Powered by Google www.ChineseStandard.net Database: 189760 (15 Jun 2024)

GB 1886.174-2016 PDF in English


GB 1886.174-2016 (GB1886.174-2016) PDF English
Standard IDContents [version]USDSTEP2[PDF] delivered inName of Chinese StandardStatus
GB 1886.174-2016English230 Add to Cart 0-9 seconds. Auto-delivery. Food additive -- Glucoamylase preparation Valid
GB 1886.174-2024English320 Add to Cart 0-9 seconds. Auto-delivery. National Food Safety Standards--Food Additives--Enzyme Preparations for Food Industry Valid

PDF Preview

Standards related to: GB 1886.174-2016

GB 1886.174-2016: PDF in English

GB 1886.174-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Food Additives - Enzyme preparation for food industry
ISSUED ON: AUGUST 31, 2016
IMPLEMENTED ON: JANUARY 01, 2017
Issued by: National Health and Family Planning Commission of the PRC
Table of Contents
Foreword ... 3 
1 Scope ... 4 
2 Terms and definitions ... 4 
3 Product classification ... 4 
4 Technical requirements ... 4 
Appendix A Method for determining enzyme activity ... 7 
Appendix B Table for comparison of absorbance and α-amylase concentration
... 44 
National Food Safety Standard -
Food Additives - Enzyme preparation for food industry
1 Scope
This Standard applies to the enzyme preparation for food industry allowed by
GB 2760.
2 Terms and definitions
2.1 Enzyme preparation for food industry
Biological products with special catalytic functions for food processing, which
are obtained by directly extracting the edible or non-edible parts of animals or
plants, or by fermentation and extraction from traditional or genetically-modified
microorganisms (including but not limited to bacteria, actinomycetes, fungal
strains).
Note: Commercial enzyme preparation products allow the incorporation of ingredients
which are suitable for the product storage and use.
2.2 Enzyme activity
The ability of an enzyme to catalyze a particular reaction under certain
conditions, i.e., enzyme activity. It is a characteristic exclusive index for the
expression of enzyme preparation products.
2.3 Antibacterial activity
The ability to inhibit or kill microorganisms.
3 Product classification
According to the product form, it is classified into two types: solid dosage form
and liquid dosage form.
4 Technical requirements
4.1 Raw material requirements
Appendix A
Method for determining enzyme activity
A.1 General
The reagents and water used in this Standard, when no other requirements are
specified, refer to the analytical reagents and the Grade 3 water specified in
GB/T 6682. Standard solutions, impurity standard solutions, preparations, and
products used in the test, when no other requirements are specified, shall be
prepared in accordance with the provisions of GB/T 601, GB/T 602, and GB/T
603. The solution used in the test, when no solvent is specified, refers to the
aqueous solution.
A.2 Determination of α-amylase activity
A.2.1 α-amylase
An enzyme capable of hydrolyzing the α-1,4 glycosidic bond in the molecular
chain of starch, and cutting the starch chain into a short-chain dextrin and a
small amount of maltose and glucose to rapidly decrease the viscosity of the
starch.
A.2.2 α-amylase activity
A.2.2.1 Medium-temperature α-amylase activity unit
1 g of solid enzyme powder (or 1 mL of liquid enzyme), at 60 °C, pH 6.0,
liquefies 1 g of soluble starch in 1 h, i.e. 1 enzyme activity unit, expressed in
U/g (U/mL).
A.2.2.2 High-temperature resistant α-amylase activity unit
1 g of solid enzyme powder (or 1 mL of liquid enzyme), at 70 °C, pH 6.0,
liquefies 1 mg of soluble starch in 1 min, i.e. 1 enzyme activity unit, expressed
in U/g (U/mL).
A.2.3 Principle
The α-amylase preparation can randomly cut the α-1,4 glycosidic bond in the
starch molecular chain to short-chain dextrin of various lengths, a small amount
of maltose and glucose, so that the characteristic reaction of starch showing
bluish violet to iodine gradually disappears, showing a brownish red. The rate
at which the color disappears is related to the enzyme activity, whereby the
mL; USE a small amount of phosphate buffer to dissolve it thoroughly; carefully
POUR the supernatant into the volumetric flask. If there is residual residue,
ADD a small amount of phosphate buffer to fully grind; TRANSFER all the final
sample into the volumetric flask; USE phosphate buffer to dilute to the mark;
SHAKE well. USE four layers of gauze to filter and leave the filtrate for use.
Note: For the enzyme activity of the medium-temperature α-amylase enzyme solution to
be tested, the enzyme concentration is controlled in the range of 3.4 U/mL~4.5 U/mL.
For the high-temperature resistant α-amylase activity to be tested, the enzyme
concentration is controlled in the range of 60 U/mL~65 U/mL.
A.2.6.2 Determination
a) PIPETTE 20.0 mL of soluble starch solution into the test tube; ADD 5.00
mL of phosphate buffer; after shaking well, PLACE in a constant-
temperature water bath at 60 °C±0.2 °C (The high-temperature resistant
α-amylase preparation is placed at 70 °C±0.2 °C) to preheat for 8 min;
b) ADD 1.00 mL of diluted enzyme solution to be tested; TIME immediately;
SHAKE well; accurately react for 5 min;
c) Immediately USE an automatic pipette to pipette 1.00 mL of reaction
solution; ADD it to a test tube preloaded with 0.5 mL of hydrochloric acid
solution and 5.00 mL of dilute iodine solution; SHAKE well. USE 0.5 mL
of hydrochloric acid solution and 5.00 mL of dilute iodine solution as blanks;
at a wavelength of 660 nm, USE a 10 mm cuvette to rapidly determine the
absorbance (A). According to the absorbance, refer to the table of
Appendix B, to obtain the concentration of the test enzyme solution.
A.2.6.3 Result calculation
A.2.6.3.1 Enzyme activity of medium-temperature α-amylase preparation
The enzyme activity X1 of medium-temperature α-amylase preparation, in U/mL
or U/g, is calculated according to formula (A.1):
Where:
c - The concentration of the test enzyme sample, in U/mL or U/g;
n - The dilution factor of the sample.
The result obtained is expressed to integer.
The test result is based on the arithmetic mean of parallel determination results.
A.3.3.2 0.05 mol/L sodium thiosulfate standard titration solution.
A.3.3.3 0.1 mol/L iodine standard solution.
A.3.3.4 0.1 mol/L sodium hydroxide solution.
A.3.3.5 2 mol/L sulfuric acid solution: PIPETTE 5.6 mL of analytical
concentrated sulfuric acid (Relative density is 1.84); slowly ADD it to the
appropriate amount of water; after cooling, USE water to dilute to 100 mL;
SHAKE well.
A.3.3.6 200 g/L sodium hydroxide solution: WEIGH 20 g of sodium hydroxide;
USE water to dissolve and dilute to 100 mL.
A.3.3.7 20 g/L soluble starch solution: WEIGH 2 g±0.001 g of soluble starch;
then USE a small amount of water to mix thoroughly; slowly POUR into boiling
water; BOIL, stir until it is transparent; COOL; USE water to dilute to 100 mL.
This solution needs to be prepared on the same day.
Note: Soluble starch shall be special starch for analysis of enzyme preparation.
A.3.4 Instruments and equipment
A.3.4.1 Analytical balance: Accuracy is 0.2 mg.
A.3.4.2 Acidimeter: Accuracy is 0.01 pH.
A.3.4.3 Analytical balance: Accuracy is 0.2 mg.
A.3.4.4 Constant-temperature water bath: 40 °C±0.1 °C.
A.3.4.5 Continuous multi-gear distributor (pipette).
A.3.4.6 Magnetic stirrer.
A.3.5 Analytical procedures
A.3.5.1 Preparation of enzyme solution to be tested
A.3.5.1.1 Liquid enzyme: USE a continuous multi-gear distributor to accurately
pipette the appropriate amount of enzyme sample; TRANSFER it into a
volumetric flask; USE a buffer solution to dilute to the mark; SHAKE well for
determination.
A.3.5.1.2 Solid enzyme: USE a 50 mL small beaker to accurately weigh the
appropriate amount of enzyme sample, accurate to 1 mg. USE a small amount
of acetic acid-sodium acetate buffer solution to dissolve; USE a glass rod to
carefully grind. Carefully POUR the supernatant into a suitable volumetric flask;
dilute to 1000 mL.
A.4.4.8.2 Sodium lactate buffer (pH=3.0, suitable for acidic protease
preparations).
TAKE 4.71 g of lactic acid (80%~90%) and 0.89 g of sodium lactate (70%); ADD
water to 900 mL; STIR well. USE lactic acid or sodium lactate to adjust the pH
to 3.0±0.05; DILUTE to 1000 mL.
A.4.4.8.3 Boric acid buffer solution (pH=10.5, suitable for alkaline protease
preparations).
WEIGH 9.54 g of sodium borate, 1.60 g of sodium hydroxide; ADD 900 mL of
water; STIR well. USE 1 mol/L hydrochloric acid solution or 0.5 mol/L sodium
hydroxide solution to adjust the pH=10.5±0.05; DILUTE to 1000 mL.
A.4.4.9 Casein solution (10.0 g/L): WEIGH 1.000 g of standard casein (NICPBP
National Drug Reference Material), accurate to 0.001 g. After using a small
amount of sodium hydroxide solution (for the acidic protease preparation, USE
2 drops~3 drops of concentrated lactic acid) to moisten, ADD about 80 mL of
the corresponding buffer solution; HEAT and boil for 30 min in a boiling water
bath; STIR occasionally until the casein is completely dissolved. After cooling
to room temperature, TRANSFER to a 100 mL volumetric flask; USE a suitable
pH buffer solution to dilute to the mark. Before diluting to the mark, CHECK and
adjust the pH to the specified value of the corresponding buffer. This solution is
stored in the refrigerator and is valid for 3 days. Before use, reconfirm and
adjust the pH to the specified value.
Note: Casein from different sources or batch numbers has an effect on the test results. If
different casein is used as a substrate, before use, the results shall be compared
with the above standard casein.
A.4.4.9.1 L-tyrosine standard stock solution (100 μg/mL): Accurately WEIGH
0.1000 g±0.0002 g of L-tyrosine previously dried at 105 °C to a constant mass;
USE 60 mL of 1 mol/L hydrochloric acid solution to dissolve and dilute to 100
mL, i.e. 1 mg/mL tyrosine solution.
PIPETTE 10.00 mL of 1 mg/mL tyrosine solution; USE 0.1 mol/L hydrochloric
acid solution to dilute to 100 mL, to obtain 100 μg/mL L-tyrosine standard stock
solution.
Note: In addition to the above protease dissolution/dilution buffer systems, producers and
users can also explore the use of other suitable buffer systems.
A.4.5 Instruments and equipment
P9135 or equivalent, accurate to 0.1 mg); ADD water to dissolve; BOIL, cool,
and filter. Adjust the pH to 3.5; USE water to dilute to 100 mL; STORE in a
refrigerator for use. The use time is no more than 3 days.
Note: The pectin substrate has a great influence on the test. If pectin powders from
different sources or batch numbers are used, it shall perform a controlled test against
the former batch number.
A.5.4.2 Sodium thiosulfate standard solution: c (Na2S2O3)=0.05 mol/L.
A.5.4.3 Sodium carbonate standard solution: c (12 Na2CO3)=2 mol/L.
A.5.4.4 Iodine standard solution: c (12 I2)=0.1 mol/L.
A.5.4.5 2 mol/L sulfuric acid solution: TAKE 5.6 mL of concentrated sulfuric acid;
slowly ADD it to the appropriate amount of water; after cooling, USE water to
dilute to 100 mL; SHAKE well and set aside.
A.5.4.6 Soluble starch indicator solution (10 g/L).
A.5.4.7 0.1 mol/L citric acid-sodium citrate buffer (pH 3.5): WEIGH 14.71 g of
citric acid (C6H8O7 • H2O), 8.82 g of trisodium citrate (C6H5Na3O7 • 2H2O); ADD
950 mL of water to dissolve; adjust the pH to 3.5; USE water to dilute to 1000
mL.
A.5.5 Instruments and equipment
A.5.5.1 Colorimetric tube: 25 mL.
A.5.5.2 Constant-temperature water bath with heating device: Temperature
control accuracy is ±0.1 °C.
A.5.5.3 Iodine flask: 250 mL.
A.5.5.4 Burette: 25 mL.
A.5.6 Analytical procedures
A.5.6.1 Preparation of sample solution
USE a 50 mL beaker of known mass to weigh 1 g~2 g of enzyme powder
(accurate to 0.0001 g) or accurately pipette 1.00 mL. USE a small amount of
citric acid-sodium citrate buffer to thoroughly dissolve; USE a glass rod to grind;
carefully POUR the supernatant into a volumetric flask. If there is residual
residue, ADD a small amount of the above buffer to fully grind; TRANSFER all
The presence of 3-hydroxy-2-butanone (acetoin) and/or diacetyl in the sample
may result in a large test result.
The acetoin, during storage, is easy to form a dimer, thus affecting the test
results. However, if the measures described in the preparation steps are
followed to prevent, this method may still be used.
A.6.3.2 Principle
The α-acetolactate decarboxylase reacts with the substrate α-acetolactate to
decarboxylate to form acetoin. The acetoin, under alkaline conditions, reacts
with a mixture of naphthol and creatine to form a red product. By determining
the absorbance of the solution at 522 nm, the amount of acetoin produced by
the reaction can be obtained from the standard curve of acetoin; so that the
enzyme activity of α-acetolactate decarboxylase can be calculated.
A.6.3.3 Reagents and materials
A.6.3.3.1 MES (9.76 g/L)-sodium chloride (35.064 g/L)-polyoxyethylene lauryl
ether1) (1.52 mL/L) buffer: WEIGH 48.80 g of 2-[N-morpholino]ethanesulfonic
acid (MES) and 175.32 g of sodium chloride in a beaker; USE about 4.5 L of
water to dissolve. Then ADD 7.60 mL of 15% polyoxyethylene lauryl ether
solution; STIR well. USE about 1 mol/L sodium hydroxide solution to adjust the
pH to 6.00 ± 0.05. Then TRANSFER to a 5000 mL volumetric flask; USE water
to dilute to the mark; STIR well. This solution, at room temperature
(15 °C~20 °C), has a storage life of one week.
A.6.3.3.2 α-acetolactate substrate (2.00 mL/L): PIPETTE 100 μL of ethyl-2-
acetoxy-2- methyl-acetoacetic acid into a 50 mL volumetric flask; ADD 6.0 mL
of about 0.50 mol/L sodium hydroxide solution. After stirring for 20 min, ADD
buffer to about 40.0 mL; USE about 1 mol/L hydrochloric acid to adjust the pH
of the solution to 6.00±0.05. Then, USE buffer to dilute to the mark. This solution
is prepared before use.
A.6.3.3.3 Naphthol (10.0 g/L)/creatine (1.0 g/L) color developer: WEIGH 5.0 g
of 1-naphthol and 0.5 g of creatine into 500 mL volumetric flask; USE about 1
mol/L sodium hydroxide solution to dissolve and dilute to the mark. This solution
is prepared before use. During the preparation, it shall be protected from light
and be in ice bath.
Warning - 1-naphthol is flammable and toxic. It is irritating to the eyes and
mucous membranes. If swallowed or absorbed through the skin,
1 ) Brij®35 is a suitable commercially-available product. Products with the equivalent
analytical effect may also be used.
A.6.3.5.1 Preparation of standard curve of acetoin
PIPETTE 400 μL of the prepared acetoin standard solution (Table A.2)
respectively into 10 mL test tube. Then, sequentially ADD 4.60 mL of color
developer to each test tube; USE a vortex oscillator to mix well. PLACE at room
temperature and start timing.
After 40.0 min of reaction, USE a spectrophotometer, at 522 nm, to determine
the absorbance of each tube solution.
A.6.3.5.2 Preparation of standard control
It is recommended to use a representative, stable sample as a standard control.
In each analysis, the standard control is tested along with the sample, to judge
the test repeatability.
A.6.3.5.3 Preparation of sample solution
A certain amount of test portion is weighed from the sample, accurate to 0.0005
g; USE the solution to dilute. The dilution factor shall be such that the final
absorbance H1 of sample falls within the range of acetoin standard curve.
A.6.3.5.4 Drawing of standard curve
USE the absorbance at a wavelength of 522 nm as the Y-axis. USE the
concentration (mg/L) of acetoin as the X-axis. DRAW a standard curve.
Calculate the slope h of the standard curve (or USE the regression equation to
calculate).
A.6.3.5.5 Reaction of enzyme sample
The sample solution and the substrate are preheated in a 30 °C water bath for
about 10 min. Then, according to the following methods, each sample solution
is treated:
a) Sample absorbance value H1: PIPETTE 200.0 μL of enzyme sample
solution into a 10 mL test tube in a water bath at 30 °C±0.1 °C; then ADD
200.0 μL of the preheated substrate; USE a vortex oscillator to mix well;
quickly PLACE back to the water bath; START timing.
b) Blank absorbance value H2: Replace the sample solution with a buffer;
operate as described above.
c) After reacting for 20.0 min, sequentially ADD 4.60 mL of color developer
to each tube; USE a vortex oscillator to mix well. PLACE at room
temperature; START timing again.
buffer, ADD 550U of glucose oxidase (5 mg of glucose oxidase “Amano”) and
125U of peroxidase (0.76 mg, 165 U/mg peroxidase “Amano”); ADD 1 mL of 4-
aminoantipyrine solution and 1.4 mL of phenol solution; USE Tris-phosphate
buffer to dilute to 50 mL. Prepared when used.
A.7.4.8 Substrate solution: DISSOLVE 2.0 g of α-methyl glucose (C7H14O6) in
50 mL of water; USE water to dilute to 100 mL (At 5 °C~15 °C, it may be stable
for two weeks).
A.7.5 Analytical procedures
A.7.5.1 Preparation of sample solution
WEIGH 1 g of enzyme sample, accurate to ±0.0001 g; or PIPETTE 1 mL of
enzyme sample, accurate to 0.01 mL. ADD it to a volumetric flask of appropriate
size; USE cooled water to dilute to the mark; MIX well.
Note: The prepared sample solution is such that 0.5 mL of A60~A0 is between 0.15~0.32.
A.7.5.2 Determination
PIPETTE 1 mL of substrate solution and 1 mL of 0.02 mol/L acetic acid-sodium
acetate solution into a 15 mm×150 mm test tube; PRESERVE heat in a
constant-temperature water bath at 40 °C±0.5 °C for 10 min. ADD 0.5 mL of
sample solution; MIX well. After accurately preserving heat in a constant-
temperature water bath at 40 °C±0.5 °C for 60 min, TRANSFER the test tube
to a boiling water bath to heat for 5 min; then USE running water to rapidly cool.
After cooling, PIPETTE 0.1 mL of this solution into a test tube; ADD 3 mL of 4-
aminoantipyrine-phenol color developer; MIX well. PLACE this test tube in a
constant-temperature water bath at 40 °C±0.5 °C to preserve heat for 20 min;
determine the absorbance A60 at 500 nm.
Blank control: PIPETTE 1 mL of 0.02 mol/L acetic acid-sodium acetate solution
and 0.5 mL of sample solution into a 15 mm×150 mm test tube; MIX well.
TRANSFER the test tube to a boiling water bath to heat for 5 min; then USE
running water to rapidly cool. After cooling, ADD 1 mL of substrate solution; MIX
well. PIPETTE 0.1 mL of this solution into an empty test tube; ADD 3 mL of 4-
aminoantipyrine-phenol color developer; MIX well. PLACE this test tube in a
constant-temperature water bath at 40 °C±0.5 °C to preserve heat for 20 min;
determine the absorbance As0 at 500 nm.
Accurately WEIGH 1000 g of glucose (C6H12O6) dried at 105 °C for 6 h;
DISSOLVE in 100 mL of water. TAKE 1 mL, 2 mL, 3 mL, 4 mL, 5 mL,
respectively; USE water to dilute to 100 mL (Each 1 mL of this solution contains
100 μg, 200 μg, 300 μg, 400 μg, and 500 μg of glucose, respectively).
The enzyme acts on the starch solution to form reducing sugars. Then,
Fehling’s reagent is added, upon heating, to quantitatively generate a cuprous
oxide precipitate. After adding potassium iodide and sulfuric acid, free iodine is
formed. At this time, immediately USE sodium thiosulfate solution to titrate.
A.7.7.2 Reagents and materials
A.7.7.2.1 30% potassium iodide solution: 150 g of potassium iodide is dissolved
in 350 mL of water and stored in a brown reagent bottle, to avoid direct sunlight.
A.7.7.2.2 25% sulfuric acid solution: 125 g of sulfuric acid is dissolved in 375
mL of water.
A.7.7.2.3 0.05 mol/L sodium thiosulfate solution: USE boiled cooling water to
dilute 500 mL of the 0.1 mol/L sodium thiosulfate solution used for quantitative
analysis. The total volume of the obtained solution is 1000 mL. After preparation,
it shall perform a calibration, to obtain the concentration correction factor f value.
A.7.7.2.4 1 mol/L acetic acid-sodium acetate buffer solution (pH 4.5): 1 mol/L
sodium acetate solution is added to 1 mol/L acetic acid solution. Adjust the pH
to 4.5.
A.7.7.2.5 Soluble starch solution (pH 4.5): The soluble starch (reagent grade)
is dried at 105 °C for 4 h and then weighed to calculate the water content. Then,
according to the water content of the soluble starch, WEIGH 0.50 g (dry basis)
of soluble starch; slowly ADD it to 50 mL of boiling water; BOIL for 5 min. After
using tap water to cool, ADD 5 mL of 1 mol/L acetic acid-sodium acetate buffer
solution; USE water to dilute to 100 mL.
A.7.7.2.6 Fehling’s reagent: WEIGH 34.66 g of copper sulfate dissolved in
water; DILUTE to 500 mL. WEIGH 173 g of potassium sodium tartrate and 50
g of sodium hydroxide dissolved in water; DILUTE to 500 mL. Before use,
accurately TAKE an equal volume of the above two solutions and mix well.
A.7.7.3 Analytical procedures
A.7.7.3.1 Preparation of sample solution
USE water to dilute enzyme sample, so that the (T0-T30)×f×1.62 value of the
obtained enzyme solution is 3 mg~10 mg of glucose.
A.7.7.3.2 Determination
ADD 10 mL of soluble starch solution to a 100 mL conical flask; PLACE in a
constant-temperature water bath at 40 °C±0.5 °C to preheat for 10 min~15 min;
ADD 1 mL of the sample diluted enzyme solution. After accurate heating for 30
A.8.1 α-amylase
An enzyme capable of hydrolyzing the α-1,4 glycosidic bond inside the
gelatinized starch, amylose, and amylopectin aqueous solution to produce
maltose, oligosaccharide, and a small amount of glucose.
A.8.2 α-amylase activity
At 40 °C, 1 g or 1 mL of enzyme sample reacts with the substrate soluble starch,
producing glucose equivalent to 10 mg in 30 min, i.e. 1 enzyme activity unit,
expressed in U/g or U/mL.
A.8.3 Principle
The enzyme acts on the starch solution to form reducing sugars. Then,
Fehling’s reagent is added, upon heating, to quantitatively generate a cuprous
oxide precipitate. After adding potassium iodide and sulfuric acid, free iodine is
formed. At this time, immediately USE sodium thiosulfate to titrate.
A.8.3.1 Reagents and materials
A.8.3.1.1 30% potassium iodide solution: 150 g of potassium iodide is dissolved
in 350 mL of water and stored in a brown reagent bottle, to avoid direct sunlight.
A.8.3.1.2 25% sulfuric acid solution: 125 g of sulfuric acid is dissolved in 375
mL of water.
A.8.3.1.3 0.05 mol/L sodium thiosulfate solution: USE boiled cooling water to
dilute 500 mL of the 0.1 mol/L sodium thiosulfate solution used for quantitative
analysis. The total volume of the obtained solution is 1000 mL. After preparation,
it shall perform a calibration, to obtain the concentration correction factor f value.
A.8.3.1.4 1 mol/L acetic acid-sodium acetate buffer solution (pH 5.0): 1 mol/L
sodium acetate solution is added to 1 mol/L acetic acid solution. Adjust the pH
to 5.0.
A.8.3.1.5 Soluble starch solution (pH 5.0): The soluble starch (reagent grade)
is dried at 105 °C for 4 h and then weighed to calculate the water content. Then,
according to the water content of the soluble starch, WEIGH 0.50 g (dry basis)
of soluble starch; slowly ADD it to 50 mL of boiling water; BOIL for 5 min. After
using tap water to cool, ADD 5 mL of 1 mol/L acetic acid-sodium acetate buffer
solution (pH 5.0); USE water to dilute to 100 mL.
A.8.3.1.6 Fehling’s reagent
- Copper solution: 34.66 g of copper sulfate is dissolved in water and diluted
to 500 mL;
f - Correction factor of concentration of 0.05 mol/L sodium thiosulfate standard
solution;
1.62 - Conversion factor;
1/10 - A constant of this analysis method (reducing sugar equivalent to 10 mg
of glucose);
n - The dilution factor of sample.
The test result is based on the arithmetic mean of parallel determination results.
The absolute difference between the two independent determination results,
obtained under repeated conditions, shall not exceed 5% of the arithmetic mean.
A.9 Determination of pullulanase activity
A.9.1.1 Pullulanase
Debranching enzyme capable of specifically cutting out the α-1,6-glycosidic
bond in the branch point of amylopectin, thereby cutting the entire lateral branch
to form amylose.
A.9.1.2 Pullulanase activity (colorimetry)
Under the given reaction conditions, 1 mL or 1 g of product reacts per minute
to produce 1 μmol of glucose, i.e. one enzyme activity unit, expressed in U/mL
or U/g.
A.9.1.3 Principle
Pullulanase, under certain conditions, catalyzes the hydrolysis of pullulan to
produce reducing sugars such as glucose. After 3,5-dinitrosalicylic acid is co-
heated with the reducing sugar solution, a brownish red amino complex may be
formed. Within a certain range, the shade of color is directly proportional to the
amount of reducing sugar produced. At a wavelength of 550 nm, determine the
absorbance of the reaction solution. The measured value is directly proportional
to the activity of pullulanase in the sample.
Note: The presence of other carbohydrases (such as glucoamylase) capable of
hydrolyzing pullulan in the sample can interfere with this method. Adding a certain
amount of acarbose can inhibit the activity of glucoamylase in the sample; and this
method is still applicable.
A.9.1.4 Reagents and materials
A.9.1.4.1 Acetic acid-sodium acetate buffer (0.2 mol/L, pH 4.5): accurately
WEIGH 4.92 g of anhydrous sodium acetate dissolved in water; ADD 4 mL of
A.9.1.6.1 Drawing of glucose standard curve
The glucose standard curve is drawn as follows:
a) PIPETTE 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, 1.2 mL, and 1.4 mL of
0.1% glucose standard solution respectively; ADD them to 7 graduated
test tubes; USE distilled water to supplement to 2.0 mL. Prepare into
standard solutions containing 100 μg, 200 μg, 300 μg, 400 μg, 500 μg,
600 μg, and 700 μg of glucose per milliliter respectively;
b) ADD 3 mL of DNS reagent to each tube; BOIL in boiling water for 7 min
(starting from boiling); TAKE out and rapidly cool to room temperature;
ADD 10 mL of distilled water and mix well;
c) USE a spectrophotometer, at 550 nm, to measure the absorbance of
solution. USE a blank tube solution to adjust the zero point. RECORD the
absorbance value. USE the absorbance as the ordinate. USE the
corresponding standard glucose concentration as the abscissa. DRAW a
standard curve.
Note: USE 0.5 mL of distilled water instead of 0.5 mL of standard glucose solution as a
blank control.
A.9.1.6.2 Sample preparation
WEIGH 1 g of enzyme sample, accurate to ±0.0001 g; or PIPETTE 1 mL of
enzyme sample, accurate to 0.01 mL. USE acetic acid-sodium acetate buffer
to dissolve; magnetically STIR and mix well; accurately dissolve and dilute to
the mark (to make the difference in absorbance of sample solution and blank
solution just fall within the range of 0.3~0.4); PLACE for 10 min for later
determination. If the sample contains glucoamylase, a buffer solution containing
acarbose must be used.
A.9.1.6.3 Determination
TAKE four 25 mL graduated test tubes with stoppers (one blank tube and three
sample tubes). Accurately ADD 1.00 mL of preheated pullulan solution to the
four tubes respectively; PLACE the test tubes in a water bath at 60 °C±0.1 °C
to preheat for 10 min. At a certain interval, ADD 1.0 mL of diluted enzyme
solution to be tested to the three sample tubes respectively (The blank tube is
not added). If the sample contains glucoamylase, 40 μL of acarbose solution
may be added to the test tubes; TIME accurately; after reacting for 30 min,
TAKE out in sequence. Immediately ADD 3.0 mL of DNS reagent to each test
tube; SHAKE and mix well; ADD 1.0 mL of enzyme solution to be tested to the
blank tube. PUT the four tubes into the boiling water bath at the same time;
after heating for 7 min, TAKE them out; COOL quickly to room temperature;
certain amount of acarbose can inhibit the activity of glucoamylase in the sample;
and this method is still applicable.
A.9.1.8.2 Reagents and materials
A.9.1.8.2.1 Buffer
According to the optimum pH provided by the product, prepare the
corresponding concentration of buffer; STORE at 4 °C. The solution is stable
for 1 month.
Note: If the sample contains glucoamylase, at a concentration of 0.03%, acarbose is added
to the buffer solution.
A.9.1.8.2.2 2% red pullulan solution: WEIGH 1.00 g of red pullulan substrate
(Megazyme lot.61201). USE buffer to dilute to 50 mL. Ensure that red pullulan
is dissolved completely. STORE at 4 °C. The solution is stable for 2 weeks.
A.9.1.8.2.3 Standard and sample preparation
A.9.1.8.2.3.1 Preparation of standard and standard curve
Accurately WEIGH a certain amount of pullulanase preparation standard; USE
the corresponding buffer to dissolve and dilute to the mark, as a standard stock
solution. USE the standard stock solution to prepare different concentrations of
enzyme standard solutions. USE the enzyme activity of the standard solutions
and their corresponding absorbance (minus blank absorbance) to make a
standard curve. It is required that the linear correlation coefficient is not less
than 0.998.
Note: According to the optimum conditions provided by the product, the enzyme a......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.