GB 5413.16-2010National food safety standard -- Determination of folic acid (folate activity) in foods for infants and young children, milk and milk products (English PDF)

4.3 Phosphate buffer

4.3.1 phosphate buffer I (0.05 mol/L). Weigh 5.85 g of potassium dihydrogen phosphate, 1.22 g potassium phosphate dibasic, with 1000 mL aqueous solution. Before use by 0.5 g/100 mL ascorbic acid.

4.3.2 II phosphate buffer (for the front cereals and their products processing). Weigh 14.2 g of disodium hydrogen phosphate, dissolved with 1000 mL of water.

Prior to use by 1.0 g/100 mL was added ascorbic acid (4.16) A pH adjustment with sodium hydroxide solution to 7.8 ± 0.1.

4.3.3 phosphate buffer III (cereals and their products for testing). Weigh 14.2 g disodium hydrogen phosphate, dissolved in 1000 mL of water.

Prior to use by 1.0 g/100 mL ascorbic acid, adjusted with sodium hydroxide solution A (4.16) pH to 6.8 ± 0.1.

4.3.4 phosphate buffer IV (0.1 mol/L) (for the preparation of cereals and cereal products standard solution). dissolve 13.61 g of potassium dihydrogen phosphate Diluted to 1000 mL in water. (4.10) with potassium hydroxide solution adjusted to pH 7.0 ± 0.1.

4.4 folic acid standard.

4.5 Ammonia (10.8%).

4.6 toluene (C7H8).

4.7 ascorbic acid (C6H8O6).

4.8 strains. Lactobacillus casei (Lactobacillus casei) ATCC 7469.

4.9 Medium

4.9.1 Lactobacillus agar. peptone of milk 15 g, yeast extract 5 g, glucose 10 g, tomato juice, 100 mL, potassium dihydrogen phosphate 2 g, polyethylene sorbitan monooleate 1 g, agar 10 g, add distilled water to 1000 mL, adjust pH to 6.8 ± 0.2 (20 °C ~ 25 °C). 121 °C Autoclave 15min, standby.

4.9.2 Lactobacillus broth medium. peptone of milk 15 g, yeast extract 5 g, glucose 10 g, tomato juice, 100 mL, potassium dihydrogen phosphate 2 g, polyethylene sorbitan monooleate 1 g, add distilled water to 1000 mL, adjust pH to 6.8 ± 0.2 (20 °C ~ 25 °C). 121 °C autoclaving Bacteria 15min, standby.

4.9.3 Folate measurement medium. Casein peptone 10 g, glucose 40 g, sodium acetate 40 g, dipotassium phosphate 1 g, potassium dihydrogen phosphate 1 g, DL- tryptophan 0.2 g, L- aspartic acid 0.6 g, L- cysteine hydrochloride 0.5 g, adenine sulfate, 10 mg, 10 guanine hydrochloride mg, uracil, 10 mg, xanthine 20 mg, polysorbate 0.1 g, glutathione 5 mg, magnesium sulfate 0.4 g, sodium chloride 20 mg, sulfur Ferrous sulfate 20 mg, manganese sulfate 15 mg, riboflavin 1 mg, p- amino benzoic acid 2 mg, vitamin B6 4mg, thiamine hydrochloride 400 µg, Calcium pantothenate 800 µg, niacin 800 µg, biotin 20 µg, add distilled water to 1000 mL, adjust pH to 6.7 ± 0.1 (20 °C ~ 25 °C).

Note. Commercially available commercial synthetic medium effect is more stable.

4.10 potassium hydroxide solution (4 mol/L). Weigh 224 g of potassium hydroxide in 1000 mL beaker with 400 mL of water to dissolve, cooled to Room temperature, transferred to a 1000 mL volumetric flask, and water volume.

4.11 papain solution. 1 g of protease (dynamic >=6000 U/mg, pH 6.0 ± 0.1,40 °C) was dissolved in 100 mL of phosphate buffer Liquid I (4.3.1) in. Pro preparation before use.

4.12 alpha- amylase solution. 1 g alpha- amylase (1.5 U/mg) was dissolved in 100 mL phosphate buffer I (4.3.1) in. Pro preparation before use.

4.13 0.22 µm filter sterilization.

Preparation of standard solution of 4.14 4.14.1 folic acid standard stock solution (500 µg/mL). Weigh 55 mg ~ 56 mg (accurate to 0.1 mg) of folic acid standard (4.4), with 50 mL of distilled water into 100 mL volumetric flask, add 2 mL of ammonia (4.5). After preparation of the solution, according to formula (1) calculate the volume of the solution, it requires mid salt stock solution at a concentration of 500 µg/mL.

Stock solution volume (mL) = ×× cm or simplified to. stock solution volume (mL) = cm × (1) where.

M-- quality standards of folic acid, in milligrams (mg);

C-- purity standards of folic acid in grams per hundred grams (g/100 g).

The solution was diluted with water to the scale, size and thoroughly mixed with water to a straw computational requirements, into a brown reagent bottle 2 °C ~ 4 °C ice Box refrigerated storage period of 4 months.

4.14.2 folic acid intermediate standard solution (50 µg/mL). 10 mL absorb folic acid stock standard solution (4.14.1) in 100 mL brown volumetric flask with water to volume, mix thoroughly, 2 °C ~ 4 °C refrigerator, shelf life of one month.

4.14.3 folic acid working standard solution (0.05 ng/mL, 0.1 ng/mL). draw 1 mL folate intermediate standard solution (4.14.2) in 100 mL Brown volumetric flask, add water to volume and mix. Then suck the liquid 1 mL to 100 mL brown volumetric flask, volume, and mix. From Liquid 5 mL were drawn in 250 mL and 500 mL brown volumetric flask, with phosphate buffer I (4.3.1) to volume, mix, it is a high concentration of working standard solution (0.1 ng/mL) and low concentrations of working standard solution (0.05 ng/mL). Pro preparation before use.

4.15 hydrochloric acid (1 mol/L). Measure 83.0 mL of hydrochloric acid dissolved in water, cool and dilute to 1000 mL.

4.16 sodium hydroxide solution A (4 mol/L). Weigh 160 g of sodium hydroxide in 1000 mL beaker, dissolved in 400 mL of water, cooled to room temperature, transferred to a 1000 mL volumetric flask, and water volume.

4.17 sodium hydroxide solution B (0.1 mol/L). draw 2.5 mL of sodium hydroxide solution A (4.16) was transferred to a 100 mL volumetric flask Water volume.

4.18 sodium hydroxide standard titration solution (0.1 mol/L ± 0.0002 mol/L). Weigh 4 g (accurate to 0.0001 g) of sodium hydroxide with water Diluted to 1000 mL, calibrated with potassium hydrogen phthalate. Save this solution containers must be sealed to prevent infiltration of carbon dioxide.

Calibration 4.18.1 sodium hydroxide standard solution. Weigh about 0.18 g (accurate to 0.0001 g) at 105 °C ~ 110 °C drying to constant weight o Potassium hydrogen phthalate with 50 mL of aqueous addition to carbon dioxide in the conical flask, add two drops of 5 g/L of phenolphthalein indicator, with a good hydroxide Sodium titration to pink, while for blank experiments. According to equation (2) to calculate the concentration of sodium hydroxide standard solution.

2042.0) (

21 × -VV

Where.

c-- the sodium hydroxide concentration, in units of moles per liter (mol/L);

m-- said mass take potassium hydrogen phthalate, in grams (g);

V1-- amount of sodium hydroxide solution, in milliliters (mL);

The amount of sodium hydroxide solution V2-- blank test, in milliliters (mL).

4.18.2 phenolphthalein solution. 0.5 g of phenolphthalein is dissolved in 75 mL volume fraction of 95% ethanol, 20 mL of water, then add hydroxide Sodium hydroxide solution (4.18), until one drop immediately turned pink, then water volume to 100 mL.

4.19 bromothymol blue indicator. Weigh 0.1 g bromothymol blue in a mortar, was added 1.6 mL of sodium hydroxide solution B (4.17) Grinding, add a little water until completely dissolved, transferred to a 250 mL volumetric flask to volume with water.

5.5 Incubator. 36 °C ± 1 °C

5.6 Buret. sub-scale value is 0.1 mL.

5.7 Vortex.

6.1 Preparation of test broth

6.1.1 Lactobacillus casei (Lactobacillus casei) ATCC 7469 lyophilized powder into Lactobacillus broth (4.9.2), and 36 °C ± 1 °C cultured for 24 h, transferred to Lactobacillus agar medium (4.9.1) in a test tube, then 36 °C ± 1 °C cultured 24 h. Develop good Culture of Lactobacillus agar (4.9.1) of the tube as reserve species.

6.1.2 From the stock transferred to three medium strain Lactobacillus agar medium, respectively (4.9.1) in a test tube, into an incubator 36 °C ± 1 °C for 24 h. Subculture monthly, as the monthly tube stored in the refrigerator. Monthly from January inoculation tube reseeded three adapters with conviction Keep new strain.

6.1.3 dated from culture tubes inoculated in a revaccination one Lactobacillus agar medium (4.9.1) tube, 36 °C ± 1 °C cultured 24 h, as a Japanese daily inoculated tube measurement.

6.1.4 inoculated a Lactobacillus broth (4.9.2) from the date of inoculation tube, 36 °C ± 1 °C cultured 24 h. From under sterile conditions the heart of the culture medium 10 min (2000 rev/min), the supernatant was discarded. With 10 mL saline (4.2) oscillation washed cells, centrifuged 10 min (2000 rev/min), the supernatant was discarded, 10 mL with physiological saline (4.2) oscillating wash. As before centrifugation, the supernatant was discarded. again Add 10 mL of normal saline (4.2), and mix. Suction 1 mL bacterial suspension in 10 mL of normal saline (4.2), and mix into the test bacteria.

6.1.5 saline (4.2) as control, using a spectrophotometer, at a wavelength at 550 nm was measured test broth (6.1.4) of the optical density Value, this value should be between 60% to 80%.

6.2 Preparation of the sample

6.2.1 Dairy products

Weigh 2 g (accurate to 0.0001 g) sample (folic acid containing about 5 µg) in 100 mL beaker, 25 mL ~ 30 mL water samples recovery Products, into 100 mL volumetric flask with water to volume, quality of folic acid concentration in the solution is about 0.05 µg/mL. Draw 1 mL the sample solution and 1 mL chicken pancreas (4.1) in a 180 mm × 15 mm screw top test tube and mixed well. Add 18 mL containing anti-Ascorbic acid phosphate buffer I (4.3.1), together with 1 mL of toluene (4.6). Also prepared ± blank, pipet 1 mL of distilled water and 1 mL Chicken pancreas (4.1) in the blank tube, add 18 mL of phosphate buffer containing ascorbic acid I (4.3.1) and 1 mL of toluene (4.6). At 37 °C Under the sample and blank tubes 16 h after incubation at 100 °C water bath 5 min. Appropriately diluted with phosphate buffer I (4.3.1), to give a concentration of about 0.1 folate solution ng/mL of.

If the sample is determined to strengthen the folic acid and folic acid compared to native large proportion, you can use 1 mL sample was added to 19 mL containing ascorbic acid Phosphate buffer I (4.3.1) was heated at 100 deg.] C water bath for 5 min, and then phosphate buffer I (4.3.1) diluted to give a concentration of about 0.1 ng/mL solution folate.

6.2.2 Cereals and cereal products

Weigh a sample containing about 1 µg of folic acid in 150 mL Erlenmeyer flask. Buffer plus 20 mL pH 7.8 phosphate solution II (4.3.2), mix Add 50 mL of water and 1.0 mL of toluene (4.6) after uniform. After capping sterilization 121 °C 15 min, followed by rapid cooling. Add 1 mL papain Enzyme solution (4.11) at 100 °C after 36 °C ± 1 °C for 3h heated 3 min, cooled. Add 1 mL alpha- amylase solution (4.12), 36 °C ± 1 °C incubated for 2 h plus 4 mL chicken pancreas (4.1), capped and heated 3 min 100 °C 36 °C ± 1 °C incubated 16 h and then cooled. With 1 mol/L Hydrochloric acid (4.15) adjusted to pH 4.5, diluted with water and dilute to 100 mL. Filtered to obtain a clear filtrate, then draw 1 mL clear filtrate with phosphorus Formate buffer III (4.3.3) and dilute to 100 mL, folate concentration of about 0.1 ng/mL in.

If the sample is determined to strengthen the folic acid and folic acid compared to native large proportion can add 20 mL 0.05 mol directly in the sample/L containing Ascorbic acid phosphate buffer II (4.3.2) and 50 mL water, sterilized at 121 °C 15 min, then draw 1mL clear filtrate, then 0.05 mol/L phosphate buffer III (4.3.3) diluted folate concentration of about 0.1 ng/mL of.

6.3 Standard curve tube production

Add distilled water in Table 1, the standard working solution (4.14.3) (standard solution Determination of cereals and cereal products with phosphate buffer IV (4.3.4) in place of phosphate buffer I (4.3.1)) and folic acid determine medium (4.9.3) in culture tubes. Table 1 for each number required Making three. Tube S2 to S10, a considerable amount of folic acid, 0.00 ng, 0.05 ng, 0.10 ng, 0.15 ng, 0.20 ng, 0.25 ng, 0.30 ng, 0.40 ng, 0.50 ng.

Table 1 Standard curve tube production

Tube No. S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 Distilled water (mL) 5 5 4 3 2 1 0 2 1 0 Standard solution (mL) 0 0 1 2 3 4 5 3 4 5 Medium (mL) 5 5 5 5 5 5 5 5 5 5

Note 1. The test tube S3 ~ S7 adding low concentrations of working standard solution.

Note 2. The test tubes S8 ~ S10 heightening concentration in the standard working solution.

6.4 Sample tube production

Add distilled water in Table 2, samples and folic acid determine medium in tubes, making the need for each number in the table 3.

Table 2 Sample tubes production

Tube No. 1234

Distilled water (mL) 4 3 2 1 Sample (mL) 1 2 3 4 Medium (mL) 5 5 5 5

6.5 Sterilization

The standard curve tubes and sterilized sample tubes 121 °C 5 min, rapidly cooled to room temperature (Commercially available media according to label directions sterilized).

Note. to ensure that the heating and cooling process conditions uniform, too sterile or too close to the number of tubes, can adversely affect the sterilization pot.

6.6 Inoculation

Under sterile conditions, each tube was added one drop (about 50 µL) of the bacterial suspension (6.1.4), capped, thoroughly shaken mix all tubes (standards Curve unvaccinated tube blank except S1).

6.7 Training

6.7.1 acidity method. 36 °C ± 1 °C cultured 72 h. Visual inspection of each tube, the tube uninoculated broth should be clear, standard Standard curve tube and sample tube broth turbidity due gradient. Uninoculated tube if turbidity and the result is invalid.

6.7.2 densitometry. at 36 °C ± 1 °C, cultured 16 h ~ 24 h. The other with 6.7.1.

6.8 Determination

6.8.1 Method acidity

6.8.1.1 with 10 mL water unvaccinated inoculated blank blank S1 and S2 of the tube culture was transferred Erlenmeyer flask with bromothymol blue (4.19) as an indicator or pH meter to pH 6.8 ± 0.2 for the titration endpoint titration with standard sodium hydroxide solution (4.18) Titration standard song Unvaccinated blank line pipe S 1 and inoculated blank tube S 2. Sodium hydroxide standard titration solution volume recorded under consumption.

Note. If the volume of the number of sodium hydroxide standard titration solution was inoculated blank titration consumption is equal to or higher than the level of non-vaccinated blank 1.5 mL, the measurement results invalid.

6.8.1.2 with 10 mL of water to the standard curve and the sample tube in tube cultures go Erlenmeyer flask with bromothymol blue (4.19) as Indicator, or a pH meter to pH 6.8 ± 0.2 for the titration endpoint titration with standard sodium hydroxide solution (4.18.1) standard titration curve and test tube Sample tube culture. Sodium hydroxide standard titration solution volume recorded under consumption.

Note. The usual standard curve tube S7 consumed 0.1 mol/sodium hydroxide standard titration solution volume number L between 8 mL ~ 12 mL.

6.8.2 Densitometry

Blank inoculate tube (Table 1 tube No. S 2) as a control, remove the highest concentration of the standard curve tube S7, oscillation 5 s, at a wavelength of 550 nm by reading the optical density values back to re-train. 2 h after the same conditions to re-measure the optical density of the tube, if the absolute difference between the results of the two optical density <=2%, then remove all the tubes measuring optical density standard solution and sample.

Draw 6.9 standard curve A standard curve for the horizontal pipe folic acid to consume the sodium hydroxide standard titration solution in milliliters or optical density value for the vertical draw standard curve line.

6.10 Calculation of folic acid in the sample tube

According to the consumption of sodium hydroxide standard titration solution in milliliters or 13.8 optical density values measured for each sample tube, check from standard curve obtained Corresponding folic acid. Three sample tube should be calculated for each number of the liquid content of folic acid per milliliter tubes measured and compared to its average value.

Tube relative deviation less than 15% of valid test tube, sample tube should be discarded invalid, the total number of valid sample tube should be greater than the sum of all sample tubes 2/3. Recalculation of the average number of valid sample tube of each solution was measured per ml of folic acid, in order to calculate the average of all the numbers again the total average of the sample tube of Cx.

Note. The sample tubes folic acid content of less than 0.05 ng, a value higher than 0.5 ng should be discarded.