GB 1886.40-2025National Food Safety Standard - Food Additive - L-Malic Acid (English PDF)

Foreword Foreword

This standard replaces GB 1886.40-2015, National Food Safety Standard - Food Additive - L-Malic Acid.

Compared with GB 1886.40-2015, the main changes in this standard are as follows: the scope has been changed; the physical and chemical indexes for moisture and for succinic acid have been added; the wording of the indexes for content and for total arsenic, and the limit for total arsenic, have been changed, and heavy metals has been deleted; the test methods for identification, content, clarity, fumaric acid, maleic acid, lead and total arsenic have been changed; a test method for succinic acid has been added; Annex A and Annex B have been changed and Annex C has been added.

1 Scope

This standard applies to the food additive L-malic acid obtained from fumaric acid or a fumarate as raw material by an enzyme engineering process, and to the food additive L-malic acid obtained from starch-based material or sugars as raw material by fermentation.

2 Chemical name, molecular formula, structural formula and relative molecular mass

2.1 Chemical name. L-hydroxysuccinic acid.

2.2 Molecular formula. C4H6O5.

2.3 Structural formula. Printed in the standard as a drawing; the digitised text does not carry it.

2.4 Relative molecular mass. 134.09, based on the 2022 international relative atomic masses.

3 Technical requirements

3.1 Sensory requirements. The sensory requirements shall comply with Table 1. Table 1 fixes the colour as white, the state as crystals or crystalline powder, and the odour as a characteristic sour odour. The test method given against all three is the same: take a suitable amount of the sample, place it in a clean, dry white porcelain dish, observe its colour and state under natural light, and smell it.

3.2 Physical and chemical indexes. The physical and chemical indexes shall comply with Table 2. Table 2 fixes the following limits, each with the test method shown against it. Content, expressed as C4H6O5, mass fraction not less than 99.0 percent, by A.4 of Annex A. Specific rotation at 20 degrees Celsius with the sodium D line, from minus 1.6 to minus 2.6 degrees times square decimetre per kilogram, by A.5 of Annex A. Clarity: shall pass the test, by A.6 of Annex A. Moisture, mass fraction not more than 1.0 percent, by the Karl Fischer method of GB 5009.3. Residue on ignition, mass fraction not more than 0.10 percent, by A.7 of Annex A. Chloride, expressed as chloride ion, mass fraction not more than 0.004 percent, by A.8 of Annex A. Sulfate, expressed as sulfate ion, mass fraction not more than 0.02 percent, by A.9 of Annex A. Fumaric acid, mass fraction not more than 0.5 percent, by A.10 of Annex A. Maleic acid, mass fraction not more than 0.05 percent, by A.10 of Annex A. Succinic acid, mass fraction not more than 2.0 percent, by A.10 of Annex A. Lead, not more than 2.0 mg/kg, by GB 5009.12 or GB 5009.75. Total arsenic, expressed as As, not more than 1.0 mg/kg, by GB 5009.11 or GB 5009.76.

A.1 Warning

Some of the procedures laid down in the test methods may give rise to hazardous situations. The operator shall take appropriate safety and health measures.

A.2 General provisions

Unless other requirements are stated, the reagents used in this standard are analytically pure reagents.

Unless other requirements are stated, the standard volumetric solutions, the standard solutions for the determination of impurities, and the preparations and products used in the tests are prepared as laid down in GB/T 601, GB/T 602 and GB/T 603.

Where the solvent used to prepare a solution is not stated, grade three water as laid down in GB/T 6682 is meant.

A.3 Identification test

A.3.1 Reagents and materials. Ammonia solution: ammonia plus water, 2 plus 3. Sulfanilic acid solution: 10 g/L. Sodium nitrite solution: 200 g/L. Sodium hydroxide solution: 40 g/L.

A.3.2.1 Identification by colour formation with the ammonium salt. Weigh 0.5 g of the sample to the nearest 0.01 g into a 50 mL test tube and add 10 mL of water to dissolve it. Neutralise with the ammonia solution, add 1 mL of the sulfanilic acid solution and heat in a boiling water bath for 5 min. Add 5 mL of the sodium nitrite solution, heat in a boiling water bath for 3 min, then add 5 mL of the sodium hydroxide solution; observed from the top downwards, a red colour shall appear at once.

A.3.2.2 Identification by infrared absorption spectrum. Following GB/T 6040, carry out a qualitative analysis by the potassium bromide pellet method and record the infrared absorption spectrum. The infrared absorption spectrum of the sample shall agree in all substantial respects with Annex B.

A.4 Determination of content, expressed as C4H6O5

A.4.1 Outline of the method. With phenolphthalein as indicator, the sample solution is titrated with sodium hydroxide standard volumetric solution, and the total acid concentration of the sample solution, expressed as C4H6O5, is calculated from the volume of sodium hydroxide standard volumetric solution consumed.

A.4.2 Reagents and materials. Sodium hydroxide standard volumetric solution, concentration of NaOH 0.5 mol/L. Phenolphthalein indicator solution, 10 g/L.

A.4.3 Apparatus and equipment. Analytical balance, readability 0.0001 g.

A.4.4 Procedure. Weigh about 1.0 g of the sample to the nearest 0.0001 g, add 20 mL of carbon dioxide free water to dissolve it, add two drops of phenolphthalein indicator solution, and titrate with the sodium hydroxide standard volumetric solution to a faint pink that does not fade within 30 s, which is the end point. Carry out a blank test by the same procedure as for the sample.

A.4.5 Calculation of the result. The content, expressed as C4H6O5, is given as the mass fraction w1 in percent and is calculated by formula (A.1). In the formula: V1 is the volume of sodium hydroxide standard volumetric solution consumed by the sample solution, in millilitres; V0 is the volume of sodium hydroxide standard volumetric solution consumed by the blank solution, in millilitres; c is the concentration of the sodium hydroxide standard volumetric solution, in moles per litre; M is the molar mass of one half of L-malic acid, in grams per mole, taken as 67.04; m is the mass of the sample, in grams; and 1000 is a conversion factor. The test result is given as the arithmetic mean of parallel determinations, and the calculated result is kept to two decimal places.

A.4.6 Precision. The absolute difference between two independent results obtained under repeatability conditions shall not be greater than 0.2 percent.

A.5 Determination of specific rotation

A.5.1 Apparatus and equipment. Polarimeter, fitted with a sodium lamp giving the sodium D line at 589.3 nm, accuracy plus or minus 0.01 degrees. Analytical balance, readability 0.01 g.

A.5.2 Procedure. Weigh 4.25 g of the sample to the nearest 0.01 g, add 20 mL of water to dissolve it and make up to 50 mL; carry out the remaining steps as laid down in GB/T 613.

A.5.3 Calculation of the result. The specific rotation is given as alpha at 20 degrees Celsius with the sodium D line, expressed in degrees times square decimetre per kilogram, and is calculated by formula (A.2). In the formula: alpha is the optical rotation of the sample solution measured at 20 degrees Celsius, in degrees; l is the length of the polarimeter tube, in decimetres; and rho is the mass concentration of the active component of the sample in the solution, in grams per millilitre.

A.6 Determination of clarity

A.6.1 Reagents and materials. Nitric acid solution: nitric acid plus water, 1 plus 2. Silver nitrate solution: 20 g/L. Dextrin solution: 20 g/L. Hydrochloric acid standard solution, concentration of HCl 0.1 mol/L. Standard working solution containing 0.01 mg/mL of chlorine: accurately transfer 14.1 mL of the hydrochloric acid standard solution, dilute with water and make up to 50 mL; accurately measure 10.0 mL of that solution, dilute with water and make up to 1000 mL.

A.6.2 Apparatus and equipment. Nessler colour comparison tubes. Analytical balance, readability 0.01 g.

A.6.3.1 Preparation of the sample solution. Weigh about 1.0 g of the sample to the nearest 0.01 g into a Nessler tube, add 20 mL of water to dissolve it and shake well.

A.6.3.2 Preparation of the standard reference solution. Accurately measure 0.20 mL of the standard working solution, add water to 20 mL, add 1 mL of the nitric acid solution, 1 mL of the silver nitrate solution and 0.2 mL of the dextrin solution, shake well and leave in the dark for 15 min.

A.6.4 Judgement of the result. Observe axially and laterally, out of direct sunlight. If the turbidity of the sample solution is not greater than the turbidity of the standard reference solution, the test is passed.

A.7 Residue on ignition

Weigh 2.5 g of the sample to the nearest 0.0001 g and carry out the remaining steps as laid down in GB/T 9741. The test result is taken as the arithmetic mean of parallel determinations, and the absolute difference between two independent results obtained under repeatability conditions shall not be greater than 0.02 percent.

A.8 Determination of chloride

Weigh 1.0 g of the sample to the nearest 0.01 g, accurately transfer 0.4 mL of the chloride standard solution, and carry out the remaining steps as laid down in GB/T 9729. If the turbidity of the sample solution is not greater than the turbidity of the standard solution, the chloride content is not more than 0.004 percent.

A.9 Determination of sulfate

A.9.1 Reagents and materials. Hydrochloric acid solution: hydrochloric acid plus water, 1 plus 4. Barium chloride solution: 250 g/L. Sulfate standard solution: 0.1 mg/mL.

A.9.2 Apparatus and equipment. Nessler colour comparison tubes, 50 mL. Analytical balance, readability 0.01 g.

A.9.3.1 Preparation of the sample solution. Weigh 1.0 g of the sample to the nearest 0.01 g into a Nessler tube, dissolve it in water to about 40 mL and shake well.

A.9.3.2 Preparation of the reference solution. Accurately transfer 2.0 mL of the sulfate standard solution into a Nessler tube, dissolve in water to about 40 mL and shake well.

A.9.3.3 Determination. To the sample solution and to the reference solution add 0.5 mL of the hydrochloric acid solution and 1 mL of the barium chloride solution, dilute to 50 mL with water, shake thoroughly and leave for 10 min.

A.9.4 Judgement of the result. Place the sample solution and the reference solution on the same black background and observe the turbidity produced, looking down from the top of the tubes. If the turbidity of the sample solution is not greater than the turbidity of the reference solution, the sulfate content is not more than 0.02 percent.

A.10 Determination of fumaric acid, maleic acid and succinic acid

A.10.1 Principle. After treatment, the sample is separated on a hydrogen form cation exchange chromatographic column, detected with an ultraviolet detector or a diode array detector, and quantified by the external standard method.

A.10.2 Reagents and materials. Water: grade one water of GB/T 6682. Sulfuric acid solution: concentration of one half H2SO4, 0.01 mol/L. Fumaric acid reference substance (C4H4O4, CAS number 110-17-8): purity not less than 99.0 percent, or a reference material certified by the state and issued with a reference material certificate. Maleic acid reference substance (C4H4O4, CAS number 110-16-7): purity not less than 99.0 percent, or a certified reference material. Succinic acid reference substance (C4H6O4, CAS number 110-15-6): purity not less than 99.0 percent, or a certified reference material. Fumaric acid standard solution, 500 micrograms per millilitre: accurately weigh 50 mg of the fumaric acid reference substance to the nearest 0.0001 g, dissolve in the sulfuric acid solution and make up to 100 mL; store in a refrigerator at 4 degrees Celsius, valid for 6 months. Maleic acid standard solution, 50 micrograms per millilitre: accurately weigh 5 mg of the maleic acid reference substance to the nearest 0.0001 g, dissolve in the sulfuric acid solution and make up to 100 mL; store in a refrigerator at 4 degrees Celsius, valid for 6 months. Succinic acid standard solution, 5 mg/mL: accurately weigh 500 mg of the succinic acid reference substance to the nearest 0.0001 g, dissolve in the sulfuric acid solution and make up to 100 mL; store in a refrigerator at 4 degrees Celsius, valid for 6 months. Microporous membrane filter: 0.22 micrometres, aqueous phase.

A.10.3 Apparatus and equipment. High performance liquid chromatograph fitted with an ultraviolet detector or a diode array detector. Analytical balances, readability 0.01 g and 0.0001 g.

A.10.4 Reference conditions for the instrument. Chromatographic column: hydrogen form cation exchange column packed with polystyrene-divinylbenzene resin, 300 mm by 7.8 mm, or another column of equivalent analytical performance. Column temperature: 45 degrees Celsius. Mobile phase: the sulfuric acid solution. Flow rate: 0.5 mL/min. Injection volume: 10 microlitres. Detection wavelength: 214 nm.

A.10.5.1 Preparation of the mixed standard solutions. Take five 100 mL volumetric flasks and accurately pipette into them 1.00 mL, 2.00 mL, 4.00 mL, 5.00 mL and 6.00 mL of the fumaric acid standard solution and of the maleic acid standard solution, and 0.40 mL, 1.00 mL, 2.00 mL, 3.00 mL and 4.00 mL of the succinic acid standard solution, then make up to the mark with the sulfuric acid solution. The mixed standard solutions so prepared have fumaric acid mass concentrations of 5.00, 10.0, 20.0, 25.0 and 30.0 micrograms per millilitre; maleic acid mass concentrations of 0.50, 1.00, 2.00, 2.50 and 3.00 micrograms per millilitre; and succinic acid mass concentrations of 20.0, 50.0, 100, 150 and 200 micrograms per millilitre. Store in a refrigerator at 4 degrees Celsius; valid for one month.

A.10.5.2 Preparation of the sample solution. Weigh 0.5 g of the sample to the nearest 0.0001 g, dissolve it in the sulfuric acid solution and make up to 100 mL (the volume to which the sample solution is made up may be adjusted as appropriate so that the mass concentrations of fumaric acid, maleic acid and succinic acid in the sample solution fall within the range of the calibration curve drawn), shake well and filter through the microporous membrane.

A.10.6.1 Drawing of the calibration curve. Under the conditions of A.10.4, inject each of the five mixed standard solutions of different concentration prepared in A.10.5.1 into the liquid chromatograph, measure the corresponding peak areas, and draw the calibration curve with the mass concentration of the mixed standard solution as the horizontal axis and the peak area as the vertical axis.

A.10.6.2 Determination of the sample solution. Under the conditions of A.10.4, inject the sample solution into the liquid chromatograph, identify the peaks by retention time, measure the peak areas of fumaric acid, maleic acid and succinic acid, and obtain from the calibration curve their mass concentrations in the sample solution.

A.10.7 Calculation of the result. The contents of fumaric acid, maleic acid and succinic acid are given as the mass fraction wi in percent and are calculated by formula (A.3). In the formula: Ci is the mass concentration of the substance to be determined in the sample solution, obtained from the calibration curve, in micrograms per millilitre; V is the volume to which the sample solution was made up, in millilitres; m is the mass of the sample, in grams; and ten to the sixth power is a conversion factor. The test result is given as the arithmetic mean of parallel determinations, kept to two significant figures.

A.10.8 Precision. The absolute difference between two independent results obtained under repeatability conditions shall not be greater than 10 percent of their arithmetic mean.

A.10.9 Detection limit and quantification limit. The detection limit for fumaric acid is 0.0040 percent and the quantification limit is 0.010 percent; the detection limit for maleic acid is 0.0020 percent and the quantification limit is 0.0050 percent; the detection limit for succinic acid is 0.080 percent and the quantification limit is 0.20 percent.

Annex B Infrared absorption spectrum of the L-malic acid reference substance

The infrared absorption spectrum of the L-malic acid reference substance is shown in Figure B.1. The figure itself is a plate and is not carried by the digitised text.

Annex C Liquid chromatogram of the mixed standard solution of fumaric acid, maleic acid and succinic acid

The liquid chromatogram of the mixed standard solution of fumaric acid, maleic acid and succinic acid is shown in Figure C.1, which is the chromatogram of a mixed standard solution containing fumaric acid at 10 micrograms per millilitre, maleic acid at 1 microgram per millilitre and succinic acid at 50 micrograms per millilitre. The figure itself is a plate and is not carried by the digitised text.