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This standard replaces "Determination of Pantothenic Acid in Foods" (GB/T 5009.210-2008).
The following technical changes have been made with respect GB/T 5009.210-2008:
——the name of this standard is revised as "National Food Safety Standard - Determination of Pantothenic Acid in Foods".
——high performance liquid chromatography method is added;
——food pretreatment method is modified;
——the detection limit and quantitation limit are added.
National Food Safety Standard - Determination of Pantothenic Acid in Foods
1 Scope
This standard specifies the methods of determining pantothenic acid and calcium pantothenate in foods.
Method I in this standard is applicable to determining pantothenic acid in foods, and Method II is applicable to determining pantothenic acid in health foods and formula foods of nutrient supplement category.
Method I Microbiological Method
2 Principle
Pantothenic acid is a necessary nutrient for growth of Lactobacillus plantarum (ATCC 8014). Under certain control condition, inoculate Lactobacillus plantarum in culture solution containing sample solution, after a certain period, determine light transmittance (or absorbance), then calculate the content of pantothenic acid in the sample according to the standard curve of pantothenic acid content and light transmittance (or absorbance).
3 Reagents and Materials
Unless otherwise specified, all reagents adopted for this method are analytically pure and water is Grade II water as specified in GB/T 6682.
3.1 Reagents
3.1.1 Hydrochloric acid (HCl).
3.1.2 Glacial acetic acid (C2H4O2).
3.1.3 Sodium hydroxide (NaOH).
3.1.4 Sodium chloride (NaCl).
3.1.5 Sodium carbonate (Na2CO3).
3.1.6 Potassium bicarbonate (KHCO3).
3.1.7 Dipotassium phosphate (K2HPO4).
3.1.8 Sodium acetate trihydrate (C2H3O2Na·3H2O).
3.1.9 Monopotassium phosphate trihydrate (KH2PO4·3H2O).
3.1.10 Magnesium sulfate heptahydrate (MgSO4·7H2O).
3.1.11 Ferrous sulfate heptahydrate (FeSO4·7H2O).
3.1.12 Manganese sulfate monohydrate (MnSO4·H2O).
3.1.13 Trihydroxymethyl aminomethane (C4H11NO3).
3.1.14 Glucose (C6H12O6).
3.1.15 Methylbenzene (C7H8).
3.1.16 Anhydrous ethanol (C2H6O).
3.1.17 Anion exchange resin Dowex 1×8: particle size 38μm~75μm.
3.1.18 Alkaline phosphatase: enzyme activity ≥23U/g.
3.1.19 Liver acetone powder, from pigeon: enzyme activity ≥0.1U/g.
3.1.20 Peptone: nitrogen content ≥10%.
3.1.21 Yeast extract: nitrogen content ≥10%.
3.1.22 Agar.
3.2 Reagent preparation
3.2.1 Acetic acid solution (0.2 mol/L): pipet 11.8 mL of glacial acetic acid, dilute it with water to 1000mL, then mix uniformly.
3.2.2 Sodium acetate solution (0.2mol/L): weigh 27.2 g of sodium acetate trihydrate, dissolve it with water, and dilute to 1000mL, then mix uniformly.
3.2.3 Hydrochloric acid solution (1mol/L): pipette 83mL of hydrochloric acid, dilute it with water to 1000mL, then mix uniformly.
3.2.4 Hydrochloric acid soak solution: pipet 100mL of hydrochloric acid to mix with 5000 mL of water.
3.2.5 Sodium hydroxide solution (1mol/L): weigh 40g of sodium hydroxide, dissolve it with water, and dilute to 1000mL, then mix uniformly.
3.2.6 Sodium hydroxide solution (0.1mol/L): weigh 4g of sodium hydroxide, dissolve it with water, and dilute to 1000mL, then mix uniformly.
3.2.7 Tris buffer solution: weigh 121.0 g of trihydroxymethyl aminomethane, dissolve it into 500mL of water, adjust the pH to 8.1 ± 0.1 with glacial acetic acid, add water to 1000mL, then mix uniformly. It may be reserved for 2 weeks in a 2℃ ~ 4℃ refrigerator.
3.2.8 Normal saline: weigh 9g of sodium chloride, dissolve it with water, and dilute to 1000mL, then mix uniformly. Conduct sterilization just before use. After 10 min of autoclaved sterilization at 121 ℃, preserve it for use.
3.2.9 Ethanol solution (20%): weigh 200 mL of anhydrous ethanol, then mix it with 800ml of water uniformly.
3.2.10 Sodium carbonate solution (0.08mol/L): weigh 8.5 g of sodium carbonate, dissolve it with water, and dilute to 1000mL, then mix uniformly.
3.2.11 Potassium bicarbonate solution (0.02mol/L): weigh 2g of potassium bicarbonate, dissolve it with water, and dilute to 1000mL, then mix uniformly.
3.2.12 Alkaline phosphatase solution: weigh 2 g of alkaline phosphatase, dissolve it with water, and diluted to 100mL. Prepare fresh solution before use, preserve it in 2℃ ~ 4℃ refrigerator for precooling.
3.2.13 Liver extract from pigeon
3.2.13.1 Activated Dowex 1×8: weigh 100g of Dowex 1×8 into a conical flask, add into 1L of hydrochloric acid solution, put it on oscillator to sufficiently shake for 10 min, filter it with Buchner funnel with filter paper. Return Dowex 1×8 to the conical flask, add into 1L of hydrochloric acid solution, repeat shaking and filtering. Add 1L of water into Dowex 1×8, shake it for 10 min, filter it, then repeat washing with water for 10 times. Add into Tris buffer solution dropwise to adjust the pH of Dowex 1×8 to 8.0 ± 0.1. Preserve it in a 2℃ ~ 4℃ refrigerator, and use it up within 2 days.
3.2.13.2 Liver extract from pigeon: put the container used into a 2℃ ~ 4℃ refrigerator for refrigeration overnight the day before preparing the reagent. Weigh 30g of liver acetone powder, from pigeon, put it inside a mortar, while keeping it in ice bath, add into 300mL of potassium bicarbonate solution in twice, grind to homogenate, then transfer it to a centrifugal tube with stopper, stopper it, then sufficiently shake it, after 10 min of refrigeration at -20℃, centrifuge it at 3000 r/min for 5 min, then transfer the supernatant to a 500mL wide-mouth bottle. Add 150 g of activated Dowex 1×8, put it inside the ice bath, mix uniformly for 5 min, pour the mixed solution into a centrifugal tube for 5min of centrifugation at 3000 r/min. Transfer the supernatant into another 500mL precooled wide-mouth bottle, after 10 min of refrigeration at -20℃, add into 150 g of activated Dowex 1×8, put it inside the ice bath, then mix uniformly for 5min, pour the mixed solution into centrifugal tube for centrifugation at 3000 r/min, put the supernatant into different test tubes with stopper (about 3mL in each tube), preserve it by freezing at -20℃. Defreeze it in 2℃ ~ 4℃ refrigerator, and preserve it until use.
3.3 Culture medium
3.3.1 Saline solution A: respectively weigh 25 g of dipotassium phosphate and 25 g of monopotassium phosphate trihydrate, dissolve it with water and dilute to 500mL, then mix uniformly. Add into 1mL of methylbenzene, it may be reserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.3.2 Saline solution B: respectively weigh 10g magnesium sulfate heptahydrate, 0.5 g of sodium chloride, 0.5 g of ferrous sulfate heptahydrate and 0.5 g of manganese sulfate monohydrate, dissolve it with water and dilute to 500mL. Add 5 drops of hydrochloric acid, and it may be reserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.3.3 Agar medium: weigh or pipet each reagent according to Table 1, add water to 100mL, mix them, heat them in boiling water bath until the agar is fully dissolved. Adjust pH to 6.8 ± 0.1 with 1mol/L hydrochloric acid solution and/or 1mol/L sodium hydroxide solution. Put it into different test tubes as soon as possible, about 3mL ~ 5mL for each test tube according to the inner diameter, the liquid level shall not be less than 2cm. Put on tampon, autoclave it at 121 ℃ for 15min. Take it out, put the test tube upright, after cooling, preserve it in refrigerator for standby.
Table 1 Preparation of Agar Medium for Strain Reserve
Reagent Consumption
Glucose 1.0g
Peptone 0.8g
Yeast extract dry powder 0.2g
Sodium acetate trihydrate 1.7g
Salt solution A 0.2mL
Salt solution B 0.2mL
Agar 1.2g
3.3.4 Culture solution for determination of pantothenic acid: culture solution for determination of pantothenic acid may be prepared according to Annex A or that with equivalent efficacy may be directly purchased from a reagent company, and prepared according to instructions before use.
3.4 Standard product
D- calcium pantothenate (C18H32CaN2O10): purity ≥99%.
3.5 Preparation of standard solution
3.5.1 Standard stock solution of pantothenic acid (40.0μg/mL): accurately weigh 43.5 mg of D-calcium pantothenate pre-dried to constant weight, dissolve it with water, then transfer it to a 1000mL volumetric flask, add 10mL of acetic acid solution and 100mL of sodium acetate solution, then dilute it with water to the scale. Store it into a brown bottle, add into 3~5 drops of methylbenzene, it may be reserved for 2 year in a 2℃ ~ 4℃ refrigerator.
3.5.2 Standard pantothenic acid intermediate solution (1.00μg/mL): accurately pipet 25.0 mL of standard pantothenic acid stock solution, put it into a 1000mL volumetric flask, add into 10mL acetic acid solution and 100mL sodium acetate solution, then dilute it with water to the scale. Add into 3~5 drops of methylbenzene, it may be reserved for 1 year in a 2℃ ~ 4℃ refrigerator.
3.5.3 Standard pantothenic acid working solution (20ng/mL): accurately pipet 2.00 mL of standard pantothenic acid intermediate solution, put it into a 100mL volumetric flask, dilute it with water to the scale, then mix uniformly. Prepare fresh solution before use.
4 Instruments and Equipment
4.1 Balance: sensibility 0.1 mg.
4.2 Thermostatic incubator: 37℃±1℃.
4.3 Pressure steam sterilizer: 121℃.
4.4 Vortex oscillator.
4.5 Centrifuger: rotation speed ≥3000r/min.
4.6 Inoculating needle and inoculating loop.
4.7 pH meter: ± 0.01 accuracy.
4.8 UV-spectrophotometer.
4.9 Superclean bench.
4.10 Ultrasonator.
5 Preparation and Preservation of Strain
5.1 Strain
Lactobacillus plantarum (ATCC 8014).
5.2 Preparation of strain for reservation
Transfer the strain Lactobacillus plantarum (ATCC 8014) to agar medium, culture in the thermostatic incubator (37℃±1℃) for 20h~24h, continuously propagate for 2~3 times. Take it out, then put it inside a 2℃ ~ 4℃ refrigerator, preserve as stocks strain. Propagate at least once each month, no more than 25 generations.
Prior to test, inoculate the stocks strain to agar medium, culture in the thermostatic incubator (37℃±1℃) for 20h~24h to activate the strains for the preparation of inoculated solution.
Note: for the stocks strain preserved for several weeks, it cannot be immediately used to prepare the inoculated solution, and prior test, continuously reproduce 2~3 generations to ensure the activity of bacteria.
Foreword i
1 Scope
2 Principle
3 Reagents and Materials
4 Instruments and Equipment
5 Preparation and Preservation of Strain
6 Analysis Procedures
7 Accuracy
8 Other
9 Principle
10 Reagents and Materials
11 Instruments and Equipment
12 Analysis Procedures
13 Expression of Analysis Results
14 Accuracy
15 Other
Annex A Preparation Method of Culture Solution for Pantothenic Acid Determination
Annex B Liquid Chromatogram of Standard Pantothenic Acid Solution
