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National food safety standard - Determination of acid value in foods
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National food safety standard - Determination of Acid Value in Foods
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GB 5009.229-2025: PDF in English GB 5009.229-2025
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard - Determination of acid value
in food
ISSUED ON. MARCH 16, 2025
IMPLEMENTED ON. SEPTEMBER 16, 2025
Issued by. National Health Commission of the People’s Republic of China;
State Administration for Market Regulation.
Table of Contents
Foreword... 3
1 Scope... 4
First method -- Cold solvent indicator titration method... 4
2 Principle... 4
3 Reagents and materials... 4
4 Instruments and equipment... 6
5 Analysis steps... 6
6 Expression of analysis results... 8
7 Precision... 9
Second method -- Cold solvent automatic potentiometric titration... 9
8 Principle... 9
9 Reagents and materials... 9
10 Instruments and equipment... 10
11 Analysis steps... 10
12 Expression of analysis results... 12
13 Precision... 12
Third method -- Hot ethanol indicator titration method... 12
14 Principle... 12
15 Reagents and materials... 12
16 Instruments and equipment... 13
17 Analysis steps... 13
18 Expression of analysis results... 14
19 Precision... 14
Forth method -- Spectrophotometry... 14
20 Principle... 14
21 Reagents and materials... 14
22 Instruments and equipment... 15
23 Analysis steps... 16
24 Expression of analysis results... 19
25 Precision... 19
26 Others... 19
Annex A Impurity removal and drying and dehydration of oil and fat samples... 21
Annex B Schematic diagram of titration end point determination by automatic
potentiometric titration method... 23
National food safety standard - Determination of acid value
in food
1 Scope
This Standard specifies the determination methods for acid value in food.
The first, second and third methods are applicable to the determination of acid value in
food [except non-dairy cream, powdered oils and fats, margarine, compound seasonings
(mayonnaise, salad dressing, oil-based chili sauce, nut and seed sauce, hot pot base and
other semi-solid seasonings)].
The fourth method is applicable to the determination of acid value in non-dairy cream,
powdered oils and fats, margarine, compound seasonings (mayonnaise, salad dressing,
oil-based chili sauce, nut and seed sauce, hot pot base and other semi-solid seasonings).
First method -- Cold solvent indicator titration method
2 Principle
Based on the principle of acid-base neutralization reaction, potassium hydroxide or
sodium hydroxide standard titration solution is used to neutralize the free fatty acids in
the sample solution, and the titration end point is determined by the acid-base indicator.
The acid value of the sample is calculated based on the consumption of the standard
titration solution.
3 Reagents and materials
Unless otherwise specified, the reagents used in this method are analytically pure, and
the water is grade 3 water specified in GB/T 6682.
3.1 Reagents
3.1.1 Isopropyl alcohol (C3H8O).
3.1.2 Anhydrous ether (C4H10O).
3.1.3 95 % ethanol (C2H5OH).
3.1.4 Phenolphthalein (C20H14O4, CAS No.. 77-09-8).
4 Instruments and equipment
4.1 Burette. capacity of 10 mL, minimum graduation value of 0.05 mL.
4.2 Burette. capacity of 25 mL or 50 mL, minimum graduation value of 0.1 mL.
4.3 Balance. the sensitivity is 0.01 g and 0.001 g respectively.
4.4 Food grinder or pounder.
4.5 Laboratory oil press. screw press, non-heating type.
4.6 Porcelain mortar.
4.7 Constant temperature water bath.
4.8 Constant temperature blast drying oven.
4.9 Centrifuge. speed ≥ 8000 r/min.
4.10 Rotary evaporator or equivalent equipment.
4.11 Soxhlet extraction device.
5 Analysis steps
5.1 Preparation of samples
5.1.1 Animal and vegetable oils and fats, edible hydrogenated oils, shortening,
cocoa butter substitutes
At room temperature, liquid samples are fully mixed and then directly sampled for
testing. For solid (or semi-solid) samples, take a representative sample and place it in a
warm water bath or a constant temperature blast drying oven to heat and melt, mix well
and then take the oil and fat for testing. If the oil and fat sample is obviously turbid,
emulsified, stratified or precipitated, it shall be treated with impurity removal and
dehydration according to Annex A.
5.1.2 Vegetable oils, nuts and seeds
Direct pressing method is used for samples susceptible to lipase, such as raw and dried
sesame. Take a representative sample, use laboratory oil press to press at room
temperature, collect the squeezed material and filter it with filter paper immediately,
and then take the oil and fat for determination.
Soxhlet extraction method is used for other samples. For samples with shells, peel off
the shell first and keep the edible part. Among them, the kernels with green inner
membrane (such as pumpkin seeds, melon seeds, etc.) shall also remove the green inner
membrane attached to the surface of the kernels (the method of removing the green
inner membrane is to spray the surface of the shelled kernels with laboratory water to
moisten, rub off the green inner membrane, and then place the kernels with the green
inner membrane removed in a 50 ℃ constant temperature blast drying oven for 45 min
to dry). The sample is fully crushed with a food grinder, pounder or porcelain mortar.
If the sample is obviously heated during the crushing process, it shall add an appropriate
amount of liquid nitrogen and carry out the crushing in a frozen state. After the sample
is prepared, it shall immediately pack the crushed sample with a filter paper tube and
place it in a Soxhlet extraction device, add anhydrous ether or petroleum ether from the
upper end of the condenser tube of the extraction device to two-thirds of the bottle
volume, and heat and extract in a water bath for 4 h. Recover the extract in a flask, place
it in a rotary evaporator with a water bath temperature not higher than 40 ℃, evaporate
the organic solvent under reduced pressure, and use the residue as an oil and fat sample
for acid value determination.
If the residue is obviously turbid, emulsified, stratified or precipitated, it shall be treated
with impurity removal and dehydration according to Annex A.
5.1.3 Other foods containing fats and oils
Take the edible part of a representative sample (samples with more water content can
be drained with gauze first), and use a food grinder, pounder or porcelain mortar to fully
crush the sample (so that the free fat in the sample can be fully extracted by petroleum
ether). If the sample is obviously heated during the crushing process, it shall add an
appropriate amount of liquid nitrogen and carry out the crushing in a frozen state.
Place the crushed sample in a wide-mouth bottle (samples with more water content can
be dehydrated by adding an appropriate amount of anhydrous sodium sulfate), add 2 ~
3 times the volume of petroleum ether, stir and mix, seal and extract for more than 12
h. After stirring, let it stand for a while, filter through a funnel filled with anhydrous
sodium sulfate, take the filtrate; in a water bath not higher than 40 ℃, use a rotary
evaporator to evaporate the petroleum ether under reduced pressure, and use the residue
as an oil and fat sample for acid value determination.
If the residue is obviously turbid, emulsified, stratified or precipitated, it shall be treated
with impurity removal and dehydration according to Annex A.
5.2 Weighing of samples
According to the estimated acid value of the sample, weigh the oil and fat sample
according to the sample weight specified in Table 1 and place it in a 250 mL conical
flask.
According to the estimated acid value of the sample, weigh the oil and fat sample
according to the sample weight specified in Table 1 and place it in a 200 mL beaker.
11.3 Determination of samples
Add 50 mL ~ 100 mL of cold solvent to the weighed oil and fat sample, then add a
polytetrafluoroethylene magnetic stirrer, and place the sample on a magnetic stirrer to
stir and dissolve. Then, insert the electrode and burette connected to the automatic
potentiometric titrator into the sample solution. Note that the glass bulb of the electrode
and the anti-diffusion head of the burette shall be completely immersed below the liquid
surface of the sample solution and it shall avoid touching the inner wall of the beaker.
Start the automatic potentiometric titrator and titrate with potassium hydroxide or
sodium hydroxide standard titration solution.
The reference conditions of the automatic potentiometric titrator are as follows.
- Minimum liquid addition volume. 0.01 mL ~ 0.06 mL.
- Maximum liquid addition volume. 0.1 mL ~ 0.5 mL.
- Signal drift. 20 mV ~ 30 mV.
- Start the automatic monitoring function to automatically plot the corresponding
pH-titration volume change curve and the corresponding first-order differential
curve in real time, as shown in Annex B.
- End point determination method. The titration end point is the point indicated by
the peak of the first-order differential curve caused by the “pH jump” on the “S”-
shape pH-titration volume real-time change curve generated when the free fatty
acids undergo neutralization reaction (as shown in Figure B.1 in Annex B). After
the titration end point, the automatic potentiometric titrator will automatically stop
titration, the titration ends, and the volume of the consumed standard titration
solution will be automatically displayed. During the titration process, if there are
multiple “pH jumps” in the oil and fat sample (such as rice bran oil, etc.), the
titration end point is the “pH jump” with the pH at the starting point of the “jump”
that is most consistent with or close to the pH range of 7.5 ~ 9.5 (as shown in Figure
B.2); if a “direct jump” type pH-titration volume change curve is generated, the
titration end point is the peak of the corresponding first-order differential curve (as
shown in Figure B.3); if multiple first-order differential peaks are generated on a
“pH jump”, the titration end point is the highest peak (as shown in Figure B.4).
After the titration of each sample ends, the electrode, burette and stirrer shall be rinsed
with solvent first and then with water before the next sample can be measured. A blank
test shall be performed at the same time.
15.2.1 Phenolphthalein indicator (10 g/L). weigh 1 g of phenolphthalein, dissolve in
95 % ethanol and dilute to 100 mL.
15.2.2 Thymolphthalein indicator (20 g/L). weigh 2 g of thymolphthalein, dissolve in
95 % ethanol and dilute to 100 mL.
15.2.3 Alkali blue 6B indicator (20 g/L). weigh 2 g of alkali blue 6B, dissolve in 95 %
ethanol and dilute to 100 mL.
15.3 Preparation of standard solutions
Same as 3.3.
16 Instruments and equipment
Same as Clause 4.
17 Analysis steps
17.1 Preparation of samples
Same as 5.1.
17.2 Weighing of samples
Same as 5.2.
17.3 Determination of samples
Take another 250 mL conical flask, add 50 mL ~ 100 mL of 95 % ethanol, then add 0.5
mL ~ 1 mL of phenolphthalein indicator, shake well and place it in a water bath to heat
until it is slightly boiling. Take out the conical flask, and while the temperature of the
ethanol solution is still above 70 ℃, titrate it with potassium hydroxide or sodium
hydroxide standard titration solution until it turns slightly red and does not fade for 15
s. Pour the solution into the conical flask containing the sample while it is still hot, then
place it in a water bath to heat until it is slightly boiling, and gently shake the sample
until it is completely melted. Take out the conical flask, and while the temperature of
the solution is still above 70 ℃, titrate it with potassium hydroxide or sodium hydroxide
standard titration solution until it turns slightly red and does not fade for 15 s, which is
the titration end point. This method does not require a blank test, and V0 = 0 mL.
NOTE. When the color of the oil and fat sample affects the determination of end point,
thymolphthalein indicator or alkali blue 6B indicator can be used instead of phenolphthalein
indicator. When using thymolphthalein indicator, the solution color turns blue, which is the titration
end point; when using alkali blue 6B indicator, the solution color fades from blue to slightly red,
21.2 Preparation of reagents
21.2.1 Copper acetate solution (50 g/L). weigh 25 g of (accurate to 0.001 g) of copper
acetate monohydrate, add 450 mL of water and stir until completely dissolved, adjust
the solution pH to 6.10 ~ 6.20 with pyridine, and finally dilute to 500 mL with water.
21.2.2 Cyclohexane-isopropyl alcohol solution (98 + 2). mix cyclohexane and
isopropyl alcohol in a volume ratio of 98. 2.
21.2.3 Petroleum ether-methyl tert-butyl ether solution (1 + 3). mix petroleum ether and
methyl tert-butyl ether in a volume ratio of 1. 3.
21.3 Standards
Oleic acid standard (C18H34O2, CAS No.. 112-80-1). purity ≥ 98 %, certified by the
state and awarded with a standard material certificate.
21.4 Preparation of standard solutions
21.4.1 Oleic acid standard stock solution (10 mg/mL). accurately weigh 1 g (accurate
to 0.0001 g) of oleic acid standard, dissolve with an appropriate amount of cyclohexane,
transfer to a 100 mL brown volumetric flask, and dilute to the mark with cyclohexane.
Store at 2 ℃ ~ 8 ℃ away from light, the shelf life is 3 months.
21.4.2 Oleic acid standard series working solutions. take different volumes of oleic acid
standard stock solution, dilute with cyclohexane to oleic acid standard working
solutions with mass concentrations of 0.0 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL,
3.0 mg/mL and 5.0 mg/mL. Prepare it before use.
21.5 Materials
21.5.1 10 mL stoppered graduated test tube.
21.5.2 15 mL centrifuge tube.
21.5.3 50 mL centrifuge tube.
21.5.4 0.22 μm or 0.45 μm nylon filter membrane.
21.5.5 Solid phase extraction column (silica gel) or equivalent purification column. 6
mL, 500 mg.
22 Instruments and equipment
22.1 Balance. the sensitivity is 0.001g and 0.0001g respectively.
22.2 Spectrophotometer. equipped with 1 cm cuvette, the maximum detection
wavelength is not less than 800 nm.
temperature not higher than 40 ℃, evaporate the organic solvent under reduced pressure,
and use the residue as an oil and fat sample for acid value determination.
If the residue is obviously turbid, emulsified, stratified or precipitated, it shall be treated
with impurity removal and dehydration according to Annex A.
23.1.3 Compound seasonings (mayonnaise, salad dressing, oil-based chili sauce,
nut and seed sauce, hot pot base and other semi-solid seasonings)
Take the edible part of a representative sample (samples with high water content can be
drained with gauze first), and use a food grinder, pounder or porcelain mortar to fully
crush the sample (so that the free fat in the sample can be fully extracted by petroleum
ether). If the sample is obviously heated during the crushing process, add an appropriate
amount of liquid nitrogen and carry out the crushing in a frozen state.
Put the crushed sample in a wide-mouth bottle (samples with high water content can be
dehydrated by adding an appropriate amount of anhydrous sodium sulfate first), add 2
~ 3 times the volume of petroleum ether, stir and mix, seal and let it stand for more than
12 h. After stirring, let it stand for a while, filter through a funnel filled with anhydrous
sodium sulfate, take the filtrate; in a water bath not higher than 40 ℃, use a rotary
evaporator to evaporate the petroleum ether under reduced pressure, and use the residue
as an oil and fat sample for acid value determination.
If the residue is obviously turbid, emulsified, stratified or precipitated, it shall be treated
with impurity removal and dehydration according to Annex A.
23.2 Purification of oil and fat samples
In order to eliminate the interference of possible stearoyl lactylate emulsifiers, the
sample shall be purified according to the following steps.
Rinse and soak the solid phase extraction column with 10 mL of cyclohexane-isopropyl
alcohol solution (98 + 2) and connect it to the solid phase extraction device for use.
Weigh 1 g (accurate to 0.001 g) of oil and fat sample into a 15 mL centrifuge tube, add
4 mL of cyclohexane-isopropyl alcohol solution (98 + 2) to dissolve (if the oil and fat
sample solidifies, it shall be placed in a warm water bath to fully dissolve, and then
cooled to room temperature), transfer all the sample solution to the solid phase
extraction column, turn on the vacuum pump and adjust the flow rate, pass through the
column at a rate of 1 drop/s ~ 2 drops/s, collect the effluent, and then rinse the centrifuge
tube with 6 mL of cyclohexane-isopropyl alcohol solution (98 + 2) and pass through
the column, combine the effluent, transfer to a flask, use a rotary evaporator at 60 ℃ to
evaporate the organic solvent under reduced pressure, the residue is used as the purified
oil and fat sample, and is treated according to 23.3.
23.3 Preparation of oil and fat sample solutions
23.3.1 Preparation of liquid oil and fat sample solutions
Weigh 1 g (accurate to 0.001 g) of liquid oil and fat sample into a 10 mL stoppered
graduated test tube, dissolve it with cyclohexane and dilute to 5 mL, then transfer all of
it to a 50 mL centrifuge tube, add 3 mL ~ 5 mL of water, cover and seal, and fully vortex
and mix it on a vortex mixer for 40 s ~ 60 s, let stand to allow it to separate into layers
(or use a centrifuge to make it to separate into layers), take the upper layer solution and
determine it according to the steps in 23.4.2.
23.3.2 Preparation of solid (semi-solid) oil and fat sample solutions
Place the solid (or semi-solid) oil and fat sample in a warm water bath or a constant
temperature blast drying oven to heat and melt, mix well and weigh 1 g (accurate to
0.001 g) of oil and fat sample into a 10 mL stoppered graduated test tube, dissolve it
with cyclohexane and dilute to 10 mL, then transfer all of it to a 50 mL centrifuge tube,
add 3 mL ~ 5 mL of water, cover and seal, fully vortex and mix on a vortex mixer for
40 s ~ 60 s, let stand to allow it to separate into layers (or use a centrifuge to make it to
separate into layers), take the upper layer solution and determine it according to the
steps in 23.4.2.
23.4 Determination
23.4.1 Plotting of standard curve
Take six 50 mL centrifuge tubes, respectively add 5 mL of oleic acid standard working
solution with mass concentrations of 0.0 mg/mL, 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL,
3.0 mg/mL and 5.0 mg/mL, add 2 mL of cupric acetate solution to each tube, cover and
place them on a vortex mixer for 30 s, then let stand to allow the solutions to separate
into layers, take the upper layer clear liquid and filter through a filter membrane, use a
1 cm cuvette, take the oleic acid standard working solution with a mass concentration
of 0.0 mg/mL as a reference, measure the absorbance at a wavelength of 710 nm, and
plot a standard curve.
23.4.2 Determination of samples
Take 4 mL ~ 5 mL of oil and fat sample solution (23.3) in a 50 mL centrifuge tube, add
2 mL of copper acetate solution, cover and place it on a vortex mixer to vortex and mix
for 30 s, then let stand to allow the solution to separate into layers (if emulsification
occurs, use a centrifuge to make the solution to separate into layers), take the upper
layer clear liquid and filter through a filter membrane, use a 1 cm colorimetric dish,
take the oleic acid standard working solution with a mass concentration of 0.0 mg/mL
as a reference, measure the absorbance at a wavelength of 710 nm, and obtain the total
free fatty acid concentration (in terms of oleic acid) in the sample solution from the
standard curve, which is converted to the acid value of the sample.
If the color of the oil and fat sample solution is darker, take an equal amount of oil and
fat sample for a blank test (except that the copper acetate solution is not added, the rest
Annex A
Impurity removal and drying and dehydration of oil and fat samples
A.1 Impurity removal of oil and fat samples
If the oil and fat sample is obviously turbid, emulsified, stratified or precipitated, it shall
be treated with impurity removal. First, place the oil and fat sample in a 50 ℃ water
bath or a constant temperature drying oven, heat the oil and fat to 50 ℃ and shake it
thoroughly to melt possible oil and fat crystals. If the oil and fat sample becomes clear
and has no precipitation at this time, it can be used as a sample. Otherwise, the insoluble
impurities shall be filtered out with filter paper while it is hot, and the filtrate is be taken
as a sample. The filtration process shall be completed as soon as possible.
If the impurity content in the oil and fat sample is high and the particles are small and
difficult to filter clean, the oil and fat sample can be centrifuged at 8000 r/min for 10
min ~ 20 min to remove impurities.
For samples with a freezing point higher than 50 ℃ or containing oil and fat
components with a freezing point higher than 50 ℃, place the oil and fat in a water bath
or constant temperature drying oven about 10 ℃ higher than its freezing point, heat the
oil and fat and shake it thoroughly to melt oil and fat crystals. If filtration is required,
place the oil and fat in a constant temperature drying oven about 10 ℃ higher than its
freezing point, filter the insoluble impurities with filter paper, and take the filtered clear
liquid oil and fat as a sample. The filtration process shall be completed as soon as
possible.
A.2 Drying and dehydration of oil and fat samples
If the oil and fat sample contains water and still cannot become clear after treatment in
A.1, it shall be dried and dehydrated. For oil and fat samples without crystallization or
coagulation, and oil and fat samples without crystallization or coagulation after
treatment in A.1 and cooling to room temperature, add 1 g ~ 2 g of anhydrous sodium
sulfate per 10 g of oil and fat sample, stir and mix thoroughly, then filter with filter
paper, and take the clear liquid oil and fat after filtration as a sample.
If the water content in the oil and fat sample is high, centrifuge the oil and fat sample
at 8000 r/min for 10 min ~ 20 min, and after it separates into layers, take the upper layer
of oil and fat sample and dehydrate it by adsorption with anhydrous sodium sulfate.
For oil and fat samples that have crystallization or solidification at room temperature,
and oil and fat samples that have obvious crystallization or solidification after treatment
in A.1 and cooling to room temperature, it can completely dissolve the oil and fat
sample with an appropriate amount of petroleum ether in a water bath at 40 ℃ ~ 55 ℃,
and then add an appropriate amount of anhydrous sodium sulfate, stir and mix
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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