This standard specifies the determination methods of niacin and nicotinamide in foods.
Method I of this standard is microbiological method, which is applicable to the determination of the total amount of niacin and nicotinamide in various foods including the fortified food with natural food as matrix; Method II is high performance liquid chromatography, which is applicable to the determination of niacin and nicotinamide in fortified food.
Method I Microbiological Method
2 Principle
Niacin (nicotinamide) is the necessary nutrient for the growth of lactobacillus plantarum (ATCC8014) and determines the content of niacin and nicotinamide under specific control conditions by utilizing the specificity of lactobacillus plantarum to niacin and nicotinamide and the optical density that is formed due to the growth of lactobacillus plantarum in the sample containing niacin and nicotinamide.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade II water (defined in GB/T 6682) are adopted for the purposes of this method. For the medium, the commercial medium complying with testing requirements may be purchased.
3.1 Strain
Lactobacillus plantarum (ATCC 8014) or other effective standard bacterial strains.
3.2 Reagents
3.2.1 Hydrochloric acid (HCl).
3.2.2 Sodium hydroxide (NaOH).
3.2.3 Sodium chloride (NaCl).
3.2.4 Concentrated sulfuric acid (H2SO4).
3.2.5 Ethanol (C2H5OH).
3.3 Reagents preparation
3.3.1 Hydrochloric acid solution (1mol/L): pipet 83mL of hydrochloric acid to a 1 000mL beaker, add into 917mL of water and mix well.
3.3.2 Hydrochloric acid solution (0.1mol/L): pipet 10mL of hydrochloric acid solution (3.3.1) and dissolve it with water to 100mL.
3.3.3 Sodium hydroxide solution (1mol/L): weigh 40g of sodium hydroxide to a 1 000mL beaker, dissolve and dilute it with water to 1 000mL and mix well.
3.3.4 Sodium hydroxide solution (0.1mol/L): pipet 10mL of sodium hydroxide solution (3.3.3) and dissolve it with water to 100mL.
3.3.5 Normal saline (0.9%): weigh 9g of sodium chloride, dissolve it into 1 000mL of water, put 10mL into the test tube and sterilize for 15min at 121℃ for future use.
3.3.6 Ethanol solution (25%): measure 200mL of absolute ethanol and mix it well with 800mL of water.
3.3.7 Sulfuric acid solution (1mol/L): inject 700mL of water into a 2 000mL beaker, pipet 56mL of sulfuric acid, slowly pour it into the water along the breaker wall and dilute to 1 000mL with water.
3.4 Medium
3.4.1 Lactobacillus agar medium: prepare according to A.1.
3.4.2 Lactobacillus broth medium: prepare according to A.2.
3.4.3 Medium for niacin determination: prepare according to A.3.
Note: Some commercialized synthetic media are effective and are prepared according to descriptions on the label.
3.5 Standards
Niacin (C6H5NO2): purity≥99.5% or the standard material approved and awarded with reference material certificate by the nation.
3.6 Preparation of standard solution
3.6.1 Niacin standard stock solution (0.1mg/mL): accurately weigh 50.0mg of niacin standard, dissolve it with ethanol solution, then transfer it to a 500mL volumetric flask, scale the volume, mix well and refrigerate it at 2℃~4℃. Each millilitre of such solution is equivalent to 100μg niacin.
3.6.2 Niacin standard intermediate solution (1μg/mL): accurately pipet 1.0mL of niacin standard stock solution of (3.6.1) into a 100mL brown volumetric flask, dilute it and bring the volume to the scale with ethanol solution, mix well and refrigerate it at 2℃~4℃. Each millilitre of such solution is equivalent to 1μg niacin.
3.6.3 Niacin standard working solution (100ng/mL): accurately pipet 5.00mL of niacin standard intermediate solution (3.6.2) into a 50mL volumetric flask, dilute it and bring the volume to the scale with water, then mix well and refrigerate it at 2℃~4℃. Each millilitre of such solution is equivalent to 100ng niacin.
4 Apparatuses
4.1 Balance: with sensitivity of 0.01g and 0.1mg respectively.
4.2 Homogeneous apparatus: it is used for the homogenization of specimen.
Note: Prior to use of glass apparatus, clean the hard glass measuring tubes and other necessary glassware with active agent (made by adding 1-dodecane sulfonic acid sodium salt or household detergent into washing water), dry them and then use them after subjecting them to dry heat (170℃) for 3h.
5 Analysis Procedures
5.1 Preparation of reserve strain
Transfer lactobacillus plantarum (ATCC 8014) to lactobacillus agar medium, culture in the thermostatic incubator (36℃±1℃) for 20h~24h, and then take it out to preserve it in the refrigerator (2℃~4℃) to serve as reserve strain, which shall be reproduced at least once a month.
Prior to test, inoculate the reserve bacterial strain to lactobacillus agar medium, culture in the thermostatic incubator (36℃±1℃) for 20h~24h to activate the bacterial strain for the preparation of inoculated solution. For the reserve strain preserved over weeks, it cannot be immediately used as inoculated preparation solution and prior test, 2~3 generations shall be continuously reproduced to ensure the activity of bacterial strain.
5.2 Preparation of inoculated solution
One day prior to test, transfer parts of strain from the lactobacillus agar medium to 10mL of sterile lactobacillus broth medium and culture them in the thermostatic incubator (36℃±1℃) for 6h~18h. Centrifuge this culture solution for 15min under aseptic condition and discard the supernatant. Add into 10mL of sterilized normal saline to redisperse cells, rapidly mix well in the vortex mixers, centrifuge for 15min and discard the supernatant. Repeat the centrifuge and washing steps for three times. Pipet 1mL of the third-time cell dispersion, add into 10mL of sterilized normal saline and mix well to make into suspension for future use. Read the transmittance of this bacterial suspension at 550nm wavelength with 721 spectrophotometer by taking 0.9% normal saline as reference; adjust the transmittance with 0.9% normal saline or the third-time cell dispersion to make its scope within 60%~80% and use it immediately.
5.3 Specimen preparation
Specimens of grain, potatoes, beans and nuts (without shell) etc. need to be pulverized, grinded and sieved (hole diameter of the sieve plate is 0.3 mm~0.5 mm); those of milk powder and rice flour need to be mixed well; those of meat, egg, fish and purtenance etc. need to be made into chyme with beating crusher; those of fruits and vegetables, and semi-solid foods etc. need to be made into homogenate and mixed well; and liquid specimens need to be shaken for mixing before use. If they cannot be detected immediately, they will be preserved in 4℃ refrigerator.
5.4 Specimen extraction
Accurately weigh niacin specimen, 2g~5g (accurate to 0.01g) of common milk and fresh fruit and vegetables specimen; 0.2g~1g (accurate to 0.01g) of grains, beans, nut fruits, viscera, raw meat and dry processed specimen and 5g of liquid specimen; for milk powder and rice flour etc., accurately weigh 2g (accurate to 0.01g) of proper specimen; 0.1g~0.5g of common nutrient replenisher and composite nutrient supplement; 0.2g~1g of foods; 5g~10g of liquid beverage or fluid and semifluid specimen into a 100mL conical flask and add into the sulfuric acid solution whose dry weight is 10 times of that of inspected substance. Hydrolyze for 30min at 121℃ and then cool to room temperature. Adjust the pH to 6.0~6.5 with 0.1mol/L sodium hydroxide solution and then adjust it to 4.5±0.1 with 0.1mol/L hydrochloric acid, bring the volume to 100mL with water, filter with ashless filter paper and the filtrate is for future use.
5.5 Dilute
Properly dilute (f) the specimen extracting solution with water according to niacin content in the specimen to make the niacin content in the specimen extracting solution after dilution stay within the range of 50.0ng~500.0ng.
5.6 Preparation of serial tubes for determination
5.6.1 Standard series tube
Respectively add 0.00mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL, 4.0mL and 5.00mL of niacin standard working solution of into test tubes and supplement to 5.0mL with water, which means that niacin content is 0.0ng, 50ng, 100ng, 150ng, 200ng, 250ng, 300ng, 400ng and 500ng in standard series tubes respectively. Add into 5.0mL of culture medium for niacin determination, which is detailed in Table 1. Mix well and prepare 3 tubes for each standard point.
Table 1 Manufacturing of Standard Curve Tube
Test tube number (No.) 1 2 3 4 5 6 7 8 9 10
Distilled water /mL 5 5 4.5 4 3.5 3 2.5 2 1 0
Standard solution */mL 0 0 0.5 1 1.5 2 2.5 3 4 5
Medium/mL 5 5 5 5 5 5 5 5 5 5
*Do not add standard solution into No.1~2 test tube; dropwise add bacterium solution into No.2 test tube;
Successively rise the solution concentration of the standards added in No.3~10 test tubes and repeat for 3 times.
5.6.2 Specimen series tube
Take 4 test tubes, add into 1.0mL, 2.0mL, 3.0mL and 4.0mL of specimen extracting solution respectively, then supplement to 5.0mL with water, and add 5.0mL culture solution for niacin determination, which is detailed in Table 2. Mix well and repeat for 3 times for each concentration.
Table 2 Manufacturing of Specimen Tube
Test tube number (No.) 1 2 3 4
Distilled water/mL 4 3 2 1
Sample/mL 1 2 3 4
Medium/mL 5 5 5 5
5.6.3 Sterilization
Plug the measuring tubes of all standard series tubes and specimen series tubes with cotton plug and autoclave for 5min at 121℃ (0.10MPa~0.12MPa). After the completion of sterilization, rapidly cool for future use.
5.7 Culture
5.7.1 Inoculation: under aseptic operation conditions, transfer the inoculated solution to sterile droppers and inoculate a drop to each measuring tube.
5.7.2 Culture: put the test tube added with bacterium solution into thermostatic incubator (36℃±1℃) and culture for 16h~24h to obtain the maximum turbidity, namely without obvious change in turbidity after another 2 hours of re-culturing. Prepare another standard 0 tube (containing 0.0g of niacin) which is not inoculated to serve as 0 control tube.
5.8 Determination
Read the optical density value at 550nm wavelength with 1cm-thick cuvette and mix the cultured measuring tube well with turbine mixer. Adjust the luminous transmittance to 100% with non-inoculated 0 control tube and then successively determine the luminous transmittance of standard series tube and specimen series tube. Take out the maximum concentration standard curve tube to shake for 5s, determine the optical density value and put it back for re-culturing. Determine the optical density of this tube 2h later under the equivalent condition; if the absolute difference of two optical density results is not greater than 2%, take out all inspection tubes to determine the optical density of standard solution and specimen.
5.9 Plotting of standard curve
Plot the standard curve with the niacin content of standard series tube as horizontal coordinate and optical density value as longitudinal coordinate and it is also permissible to plot the fitting curve for each standard point. The relative standard deviation in optical density values of 3 tubes for each standard point shall be less than 10%; if 2 tubes among these 3 tubes for certain standard point have niacin content within the range of 50ng~500ng and the deviation of niacin content of specimen extracting solution per millilitre converted from two tubes is less than 10%, then this result is useable. If the relative standard deviation of the niacin content in 3 specimen tubes is greater than 10%, then this point is rejected and it is not involved with the plotting of standard curve.
Contents Foreword i 1 Scope 2 Principle 3 Reagents and Materials 4 Apparatuses 5 Analysis Procedures 6 Expression of Analysis Results 7 Accuracy 8 Others 9 Principle 10 Reagents and Materials 11 Apparatuses 12 Analysis Procedures 13 Expression of Analysis Results 14 Accuracy 15 Others Appendix A Medium and Reagents Appendix B Calibration Method for Concentration of Standard Solution Appendix C Liquid Chromatogram of Niacin and Nicotinamide Standard Solutions
National Food Safety Standard
Determination of Niacin and Nicotinamide in Foods
食品安全国家标准
食品中烟酸和烟酰胺的测定
1 Scope
This standard specifies the determination methods of niacin and nicotinamide in foods.
Method I of this standard is microbiological method, which is applicable to the determination of the total amount of niacin and nicotinamide in various foods including the fortified food with natural food as matrix; Method II is high performance liquid chromatography, which is applicable to the determination of niacin and nicotinamide in fortified food.
Method I Microbiological Method
2 Principle
Niacin (nicotinamide) is the necessary nutrient for the growth of lactobacillus plantarum (ATCC8014) and determines the content of niacin and nicotinamide under specific control conditions by utilizing the specificity of lactobacillus plantarum to niacin and nicotinamide and the optical density that is formed due to the growth of lactobacillus plantarum in the sample containing niacin and nicotinamide.
3 Reagents and Materials
Unless otherwise specified, analytically-pure reagents and Grade II water (defined in GB/T 6682) are adopted for the purposes of this method. For the medium, the commercial medium complying with testing requirements may be purchased.
3.1 Strain
Lactobacillus plantarum (ATCC 8014) or other effective standard bacterial strains.
3.2 Reagents
3.2.1 Hydrochloric acid (HCl).
3.2.2 Sodium hydroxide (NaOH).
3.2.3 Sodium chloride (NaCl).
3.2.4 Concentrated sulfuric acid (H2SO4).
3.2.5 Ethanol (C2H5OH).
3.3 Reagents preparation
3.3.1 Hydrochloric acid solution (1mol/L): pipet 83mL of hydrochloric acid to a 1 000mL beaker, add into 917mL of water and mix well.
3.3.2 Hydrochloric acid solution (0.1mol/L): pipet 10mL of hydrochloric acid solution (3.3.1) and dissolve it with water to 100mL.
3.3.3 Sodium hydroxide solution (1mol/L): weigh 40g of sodium hydroxide to a 1 000mL beaker, dissolve and dilute it with water to 1 000mL and mix well.
3.3.4 Sodium hydroxide solution (0.1mol/L): pipet 10mL of sodium hydroxide solution (3.3.3) and dissolve it with water to 100mL.
3.3.5 Normal saline (0.9%): weigh 9g of sodium chloride, dissolve it into 1 000mL of water, put 10mL into the test tube and sterilize for 15min at 121℃ for future use.
3.3.6 Ethanol solution (25%): measure 200mL of absolute ethanol and mix it well with 800mL of water.
3.3.7 Sulfuric acid solution (1mol/L): inject 700mL of water into a 2 000mL beaker, pipet 56mL of sulfuric acid, slowly pour it into the water along the breaker wall and dilute to 1 000mL with water.
3.4 Medium
3.4.1 Lactobacillus agar medium: prepare according to A.1.
3.4.2 Lactobacillus broth medium: prepare according to A.2.
3.4.3 Medium for niacin determination: prepare according to A.3.
Note: Some commercialized synthetic media are effective and are prepared according to descriptions on the label.
3.5 Standards
Niacin (C6H5NO2): purity≥99.5% or the standard material approved and awarded with reference material certificate by the nation.
3.6 Preparation of standard solution
3.6.1 Niacin standard stock solution (0.1mg/mL): accurately weigh 50.0mg of niacin standard, dissolve it with ethanol solution, then transfer it to a 500mL volumetric flask, scale the volume, mix well and refrigerate it at 2℃~4℃. Each millilitre of such solution is equivalent to 100μg niacin.
3.6.2 Niacin standard intermediate solution (1μg/mL): accurately pipet 1.0mL of niacin standard stock solution of (3.6.1) into a 100mL brown volumetric flask, dilute it and bring the volume to the scale with ethanol solution, mix well and refrigerate it at 2℃~4℃. Each millilitre of such solution is equivalent to 1μg niacin.
3.6.3 Niacin standard working solution (100ng/mL): accurately pipet 5.00mL of niacin standard intermediate solution (3.6.2) into a 50mL volumetric flask, dilute it and bring the volume to the scale with water, then mix well and refrigerate it at 2℃~4℃. Each millilitre of such solution is equivalent to 100ng niacin.
4 Apparatuses
4.1 Balance: with sensitivity of 0.01g and 0.1mg respectively.
4.2 Homogeneous apparatus: it is used for the homogenization of specimen.
4.3 Thermostatic incubator: 36℃±1℃.
4.4 Vortex oscillator.
4.5 Pressure-steam sterilizer:121℃ (0.10MPa~0.12MPa).
4.6 Centrifuger: rotation speed ≥ 2 000r/min.
4.7 PH meter: with the accuracy of ±0.01.
4.8 Spectrophotometer.
4.9 Superclean bench.
4.10 Ultrasonic vibrator.
Note: Prior to use of glass apparatus, clean the hard glass measuring tubes and other necessary glassware with active agent (made by adding 1-dodecane sulfonic acid sodium salt or household detergent into washing water), dry them and then use them after subjecting them to dry heat (170℃) for 3h.
5 Analysis Procedures
5.1 Preparation of reserve strain
Transfer lactobacillus plantarum (ATCC 8014) to lactobacillus agar medium, culture in the thermostatic incubator (36℃±1℃) for 20h~24h, and then take it out to preserve it in the refrigerator (2℃~4℃) to serve as reserve strain, which shall be reproduced at least once a month.
Prior to test, inoculate the reserve bacterial strain to lactobacillus agar medium, culture in the thermostatic incubator (36℃±1℃) for 20h~24h to activate the bacterial strain for the preparation of inoculated solution. For the reserve strain preserved over weeks, it cannot be immediately used as inoculated preparation solution and prior test, 2~3 generations shall be continuously reproduced to ensure the activity of bacterial strain.
5.2 Preparation of inoculated solution
One day prior to test, transfer parts of strain from the lactobacillus agar medium to 10mL of sterile lactobacillus broth medium and culture them in the thermostatic incubator (36℃±1℃) for 6h~18h. Centrifuge this culture solution for 15min under aseptic condition and discard the supernatant. Add into 10mL of sterilized normal saline to redisperse cells, rapidly mix well in the vortex mixers, centrifuge for 15min and discard the supernatant. Repeat the centrifuge and washing steps for three times. Pipet 1mL of the third-time cell dispersion, add into 10mL of sterilized normal saline and mix well to make into suspension for future use. Read the transmittance of this bacterial suspension at 550nm wavelength with 721 spectrophotometer by taking 0.9% normal saline as reference; adjust the transmittance with 0.9% normal saline or the third-time cell dispersion to make its scope within 60%~80% and use it immediately.
5.3 Specimen preparation
Specimens of grain, potatoes, beans and nuts (without shell) etc. need to be pulverized, grinded and sieved (hole diameter of the sieve plate is 0.3 mm~0.5 mm); those of milk powder and rice flour need to be mixed well; those of meat, egg, fish and purtenance etc. need to be made into chyme with beating crusher; those of fruits and vegetables, and semi-solid foods etc. need to be made into homogenate and mixed well; and liquid specimens need to be shaken for mixing before use. If they cannot be detected immediately, they will be preserved in 4℃ refrigerator.
5.4 Specimen extraction
Accurately weigh niacin specimen, 2g~5g (accurate to 0.01g) of common milk and fresh fruit and vegetables specimen; 0.2g~1g (accurate to 0.01g) of grains, beans, nut fruits, viscera, raw meat and dry processed specimen and 5g of liquid specimen; for milk powder and rice flour etc., accurately weigh 2g (accurate to 0.01g) of proper specimen; 0.1g~0.5g of common nutrient replenisher and composite nutrient supplement; 0.2g~1g of foods; 5g~10g of liquid beverage or fluid and semifluid specimen into a 100mL conical flask and add into the sulfuric acid solution whose dry weight is 10 times of that of inspected substance. Hydrolyze for 30min at 121℃ and then cool to room temperature. Adjust the pH to 6.0~6.5 with 0.1mol/L sodium hydroxide solution and then adjust it to 4.5±0.1 with 0.1mol/L hydrochloric acid, bring the volume to 100mL with water, filter with ashless filter paper and the filtrate is for future use.
5.5 Dilute
Properly dilute (f) the specimen extracting solution with water according to niacin content in the specimen to make the niacin content in the specimen extracting solution after dilution stay within the range of 50.0ng~500.0ng.
5.6 Preparation of serial tubes for determination
5.6.1 Standard series tube
Respectively add 0.00mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL, 4.0mL and 5.00mL of niacin standard working solution of into test tubes and supplement to 5.0mL with water, which means that niacin content is 0.0ng, 50ng, 100ng, 150ng, 200ng, 250ng, 300ng, 400ng and 500ng in standard series tubes respectively. Add into 5.0mL of culture medium for niacin determination, which is detailed in Table 1. Mix well and prepare 3 tubes for each standard point.
Table 1 Manufacturing of Standard Curve Tube
Test tube number (No.) 1 2 3 4 5 6 7 8 9 10
Distilled water /mL 5 5 4.5 4 3.5 3 2.5 2 1 0
Standard solution */mL 0 0 0.5 1 1.5 2 2.5 3 4 5
Medium/mL 5 5 5 5 5 5 5 5 5 5
*Do not add standard solution into No.1~2 test tube; dropwise add bacterium solution into No.2 test tube;
Successively rise the solution concentration of the standards added in No.3~10 test tubes and repeat for 3 times.
5.6.2 Specimen series tube
Take 4 test tubes, add into 1.0mL, 2.0mL, 3.0mL and 4.0mL of specimen extracting solution respectively, then supplement to 5.0mL with water, and add 5.0mL culture solution for niacin determination, which is detailed in Table 2. Mix well and repeat for 3 times for each concentration.
Table 2 Manufacturing of Specimen Tube
Test tube number (No.) 1 2 3 4
Distilled water/mL 4 3 2 1
Sample/mL 1 2 3 4
Medium/mL 5 5 5 5
5.6.3 Sterilization
Plug the measuring tubes of all standard series tubes and specimen series tubes with cotton plug and autoclave for 5min at 121℃ (0.10MPa~0.12MPa). After the completion of sterilization, rapidly cool for future use.
5.7 Culture
5.7.1 Inoculation: under aseptic operation conditions, transfer the inoculated solution to sterile droppers and inoculate a drop to each measuring tube.
5.7.2 Culture: put the test tube added with bacterium solution into thermostatic incubator (36℃±1℃) and culture for 16h~24h to obtain the maximum turbidity, namely without obvious change in turbidity after another 2 hours of re-culturing. Prepare another standard 0 tube (containing 0.0g of niacin) which is not inoculated to serve as 0 control tube.
5.8 Determination
Read the optical density value at 550nm wavelength with 1cm-thick cuvette and mix the cultured measuring tube well with turbine mixer. Adjust the luminous transmittance to 100% with non-inoculated 0 control tube and then successively determine the luminous transmittance of standard series tube and specimen series tube. Take out the maximum concentration standard curve tube to shake for 5s, determine the optical density value and put it back for re-culturing. Determine the optical density of this tube 2h later under the equivalent condition; if the absolute difference of two optical density results is not greater than 2%, take out all inspection tubes to determine the optical density of standard solution and specimen.
5.9 Plotting of standard curve
Plot the standard curve with the niacin content of standard series tube as horizontal coordinate and optical density value as longitudinal coordinate and it is also permissible to plot the fitting curve for each standard point. The relative standard deviation in optical density values of 3 tubes for each standard point shall be less than 10%; if 2 tubes among these 3 tubes for certain standard point have niacin content within the range of 50ng~500ng and the deviation of niacin content of specimen extracting solution per millilitre converted from two tubes is less than 10%, then this result is useable. If the relative standard deviation of the niacin content in 3 specimen tubes is greater than 10%, then this point is rejected and it is not involved with the plotting of standard curve.
Contents of GB 5009.89-2016
Contents
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Apparatuses
5 Analysis Procedures
6 Expression of Analysis Results
7 Accuracy
8 Others
9 Principle
10 Reagents and Materials
11 Apparatuses
12 Analysis Procedures
13 Expression of Analysis Results
14 Accuracy
15 Others
Appendix A Medium and Reagents
Appendix B Calibration Method for Concentration of Standard Solution
Appendix C Liquid Chromatogram of Niacin and Nicotinamide Standard Solutions
