength has been changed from “492 nm” to “490 nm”;

— in 7.1, the identification of enzyme activity units for diaphorase and glutamate dehydrogenase has

been supplemented; the concentration of KOH, NAD has been modified; the NAD and diaphorase

have been mixed into a solution; and the buffer, NAD and enzymes have been labelled with R1, R2,

and R3;
— the apparatus list (7.2) has been updated;

— in 7.3, the procedure of the test method of spectrophotometer has been modified by halving the

sample mass and solution volume;

— in 7.3.4, the method of judging the absorbance of the reaction end point has been modified and, as a

result, the previous Annex B “Example of plotting and extrapolation of absorbance values” has been

deleted;
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ISO 4134:2021(E)

— in 8.4, the formula and symbol description of spectrophotometer has been modified;

— the previous Annex C “Derivation of equation for calculation of L-(+)-glutamic acid content” has

been deleted;
— the Bibliography has been updated.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
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INTERNATIONAL STANDARD ISO 4134:2021(E)
Meat and meat products — Determination of L-(+)-
glutamic acid content — Reference method
1 Scope

This document specifies the spectrophotometer method and the light absorption microplate reader

method for the determination of the free L-(+)-glutamic acid content of meat and meat products.

This document is applicable to meat and meat products, including livestock and poultry products.

2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 648, Laboratory glassware — Single-volume pipettes
ISO 1042, Laboratory glassware — One-mark volumetric flasks

ISO 1442, Meat and meat products — Determination of moisture content (Reference method)

ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 8655-2, Piston-operated volumetric apparatus — Part 2: piston pipettes
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
free L-(+)-glutamic acid

L-(+)-glutamic acid and glutamate existing in meat and meat products in the form of free state

4 Principle

The free L-(+)-glutamic acid present in a test portion is extracted with perchloric acid solution. The

extract is centrifuged, decanted and filtered, and diluted to appropriate concentration with water, and

the pH is adjusted to 10. Nicotinamide adenine dinucleotide (NAD) is reduced by the L-(+)-glutamic

acid in the presence of glutamate dehydrogenase, see Formula (1). The resultant reduced nicotinamide

adenine dinucleotide (NADH) reacts with iodonitrotetrazolium chloride in the presence of diaphorase,

see Formula (2). The resulting formazane is measured at a wavelength of 490 nm and the free L-(+)-

glutamic acid content of the test sample is calculated.
glutamatedehydrogenasee
Lg--()++lutamicacid/glutamateNAD +HO←→
(1)
α-ketoglutamate++NADH NH +H
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ISO 4134:2021(E)
diaphorase
+ +
NADH + iodonitrotetrazolium chloride +H ←→ NAD + formazane (2)
5 Sampling

Sampling is not part of the method specified in this document. A recommended sampling method is

given in CAC/GL 50-2004.

It is important that the sample received by the laboratory is truly representative and has not been

damaged or changed during transport or storage.

Start from a representative sample of at least 200 g. Store the sample at a temperature of 4 °C or frozen

at −18 °C if not immediately analysed, so that deterioration and change in composition are prevented.

6 Preparation of test sample

Homogenize the laboratory sample with the appropriate equipment (see 7.2.1). Take care that the

temperature of the sample does not exceed 25 °C. If a mincer is used, pass the sample at least twice

through the equipment.

Fill a suitable airtight container with the prepared sample. Close the container and store at a

temperature of 4 °C or frozen at −18 °C if not immediately analysed, so that deterioration and change

in composition are prevented. Analyse the sample as soon as practicable, but always within 24 h after

homogenization.
7 Test method of spectrophotometer
7.1 Reagents

Only reagents of recognized analytical grade and only water of at least grade 2 purity as defined in

ISO 3696 shall be used. Except for the solutions of inorganic compounds (7.1.1 and 7.1.2), store all

solutions in stoppered brown glass bottles which have been scrupulously cleaned and steamed or

sterilized.
7.1.1 Dilute perchloric acid, c = 1,0 mol/l.

WARNING — Contact with oxidizable or combustible materials or with dehydrating or reducing

agents can result in fire or explosion. Persons using this acid should be thoroughly familiar with

its hazards. See the safety practices listed in Annex A.

Add 8,6 ml of the perchloric acid (70 g/ 100 g, ρ = 1,67 g/ml) to the bulk of water, diluting to 100 ml.

7.1.2 Potassium hydroxide solution, c = 4 mol/l, 2 mol/l, 0,5 mol/l and 0,02 mol/l.

Dissolve 22,4 g of potassium hydroxide in water. Dilute the solution to 100 ml, c = 4 mol/l, and mix

evenly after cooling.

Dissolve 11,2 g of potassium hydroxide in water. Dilute the solution to 100 ml, c = 2 mol/l, and mix

evenly after cooling.

Transfer 2,5 ml of 2 mol/l potassium hydroxide solution to 10 ml volumetric flask, dilute to the mark

with water and mix, c = 0,5 mol/l.

Transfer 0,1 ml of 2 mol/l potassium hydroxide solution to 10 ml volumetric flask, dilute to the mark

with water and mix, c = 0,02 mol/l.
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ISO 4134:2021(E)
7.1.3 Solution R1, triethanolamine phosphate buffer solution, pH = 8,6.

Dissolve 1,86 g of triethanolamine hydrochloride in approximately 25 ml of water, adjust the pH to 8,6

with 2 mol/l potassium hydroxide solution (7.1.2), detected by a pH-meter. Add 0,68 g of octylphenol

decaethyleneglycol ether (e.g. Triton X-100). Dilute to 100 ml with water and mix (solution A).

Dissolve 0,86 g of dipotassium hydrogen phosphate (K HPO ) and 7 mg of potassium dihydrogen

2 4

phosphate (KH PO ) in water. Dilute to 100 ml with water and mix evenly (solution B).

2 4
Mix 20 ml of solution A with 5 ml of solution B.

The solution is stable for two months when stored at a temperature of between 0 °C and 6 °C.

7.1.4 Solution R2, the mixed solution of NAD and diaphorase (lipoamide dehydrogenase EC

1.8.1.4), ρ = 11 mg/ml, diaphorase, approximately 4 IU/ml.
NAD

Weigh 110 mg of NAD, and approximately 8 mg (approximately 40 IU) of diaphorase in a stoppered

flask. Add 10,0 ml water and mix evenly.

The solution is stable for one week when stored in the dark at a temperature of between 0 °C and 6 °C.

7.1.5 Solution R3, iodonitrotetrazolium chloride (INT) solution, 2-(4-iodophenyl)-3-(4-

nitrophenyl)-5-phenyltetrazolium chloride, ρ = 0,6 mg/ml.

Weigh 6 mg of INT in a small, stoppered brown flask. Add 10 ml of water and mix evenly.

The solution is stable for four weeks when stored in the dark at a temperature of between 0 °C and 6 °C.

7.1.6 Solution R123, the mixed solution of solution R1, solution R2 and solution R3.

Pipette 6 ml of the solution R1, 2 ml of the solution R2 and 2 ml of the solution R3 in a stoppered brown

glass bottle, and mix evenly before the test.

The mixed solution is stable for 1 h in stoppered brown glass bottles at room temperature.

7.1.7 Glutamate dehydrogenase (GLDH) solution (EC 1.4.1.3), approximately 900 IU/ml.

Weigh 10 mg (approximately 900 IU) of lyophilized glutamate dehydrogenase (GLDH) in a small

stoppered flask. Add 1 ml water and mix.

Free from ammonium sulfate, ethylene-dinitrilotetraacetic acid (EDTA) and glutaminase, this solution

is stable for 12 months when stored at a temperature of between 0 °C and 6 °C.
7.1.8 L-(+)-glutamic acid standard stock solution, ρ = 1 000 mg/l.

Weigh, to the nearest 0,000 1 g, approximately 50,0 mg of L-(+)-glutamic acid (C H O N). Dissolve it in

5 9 4
approximately 25 ml of water.

Adjust the pH to 5 to 6 with a few drops of 2 mol/l potassium hydroxide solution (7.1.2). Then adjust the

pH to 7,0 slowly with 0,02 mol/l potassium hydroxide solution (7.1.2). Dilute to 50 ml with water and

mix evenly.

The solution is stable for six months when stored at a temperature of between 0 °C and 6 °C.

1) The EC number refers to the Enzyme Classification number as given in: The International Union of Biochemistry,

Enzymenomenclature, Elsevier, Amsterdam, 1965.
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ISO 4134:2021(E)
7.1.9 L-(+)-glutamic acid standard solution, ρ = 100 mg/l.

Pipette accurately 5,0 ml of L-(+)-glutamic acid standard stock solution (7.1.8) into a 50 ml volumetric

flask (7.2.8), dilute to the mark with water and mix evenly.
The solution is with the current use.

7.1.10 L-(+)-glutamic acid series standard solution, ρ = 5 mg/l, 10 mg/l, 15 mg/l, 20 mg/l, 30 mg/l

and 40 mg/l.

Pipette accurately 0,50 ml, 1,00 ml, 1,50 ml, 2,00 ml, 3,00 ml and 4,00 ml of L-(+)-glutamic acid standard

solution (7.1.9) into each of six 10 ml volumetric flasks (7.2.8) separately, dilute to the mark with water

and mix evenly.
The solution is with the current use.
7.2 Apparatus
The usual laboratory equipment and, in particular, the following shall be used.

7.2.1 Mechanical or electrical equipment, capable of homogenizing the laboratory sample.

This includes a high-speed rotational cutter, or a mincer fitted with a plate with apertures not exceeding

4,0 mm in diameter.
7.2.2 Laboratory mixer, stirrer or oscillator.

7.2.3 Laboratory centrifuge, with 50 ml or 100 ml centrifuge tubes, operating at a radial acceleration

of about 2 000 gn or equivalent speed (e.g. 3 500 r/min to 4 000 r/min).
7.2.4 Analytical balance, capable of weighing to the nearest 0,001 g, 0,000 1 g.
7.2.5 Constant temperature drying box.
7.2.6 pH-meter.
7.2.7 Filter papers, diameter of about 15 cm, high or moderate speed.

7.2.8 One-mark volumetric flasks, capacities of 10 ml, 50 ml and 100 ml, conforming to ISO 1042,

class B standard.

7.2.9 Single-volume pipettes, capacities of 50 ml, 25 ml and 1 ml conforming to ISO 648, class B

standard.

7.2.10 Single channel or multi-channel transferring pipettes and tips, of 5 ml, 1 000 μl, 200 μl and

100 μl, conforming to ISO 8655-2.

7.2.11 Small plastic spatula or lid, for mixing the content evenly by stirring with the spatula in the

cuvette or by shaking the cuvette covered with the lid.

7.2.12 Photoelectric colorimeter, provided with a filter having a transmittance maximum at a

wavelength of 490 nm, or spectrometer.
7.2.13 Cuvettes, of 10 mm optical path length.
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ISO 4134:2021(E)
7.3 Procedure
7.3.1 General

If it is required to check whether the repeatability requirement is met, two individual determinations

should be performed.
7.3.2 Test portion

Weigh, to the nearest 0,001 g, approximately 25 g, or other appropriate mass (m ) of the test sample

(see Clause 6).
7.3.3 Preparation of extract

7.3.3.1 Add 50 ml of dilute perchloric acid solution (7.1.1) to the test sample, homogenize the mixture

with the laboratory mixer (7.2.2).

7.3.3.2 Transfer the homogenized sample to centrifuge tube (7.2.3). Centrifuge for 10 min at 10 °C,

2 000 gn or equivalent speed (e.g. 3 500 r/min to 4 000 r/min). Carefully move aside the fat layer and

decant all the supernatant liquid through a filter paper (7.2.7) into a 100 ml conical flask. Discard the

first 10 ml of the filtrate.

7.3.3.3 Transfer 25 ml of the solution (which should be only slightly turbid) with pipette (7.2.9) into

centrifuge tube (7.2.3). With detected by the pH-meter (7.2.6), adjust the pH to 7 to 8 with 4 mol/l

potassium hydroxide solution (7.1.2), then adjust the pH to 10,0 slowly with 2 mol/l and 0,5 mol/l

potassium hydroxide solution (7.1.2). Centrifuge for 3 min at 2 000 gn or equivalent speed (e.g. 3 500 r/

min to 4 000 r/min).

NOTE If the pH is slightly above 10,0, it can be adjusted with dilute perchloric acid back to the required pH

value (7.1.1).

7.3.3.4 Transfer all the supernatant into a 50 ml volumetric flask (7.2.8). Dilute to the mark with water

and mix.

7.3.3.5 Cool the solution in ice for 10 min, and filter through a filter paper (7.2.7). Discard the first

10 ml of the filtrate.

7.3.3.6 Pipet 5 ml, or some other appropriate volume (V ) of the filtrate into a 50 ml volumetric flask

(7.2.8). Dilute to the mark with water and mix. The solution obtained will be used to determine the

content of free L-(+)-glutamic acid in the test portion.

The volume V should be chosen so that the L-(+)-glutamic acid content of the solution is between 8 mg/l

and 40 mg/l.
7.3.4 Determination
7.3.4.1 Preparation of detection instrument

Set up the spectrophotometer (7.2.12) and preheat the instrument according to the instrument

specification until equilibrium conditions are achieved. Set the detection wavelength to 490 nm. Adjust

the baseline of the equipment to zero with pure water.
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ISO 4134:2021(E)
7.3.4.2 Absorbance determination of L-(+)-glutamic acid test solution

7.3.4.2.1 Keep the temperature of the solution R123 (7.1.6), water and the L-(+)-glutamic acid test

solution (7.3.3.6) at between 20 °C and 25 °C.

Pipette 1,0 ml of the solution R123 (7.1.6) into a cuvette (7.2.13) and add 2,0 ml of water. The solution

obtained is the blank solution.

Pipette 1,0 ml of the solution R123 (7.1.6), 1,8 ml of water and 200 μl of the L-(+)-glutamic acid test

solution (7.3.3.6) into another cuvette. The solution obtained is the test solution.

Mix the solutions with spatula or lid (7.2.11), put into spectrophotometer and read the absorbance A of

the test solution and A of the blank solution at a wavelength of 490 nm against water.

7.3.4.2.2 Pipette 50 μl of the GLDH solution (7.1.7) into each of the cuvettes. Mix the contents of the

cuvettes evenly (7.2.11).

Read the absorbance A ′ of the test solution and A ′ of the blank solution at a wavelength of 490 nm

1 b1

against water after kept still for from 10 min to 15 min, and record every 2 min until a constant

absorbance is obtained. Take the constant absorbance value as the test result.

The exposure of the reaction solution in light should be avoided as much as possible. The temperature

of the solution should be maintained at between 20 °C and 25 °C.
7.3.4.3 Absorbance determination of L-(+)-glutamic acid standard solution

Repeat the operations described in 7.3.3.3.1, but replace the L-(+)-glutamic acid test solution (7.3.3.6)

with the L-(+)-glutamic acid series standard solution (7.1.10). Read the absorbance A of the L-(+)-

glutamic acid standard solution and A of the blank solution. Then, repeat the operations described

in 7.3.3.3.2. Read the absorbance A ′ of the L-(+)-glutamic acid standard solution and A ′ of the blank

s1 b2
solution.

NOTE If 7.3.3.3 and 7.3.4.3 are detected at the same batch, the blank solution determination of 7.3.4.3 can be

omitted. Take the value of A for A and take the value of A ′ for A ′ directly.
b1 b2 b1 b2
7.3.4.4 Moisture content determination of test sample
Determine the moisture content of the test sample in accordance with ISO 1442.
7.4 Calculation and results
7.4.1 Absorbance difference of L-(+)-glutamic acid standard solution

Calculate the absorbance difference for the L-(+)-glutamic acid standard solution using Formula (3):

′ ′
ΔAA= − AA− − A (3)
ss11()1s ()bb22
where
ΔA is the absorbance difference for the L-(+)-glutamic acid standard solution;
A is the absorbance of the standard solution, measured in 7.3.4.3;
A ’ is the absorbance of the standard solution, measured in 7.3.4.3;
A is the absorbance of the blank solution, measured in 7.3.4.3;
A ’ is the absorbance of the blank solution, measured in 7.3.4.3.
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ISO 4134:2021(E)
7.4.2 Absorbance difference of L-(+)-glutamic acid test solution

Calculate the absorbance difference for the L-(+)-glutamic acid test solution using Formula (4):

′ ′
ΔAA= − AA− − A (4)
() ()
11 11bb1
...