ISO/DIS 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

Standards Content (sample)

DRAFT INTERNATIONAL STANDARD
ISO/DIS 4134
ISO/TC 34/SC 6 Secretariat: SAC
Voting begins on: Voting terminates on:
2020-12-01 2021-02-23
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
ICS: 67.120.10
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
This document is circulated as received from the committee secretariat.
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Reference number
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ISO/DIS 4134:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
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PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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ISO/DIS 4134:2020(E)
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ii © ISO 2020 – All rights reserved
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ISO/DIS 4134:2020(E)
Contents Page

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

Introduction ................................................................................................................................................................................................................................vi

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

7.3.2 Preparation of extract ........................................................................................................................................... ....... 5

7.3.3 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 Equation of linear regression of L-(+)-glutamic acid standard curve ................................ 7

7.4.4 L-(+)-glutamic acid concentration of the test solution ................................................................... 8

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

7.5 Precision ....................................................................................................................................................................................................... 9

7.5.1 Interlaboratory test ....................................................................................................................................................... 9

7.5.2 Repeatability ....................................................................................................................................................................... 9

7.5.3 Reproducibility .................................................................................................................................................................. 9

7.6 Detection limit ......................................................................................................................................................................................... 9

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

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

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

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

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

8.3.2 Determination .................................................................................................................................................................10

8.4 Calculation and results ..................................................................................................................................................................11

8.4.1 Absorbance difference for L-(+)-glutamic acid standard solution ....................................11

8.4.2 Absorbance difference for L-(+)-glutamic acid test solution..................................................11

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

8.4.4 L-(+)-glutamic acid concentration of the test solution ................................................................12

8.4.5 L-(+)-glutamic acid content of test sample ............................................................................................12

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

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

8.5.2 Repeatability ....................................................................................................................................................................13

8.5.3 Reproducibility ...............................................................................................................................................................13

8.6 Detection limit ......................................................................................................................................................................................13

9 Test report ................................................................................................................................................................................................................13

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

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

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ISO/DIS 4134:2020(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 to the previous edition are as follows:
— A new test method - light absorption microplate reader method is added.

— The determine scope of the document has been further clearly defined as free L-(+)-glutamic acid in

meat and meat products.
— The clauses order of the document has been rearranged.

— The introductory texts for “Foreword”, “Normative references” and “Terms and definitions” have

been modified in accordance with ISO/IEC Directives, Part 2.
— The title of the document has been modified.
— The Normative references has been updated.

— The terms and definitions has been modified with adding the terms of “Free L-(+)-glutamic acid”.

— 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”.(Clause 4)

— 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; the buffer, NAD and enzymes have been labelled with R1, R2, and

R3.(Clause 7.1)
— The apparatus list has been updated.
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ISO/DIS 4134:2020(E)

— The “Procedure” of “Test method of spectrophotometer” has been modified with halving the sample

weight and solution volume.

— The method of judging the absorbance of the reaction end point has been modified. Accordingly, the

previous Annex B “Example of plotting and extrapolation of absorbance values” has been deleted.

(Clause 7.3.3)

— In the “Calculation and results”, the equation 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 documents list of “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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ISO/DIS 4134:2020(E)
Introduction
This document does not involve the use of patents.
vi © ISO 2020 – All rights reserved
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DRAFT INTERNATIONAL STANDARD ISO/DIS 4134:2020(E)
Meat and meat products — Determination of L-(+)-
glutamic acid content — Reference method
1 Scope

This document specifies spectrophotometer method and 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

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

3.2
L-(+)-glutamic acid content of meat and meat products

The mass fraction of the free L-(+)-glutamic acid in meat and meat products determined according to

the procedure described in this International Standard
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 [Formula (1)]. The resultant reduced nicotinamide

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

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ISO/DIS 4134:2020(E)

[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.
glutamatedehydrogenase
L-(+)-glutamiacid/glutamate + NAD +H O←→
α-ketoglutamate + NADH +NH + H (1)
diaphorase
+ +
NADH + iodonitrotetrazolium chloride +H ←→ NAD + formazane (2)
5 Sampling

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

method is given in CAC/GL 50-2004.

It is important that the laboratory-received sample 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 such a temperature of 4°C or

lower that deterioration and change in composition are prevented.
6 Preparation of test sample

Homogenize the laboratory sample with the appropriate equipment (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 such a

temperature of 4°C or lower 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 to 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(HClO4) = 1.0 mol/L

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

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

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

Add 8.6 mL of the perchloric acid (70 % (by mass), ρ = 1.67 g/mL) to the bulk of water, diluting to 100 mL.

7.1.2 Potassium hydroxide solution, c(KOH) = 4 mol/L, 2 mol/L, 0.5 mol/L, 0.02 mol/L

Dissolve 22.4 g of potassium hydroxide in water. Dilute the solution to 100 mL, c(KOH) = 4 mol/L, and

mix evenly after cooling.

Dissolve 11.2 g of potassium hydroxide in water. Dilute the solution to 100 mL, c(KOH) = 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(KOH) = 0.5 mol/L.
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ISO/DIS 4134:2020(E)

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(KOH) = 0.02 mol/L.
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 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 2 months when stored at a temperature of between 0 °C and 6 °C.

7.1.4 Solution R2, the mixed solution of nicotinamide adenine dinucleotide (NAD) and

diaphorase (lipoamide dehydrogenase EC 1.8.1.4): c(NAD) = 11 mg/mL, c(diaphorase)

approximately 4 U/mL.

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

Add 10.0 mL water and mix evenly.

The solution is stable for 1 week when stored in 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, c(INT) = 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 4 weeks when stored in 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 appropriate volume of the solution R1, the solution R2 and the solution R3, and mix evenly at a

volume ratio of 3:1:1 before 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): c(GLDH) approximately 900 U/mL.

Weigh 10 mg (approximately 900 U) 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, c(C H O N) = 1000 mg/L
5 9 4

Weigh, to the nearest 0.0001 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 5-6 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 6 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/DIS 4134:2020(E)
7.1.9 L-(+)-glutamic acid standard solution, c(C H O N) = 100 mg/L.
5 9 4

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, c(C H O N) = 5 mg/L, 10 mg/L, 15 mg/L,20 mg/L,

5 9 4
30 mg/L, 40 mg/L.

Pipette accurately 0.50 mL, 1.00 mL, 1.50 mL, 2.00mL, 3.00 mL, 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.

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. 3 500-4 000 rpm).
7.2.4 Analytical balance, capable of weighing to the nearest 0.001g.
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, complying with ISO 1042,

class B standard.

7.2.9 Single-volume pipettes, capacities of 50 mL, 25 mL and 1mL complying with 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 complying with ISO 8655-2.

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

cuvette or shaking the cuvette covered with 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/DIS 4134:2020(E)
7.3 Procedure

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

should be performed.
7.3.1 Test portion

Weigh, to the nearest 0.001 g, approximately 25 g, or other appropriate weight (m ) of the test sample

(see Clause 6).
7.3.2 Preparation of extract

7.3.2.1 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).

7.3.2.2 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. 3 500~4 000 rpm). 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.

7.3.2.3 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 7-8 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.

3 500~4 000 rpm).

NOTE If the pH is slightly above 10.0, it could be adjusted with dilute perchloric acid back to the required pH

value (7.1.1).

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

and mix.

7.3.2.5 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.

7.3.2.6 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.

NOTE 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.
7.3.3 Determination
7.3.3.1 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/DIS 4134:2020(E)
7.3.3.2 Absorbance determination of L-(+)-glutamic acid test solution

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

solution (7.3.2.6) at between 20 °C and 25 °C, respectively.

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

Mix the solutions with spatula or lid, respectively (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,

1 b1
respectively.

7.3.3.2.2 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 between 20 °C and 25 °C.
7.3.3.3 Absorbance determination of L-(+)-glutamic acid standard solution

Repeat the operations described in 7.3.3.2.1, but replace the L-(+)-glutamic acid test solution (7.3.2.6)

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. And then, repeat the operations described

in 7.3.3.2.2. Read the absorbance A ’ of the L-(+)-glutamic acid standard solution and A ’ of the blank

s1 b2
solution.

Note: If 7.3.3.2 and 7.3.3.3 are detected at the same batch, the blank solution determin

...