ISO/DIS 4134

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

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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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bodies (ISO member bodies). The work of preparing International Standards is normally carried out

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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).

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

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

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

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

— 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

— 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

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

— 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.

— In the “Calculation and results”, the equation and symbol description of spectrophotometer has

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

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

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.

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

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

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

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

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

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

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

It is important that the laboratory-received sample is truly representative and has not been damaged or

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

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

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

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.

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

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

Dissolve 11.2 g of potassium hydroxide in water. Dilute the solution to 100 mL, c(KOH) = 2 mol/L, and

Transfer 2.5 mL of 2 mol/L potassium hydroxide solution to 10 mL volumetric flask, dilute to the mark

Transfer 0.1 mL of 2 mol/L potassium hydroxide solution to 10 mL volumetric flask, dilute to the mark

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

phosphate (KH PO ) in water. Dilute to 100 mL with water and mix evenly (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)

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

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-

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

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

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

Weigh, to the nearest 0.0001 g, approximately 50.0 mg of L-(+)-glutamic acid (C H O N). Dissolve it in

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

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,

Pipette accurately 5.0 mL of L-(+)-glutamic acid standard stock solution (7.1.8) into a 50 mL volumetric

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,

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

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

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

7.2.8 One-mark volumetric flasks, capacities of 10 mL, 50 mL and 100 mL, complying with ISO 1042,

7.2.9 Single-volume pipettes, capacities of 50 mL, 25 mL and 1mL complying with ISO 648, class B

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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,

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

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

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

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

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

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