QB/T 1803-1993 General test methods for industrial enzyme preparations
Some standard content:
Light Industry Industry Standard of the People's Republic of China
General methods of determinationfor industral enzymes
1 Subject content and scope of application
This standard specifies the test methods for industrial enzyme preparations. This standard is applicable to the detection of industrial enzyme preparations. 2 Reference standards
GB 601
GB1250
Chemical reagents-Preparation of standard solutions for titration analysis (volumetric analysis)-Preparation of preparations and products used in test methods Chemical reagents
Chemical reagents-Acid-base indicators-General method for determination of pH color range-Expression and determination method of limit values GB 4789.2
GB 4789. 3
GB 4789. 4
GB 4789. 15
GB 6004
GB 6682
GB 8170
GB 8451
GB 9721
Microbiological examination of food hygieneDetermination of total colony countMicrobiological examination of food hygieneDetermination of coliform groupMicrobiological examination of food hygieneSalmonella examinationMicrobiological examination of food hygiene
Determination of mold and yeast count
Wire woven square hole mesh for test sieve
Specification of water for laboratory
Rules for rounding off values
Test method for limit of heavy metals in food additivesGeneral rules for molecular absorption spectrophotometry of chemical reagents (ultraviolet and visible light part) 3 General principles and basic requirements
3.1 General principles
3.1.1 The terms and measurement units used in the methods shall comply with the standards prescribed by the state. QB/T1803—1993
3.1.2 The various analytical instruments used in the method (such as analytical balance, acidity meter, spectrophotometer and ultraviolet spectrophotometer, etc.) must be calibrated on time; the burettes, pipettes, volumetric flasks and other instruments used should be calibrated regularly according to the relevant verification procedures. 3.1.3 The "instruments" used in the method are special instruments that must be used in the test, and general experimental instruments are no longer listed. 3.1.4 The water used in the method, unless otherwise specified, shall comply with the third-level (or above) specification requirements in GB6682. 3.1.5 The reagents used in the method, unless otherwise specified, are all analytically pure (AR). 3.1.6 "Solution" in the method refers to aqueous solution unless otherwise specified. "Dilution to scale" refers to dilution to scale with water. When there are two or more test methods for the same test item, each laboratory can choose according to its own conditions, but the first method shall be the secondary method. 3.1.7 "
3.1.8 Physical and chemical requirements The test results shall be reported with actual measured data. The calculation and selection of data shall comply with GB8170; Determination of limit values Approved by the Ministry of Light Industry of the People's Republic of China on July 29, 1993 and implemented on March 1, 1994
QB/T 1803- 1993
The comparison method of rounded values in GB1250 shall be followed, and the significant figures of the results shall be consistent with the technical requirements. 3.2 Basic requirements
3.2.1 Parallel tests must be conducted for the determination of samples. 3.2.2 The concentration of the standard solution is generally expressed in terms of molar concentration, in units of moles per liter or moles per cubic meter, such as: hydrochloric acid standard solution Cc(HCI)-0. 1mo1/L1.
3.2.3 The glassware used in the test should be soaked in chromic acid cleaning solution or synthetic detergent, rinsed with tap water, and then cleaned with distilled water.
3.2.4 The significant figures when aspirating or weighing in the test method indicate the required accuracy. 4 Appearance and odor inspection
Take 2g of solid enzyme sample (or 10ml. liquid enzyme) and pour it into a 25mL dry beaker, place it under the nose to smell it, and then visually inspect the appearance and make inspection records.
5 Test method for enzyme activity
For the determination of enzyme activity, see Appendix A (Supplement). 6 Test method for loss on drying
Under normal pressure, place the sample in an electric drying oven at 103°C ± 2°C and dry it for 2 hours. Determine the mass of volatile matter lost by weighing and express it as a percentage.
6.2 Instruments and equipment
6.2.1 Electric drying oven 103°C ± 2°C
6.2.2 Weighing bottle 25mmX40mm.
6.2.3 Dryer with color-changing silica gel as desiccant . 6.2.4 The sensitivity of the analytical balance is 0.1mg.
6.3 Analysis steps
Use a weighing bottle that has been dried to constant weight to weigh about 0.2g of the enzyme sample, accurate to 0.0002g, and place it in a 103C2C electric drying oven, remove the cover, and place it on its side next to the weighing bottle. Dry for 2h, take it out, cover it, put it in a dryer and cool it to room temperature (about 30min), and weigh it. 6.4 Calculation
m2×100
Wu Zhong: X The drying loss of the sample, % (m/m) m.-Before drying, add the sample to the weighing bottle The mass of the sample, g; m2\After drying, weigh the mass of the weighing bottle plus the sample, gm the mass of the weighing bottle. .
The result is expressed to one decimal place.
6.5 The allowable difference of the result
The relative error of parallel tests shall not exceed 0.5%. 7 Test method for fine (granular)
7.1 Principle
Take a certain amount of enzyme sample, sieve it with the specified standard sieve, weigh it, and calculate the percentage of the mass of the enzyme sample passing through the standard sieve to the total sampled amount (or calculate the percentage of the mass of the intermediate of the double-layer sieve to the total sampled amount). 118
7.2 Instruments and equipment
7.2.1 Standard test sieve
QB/T 1803-1993
SSW1.18/0.630mmGB6004 (equivalent to the original 14 mesh)SSW0.80/0.450mmGB6004 (equivalent to the original 20 mesh)SSW0.40/0.250mmGB6004 (equivalent to the original 39 mesh)SSW0.25/0.160mmGB6004 (equivalent to the original 62 mesh)7.2.2 The sensitivity of the physical balance is 0.2g.
7.3 Analysis steps
7.3.1 Weigh 100g of solid enzyme sample, accurate to 0.2g. 7.3.2 Install the specified standard sieve on the sieve bottom plate, then transfer all the weighed samples onto the standard sieve, cover it, oscillate and sieve for 5 minutes (and knock the sieve from time to time), let it stand for 2 minutes, remove the cover, carefully transfer all the sieve material into a beaker of known mass, weigh it with a balance, and calculate according to formula (2).
7.3.3 Enzymes for detergents are sieved according to the particle size specified in the product standard using upper and lower limit double-layer sieves, weigh the mass of the middle material of the double-layer sieve (i.e. the sieve material on the lower sieve), and calculate according to formula (3). 7.4 Calculation
100m×100=100-m
Where: X, - - sample fineness, % (m/m); m - sample particle size, % (m/m);
mass of the sieve material (or middle material), g; - sample size, g.
The result is expressed as an integer.
7.5 Allowable error of results
The relative error of parallel tests shall not exceed 1%. 8 Test method for bulk density
8.1 Principle
(3)
Measure 100 mL of solid enzyme sample (or liquid enzyme) at 20°C, weigh it, and calculate the mass of the enzyme per unit volume, which is the bulk density of the sample, expressed in g/mL.
8.2 Instruments and equipment
8.2.1 Constant temperature water bath
20°C±0.1°C.
8.2.2 The sensitivity of the analytical balance is 0.1 mg.
8.2.3 Volumetric flask 100 mL (accurately calibrated). 8.3 Analysis steps
Use a clean, dry, 100mL volumetric flask of known mass, remove the stopper, place a triangular glass funnel at the top, and naturally and slowly pour the enzyme sample (20℃) into the volumetric flask (not necessary) until the scale. Remove the triangular funnel, cover the stopper, and weigh. 8.4 Calculation
Where: X
The bulk density of the sample, g/mL;
The mass of the volumetric flask plus the sample, 8;
The mass of the volumetric flask, g;
(4)
100--the sampling volume, mL.
The result is expressed to two decimal places.
8.5 Permissible difference in results
The relative error of parallel tests shall not exceed 5%. 9 Test method for pH value
9.1 Principle
QB/T 1803-1993
Immerse the indicating (glass) electrode and the reference (calomel) electrode in the sample solution to form a primary cell, whose electromotive force is related to the pH value of the solution. The pH value of the solution can be obtained by measuring the electromotive force of the primary cell. 9.2 Reagents and solutions
9.2.1 Tartrate standard buffer solution Dissolve racemic potassium hydrogen tartrate (KHC,H,O) in carbon dioxide-free water and shake vigorously to a saturated solution. At 25℃, pH=3.56. 9.2.2 Phosphate standard buffer solution Weigh 3.40g of potassium dihydrogen phosphate (KHzPO,) and 3.55g of disodium hydrogen phosphate (Na2HPO.,) and dissolve them in carbon dioxide-free water and dilute to 1000mL. At 25℃, pH=6.86. Potassium dihydrogen phosphate and disodium hydrogen phosphate must be dried at 120℃±10℃ for 2h in advance.
A mixed phosphate pH buffer (pH=6.864 at 25℃) specially used for calibration can also be used. 9.2.3 Borate standard buffer solution Weigh 3.81g of sodium tetraborate (Na2B,O, 10H20), dissolve it in carbon dioxide-free water, and dilute to 1000mL. The concentration of this solution is c(NazB,O,·10H,0)=0.01mol/L. At 25℃, the pH is 9.18. When storing, prevent carbon dioxide from the air from entering.
It can also be prepared with a borax pH buffer (pH=9.18 at 25℃) specially used for calibration. 9.2.4 Calcium hydroxide standard buffer solution At 25℃, dissolve calcium hydroxide in carbon dioxide-free water to make a saturated solution, and its concentration c[1/2Ca(OH)23 should be between 0.0400 and 0.0412 mol/L. Prevent carbon dioxide from entering the air during storage. Once turbidity occurs, it should be re-prepared. At 25℃, pH=12.45.
9.2.5 Water meets the third-grade water specifications in GB6682. 9.3 Instruments
9.3.1 The pH meter has a graduation value of 0.02 pH units and should be equipped with an electromagnetic stirrer. 9.3.2 The indicating (glass) electrode must be soaked in water for more than 24 hours before use, and should be cleaned immediately after use and immersed in water. 9.3.3 When using the reference (saturated calomel) electrode, the rubber plug of the small hole at the top of the electrode must be pulled out to prevent the generation of diffusion potential and affect the measurement results. There must be no bubbles in the potassium fluoride solution in the electrode to prevent short circuit. 9.4 Analysis steps
9.4.1 Adjust the temperature compensation knob to 25℃, calibrate the pH meter with two standard buffer solutions close to the pH value of the solution to be tested, and position it. 9.4.2 Rinse the electrode with water, then wash the electrode with the sample solution, adjust the sample solution temperature and adjust the temperature compensation to 25℃, and measure the pH value of the sample solution. Repeat the measurement operation until the pH value reading is stable for 1 minute. The result is expressed to one decimal place.
9.4.3 Allowable difference of results
The difference between parallel tests shall not exceed 0.1pH. 10 Test method for decomposition (dissolution) time of enzymes for detergents 10.1 Principle
Weigh 1g of solid enzyme sample, put it into a certain amount of water, and record the time required for it to completely disintegrate (dissolve) in water at the specified temperature and stirring speed.
10.2 Instrument
10.2.1 Constant temperature water bath 30℃±0.2℃.
10.2.2 Electromagnetic stirrer (with temperature control). 10.2.3 The sensitivity of the analytical balance is 0.1mg.
10.2.4 Stopwatch.
10.3 Analysis steps
QB/T 1803—1993
Weigh 1.000g of solid enzyme sample, accurate to 0.001g, and place it in a 150mL beaker filled with 100mL of 30℃±0.2℃ water in advance, put a rotor in it, and place it on a 30℃ electromagnetic stirrer. Start the time immediately, stir at medium speed, and record the time it takes for the sample to completely dissolve (dissolve) in water. First, make a preliminary test, stop stirring and observe once every 30s, and record the time required until the whole dissolution (dissolution) is completed. In the formal test, according to the results of the preliminary test, stop stirring at the predicted time, observe and record the time it takes for the whole disintegration (dissolution) in water. The results are expressed to seconds.
10.4 Allowable difference of results
The difference between parallel tests shall not exceed 30s.
11 Calculation method of enzyme activity preservation rate
11.1 Principle
According to the enzyme activity indicated on the label and the measured enzyme activity, calculate the percentage of the measured enzyme activity to the indicated enzyme activity. 11.2 Calculation of enzyme activity preservation rate
Where: X—enzyme activity preservation rate of the sample, %; X
E,--—measured enzyme activity of the sample, u/g (u/mL) E——labeled enzyme activity of the sample, u/g (u/mL). The obtained results are expressed to integers.
12 Testing of hygiene requirements
12.1 Determination of heavy metals shall be carried out in accordance with GB8451. 12.2 Determination of total bacterial count shall be carried out in accordance with GB4789.2. 12.3 The determination of coliform group shall be carried out in accordance with GB4789.3. ×100
12.4 The determination of bacteria and alcohol mother bacteria shall be carried out in accordance with GB4789.15. 12.5 The inspection of Salmonella shall be carried out in accordance with GB4789.4. (5)
A1~-Amylase
A1.1 Definition
QB/T18031993
Appendix A
Test method of enzyme activity
(Supplement)
1g solid enzyme powder (or 1mL liquid enzyme), at 60℃, pH-6.0, liquefies 1g soluble starch in 1h: 1 activity unit, expressed as u/g (u/mL).
A1.2 Principle
α-Amylase can mechanically cut the α1,4 glucosidic bonds in the starch molecular chain into long and short chain dextrins, reducing maltose and glucose, and making the blue-purple characteristic reaction of starch to iodine gradually disappear and turn reddish brown. The speed of color disappearance is related to the enzyme activity, so the enzyme activity can be calculated by the absorbance after the fixed reaction. A1.3 Reagents and solutions
A1.3.1 Original iodine solution Weigh 11g iodine (I) and 22g iodine (KI), dissolve the iodine completely with a small amount of water, then make up to 500mL and store in a brown bottle.
A1.3.2 Dilute iodine solution Take 2.00mL of the original iodine solution (A1.3.1), add 20g potassium iodide, dissolve it with water and make up to 500mL, and store in a brown bottle.
A1.3.3 20g/L soluble starch solution Weigh 2.000g of soluble starch (on an absolute dry basis) accurately to 0.001g, mix with water to make a slurry, slowly pour into 70ml boiling water under stirring, then rinse the beaker containing starch with 30mL of water several times, add the washing liquid into it, heat until completely transparent, cool, and dilute to 100mL. This solution needs to be prepared on the same day. Note: 1) Use soluble starch produced by Zhejiang Huilingchao Food Chemical Joint Company. A1.3.4 Phosphate buffer (pH6.0) Weigh 45.23g of disodium hydrogen phosphate (NazHPO.·12H2O) and 8.07g of citric acid (CHO*H,0), dissolve in water and dilute to 1000mL. After preparation, calibrate with a pH meter. A1.4 Instruments and equipment
A1.4.1 The spectrophotometer should comply with the relevant provisions of GB9721. A1.4.2 Warm water bath 60℃±0.2℃.
A1.4.3 Stopwatch.
A1.4.4 Test tube 25mmX200mm,
A1.5 Analysis steps
A1.5.1 Preparation of enzyme solution to be tested
Weigh 1~2g of enzyme powder, accurate to 0.0002g (or absorb liquid enzyme 1.00mL), first dissolve it with a small amount of phosphate buffer (A1.3.4), and pound it with a glass stirring rod, carefully pour the supernatant into a volumetric flask, add a small amount of buffer to the precipitated part, pound it 3~4 times, and finally transfer all of it into a volumetric flask, dilute it to the scale with buffer (divide the estimated enzyme activity by 4, that is, the enzyme activity should be within the range of 3.75.6u/mL), shake it. Filter through four layers of gauze, and the filtrate is used for determination. A1.5.2 Determination
A1.5.2.1 Pipette 20.0M soluble starch solution (A1.3.3) into a test tube (A1.4.4), add 5.00ml buffer solution (A1-3.4), shake well, and preheat in a 60℃±0.2℃ constant temperature water bath for 5min. A1.5.2.2 Add 1.00mL of the diluted enzyme solution to be tested (A1.5.1), immediately start timing, mix well, and ensure the reaction lasts for 5min. A1.5.2.3 Immediately pipette 1.00mL of the reaction solution (A1.5.2.2) into the dilute iodine solution (A1.3.2) 5.00mL, shake well, and use dilute iodine solution as a blank, and quickly measure its absorbance (A) at a wavelength of 660nm with 10mm colorimetric blood. According to its absorbance, look up Table A1 to obtain the concentration of the test enzyme solution ()
Absorbance (4)
QB/T 1803--1993
, absorbance and test α-amylase enzyme concentration comparison table Table A1
Enzyme concentration (e)
4, 669
Absorbance (A)
Enzyme concentration (c)
Satin gloss (A)
Concentration ()
4, 244
4, 140
Absorbance spot A )
Enzyme concentration (c)
QB/T1803--1993
Table A1 (continued)
Absorbance (A)
Enzyme concentration (c)
Luminescence (A)
Enzyme concentration (t)
3,744
Absorbance (A)
Enzyme concentration (e))wwW.bzxz.Net
QB/T 1803-1993
Table A1 (continued)
Absorbance (A)
Enzyme concentration (c)
Absorbance (A)
Enzyme concentration (c)
3,364
Absorbance (A)
Enzyme concentration (c)）
QB/T 1803--- 1993
Table A1 (continued)
Absorbance (A))
Enzyme concentration (c)
Absorbance (A)
Enzyme concentration (c)
Absorbance (A)
A1.6 Calculation
Where: X
Enzyme concentration (c)
QB/T1803—1993
Table A1 (end)
Absorbance (A)
Enzyme activity of the sample, u/g (u/mL);
Concentration of the test enzyme solution, u/mL;
-dilution factor of the sample.
The result is expressed to an integer.
Enzyme concentration (c)
Absorbance (A)
Enzyme concentration (c)
.....(A)
A1.7 Permissible difference of results
The relative error of parallel tests shall not exceed 2%. A2 Glucoamylase
A2.1 Definition
QB/T1803-1993
1g solid enzyme powder (or 1mL liquid enzyme), at 40℃, pH-4.6, decomposes soluble starch for 1h to produce 1mg glucose, which is one enzyme activity unit, expressed in u/g (u/mL). A2.2 Principle
Glucoamylase has the function of catalyzing starch hydrolysis, and can decompose α-1,4-glucosidic bonds to produce glucosyltransferase starting from the non-reducing end of starch molecules. Glucose molecules contain aldehyde groups, which can be oxidized by sodium hypoiodite. Excess sodium hypoiodite is acidified to precipitate iodine, which is then titrated with sodium thiosulfate standard solution to calculate the enzyme activity.
A2.3 Reagents and solutions
A2.3.1 Acetic acid-sodium acetate buffer solution (pH=4.6) Weigh 6.7g of sodium acetate (CH:COONa·3H2O), dissolve it in water, add 2.6mL of glacial acetic acid (CH.COOH), and dilute to 1000mL with water. After preparation, calibrate with a pH meter.
A2.3.2 Sodium thiosulfate standard solution c(NazS,0,)=0.05mol/L Prepare and calibrate according to GB601.
A2.3.3 Iodine solution c(1/2I2)0.1mol/L Prepare and calibrate according to GB601.
A2.3.4 Sodium hydroxide solution c(NaOH)=0.1mol/L Prepared according to GB601.
A2.3.5 200g/L Sodium hydroxide solution
Weigh 20g of sodium hydroxide, dissolve it in water and make up to 100mL. A2.3. 6 Sulfuric acid solution c(1/2H,SO,)2mol/L Take 5.6mL of concentrated sulfuric acid (density is 1.84), slowly inject it into 80mL of water, cool it, and make up to 100mL. A2.3.7 20g/L Soluble starch 2 liquid solution
Same as A1.3.3.
Note: 2) Use the soluble starch produced by Jianjiang Lingchao Food Chemical Joint Company. A2.3.8 10g/L Starch indicator solution
Prepared according to GB 604. Or dilute the 20g/L soluble starch solution by half. A2.4 Instruments and equipment
A2.4.1 Constant temperature water bath
A2.4.2 Stopwatch.
40℃±0.2℃.
50mL.
A2.4.3 Colorimetric tube
A2.5 Analysis steps
A2.5.1 Preparation of enzyme solution to be tested
Weigh 1~2g of enzyme powder, accurate to 0.0002g (or absorb 1.00mL of liquid enzyme), first dissolve it with a small amount of acetic acid buffer (A2.3.1), and pound it with a glass stirring rod, and carefully pour the supernatant into a volumetric flask. Add a small amount of buffer to the sediment, pound it 3~4 times, and finally transfer all of it to a volumetric flask, dilute it to the scale with buffer (estimate the enzyme activity according to the corresponding dilution multiple in Table A2, so that the enzyme activity is within the range of 100~250u/mL), and shake it. Filter through four layers of gauze and use the filtrate for determination.05mol/LPrepare and calibrate according to GB601.
A2.3.3Iodine solution c(1/2I2) 0.1mol/LPrepare and calibrate according to GB601.
A2.3.4Sodium hydroxide solution c(NaOH) = 0.1mol/LPrepare according to GB601.
A2.3.5200g/LSodium hydroxide solution
Weigh 20g of sodium hydroxide, dissolve it in water and make up to 100mL. A2.3. 6Sulfuric acid solution c(1/2H,SO,) 2mol/LMeasure 5.6mL of concentrated sulfuric acid (density is 1.84), slowly inject it into 80mL of water, cool it, and make up to 100mL. A2.3.720g/LSoluble starch 2 liquid solution
Same as A1.3.3.
Note: 2) Use the soluble starch produced by Jianjiang Lingchao Food Chemical Joint Company. A2.3.8 10g/L starch indicator solution
Prepare according to GB 604. Or dilute 20g/L soluble starch solution by half. A2.4 Instruments and equipment
A2.4.1 Constant temperature water bath
A2.4.2 Stopwatch.
40℃±0.2℃.
50mL.
A2.4.3 Colorimetric tube
A2.5 Analysis steps
A2.5.1 Preparation of enzyme solution to be tested
Weigh 1~2g of enzyme powder, accurate to 0.0002g (or absorb 1.00mL of liquid enzyme), first dissolve it with a small amount of acetic acid buffer (A2.3.1), and pound it with a glass stirring rod, and carefully pour the supernatant into a volumetric flask. Add a small amount of buffer to the sediment, pound it 3~4 times, and finally transfer it all to a volumetric flask, dilute it to the scale with buffer (estimate the enzyme activity according to the corresponding dilution multiple in Table A2, so that the enzyme activity is within the range of 100~250u/mL), and shake it. Filter through four layers of gauze, and the filtrate is used for determination. 12805mol/LPrepare and calibrate according to GB601.
A2.3.3Iodine solution c(1/2I2) 0.1mol/LPrepare and calibrate according to GB601.
A2.3.4Sodium hydroxide solution c(NaOH) = 0.1mol/LPrepare according to GB601.
A2.3.5200g/LSodium hydroxide solution
Weigh 20g of sodium hydroxide, dissolve it in water and make up to 100mL. A2.3. 6Sulfuric acid solution c(1/2H,SO,) 2mol/LMeasure 5.6mL of concentrated sulfuric acid (density is 1.84), slowly inject it into 80mL of water, cool it, and make up to 100mL. A2.3.720g/LSoluble starch 2 liquid solution
Same as A1.3.3.
Note: 2) Use the soluble starch produced by Jianjiang Lingchao Food Chemical Joint Company. A2.3.8 10g/L starch indicator solution
Prepare according to GB 604. Or dilute 20g/L soluble starch solution by half. A2.4 Instruments and equipment
A2.4.1 Constant temperature water bath
A2.4.2 Stopwatch.
40℃±0.2℃.
50mL.
A2.4.3 Colorimetric tube
A2.5 Analysis steps
A2.5.1 Preparation of enzyme solution to be tested
Weigh 1~2g of enzyme powder, accurate to 0.0002g (or absorb 1.00mL of liquid enzyme), first dissolve it with a small amount of acetic acid buffer (A2.3.1), and pound it with a glass stirring rod, and carefully pour the supernatant into a volumetric flask. Add a small amount of buffer to the sediment, pound it 3~4 times, and finally transfer it all to a volumetric flask, dilute it to the scale with buffer (estimate the enzyme activity according to the corresponding dilution multiple in Table A2, so that the enzyme activity is within the range of 100~250u/mL), and shake it. Filter through four layers of gauze, and the filtrate is used for determination. 128
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