National Standard of the People’s Republic of China
GB 5413.19—2010
National food safety standard
Determination of free biotin in foods for infants and young children,milk and milk products
Issue Date: 2010-03-26Implementation Date: 2010-06-01
Issued by Ministry of Health of the People’s Republic of China
GB5413.19—2010
Preface
This standard has substituted GB/T 5413.19-1997 Determination of Free Biotin Content in Formulated Foods for Infants and Young Children and Milk Powder.
The appendix A is the normative appendix.
Previous standard editions substituted by this edition are as follows:
-- GB 5413 – 1985; GB/T 5413.19 – 1997
National food safety standard
Determination of free biotin in foods for infants and young children,milk and milk products
1Scope
This Standard specifies the method for the determination of free biotin in foods for infants and young children, milk and milk products.
This standard applies to determination of free biotin content in formulated food for infants and young children and milk powder.
2Normative reference
The following normative documents contain provision which, through reference in this text, constitute provisions of This Standard. For dated reference, subsequent amendments to, or revisions of, (excluding mistakes) any of these publications do not apply. However, parties to agreements based on this standard are encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies.
3Principle
Themethod is based on growth of a biotin-dependentbacterium Lactobacillus planetarum in a medium including all essential nutrientswith the exception of biotin. Biotin is the limitingfactor for growth and can thus be determinedquantitatively by comparing sample extracts withknown standard concentrations. The addition of abiotin standard in specified increasing concentrations givesa growth response by this organism that can be measuredtitrimetrically or turbidimetrically.
4Reagents, strains and culture medium
Unless otherwise specified, the reagents used in the method are the ones of analytically pure, and the water used is the Level 2 Water regulated in GB/T 6682.
4.1Strain
Lactobacillus plantarum,ATCC 8014
4.2Biotin (d-Biotin or Vitamin H) Standard (C10H16N2O3S), purifty≥99 %
4.3Culture medium
4.3.1Lactobacillus agar culture medium, refer to the appendix A
4.3.2Lactobacillus broth culture medium,refer to the appendix A
4.3.3Culture medium for determination of biotin,refer to the appendix A
Note: If use the commercial culture medium, please prepare it according to label instructions.
4.4Ethanol solution (50%,v/v)
4.5Sulfuric Acid (H2SO4) solution (3%)
Pipette 3 mL Sulfuric Acid and dilute to 100 mL with distilled water.
4.6Sodium Hydroxide (NaOH)solution (0.5 mol/L)
Dissolve 20 g Sodium Hydroxide into 1000 mL distilled water.
4.7Sodium Chloride (NaCl) (9 g/L)
Dissolve 9.0 g Sodium Chloride into 1000 mL distilled water, and pipette 10 mL above solution to each tube. And then sterilization at 121℃for 15 min.
4.8Preparation of standard solution
4.8.1Standard biotin stock solution (100μg/mL)
Dissolve standard biotin sample (4.2) with ethanol (4.4), and dilute it to the biotin concentration of 100μg/mL, and store it in a refrigerator.
4.8.2Standard biotin medium solution(1μg /mL)
Pipette 1mL standard stock solution (4.8.1) and dilute it to 100mL with Ethanol (4.4).
4.8.3Standard biotin working solution (10 ng/mL)
Pipette 1mLmedium solution (4.8.2), dilute it to 100ml with Ethanol (4.4).
4.8.4Standard curve working solution
Prepare it for 2 concentrations. The higher concentration is 0.2ng/mL and the lower concentration is 0.1 ng/mL. Pipette 5 mL and dilute to 250 mL and 500 mL separately.
Note: All the standard solution should be kept in the refrigerator. 4.8.1, 4.8.2 and 4.8.3 can be store for 3 month and the 4.8.4 should be prepared before use.
5Instruments and Equipments
Including the common sterilization and culture apparatus of the microbiological lab, other equipments and materials are following:
5.1Balance (sensitive to 0.1 mg)
5.2pH meter: accurate≤0.02
5.3Spectrophotometer
5.4Vortex mixer
5.5Centrifuge: speed ≥2000 rpm
5.6Constant temperature incubator: 36 ℃±1 ℃
5.7Refrigerator：2 ℃～5 ℃
5.8Sterilized straw: 10 mL (with 0.1 mL Calibration) or Micropipettor or Micropipettor tips
5.9Bottle Top Dispenser: 0 mL～10 mL
5.10Flask:200 mL
5.11Volumetric flask (A grade)：100mL, 250mL, 500mL.
5.12One-markpipette (A grade): 5 mL
5.13Funnel: diameter 90 mm
5.14Quantitative filter papers: diameter 90 mm
5.15Tubes:18mm×180 mm
Note: before use, wash the hard glass pipes and other necessary glassware with the active agent (lauryl sodium or household detergent to wash water) and dry heat at 200 ℃for 2 h.
6Analysis Procedures
6.1Preparation of microorganisms
6.1.1Preparation of liquid culture medium: Stock cultures of the test organism, plantarum L.ATCC 8014 (4.1),are prepared by stab inoculation of Lactobacilli Agar Medium (4.3.1) and thenincubated at 36°C±1°C overnight. Transfers are made 2-3 times to increase the activities. And then transfer theLactobacillus plantarum(4.1) to the Lactobacillus broth culture medium (4.3.2) to incubate.
6.1.2The cells are centrifugedunder aseptic conditions and the supernatant liquid decanted.The cells are washed three times with 10 mL sterile 0.9%Sodium Chloride(4.7). After the third or fourth wash, the cells are re-suspended in 10mL sterile 0.9%Sodium Chloride(4.7) for test.
6.1.30.9%Sodium Chloride(4.7) was test as the blank, the transmittance of the cell suspension (6.1.2) was determined by spectrometer under wavelength of 550 nm.Try to test the transmittances of the samples between 60% and 80%.
6.2Treatment of samples
6.2.1Dry powder sample
Precisely weigh a certain amount of sample, which should contain 0.2 mg to 0.5mg biotin, and put it into a 250 mL flask. Andcontinue the step as stated in 6.2.3.
6.2.2Liquid sample
Pipette a certain amount of liquid sample into a 250 mL flask, which should contain 0.2 mg to 0.5mg biotin, and continue the step as stated in 6.2.3.
6.2.3Extracting sample
Add Sulfuric Acid solution (4.5) into the flask, autoclave at 121 ℃for 30 min. After cool down to the room temperature, adjust to pH4.5±0.2 with Sodium Hydroxide solution (4.6). Transfer it quantitatively to a 250ml bottle, dilute it with water to volume and fully mix it.Filter above solution with filtering paper, abandon the first several milliliters, pipette 5ml filtered solution,add 20 mL water, and adjust to pH6.8±0.2 with sodium hydroxide solution (4.6) and dilute it to 100Ml with water.
6.3Preparation of standard curve
Add distilled water, standard solution （4.8.4）and culture medium for Biotin determination（4.3.3）in culture tubes in triplicate in sequence as stated in Table 1.
Table 1 Preparation of Standard Curve
Test tube No. / S1 / S2 / S3 / S4 / S5 / S6 / S7 / S8 / S9 / S10Distilled water, mL / 5 / 5 / 4 / 3 / 2 / 1 / 0 / 2 / 1 / 0
Standard solution, mL
(0.1 ng/Ml) / 0 / 0 / 1 / 2 / 3 / 4 / 5 / 0 / 0 / 0
Standard solution, mL
(0.2 ng/mL) / 0 / 0 / 0 / 0 / 0 / 0 / 3 / 4 / 5
Culture medium, mL / 5 / 5 / 5 / 5 / 5 / 5 / 5 / 5 / 5 / 5
6.4Preparationof the tested liquid samples
Add distilled water, sample solution and culture medium for Biotin determination in culture tubes in triplicate in sequence as stated in Table 2.
A blank tube should be prepared in each portion of sample and 5 mL tested liquid sample and 5 mL culture medium should be added into each tube.
Table 2 Preparation of the Tested Liquid Samples
Test tube No. / 1 / 2 / 3 / 4Distilled water, mL / 4 / 3 / 2 / 1
Sample, mL / 1 / 2 / 3 / 4
Culture medium, mL / 5 / 5 / 5 / 5
6.5Sterilization
The tubes of 6.3 and 6.4 are all covered by caps and then be autoclaved under 121℃ for 5 minutes. The sterilization of the commercial medium should be treated according the labeling.
6.6Inoculation
The tubes were taken out of the autoclave and rapidly cool down to 30℃lower. Pipette a drop of the cell suspension (6.1.2) (about 50 μL) into above tubes, excluding the tubes of S1 and sample blank.
6.7Culturing
Incubate the tubes in a constant temperature (±0.5℃) between36℃±1℃ for19h~20h.
6.8Determination
It will be determined as ineffective if a test tube is polluted by any foreign microorganism. Carry out response prediction by visually inspecting every test tube: the non-inoculated tubes should be clear and no other microorganisms should grow in the standard solution and sample.
6.8.1The blank tube S1 as the control, the highest concentration tube of standard curve was tested, and repeated the test 2 h later. If the difference of transmittance is lower than 2%, take out of the tested tubes to test the absorbance A.
6.8.2Take the blank tube S2 as the blank, adjust the absorbance to 0, and test theabsorbance values of other samples and blank.
6.8.3Mix the samples with the Vortex mixer (a drop of defoamer can be added) and test the absorbance of the samples in the spectrophotometer cell under wavelength of 550 nm. After 30s, record the absorbance A. the stable time for each tube should be equal. Draw the standard curve and the abscissa presents content of biotin standard, the ordinate presents the absorbance.
6.8.4Among the tested tubes, the absorbance of the sample blank should be lower than 0.05. If the absorbance is above 0.02, pipette 1 mL into the cell and the absorbance value should be subtracted 1/5 absorbance of sample blank; pipette 2 mL into the cell and the absorbance value should be subtracted 2/5 absorbance of sample blank; and so on. According the absorbance value A of the tested sample, check the biotin concentration from the standard curve, and according to the dilute factor and sample weight, to calculate the biotin content of the sample. The absorbance values beyond the range of S3-S10 should be truncated.
6.8.5For each tested samples, to calculate the biotin contentsand average values from the absorbance values. In each sample, Difference between the concentration of each tube and the average value, should not be over ±15%. Otherwise it should be truncated. If the number of the tubes which meet above requirement is less than 2/3, this sample should be determined repeat.If the number of the tubes is more than 2/3, recalculate each sample’s valid average absorbance, and than calculate the total average value Cx which can be used for the biotin content calculation.
7Presentation of analytical result
Biotin content of the samples can be calculated according to formula (1)
Where: X ------Bbiotin content, μg/100g
Cx----- the total average value from 6.8.5, ng
m -----Weight of sample, g
f ------Diluting factor
The results expressed by the Arithmetic mean from two independent data which got in repeatable condition, and was Retained two significant figures.
8Allowable difference
Difference between two determined values of a same sample should be no greater than 10% of the average of the two determined values.
9Others
The Detection limit of this standard is 20.0μg/kg.
Appendix A
(Informative)
Culture Medium and Agents
A.1 Lactobacillus agar culture medium
A.1.1 Component
Potolytic peptone,15g; yeast extract, 5g; glucose10g; tomato juice,100ml; potassium dihydrogen phosphate (KDP), 2g;sorbitan monooleate, 1g; agar, 10g, added with distilled water to 1000ml, pH 6.8 ± 0.2 (25℃±5℃).
A.1.2 Procedure
Add 10.0g agar into A.1.1, heat it to boiling and make the agar dissolved. Mix evenly and pipette 10 mL of the mixture into each tube and autoclave at 121℃for 15 min.
A.2Lactobacillus broth culture medium
A.2.1 Component
Potolytic peptone,15g; yeast extract, 5g; glucose10g; tomato juice,100ml; potassium dihydrogen phosphate (KDP), 2g; sorbitan monooleate, 1g; agar, 10g, added with distilled water to 1000ml, pH 6.8 ± 0.2 (25℃±5℃).
A.2.2 Procedure
Heat the component A.2.1 to boiling and make the agar dissolved. Mix evenly and pipette 10 mL of the mixture into each tube and autoclave at 121℃for 15 min.
A.3Lactobacillus broth culture medium
A.3.1 Component
Casein hydrolyzed Amino Acids for vitamins determination,12.0 g; glucose, 40.0 g; Sodium acetate, 20.0 g; L-Cystine, 0.2g, DL-Tryptophan, 0.2g; Adenine sulphate, 20.0 mg; Guanine hydrochloride, 20.0 mg; Uracil, 20.0 mg; Thiamine hydrochloride, 2.0 mg;Riboflavin, 2.0 mg;Nicotinic acid, 2.0 mg; Calcium Pantothenate2.0 mg; pyridoxine hydrochloride, 4.0 mg; ρ-aminobenzoic acid,200.0 μg; Potassium hydrogen phosphate,1.0 g; Potassium dihydrogen phosphate,1.0 g; Magnesium sulfate,0.4 g; Sodium chloride,20.0 mg; Ferrous sulfate,20.0 mg; Manganese sulfate,20.0 mg; add water to 1000 mL and the pH 6.8±0.2（25 ℃±5 ℃）。
A.3.2 Procedure
Dissolve above component with water and adjust the pH ready for use.