GB 5413.17-2010National food safety standard -- Determination of pantothenic acid in foods for infants and young children, milk and milk products (English PDF)

4.7 Medium

4.7.1 Lactobacillus agar medium. light tryptone 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).

4.7.2 Lactobacillus broth. light tryptone 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).

4.7.3 pantothenic acid measurement medium. Glucose 40 g, sodium acetate, 20 g, no vitamin acid hydrolysis of casein 10 g, dipotassium phosphate 1 g, Potassium dihydrogen phosphate 1 g, L- cystine 0.4 g, L- tryptophan 0.1 g, magnesium sulfate 0.4 g, sodium chloride 20 mg, ferrous sulfate 20 mg, sulfuric acid Manganese 20 mg, adenine sulfate 20 mg, guanine hydrochloride 20 mg, uracil, 20 mg, carotene, 400 µg, thiamin hydrochloride 200 µg, Biotin 0.8 µg, p- amino acid 200 µg, nicotinic acid 1 mg, pyridoxine hydrochloride 800 µg, polyethylene sorbitan monooleate 0.1 g, plus steamed Distilled water to 1000 mL, adjust pH to 6.7 ± 0.1 (20 °C ~ 25 °C).

4.8 Tris buffer solution. Weigh 24.2 g Trizma Base in the beaker, add 200 mL of water to dissolve.

4.9 hydrochloric acid (0.1 mol/L). draw 8.3 mL of hydrochloric acid, diluted with water to 1000 mL.

4.10 Standard solution

4.10.1 pantothenic acid stock standard solution (40 µg/mL). Weigh 45 mg ~ 55 mg calcium pantothenate standard (4.2), dissolved in 500 mL Distilled water, add 10 mL of acetic acid solution (4.3), added to 100 mL of sodium acetate (4.5), diluted with water to a precise concentration of calcium pantothenate 43.47 µg/mL (ie, pantothenic acid at a concentration of 40 µg/mL), add 0.5 mL of toluene (4.4) stored at 2 °C ~ 4 °C refrigerator shelf life of 4 Months.

4.10.2 pantothenic acid intermediate stock solution (1 µg/mL). 25 mL of stock standard solution (4.10.1), distilled water was added 500 mL, in acetic acid Solution 10 mL (4.3), sodium acetate 100 mL (4.5), and diluted with water to 1 L. Add 0.5 mL of toluene (4.4) stored in a refrigerator (2 °C ~ 4 °C), storage period of one month.

4.10.3 pantothenic acid working standard solution (10 ng/mL, 5 ng/mL). 5.0 mL twice withdrawing intermediate stock solution (4.10.2) were given water the volume to 500 mL and 1000 mL, prepared before use.

6.1 Preparation of test broth

6.1.1 The Lactobacillus (Lactobacillus plantarum) ATCC 8014 lyophilized powder into Lactobacillus broth (4.7.2) Tubes, 36 °C ± 1 °C cultured 24 h. Then transfer to Lactobacillus agar medium (4.7.1) in a test tube, 36 °C ± 1 °C cultured 24 h.

Develop good Lactobacillus agar medium (4.7.1) in vitro cultures of bacteria as a reserve.

6.1.2 from stock strains were transferred to three medium Lactobacillus agar medium (4.7.1) in a test tube, into an incubator 36 °C ± 1 °C cultured 24 h. Subculture monthly, as the monthly tube stored in the refrigerator. Monthly from monthly tube re-inoculated 3 adapter Save the new strain of tubes.

6.1.3 dated from culture tubes inoculated in a revaccination one Lactobacillus agar medium (4.7.1) tube, 36 ± 1 °C °C cultured 24 h, as a Japanese daily inoculated tube measurement.

6.1.4 inoculated a Lactobacillus broth (4.7.2) 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 (4.1) oscillation washed cells, centrifuged 10 min (2000 rev/min), the supernatant was discarded, with 10 mL of normal saline (4.1) oscillation cleaning. As before centrifugation, the supernatant was discarded. again Add 10 mL of normal saline (4.1), and mix. Suction 1 mL bacterial suspension in 10 mL of normal saline (4.1), and mix into the test bacteria.

6.1.5 saline (4.1) as control at 550 nm wavelength spectrophotometer measured test broth (6.1.4) of the optical density, this value should be between 60% to 80%.

6.2 Sample handling

Weigh 2 g (accurate to 0.0001 g) or solid sample 5 g (accurate to 0.0001 g) a liquid sample (containing about pantothenic acid 0.1 mg) in 250 Triangle mL beaker. Was added 10 mL Tris buffer (4.8), then add a small amount of water, 121 °C hydrolysis 15 min, cooled. With hydrochloric acid (4.9) PH was adjusted to 4.5 ± 0.2, into 250 mL volumetric flask to volume with water. Filtered, the filtrate was suction 4 mL, diluted to a concentration of pantothenic acid is about 5 ng/mL.

6.3 Standard curve

Add distilled water in Table 1, the standard solution and the medium in tubes, Table 1, each of the numbers required to make three. Tube S2 to S10 , A considerable amount of pantothenic acid 0 ng, 5 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 40 ng, 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 tubes plus low concentration of the standard solution S3 ~ S7.

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

6.4 Sample tube production

Add distilled water in Table 2, the sample and the medium in tubes, in triplicate.

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 to each tube was added one drop (about 50 µL) test bacteria solution (6.1.4), capped, thoroughly shaken mix all tubes (superscript Standard curve except unvaccinated blank tube S1).

6.7 Training

36 °C ± 0.5 °C cultured 16 h ~ 24 h. Visual inspection of each tube, the tube unvaccinated broth should be clear, standard curve Ray tube and the sample tube broth turbidity due gradient. Uninoculated tube if turbidity and the result is invalid.

6.8 Determination

Blank inoculate tube (Table 1 test tube number S2) as control, remove the highest concentration of the standard curve tube S7, oscillation 5 s, at a wavelength of 550 nm Read the optical density conditions, culture back again. 2 h after the same conditions to re-measure the optical density of the tube, if the optical density of the absolute difference between the two Results <=2%, then remove all the tubes measuring optical density of the sample and standard solutions.

Draw 6.9 standard curve Pantothenic acid content of a standard curve for the horizontal tube to the optical density value for the vertical standard curve.

6.10 Calculation of pantothenic acid content of the sample tube

6.8 optical density values in accordance with each sample tube assay, Richard corresponding pantothenic acid content from the standard curve. Three samples of each tube numbered it should be calculated per ml tube pantothenic acid content was measured and compared with the average of the three. Relative standard deviation of less than 15% of the tube is effectively tube, Invalid sample tube should be discarded. The total number of valid sample tube should be greater than two-thirds of the total number of sample tubes. Recalculated effective sample tube in each numbered Determination of the average per milliliter of liquid pantothenic acid content, calculated as the average of the average of the total number of all sample tubes Cx.

Note. The sample tubes value pantothenic acid content of less than 5 ng, 50 ng of the above should be discarded.

10.8 Pantothenic acid standard solution

10.8.1 pantothenic acid standard stock solution (1 mg/mL). Weigh accurately calcium pantothenate 1.087 g, dissolved in water and dilute to 1000 mL.

Pantothenic acid concentration of calcium pantothenate concentration × 0.920 = 10.8.2 pantothenic acid intermediate standard solution (0.1 mg/mL). draw standard stock solution (10.8.1) 10 mL to 100 mL volumetric flask, add water Volume. Pro preparation before use.

12.1 Sample processing

12.1.1 Free starch sample processing

Weigh mixed solid sample of about 5 g (accurate to 0.0001 g) or a liquid sample of about 20 g (accurate to 0.0001 g) in 150 mL Flask, solid sample was added about 30 mL 40 °C ~ 50 °C warm water, dissolve after shaking ultrasonic extraction 20 min.

12.1.2 Starch-containing sample processing

If the sample contains starch, mixing solid weighed sample of about 5 g (accurate to 0.0001 g) or a liquid sample of about 20 g (accurate to 0.0001 g) in 150mL flask, adding amylase (10.1) approximately 0.2 g, solid sample was added about 30 mL 40 °C ~ 50 °C warm water Shaking dissolved, covered with cork, hydrolysis 30 min at 50 °C ~ 60 °C conditions.

12.2 Preparation of the test liquid

After the sample solution was cooled to room temperature, with hydrochloric acid (10.5) adjusting the pH to 4.5 ± 0.1, add 5 mL zinc sulfate solution (10.6), fully mixing. Transferred to 50 mL volumetric flask to volume with water and mix thoroughly with a filter paper. The filtrate after 0.45 µm filter membrane, that is, the sample test solution.

12.3 Reference chromatographic conditions

Column. ODS-C18 (particle size 5 µm, 250 mm × 4.6 mm) or equivalent in performance to the column.

Mobile phase. take potassium dihydrogen phosphate solution (10.7) 900 mL, Methanol (10.2) 100 mL, and mix through 0.45 µm microporous membrane Pressure filtration.

Flow rate. 1.0 mL/min.

Detection wavelength.200 nm.

Column temperature. 30 °C ± 1 °C.

Injection volume. 10 µL.

12.4 Determination

12.4.1 Determination of a standard curve

Imbibe respectively pantothenic acid intermediate standard solution (10.8.2) 1.0 mL, 2.0 mL, 4.0 mL, 8.0 mL, 12.0 mL in 100 mL capacity Bottle, add water to volume, to give concentrations of 1.0 µg/mL, 2.0 µg/mL, 4.0 µg/mL, 8.0 µg/mL, 12.0 µg/mL of Pantothenic acid working standard solution, prepared before use.

The above-described pantothenic acid standard working sequentially chromatography (standard sample chromatogram in Appendix A in Figure A.1), recording chromatographic peak height (or Peak area). Peak height (or peak area) for the vertical axis, the standard working solution concentration as the abscissa standard curve.

12.4.2 Determination of the sample solution

Draw a sample test solution (12.2) 10 µL, the sample to be tested liquid chromatography, Richard pantothenic acid concentration in the sample solution from the standard curve degree.