Some standard content:
QB/T2306--1997
Thermostable α-amylase preparation is an endonuclease with high heat resistance, and its preparation is different from ordinary α-amylase preparation. It is now widely used in food and other industries.
This standard divides the product into two dosage forms and three quality grades. Among them, the superior and first-class products are used in the food industry, so the sanitary indicators are not equivalent to the "Sanitary Requirements for Enzymes for Food Industry" formulated by the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO) in Geneva in 1972. Since qualified products are used in other industries, no sanitary indicators are set. The physical and chemical indicators are formulated by referring to the advanced enterprise standards of major foreign companies and combined with my country's production practice. The first method for determining enzyme activity is spectrophotometry, and the absorbance and enzyme concentration comparison table is listed in Appendix A. Appendix A is the "Standard Appendix.
When determining enzyme activity, the quality of the substrate has a great influence on the analysis results. Therefore, this standard stipulates that the "soluble powder" reagent marked with "specially for enzyme preparation determination" produced by Jiangling Chao Food Chemical Industry United Company shall be used as the substrate for enzyme activity analysis. If it is temporarily unavailable and other widely produced "soluble starch" is used, a control test must be conducted. This standard is proposed by the Food and Papermaking Department of the China Light Industry Association. This standard is technically managed by the National Food Fermentation Standardization Center. The drafting units of this standard are: Wuxingda Bioengineering Co., Ltd., Shanghai Institute of Industrial Microbiology, China Institute of Food Fermentation Industry, Tianjin Institute of Light Industry, Yantai Xingda Bioengineering Co., Ltd. The main drafters of this standard are: Hui Xingyun, Wu Bingyan, Hu Xuezhi, Tian Qijing, Wang Furong, and Shan Shoushui. 97
1 Scope
Light Industry Industry Standard of the People's Republic of China Standard
Thermoresistant α-amylase preparations
QB/T2306--1997
This standard specifies the terms and definitions, technical requirements, test methods, inspection rules and markings, packaging, transportation and storage requirements of thermoresistant α-amylase preparations.
This standard applies to thermoresistant α-amylase preparations prepared by deep fermentation and purification of Bacillus licheniformis (or other variant strains).
2 Cited standards
The provisions contained in the following standards constitute the provisions of this standard through the use of reference signs in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest versions of the following standards. GB601-1988 Preparation of standard solutions for titration analysis (volume analysis) of chemical reagents GB4789-1994 Food hygiene microorganisms Physical and chemical inspection GB/T5009.11-1996 Food hygiene physical and chemical inspection Method for determination of total tartar in food GB/T5009.12—1996 Food hygiene physical and chemical inspection Method for determination of lead in food GB8451-1987 Test method for limits of heavy metals in food additives QB/T1803-1993 General test method for industrial enzyme preparations QB/T18041993 General inspection rules and marking, packaging, transportation and storage of industrial enzyme preparations 3 Terms and definitions
3.1 Heat-resistant α-amylase
An α-amylase preparation with high heat resistance prepared by fermentation and purification of selected Bacillus licheniformis. 3.2 Enzyme activity unit
The amount of enzyme required to liquefy 1 mg of soluble starch into dextrin in 1 min at 70°C and pH 6.0 is 1 activity unit, expressed as u/mL (u/g). ||tt ||4 Technical requirements
4.1 Appearance requirements
Liquid dosage form: beige liquid, with the enzyme's unique smell, a small amount of polymers are allowed. Solid dosage form: yellow-brown powder, easily soluble in water. With the enzyme's unique smell, no deliquescent agglomeration. 4.2 Physical and chemical hygiene requirements
Should meet the requirements of Table 1.
Approved by China Light Industry Association on June 20, 1997
1998-04- 01 Implementation
Enzyme activity preservation rate,
Enzyme heat resistance survival rate
95℃.60min
Drying weight loss,
Bulk density,
Preservation rate
Fineness [0.4mm (39 mesh standard sieve) passing rate],
Heavy metal (in terms of Pb),
Lead (in terms of Pb),
Arsenic (in terms of As Count),
Total colony count, pieces/mL(g)
Escherichia coli.MPN/100mL(g)≤
Salmonella (25g sample)
Mold,
Yeast,
pieces/mL(g)
pieces/mL(g)
Superior product
QB/T2306—1997
Liquid dosage form
First-class product
Enzyme activity
Store at 25℃
6 months
Store at 5℃
12 months
Labeled enzyme activity
Qualified product
20 000u/mL
Store at 25℃
3 months
1.15~1.25
5×104
3×103
Not detectable
Superior product
Solid dosage form
Enzyme activity
Store at 25℃Store at 25℃
3 months
Qualified products shall not be used in other food industries except for distilled liquor. ②In addition to 20 000u/mL (u/g), the enzyme activity specifications of the product can also be implemented according to the supply and demand contract. 5 Test method
6 months,
Store at 5℃
12 months
Labeled enzyme activity
20000u/g
Store at 25℃
6 months
5×104
3×103
Not detectable
The water used in this method is distilled water or deionized water. All reagents used are analytical grade unless otherwise specified. 5.1 Determination of enzyme activity
5.1.1 Spectrophotometer method (first method)
5.1.1.1 Principle
Qualified products
Store at 25℃
6 months
α-Amylase can randomly cut the α-1,4 glucosidic bonds in the starch molecular chain into short-chain dextrins of varying lengths, a small amount of maltose and glucose, making the starch react specifically to iodine with a blue-black color. As the starch degrades, the blue color gradually fades and turns into reddish brown. The speed at which the color disappears is related to the enzyme activity. Therefore, the enzyme activity can be calculated under standard conditions by measuring the photoluminescence of the solution after the reaction. 5.1.1.2 Reagents and solutions
a) Stock solution
Weigh 22.0g potassium iodide and dissolve it in 300ml water, add 11.0g iodine, stir to dissolve, then transfer to a 500ml volumetric flask, make up to volume with water, store in a brown bottle for later use (prepare once a month). b) Dilute iodine solution
Weigh 20.0g potassium iodide and dissolve it in about 300mL water, accurately add 2.00mL of the original iodine solution, transfer all to a 500mL volumetric flask, make up to volume with water, store in a brown bottle for later use (must be prepared on the same day). c) 20g/L soluble starch solution
Weigh 2.0000g of soluble starch (on an absolute dry basis) to an accuracy of 0.0001g, mix with water to make a slurry, slowly pour into 70mL boiling water while stirring, then rinse the small beaker containing starch with 30mL of water several times, add the washing liquid into it, heat and boil for 2min until completely transparent, cool to room temperature, transfer all to a 100mL volumetric flask, and dilute with water to the mark. This solution must be prepared on the same day. d) Phosphate buffer (pH-6.0)
Weigh 45.23g of sodium hydrogen phosphate (Na2HPO.·12H20) and 8.07g of citric acid (CHO,·H,O), dissolve in water and make up to 1000ml. After preparation, calibrate with a pH meter. e) Hydrochloric acid solution c (HC) = 0.1mol/, prepared according to GB601. 5.1.1.3 Instruments and equipment
Spectrophotometer;
Constant overflow water bath 50℃～100℃ with accuracy of ±0.1℃, test tube 25mm×200mm;
Volume flask,
Automatic pipette,
Stopwatch.
5.1.1.4 Preparation of enzyme solution to be tested
a) Liquid enzyme preparation: Prepare directly with buffer solution (5.1.1.2d) to control the final enzyme solution concentration within the range of 65~70u/mL. b) Solid enzyme preparation: Weigh 1~2g of enzyme powder with an accuracy of 0.0002g, first dissolve with a small amount of phosphate buffer, and pound with a glass stirring rod (or stir with a magnetic stirrer) until the solid is completely dissolved (about 10-15mm), carefully pour the supernatant into a volumetric flask, add a small amount of buffer to the residue, pound 3-4 times, and finally transfer all to a volumetric flask, dilute with buffer to the scale (to control the enzyme concentration within the range of 65-70u/mL), shake. Filter through four layers of gauze and collect the filtrate for determination. 5.1. 1.5 Determination
a) Absorb 20.01mL of soluble starch solution (5.1.1.2c) and 5.0mL of buffer (5.1.1.2.4) into a test tube, preheat and balance in a 70C constant temperature water bath for 5 minutes.
b) Add 1.00mL of the diluted enzyme solution to be tested (5.1.1.4), immediately record the time with a stopwatch, spread evenly, and react accurately for 5 minutes. c) Immediately use an automatic pipette to draw 1.00ml of reaction solution b) and add it to a test tube pre-filled with 0.5ml of 0.1mol/L hydrochloric acid and 5.00mL of dilute iodine solution (5.1.1.2b), and shake well.
d) Use a mixture of 0.5mL of 0.1mol/L hydrochloric acid and 5.00mL of dilute iodine solution as a reagent blank, and quickly measure its absorbance (A) at a wavelength of 660nrm using 10mm colorimetric blood. According to the absorbance, check Appendix A (Standard Appendix) to obtain the concentration (c) of the test enzyme solution. 5.1.1.6 Calculation
X=mcXnX16.67
Note: 1) Use the "compatible starch" produced by Jianjiang Linghu Food Chemical Joint Company, marked with the words "Specially for determination of transdermal preparations" and batch number. 100
(1)
Where: X-
QB/T 2306--1997
Enzyme activity of a sample, u/ml (u/g); concentration of the test enzyme, u/mL
nDilution factor of the sample;
16.67——conversion constant.
The result is expressed to the integer.
5.1.1.7 Allowable difference of results
The relative error of parallel tests shall not exceed 2%. 5.1.2 Visual colorimetry (second method)
5.1.2.1 Principle
Same as 5.1.1.1.
5.1.2.2 Reagents and solutions
a) Original iodine solution is the same as 5.1.1.2a.
b) Diluted iodine solution is the same as 5.1.1.2b.
c) 20g/L soluble starch solution is the same as 5.1.1.2c . d) Phosphate buffer (pH=6.0) Same as 5.1.1.2d. e) Standard endpoint color solution:
Solution A: Weigh 0.2439g of cobalt chloride (CoClz·6HzO) and 0.4878g of potassium dichromate, dissolve in water and make up to 500mL. Solution B: Weigh 40mg of chrome black T (C,OHz2N,N·O,S), dissolve in water and make up to 100mL. Solution C (standard endpoint color solution: Take 40mL of solution A and 5.0mL of solution B, mix thoroughly and set aside. This mixture should be stored in the refrigerator and needs to be re-prepared after 15 days.
5.1.2.3 Instruments and equipment
Warm water bath 50~100℃
Accuracy ±0.1℃,
Volumetric flask,
Test tube 25mmX200 mm;
stopwatch. bZxz.net
5.1.2.4 Analysis steps
Preparation of the enzyme solution to be tested: Prepare according to 5.1.1.4, but the final concentration of the solution should be slightly controlled within the range of 300~350u/mL. Pipette 6mL of the standard endpoint color solution into a test tube as a colorimetric standard. Pipette 20mL of 20g/L soluble gel solution and 5mL of pH 6.0 phosphate buffer solution into a 25mmX200mm test tube, preheat and equilibrate in a 70C constant temperature water bath for 5min, then add 0.5mL of the pre-diluted enzyme solution, immediately record the time with a stopwatch, shake well, and when the reaction is close to 2min, start to take out 1.00mL of the reaction solution from time to time with a pipette, and add it to a test tube pre-prepared with 5.00mL of dilute iodine (5.1.1.2b). When the color of the solution in the test tube changes from purple to reddish brown and gives the same color as the standard endpoint solution, it is the end point of the reaction, and the time required to reach the end point (minutes) is recorded. The total time of the enzyme reaction is controlled within 2~2.5min. 5.1.2.5 Calculate
±×20×20/1000Xn)-0.5×10*..Where: X is the enzyme activity of the sample, u/mL (u/g); T is the time required to reach the end point of the reaction, miR.
20—volume of soluble starch solution, mL;
20/1000-
concentration of soluble starch solution, g/mL;
1-sample dilution multiple;
103——1g is equal to 10°mg;
0.5-measurement enzyme solution dosage, mlL.
5.2 Determination of enzyme activity preservation rate
QB/T 2306 --- 1997
According to the method in Chapter 11 of QB/T1803. 5.3 Determination of enzyme heat resistance survival rate
5.3.1 Reagents and solutions
5.3.1.1 Sodium hydroxide solution: c (NaOH) = 0.1 mol/L Prepared according to GB601. 5.3.1.2 Dextrin solution: Weigh 100g of dextrin in a beaker, add 300mL of water, stir well, add 1300 units of thermostable α-amylase (13 units per gram of dextrin), heat on an electric stove until boiling, cool, adjust pH to 6.0-7.0 with 0.1mol/L sodium hydroxide solution, then transfer to a 500ml volumetric flask, dilute to the mark with water, shake well, and set aside. 5.3.2 Instruments and equipment
Acidity meter,
Constant temperature water bath 50~~100℃
Accuracy ±0.1℃;
Electric furnace 1000W;
Precision overflow meter with graduation value 0.1C;
Spectrophotometer;
Colorimetric tube 50ml.
Stopwatch.
5.3.3 Analysis steps
5.3.3.1 Preparation of test enzyme solution
When preparing the test enzyme solution, use dextrin solution (5.3.1.2) instead of buffer solution (5.1.1.2d). The control range of the final enzyme solution concentration is the same as that of 5.1.1.4 (or 5.1.2.4).
5.3.3.2 Heat treatment
Put 25mL of the enzyme solution to be tested into a 50mL colorimetric tube, place it in a 95C constant temperature water bath for heat treatment for 60min, cool it, make up the volume to the original enzyme solution with water, and shake it well.
5.3.3.3 Determination of enzyme activity
Use the method 5.1.1 (or 5.1.2) corresponding to 5.3.3.1. 5.3.3.4 Calculation of heat resistance survival rate
Where: X is the heat resistance survival rate of the sample, %; E, the enzyme activity measured after the sample is heat treated, u/mL (u/g) E-the enzyme activity measured before the sample is heat treated, u/mL (u/g). The results are expressed to integers.
5.4 Determination of pH value
Carry out the determination of pH value according to the method of QB/T1803. 5.5 Determination of loss on drying
According to the determination method of dry coal loss in QB/T1803. 5.6 Determination of bulk density
According to the determination method of bulk density in QB/T1803. 5.7 Determination of fineness
According to the determination method of fineness (particle) in QB/T1803. 5.8 Determination of heavy metals
According to the method of G8451.
(3)
5.9 Determination of lead
QB/T 2306-1997
According to the dithiamine monochromatic method or atomic absorption spectrophotometry in GB/T5009.12. Sample treatment adopts nitric acid-sulfuric acid method. 5.10 Determination
According to the silver salt method in GB/T5009.11. Sample treatment adopts nitric acid-sulfuric acid method. 5.11 Determination of total colony count
Perform according to GB4789.2.
5.12 Determination of coliform bacteria
Perform according to GB4789.3.
5.13 Inspection of Salmonella
Perform according to GB4789.4.
5.14 Determination of body bacteria and yeast
Perform according to GB4789.15 method.
6 Inspection rules
According to Chapter 2 of QB/T1840.
Marking, packaging, transportation and storage
According to Chapter 3 of QB/T1840.
Absorbance (A)
QB/T2306-1997
Appendix A
(Appendix of the standard)
Table A1 Comparison table of absorbance and enzyme concentration of tested α-amylase Enzyme concentration (c)
Absorbance (A)
Enzyme concentration (c)
Absorbance (A)
Enzyme concentration (c))
Absorbance (A)
Enzyme concentration (c)
QB/T 2306—1997
Continued Table A1
Absorbance (A)
Enzyme concentration (c)
Absorbance (A)
Acid concentration (c)
Absorbance (A)
Phosphorus concentration (c)
QB/T 2306 --- 1997
Continued Table A1
Luminosity (A)
Enzyme concentration (e)
Luminosity (A)
Enzyme concentration (e)
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