This standard specifies the determination of vitamin B6 in food by microbiological method. This standard is applicable to the determination of vitamin B' in various foods. This standard is also applicable to the determination of vitamin B6 in feed. The detection limit of this method is 0.1ng, and the linear range is 0.1ng-6ng. GB/T 5009.154-2003 Determination of vitamin B6 in food GB/T5009.154-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.154—2003
Replaces GB/T17407-1998
Determination of vitamin B6 in foods
Determination of vitamin B in foodsPublished on August 11, 2003
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
Implemented on January 1, 2004
GB/T5009.154—2003
This standard corresponds to AOAC.45 Vitamins and Nutrients Part 43.229 Microbiological Determination of Vitamin B6 in Foods (1995 Edition).
The consistency between this standard and AOAC43.229 is non-equivalent. This standard replaces GB/T17407-1998 "Determination of Vitamin B6 in Food". This standard modifies the structure of the original standard according to GB/T20001.4-2001 "Standard Abbreviation Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting unit of this standard is: Institute of Nutrition and Food Hygiene, Chinese Academy of Preventive Medicine. The main drafters of this standard are: Shen Xiang, Zhou Ruihua, Yang Xiaoli, Wang Guangya. The original standard was first issued in 1998, and this is the first revision. 292
1 Scope
Determination of Vitamin B6 in Food
This standard specifies the determination of Vitamin B6 in food by microbiological method. This standard is applicable to the determination of Vitamin B6 in various foods. This standard is also applicable to the determination of Vitamin B6 in feed. The detection limit of this method is 0.1ng, and the linear range is 0.1ng～6ng. 2 Principle
GB/T5009.154—2003
The growth of a certain kind of bacteria in food must have the presence of a certain kind of vitamin. Carlsberg (Saccharomyces Carlsbrgensis) can only grow in the presence of vitamin B. Under certain conditions, the amount of vitamin B. is proportional to its growth. The turbidity of the bacteria in the sample solution is determined by turbidimetry, and the content of vitamin B. in the sample is obtained by comparing it with the standard curve. 3 Reagents
3.1 Agar.
3.2 Pyridoxine Y culture medium.
3.3 0.22mol/L sulfuric acid solution, add 700mL water and 12.32mL sulfuric acid (HzSO) to a 2000mL beaker, and dilute to 1000mL with water.
3.40.5mol/L sulfuric acid solution, add 700mL water to a 2000mL beaker, and dilute 28mL sulfuric acid (H,SO) to 1000mL with water.
3.510mol/L sodium hydroxide solution, dissolve 200g sodium hydroxide in water and dilute to 500mL. 3.60.1mol/L sodium hydroxide solution, take 10mL10mol/L sodium hydroxide and dilute to 1000mL with water. 3.7 Culture medium: weigh 5.3g of pyridoxine Y culture medium and dissolve it in 100mL of distilled water. Notice: The pyridoxine Y culture medium used in this standard shall not contain vitamin B. Growth factor. 3.8 Pyridoxine standard stock solution (100μg/mL): weigh 122mg of pyridoxine hydrochloride standard and dissolve it in 1L25% ethanol, keep it in a refrigerator at 4℃, and it is stable for 1 month.
3.9 Pyridoxine standard intermediate solution (1μg/ml), take 1mL pyridoxine standard stock solution and dilute to 100mL. 3.10 Agar medium: 5.3g pyridoxine Y medium, 1.2g agar. Dilute to 100mL. 3.11 Physiological saline: Take 9g sodium nitride and dissolve it in 1000mL water. 3.12 Bromocresol green (0.4g/L): Solution, weigh 0.1g bromocresol green in a mortar, add 1.4mL 0.1mol/L sodium hydroxide to grind, add a little water and continue grinding until it is completely dissolved, and dilute to 250mL with water. 4 Instruments and equipment
4.1 Electric constant temperature incubator.
4.2 High pressure swab.
4.3 Liquid rapid mixer.
4.4 Centrifuge.
4.5 Grating spectrophotometer.
4.6 Hard glass test tube.
GB/T5009.154-2003
5 Analysis steps
5.1 Preparation and preservation of bacterial strains (light-proof treatment) 5.1.1 Inoculate pure strains of Carlsberg yeast (Saccharomyces Carlsbergensis ATCC No. 9080, abbreviated as SC) into 2 or more agar culture tubes, keep them in a constant temperature box at 30℃±0.5℃ for 18h~20h, take them out and store them in a refrigerator for no more than two weeks. Strains that have been stored for more than a few weeks cannot be used immediately for the preparation of inoculation liquid. They must be transplanted once a day before use for 2d~3d in a row before use, otherwise they will not grow well. 5.1.2 Preparation of seed culture medium
Add 0.5mL 50ng/mL vitamin B: standard application solution to a pointed tube, add 5.0mL basic culture medium, plug with cotton, sterilize in autoclave at 121℃ for 10min, take out, and place in refrigerator. This tube can be kept for several weeks. 2 to 4 tubes can be prepared each time. 5.2 Sample treatment (the whole step needs to be protected from light) 5.2.1 Weigh 0.5g~10.0g of sample (vitamin Bs content does not exceed 10ng) and put it into a 100mL conical flask, add 72mL 0.22mol/L sulfuric acid. Put it in autoclave at 121℃ for hydrolysis for 5h, take it out and cool it, adjust the pH value to 4.5 with 10.0mol/L sodium hydroxide and 0.5mol/L sulfuric acid, and use bromocresol green as indicator. (The indicator changes from yellow to yellow-green), transfer the solution in the triangular flask to a 100mL volumetric flask, dilute to 100mL with distilled water, filter with filter paper, and store the filtrate in the refrigerator for later use (storage period does not exceed 36h). 5.2.2 Preparation of inoculation solution
The day before use, transfer the Carlsberg yeast strain from the reserve strain tube to the sterilized seed culture solution. Two tubes can be prepared at intervals and cultured in a constant temperature box at 30℃±0.5℃ for 18h~20h. Take out and centrifuge for 10min (3000/min), pour off the upper liquid, rinse twice with sterilized saline, add 10mL of sterilized saline, place the centrifuge tube on a liquid rapid mixer to mix, make the strain into a suspension, pour this liquid into a sterilized syringe, and use immediately. 5.2.3 Preparation of standard curve
Take 2.00mL of standard stock solution and dilute it to 200mL to form the intermediate solution. Take 5.00mL of the intermediate solution and dilute it to 100mL as the working solution. The concentration is 50ng/mL. Add 0.00, 0.02, 0.04, 0.08, 0.12 and 0.16mL of working solution to each of the three test tubes, then add 5.00mL of pyridoxine Y medium, mix well, and add cotton plugs. 5.2.4 Preparation of sample tubes
Add 0.05, 0.10, 0.20mL of sample solution to the test tubes respectively, then add 5.00mL of pyridoxine Y medium, plug the test tubes with cotton plugs, put the prepared standard curve and sample test tubes into a pressure cooker at 121℃ for 10 minutes, cool to room temperature for use. 5.2.5 Inoculation and culture
Inoculate one drop of inoculation solution in each tube and culture it in a constant temperature box at 30℃±0.5℃ for 18h~22h. 5.3 Determination
After taking out the standard tube and sample tube from the constant temperature box after culture, use a spectrophotometer at a wavelength of 550nm, adjust the zero value with the zero tube of the standard tube, and measure the absorbance value of each tube. Use the concentration of vitamin B in the standard tube as the horizontal axis and the absorbance value as the vertical axis to draw the standard working curve of vitamin B. Use the absorbance value obtained by the sample tube to find the content of vitamin B in the sample tube on the standard curve. 6 Calculation of results
The content of vitamin B in the sample is calculated according to the following formula: X=cxVx100
m×100wwW.bzxz.Net
Where:
X—the content of vitamin B in the sample, in mg/100g. C—Concentration of vitamin B in the sample extract, in nanograms per milliliter (ng/mL). 294
Concentration of vitamin B in each sample measuring tube, in nanograms per milliliter (ng/mL). 一The sum of the fixed volume and dilution volume of the sample extract, in milliliters (mL). 一The mass of the sample, in grams (g).
100/10°——Converted into milligrams of vitamin B per 100g of sample. The calculation result is expressed to two decimal places. Precision
GB/T5009.154—2003
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 295
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.