Some standard content:
QB/T2491--2000
Isomaltose is a kind of functional oligosaccharide, and its main functional components are isomaltose, panose, isomaltotriose and low sugars above tetrasaccharide.
In the test method of this standard, the sensory requirements of dry matter (solids), pH and transmittance are determined by QB/T23191997 "Liquid Glucose"; the determination of water content and solubility is determined by QB/T 2320-1997 "Maltodextrin". Glucose transaminase is one of the important raw and auxiliary materials. There is no unified method for the determination of activity, and the quality cannot be correctly evaluated. Therefore, the definition and determination method of glucose transaminase are given in Appendix A. Appendix A of this standard is a suggestive appendix. This standard is proposed by the State Bureau of Light Industry.
This standard is under the jurisdiction of the National Food Standardization Center. The drafting units of this standard are: China Food Fermentation Industry Research Institute, Jiangsu Institute of Microbiology, Shandong Huanyu Group Co., Ltd., Shanghai Zhengguanghe Portuguese Pharmaceutical Sugar Co., Ltd., Xinjiang Zongheng Co., Ltd., Shudongtian Green Source Bioengineering Co., Ltd. The main drafters of this standard are: Bao Yuanxing, Guo Xinguang, Yang Weiya, Liu Zongli, Ding Shengjin, Sun Zhaolong, Liu Yong. 205
1 Scope
Light Industry Standard of the People's Republic of China
Isomaltooligosaccharides
QB/T 2491--2000
This standard specifies the definition and classification, technical requirements, test methods, inspection rules and marking, packaging, transportation and storage requirements of isomaltooligosaccharides.
This standard applies to IMO-50 type isomaltooligosaccharides produced by enzymatic method using starch as raw material. 2 Referenced Standards
The provisions contained in the following standards constitute the provisions of this standard through reference in this standard. When this standard is published, the versions shown are valid. All standards are subject to revision, and parties using this standard should explore the possibility of using the latest versions of the following standards. GB191-2000 Pictorial marking on packaging
GB/T601-1988 Preparation of standard solutions for titration analysis (volume analysis) of chemical reagents GB/T603-1988 Preparation of preparations and products used in test methods for chemical reagents GB4789.2-1994 Microbiological examination of food hygiene Determination of total colony count GB4789.3-1994 Microbiological examination of food hygiene Determination of coliform group GB 4789.4--1994
Microbiological examination of food hygiene Test for salmonella GB/T5009.11-1996 Method for determination of total arsenic in food GB/T 5009.12—1996
Determination method of lead in food
GB/T6682—1992Specifications and test methods for water for analytical laboratoriesGB7718—1994General standard for food labelingQB/T2319~1997Liquid glucose
QB/T 2320-1997
3Definition and classification
3.1Definition
Maltodextrin
3.1.1Isomaltooligosaccharide, abbreviated as IMO, is a type of starch sugar, the main components of which are isomaltose (TG2), panose (P), isomaltotriose (IG:) and low mulberry sugar with tetrasaccharides or above (G,) linked by α-1,6 glycosidic bonds.
3.1.2IMO-50 type
IG2+P+IG:+G,≥50% (total sugar) products. 3.2 Classification
Divided into syrup and powdered sugar according to the form.
4 Technical requirements
4.1 Sensory requirements
Syrup is a colorless or light yellow, transparent viscous liquid. It has a mild sweet taste, no peculiar smell, and no impurities visible to the naked eye. Approved by the State Bureau of Light Industry on October 31, 2000
Implemented on April 1, 2001
QB/T 2491--2000
Powdered sugar is a white amorphous powder. It has a mild sweet taste, no peculiar smell, and no impurities visible to the naked eye. 4.2 Physical and chemical requirements
Should meet the requirements of Table 1.
Moisture, %
Dry matter (solids), %
Transmittance, %
Solubility, %
Dextrin, %
Sulfated ash, %
IMO content (total sugar), %
IGz+P+IG content (total sugar), %
4.3 Hygiene requirements
Shall comply with the requirements of Table 2.
Monument (as As), mz/kg
Lead (as Pb), mg/kg
Total colony count (cfu/or mL)
Eliform group, MPN/(100g or 100mL)
Pathogenic bacteria (Salmonella)
Test method
Shall not be detected
The water used in the test method shall comply with the water of Grade III (including Grade III) or above in GB/T6682, and all reagents used shall be analytical grade unless otherwise specified.
5.1 Infection test
5.1.1 Syrup
Put 30ml of the sample in a colorless, clean and dry sample cup (or a 50mL small beaker) in a bright place, use your mouth to observe its color and density, check whether there are any impurities visible to the naked eye, and use a glass rod to take an appropriate amount of sample and put it in your mouth to taste its taste (before tasting the second sample, you must rinse your mouth with water). Keep a good record. 5.1.2 Powdered sugar
Take an appropriate amount of sample, and observe the color and shape of the sample under natural light, whether there are any impurities: take a small amount of sample, put it in your mouth, taste it carefully (before tasting the second sample, you must rinse your mouth with water), and keep a good record. 5.2 Water
Determine according to 6.2.1 of QB/T2320--1997. 207
5.3 Dry matter (solid content)
QB/T 2491—2000
Determined according to 5.2.2 of QB/T2319-1997. 5.4 pH
Determined according to 5.2.3 of QB/T2319-1997. 5.5 Transmittance
Determined according to 5.2.4 of QB/T2319-1997. 5.6 Solubility
Determined according to 6.2.3 of QB/T 2320--1997. 5.7 Dextrin
5.7.1 Method Summary
After dextrin is hydrolyzed into dextrose by hydrochloric acid, it is directly titrated with the calibrated Fehling solution under heating conditions, using methylene blue as an indicator. According to the consumed volume of the sample solution, the dextrose content is calculated and then converted into dextrin content. 5.7.2 Reagents
5.7.2.11 g/L glucose standard solution: Accurately weigh 1 g of standard anhydrous glucose dried to constant weight at 100°C ± 2°C, weigh to 0.0001 g, add water to dissolve, then add 5 mL of hydrochloric acid, transfer to a 1000 mL volumetric flask and dilute to the scale, spread evenly, and set aside. 5.7.2.2 Fehling A solution: Weigh 15 g of copper sulfate and 0.05 g of methylene blue, add water to dissolve, transfer to a 1000 mL volumetric flask and dilute to the scale with water, shake the hook, let it stand for two days, and then filter into a brown bottle. 5.7.2.3 Zanlin B solution: Weigh 50g potassium sodium tartrate and 75g sodium hydroxide, dissolve in water, then add 4g potassium ferrocyanide. After complete dissolution, transfer to a 1000mL volumetric flask and dilute to the scale with water. Store in a rubber-stoppered glass bottle. 5.7.2.4 Anhydrous ethanol
5.7.2.5 80% ethanol aqueous solution: prepare with anhydrous ethanol; 5.7.2.6 Concentrated hydrochloric acid;
5.7.2.7 200g/L sodium hydroxide solution: Weigh 20g sodium hydroxide and make up to 100mL with water. 5.7.3 Analysis steps
5.7.3.1 Standardization of Fehling's solution
First, take 5.0 mL of Fehling's solution B and then 5.0 mL of Fehling's solution A into a 150 mL conical flask, add 10 mL of water, add 3 glass beads, and heat on an electric stove covered with asbestos mesh. Control the liquid in the flask to boil within 120s±15s and keep it boiling slightly. Titrate the glucose standard solution until it becomes colorless. The entire titration operation should be completed within 3 minutes. Record the volume of glucose standard solution consumed. 5.7.3.2 The mass of glucose equivalent to 5 mL of Fehling's solution A and B is calculated according to formula (1): RP=mXu
Where: RP. The mass of glucose equivalent to 5 mL of Fehling's solution A and B, g; m1--the amount of standard anhydrous glucose weighed, g; --the volume of glucose standard solution consumed, mL; 1000--the total volume of glucose standard solution prepared, mL. 5.7.3.3 Preparation of sample
: (i)
Accurately weigh 1~2g of sample to the nearest 0.01g, put it in a beaker, dissolve it with 16mL warm water, slowly add 64mL of anhydrous ethanol under stirring, leave it for more than 12h to precipitate dextrin, filter it with filter paper, and wash the filter paper and precipitate with a small amount of 80% ethanol twice. Wash the filter paper and precipitate with 70ml. hot water into a 250mL conical flask, add 5mL of concentrated hydrochloric acid, and cook it in a pressure cooker at 121℃ (100kPa pressure) for 30min to hydrolyze dextrin into glucose. After natural cooling, take it out, filter it, neutralize it with 200g/L sodium hydroxide solution, use pH test paper to judge the end point, wash it into a 250mL volumetric flask, and dilute it to the scale with water. 5.7.3.4 Determination of sample
Replace the glucose standard solution with the sample, and titrate it according to 5.7.3.1 to determine its glucosamine content. 208
5.7.3.5 Expression of analysis results
The dextrin content of the sample is calculated according to formula (2):
QB/T 2491--2000
X100X0. 9
m2×250
Wherein: X,——dextrin content of the sample, g/100g; RP-Fehling solution A, B 5mL each is equivalent to the mass of glucose, nominal?m2——sampling volume g;
250——total volume of sample solution prepared, mL; V2——volume of sample consumed in titration, mL; 0.9——conversion coefficient
The calculation result is rounded to one decimal place.
5.7.3.6 Permissible difference
The difference between two determination results of the same sample shall not exceed 5% of the average value. 5.8 Sulfated ash
Determine according to 5.2.8 of QB/T 2319-1997. 5.9 IMO content
5.9.1 High performance liquid chromatography (HPLC)
5.9.1.2 Method summary
Each component entering the chromatographic column at the same time is repeatedly distributed between the two phases of the chromatographic column with the mobile phase due to the different dissolution, adsorption, penetration or ion exchange between the mobile phase and the stationary phase. Due to the different moving speeds of each component in the chromatographic column, after passing through a certain length of the chromatographic column, they are separated from each other, flow out of the chromatographic column in sequence, enter the signal detector, and the peak value of each component is displayed on the recorder or data processing device. According to the retention time, the normalization method or external standard method is used for quantification. 5.9.1.3 Instruments
5.9.1.3.1 High performance liquid chromatograph (equipped with a differential refractometer and a column thermostat system), 5.9.1.3.2 Vacuum filtration and degassing device for mobile phase and 0.2μm and 0.45um microporous membranes; 5.9.1.3.3 Chromatographic columns
a) Calcium type cation exchange resin column: Aminex HPX-42A (BIO-RAD), filler particle size: 5um; column size; Φ7.8mm×300mm, or other chromatographic columns with similar analytical effects; b) Amino bonded column: TSKgelAmide-80, filler particle size: 5μm; column size: Φ4.6mm×250mm or other chromatographic columns with similar analytical effects;
5.9.1.3.4 Analytical balance: sensitivity 0.0001g; 5.9.1.3.5 Micro-injector: 10L.
5.9.1.4 Reagents
5.9.1.4.1 Water: double distilled water or ultrapure water; 5.9.1.4.2 Acetonitrile: chromatographic grade;
5.9.1.4.3 Standard products of glucose, maltose, isomaltose, maltotriose, panose, isomaltotriose, maltotetraose, isomaltotetraose, maltopentaose, and maltohexaose are all Sigma reagents. Prepare 0.5% aqueous solution with water respectively. 5.9.1.5 Analysis steps
5.9.1.5.1 Double column method (arbitration method)
a) Sample preparation
Weigh 5g bran slurry or sugar powder sample, weigh to 0.0001g, add water to dissolve, transfer to a 100mL volumetric flask and dilute to the mark with water, filter with a 0.2μm or 0.45um aqueous microporous membrane, and set aside the filtrate. 209
b) Sample determination
QB/T2491—2000
Calcium type cation exchange resin column: The mobile phase is pure water. The day before the determination, turn on the power of the differential refractive index detector, preheat and stabilize it, install the chromatographic column, adjust the column temperature to 85℃, and let the mobile phase flow through at a flow rate of 0.1ml/min to equilibrate overnight. Before the formal injection analysis, the mobile phase used is introduced into the reference cell for more than 20 minutes, and then the normal flow path is restored to allow the mobile phase to pass through the sample cell, and the flow rate is adjusted to 0.6mL/min, and the baseline is taken. After the baseline is stable, the sample can be injected, and the injection volume is 5~~10μL. The standard solutions of glucose, maltose, maltotriose, maltotetraose, maltopentaose, and maltohexaose and the prepared samples are injected respectively. According to the retention time of the standard, the chromatographic peaks of various sugar components in the sample are qualitatively identified. According to the peak area of the sample, the percentage content of various sugar components is calculated by the normalization method.
base bonded column: the mobile phase is acetonitrile: water = 67:33. The power of the differential refractive index detector is turned on the day before the determination, preheated and stabilized, the chromatographic column is installed, the column temperature is adjusted to 75℃, and the mobile phase is passed through at a flow rate of 0.1mL/min for equilibrium overnight. Before formal sample injection analysis, the mobile phase used is introduced into the reference cell for more than 20 minutes, and then the normal flow path is restored to allow the mobile phase to pass through the sample cell, and the flow rate is adjusted to 1.0mL/min, and the baseline is taken. After the baseline is stable, the sample can be injected, and the injection volume is 5-10μL. The standard solutions of glucose, maltose, maltotriose, isomaltose, panose, isomaltotriose, maltotetraose, isomaltotetraose, maltopentaose, and maltohexaose and the prepared samples are injected separately. The chromatographic peaks of various sugar components in the sample are qualitatively determined according to the retention time of the standard. According to the peak area of the sample, the percentage content of various sugar components is calculated by normalization method. c) Expression of analysis results
Calcium type cation exchange resin column, sample component DP: the percentage of total sugars is calculated according to formula (3): DP,=
Wherein: DP, is the percentage of component i in the sample in the total sugars, %, A,—the peak area of component i in the sample,
ZA;-——the sum of the peak areas of each component in the sample. Amino bonded column, the percentage of glucose in the sample to the total sugar is calculated according to formula (4): G=DP
The percentage of isomaltose in the sample to the total sugar is calculated according to formula (5): AG2
IG,Acz+Aice
The percentage of panose in the sample to the total sugar is calculated according to formula (6): Ap
P=Aca+Ap+Aics
The percentage of isomaltotriose in the sample to the total sugar is calculated according to formula (7): AiG3
AG3+Ap+AiG3
The percentage of tetrasaccharides and above in the sample to the total sugar is calculated according to formula (8): G,=100-DP1-DP2-DP:
Wherein: G(DP,)
The percentage of glucose in the sample to the total sugar, %; IG2 --
The percentage of isomaltose in the sample to the total sugar content, %; AIG2—-The peak area of isomaltose in the sample; Ac2
The peak area of maltose in the sample;
The percentage of disaccharides in the sample to the total sugar content, %; P—-The percentage of panose in the sample to the total sugar content, %; Ap
-The peak area of panose in the sample;
Ac3——-The peak area of maltotriose in the sample; 210
（3）
（4）
（5）
（6）
QB/T 2491--2000
AIG3—Peak area of isomaltotriose in the sample; DP:-—Percentage of trisaccharides in the total sugar content of the sample, %; Percentage of isomaltotriose in the total sugar content of the sample, %; IG3
G—Percentage of tetrasaccharides and above in the total sugar content of the sample, %. The calculation result is rounded to the nearest integer.
d) Allowable difference
The difference between two determination results of the same sample shall not exceed 5% of the average value. 5.9.1.5.2 Single column method
This method is applicable to low-saturation isomaltose with very low penta-, hexa-saccharide and dextrin content. a) Sample preparation
Same as 5. 9.1.5.1a).
b) Determination of samples
1) Standard solution and standard curve
Use the standard of each sugar to prepare 6 standard solutions with different concentrations in the range of 0.5-10 mg/mL. After injection, draw a standard curve based on the standard concentration versus the peak area. The linear correlation coefficient should be above 0.9990. 2) Determination of samples
Same as 5.9.1.5.1b) Determination of amino bonded column, and finally calculate the percentage of various sugar components by external standard method. c) Expression of analysis results
The content of various sugars in the sample is calculated according to formula (9): A x
The percentage of a certain sugar in the sample, g/100g (or mL); where ·X.—
A,-——The peak area of a certain sugar in the sample; m.
-The mass of a certain sugar standard in the standard sample, g; V.—The dilution volume of the standard sample, mL;
A, The peak area of a certain sugar standard in the standard sample; m—The mass of the sample·g;
V—The dilution volume of the sample, mL.
The calculation result is retained to the integer.
d) Permissible difference
The difference between the two determination results of the same sample shall not exceed 5% of the average value. 5.9.2 Thin layer chromatography
5.9.2.1 Apparatus
5.9.2.1.1GF254 silica gel plate: specification 100×100mm;5. 9. 2. 1.2
Chromatography cylinder: 100mmX200mm;
5.9.2.1.3
Micro-injector: 5μl
5.9.2.1.4 Oven;
5.9.2.1.5
CS-930 dual-wavelength chromatographic scanner;
Dryer: use color-changing silica gel as desiccant;5. 9.2.1. 6
Electric hair dryer: 300～500W;
5. 9. 2. 1.7
5.9.2.1.8 Culture m: 15cm.
5.9.2.2 Reagents
QB/T 2491-2000
5.9.2.2.1 Standards of glucose, maltose, isomaltose, maltose, panose, maltose, maltotetraose, isomaltotetraose and maltopentaose are all Siga reagents:
5. 9. 2.2. 285% phosphorus
5.9.2.2.3 Developing agent, n-butanol: glacial acetic acid*water = 2#1:15.9.2.2.4 Color developer A solution: 4g diphenylamine + 100mL diketone, 5.9.2.2.5 Color developer B solution: 4mL aniline + 100mL diketone; before color development, add 20mL of 85% phosphoric acid to 100mL of A solution and 100mL of B solution, shake while adding to dissolve all the sediments, and prepare the color developer solution for use.
5.9.2.3 Preparation of standard solutions
5.9.2,3.1 Standard solution 1: Prepare 10g/1 of each standard substance (5.9.2.2.1). 5. 9.2.3.2 Standard bath solution I: Mix equal volumes of standard solutions of maltose, maltose, maltotriose, maltotetraose and maltopentaose (ml). 5.9.2.3.3 Standard solution 1: Mix equal volumes of standard solutions of maltose, isomaltose, maltotriose, isomaltotriose and panose (m2). 5.9.2.4 Analysis steps
a) Place the GF254 silica gel plate in a 105℃ oven for activation for 1.5h, and cool it to room temperature in a dryer. 6) Prepare the sample into a 10~20g/L solution, and use a microinjector to spot 0.51L at a distance of 1cm from the edge of the silica gel plate. Use the same method to spot ml and m2 standard solutions. c) Pour the developing agent into the chromatography cylinder, place the silica gel plate in the chromatography cylinder for development, and after development, place it in the fume hood to dry in the shade or use a hair dryer to dry it. Repeat the development twice (a total of 10 times). d) Immerse the silica gel plate in the color developer, take it out immediately, wipe off the color developer on the back, and blow it dry. Then put the silica gel plate in an 80℃ oven for 15min~30min to develop color.
e) Scan the colored silica gel plate with a CS-930 dual-wavelength chromatographic scanner at 640nm, and quantify the standard according to the retention time.
Determine according to GB/T 5009.11.
Determine according to GB/T 5009.12.
5.12 Total colony count
Determine according to GB/T 4789.2
5.13 Coliform bacteria
Determine according to GB/T4789.3.
5.14 Salmonella
Determine according to GB/T 4789. 4.
6 Inspection rules
6.1 Products are batched at one time, and the maximum batch shall not exceed the shift output. 6.2 Each batch of products must be inspected by the inspection department of the manufacturer before leaving the factory, and a product quality certificate must be attached. 6.3 Sampling method
6.3.1 Samples are drawn according to the provisions of Table 3 and Table 4. 212
Batch model, box
100 or less
100~250
251~500
500 or more
Batch range, barrel
50 or less
50~100
100 or more
6.3.2 Each tank truck must be inspected for products.
QB/T2491—2000
Number of samples to be drawn, box
Number of unit packages to be drawn, bottle
Number of samples to be drawn, barrel
6.3.3 Samples for barreled and tank truck products must be drawn from 10 cm below the liquid surface. The sampler must meet food hygiene standards. 6.3.4 The sample volume of each tank truck product shall not be less than 2 kg, the sample volume of each barrel product shall not be less than 1 kg, and the total sample volume of bottled products shall not be less than 600 g.
6.3.5 After mixing the drawn samples, divide them into two parts and seal them. Paste labels and indicate the product name, manufacturer name and address, batch number, sampling date and location, and sampler name on the labels. One part will be sent to the laboratory for inspection, and the other will be sealed and kept for half a month for future reference. When microbiological testing is required, the sampler and glass bottle should be sterilized in advance (the sample must not touch the bottle mouth). 6.4 Factory inspection
Sensory properties, moisture, dry matter (solids), pH, transmittance, solubility, dextrin, IG2+P+IG:, IMO content. 6.5 Type inspection
6.5.1 In addition to the factory inspection items, it also includes sulfated ash, stele, lead, total colony count, coliform group and pathogenic bacteria. 6.5.2 Under normal circumstances, formal inspection needs to be carried out once a quarter. It is also required in one of the following situations. a) Changes in major raw and auxiliary materials;
b) Changes in key processes and equipment;
c) When new trial-produced products or normal production products are suspended for more than 3 months and then resumed, d) When the national quality supervision agency conducts random inspections. 6.6 Judgment rules
6.6.1 If the inspection results show that the sensory or 1~2 physical and chemical indicators are unqualified, the batch of products can be doubled and sampled for re-inspection. If one of the re-inspection results is unqualified, the batch of products will be unqualified. 6.6.2 If one of the hygiene indicators is unqualified, the batch of products will be judged as unqualified and shall not be re-tested. 7 Marking, packaging, transportation and storage
7.1 The label of products for direct consumption must comply with the provisions of GB7718. 7.2 The packaging storage and transportation graphic signs should comply with the provisions of GB191. The outside of the container must be marked with the product name, manufacturer name, factory address, net content, batch number, production date, shelf life, implementation standard number and quality grade. 7.3 The packaging and containers must be clean, hygienic, without damage, and comply with the relevant provisions of the "Food Hygiene Law of the People's Republic of China". 7.4 During transportation, dust and flies must be prevented, and exposure to the sun and rain must be strictly prevented. It is strictly forbidden to mix and transport with toxic and harmful substances. Measures should be taken to prevent exposure to sunlight and rain. Loading and unloading during transportation should comply with the provisions of the packaging storage and transportation diagram on the outer packaging. 7.5 Finished products should be stored in a dry, ventilated and clean warehouse, and the principle of first-in-first-out should be followed. 7.6 Product shelf life: syrup should not be less than 6 months in the first and fourth quarters, not less than 3 months in the second and third quarters, and not less than 6 months in powdered sugar. 213
A1 Definition
QB/T2491-2000
Appendix A
(Appendix for illustration)
Definition and determination of glucosyltransferase activity 1mL of liquid enzyme, at 40C+PH=5.0, decomposes α-amino-D-glucosidase for 1h to produce 1 glucosidase as an enzyme activity unit, expressed as u/mL.
A2 Test method
A2.1, Method summary
Glucosyltransferase acts on the substrate α-methyl-D-glucose to generate D-glucose and methanol. The content of D-glucose can be determined by a color reaction with 4-aminoantipyrine and phenol reagent containing glucose oxidase and peroxidase. α-Methyl-D-glucose. Glucose transamination, D-glucose + methanol glucose amination, gluconic acid + H,02
D-glucose-
peroxidase = benzophenone
H,O4-aminoantipyrine phenol
A2.2 Instrument
A2.2.1 Oven: control accuracy ±2℃;
A2.2.2 Overflow water bath, accuracy 0.1℃
A2.2.3 Spectrophotometer: wavelength 360~800mm, A2.2.4 Test tube: Φ15mmX150mm.
A2.3 Reagents
A2.3.1 Anhydrous glucose fondant, analytical grade;
A2.3.2 20g/L α-methyl-D-glucose glycoside solution: weigh 2g α-methyl-D-glucose glycoside solution to 0.1g, add water to dissolve, and dilute to 100mL. Place in refrigerator (515℃) for 2 weeks before use; A2.3.3 4g/L 4-aminoantipyrine solution: weigh 0.2g 4-aminoantipyrine, add 10~20mL water to dissolve, and then dilute to 50mL with water
A2.3.4 2mol/L caustic soda solution: prepare according to GB601; A2.3.5 1mol/L sodium nitrite solution: prepare according to GB601 A2.3.6 Tris-phosphate buffer (pH=7.2): weigh 36.3g 2-amino-2-hydroxymethyl-1,3-propanediol LNH,C(CH2OH), J and 50.0g sodium dihydrogen phosphate (NaHzPO 4.2H2O) in beaker A, add about 900mL of water to dissolve it, adjust the pH to 7.2 with 2mol/L hydrochloric acid solution (if excessive amount is added, adjust it back with 1mol/L NaOH solution), transfer this solution into a 1000mL volumetric flask, and make up to volume with water: A2.3.750g/L phenol solution: weigh 5g phenol in a beaker, weigh to 0.01g, add 50mL of water, heat to dissolve, cool to room temperature, transfer into a 100rL volumetric flask, and make up to volume with water; A2.3.84-aminoantipyrine solution-phenol colorimetric solution: weigh 12.5mg of glucose loading enzyme and phosphine peroxide in a beaker, add about 40mL Tris-phosphate buffer (pH=7.2), dissolve it, then add 1mL 4-aminoantipyrine solution (A2.3.3) and 1,4mL phenol solution (A2.3.7), transfer this solution into a 50mL volumetric flask, and dilute to volume with Tris-phosphate buffer A2.3.9 Acetic acid-sodium acetate buffer solution (pH=5.0): Prepare according to GB/T603. A2.4 Analysis steps
A2.4.1 Preparation of sample
Absorb 1mL of glucose transaminase and dilute to 500mL with water. 214
A2.4.2 Preparation of standard curve
QB/T 2491-
—2000
A2.4.2.1 Weigh 1g of anhydrous glucose dried to constant weight at 105°C±2°C, weigh to 0.0001g, dissolve in water, and make up to 100ml. Take 1.0, 2.0, 3.0, 4.0, 5.0mL of this solution respectively, and make up to 100mL. This solution is equivalent to glucose solution containing 0.1, 0.2, 0.3, 0.4 and 0.5 μg/L.
A2.4.2.2 Take 0.1ml of the above glucose solution in 5 test tubes, add 3mL of 4-amino-antipyrine solution-phenol colorimetric solution (A2.3.8), and after sufficient mixing, accurately keep warm in a warm water bath at 40C±0.5℃ for 20min, then take it out and immediately cool it to room temperature. Use water as a control and measure its absorbance (Am10, As20~A30~Aa40As50) at a wavelength of 500nm. At the same time, use water instead of glucose solution for a blank test and measure its absorbance (A). A2.5 Expression of analytical results
When the absorbance difference is 1.000, the glucose micro-glucose obtained from the glucose standard curve is calculated according to formula (A1): 20
A0-Ago
A30~Ag.
A.40-As0 A50-A0
（A1）
Wherein: G—the amount of medicinal sugar obtained from the glucose standard curve when the absorbance difference is 1.000, μgAs10, Am20~As30, A40, A50~glucose—the absorbance when the concentration of glucose solution is 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L and 0.5g/L respectively;
A2.6 Determination of sample
Absorbance of blank solution. www.bzxz.net
A2.6.1 Add 1mL of methyl-D-glucoside bath solution and 1mL of acetic acid-sodium acetate buffer solution (A2.3.9) to a test tube, place it in a constant temperature water bath at 40℃0.5℃, and place it for 1015min. Then, add 0.5mL of sample solution (A2.4.1), shake it thoroughly, and place it in a warm water bath at 40℃0.5C for 60min. Then, move it to a boiling water bath and heat it for 5min, and cool it in running water. A2.6.2 Take 0.1mL of the above reaction solution in a test tube, and perform the same operation as A2.4 and 2 to determine its aurora (A). A2.6.3 Blank determination:
Add 1mL of acetic acid-sodium acetate buffer solution (A2.3.9) and 0.5mL of sample solution to a test tube, heat it in a boiling water bath for 5min, and cool it in running water. Add 1mL of 1-methyl-D-glucoside solution, and then perform the same operation as A2.4.2 to determine its absorbance (A.).
A2.6.4 Expression of analysis results:
The activity of glucosyltransferase is determined according to formula (A2): 2.5
X -(AA)XGX
=(A,-A)XGX50Xn
Wherein: X-glucosyltransferase activity, u/mL; A. absorbance of sample reaction solution;
A.-absorbance of blank solution;
2.5-volume of reaction system, m.,
0.1-sample volume of reaction solution, mL
-sample dilution multiple.
The obtained result is retained to an integer.
(A2)2 Take 0.1ml of the above glucose solution in 5 test tubes, add 3mL.4-amino-antipyrine solution-phenol colorimetric solution (A2.3.8), after fully mixing, accurately keep warm in a warm water bath at 40C±0.5℃ for 20min, then take it out and immediately cool it to room temperature. Use water as a control and measure its absorbance (Am10, As20~A30~Aa40As50) at a wavelength of 500nm. At the same time, use water instead of glucose solution for a blank test and measure its absorbance (A) A2.5 Expression of analytical results
When the absorbance difference is 1.000, the glucose micro-glucose obtained from the glucose standard curve is calculated according to formula (A1): 20
A0-Ago
A30~Ag.
A.40-As0 A50-A0
（A1）
Wherein: G—the amount of medicinal sugar obtained from the glucose standard curve when the absorbance difference is 1.000, μgAs10, Am20~As30, A40, A50~glucose—the absorbance when the concentration of glucose solution is 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L and 0.5g/L respectively;
A2.6 Determination of sample
Absorbance of blank solution.
A2.6.1 Add 1mL of methyl-D-glucoside bath solution and 1mL of acetic acid-sodium acetate buffer solution (A2.3.9) to a test tube, place it in a constant temperature water bath at 40℃0.5℃, and place it for 1015min. Then, add 0.5mL of sample solution (A2.4.1), shake it thoroughly, and place it in a warm water bath at 40℃0.5C for 60min. Then, move it to a boiling water bath and heat it for 5min, and cool it in running water. A2.6.2 Take 0.1mL of the above reaction solution in a test tube, and perform the same operation as A2.4 and 2 to determine its aurora (A). A2.6.3 Blank determination:
Add 1mL of acetic acid-sodium acetate buffer solution (A2.3.9) and 0.5mL of sample solution to a test tube, heat it in a boiling water bath for 5min, and cool it in running water. Add 1mL of 1-methyl-D-glucoside solution, and then perform the same operation as A2.4.2 to determine its absorbance (A.).
A2.6.4 Expression of analysis results:
The activity of glucosyltransferase is determined according to formula (A2): 2.5
X -(AA)XGX
=(A,-A)XGX50Xn
Wherein: X-glucosyltransferase activity, u/mL; A. absorbance of sample reaction solution;
A.-absorbance of blank solution;
2.5-volume of reaction system, m.,
0.1-sample volume of reaction solution, mL
-sample dilution multiple.
The obtained result is retained to an integer.
(A2)2 Take 0.1ml of the above glucose solution in 5 test tubes, add 3mL.4-amino-antipyrine solution-phenol colorimetric solution (A2.3.8), after fully mixing, accurately keep warm in a warm water bath at 40C±0.5℃ for 20min, then take it out and immediately cool it to room temperature. Use water as a control and measure its absorbance (Am10, As20~A30~Aa40As50) at a wavelength of 500nm. At the same time, use water instead of glucose solution for a blank test and measure its absorbance (A) A2.5 Expression of analytical results
When the absorbance difference is 1.000, the glucose micro-glucose obtained from the glucose standard curve is calculated according to formula (A1): 20
A0-Ago
A30~Ag.
A.40-As0 A50-A0
（A1）
Wherein: G—the amount of medicinal sugar obtained from the glucose standard curve when the absorbance difference is 1.000, μgAs10, Am20~As30, A40, A50~glucose—the absorbance when the concentration of glucose solution is 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L and 0.5g/L respectively;
A2.6 Determination of sample
Absorbance of blank solution.
A2.6.1 Add 1mL of methyl-D-glucoside bath solution and 1mL of acetic acid-sodium acetate buffer solution (A2.3.9) to a test tube, place it in a constant temperature water bath at 40℃0.5℃, and place it for 1015min. Then, add 0.5mL of sample solution (A2.4.1), shake it thoroughly, and place it in a warm water bath at 40℃0.5C for 60min. Then, move it to a boiling water bath and heat it for 5min, and cool it in running water. A2.6.2 Take 0.1mL of the above reaction solution in a test tube, and perform the same operation as A2.4 and 2 to determine its aurora (A). A2.6.3 Blank determination:
Add 1mL of acetic acid-sodium acetate buffer solution (A2.3.9) and 0.5mL of sample solution to a test tube, heat it in a boiling water bath for 5min, and cool it in running water. Add 1mL of 1-methyl-D-glucoside solution, and then perform the same operation as A2.4.2 to determine its absorbance (A.).
A2.6.4 Expression of analysis results:
The activity of glucosyltransferase is determined according to formula (A2): 2.5
X -(AA)XGX
=(A,-A)XGX50Xn
Wherein: X-glucosyltransferase activity, u/mL; A. absorbance of sample reaction solution;
A.-absorbance of blank solution;
2.5-volume of reaction system, m.,
0.1-sample volume of reaction solution, mL
-sample dilution multiple.
The obtained result is retained to an integer.
(A2)
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