Vitamin B12 (Cyanocobalamin) Test Kit

【Principles】

Vitamin B12 (Cyanocobalamin) Test Kit used microbiology method to detect the volume of VB12 in food matrices. VB12 supplies the growth of Lactobacillus delbrueckiikit. Its growth rate associated with the volume of VB12 within the sample. Take the VB12 medium (Lactobacillus delbrueckiikit added), samples or Standards and add to the microwell. Incubate at 37℃ and avoid light exposure. Lactobacillus delbrueckiikit will start growing until VB12 are consumed entirely. It takes 44-48 hours to complete incubation. Use microplate reader to read the sample turbidity with 610-630nm or 540-550nm filter. The volume of VB12 in sample can be determined by comparing the sample turbidity with the Standards.

【Provided Materials and Reagent】

【Materials Required But Not Provided】

1. Devices

—- Clean bench

—- Microplate Reader (610-630nm or 530-540nm filter)

—- Water Bath: 95℃

—- Incubator: 37℃

—- Centrifuge (≥8000g)

—- Sterilized tips: 20-200μL, 100-1000μL

—- Sterilized centrifuge tubes: 15mL, 50mL

—- Sterilized reaction tubes: 1.5mL / 2mL

—- Vortex

—- Sterilized syringe and 0.22μm sterilized filter

—- Sterilized tweezers

2. Reagent for native VB12 detection

—- Sterile water

—- 1% NaCH or KCN Solution:1g NaCN or KCN dissolved in 100mL redistilled water

—- 1M NaOH: 4g NaOH dissolved in 100mL redistilled water

—- Taka-Diastase from Aspergillus oryzae

—- Acetate buffer (pH 4.0): weigh 20.5g C₂H₃NaO₂ (Sodium Acetate) in a 1000mL beaker, add 950mL redistilled water and dissolve on a magnetic stirrer. Adjust pH to 4.0 with CH₃COOH (Acetic Acid, >99%). Transfer the solution to a 1000mL volumetric flask and fill up with redistilled water to 1000mL. The buffer can be stored at 2-8˚C for 1 week.

—-VB12 Extraction Buffer: 1.3g Na₂HPO₄ (Sodium Hydrogen Phosphate), 1.0g Na₂S₂O₅ (Sodium Pyrosulfite) and 1.2g C₆H₈O₇·H₂O (Citric Acid Monohydrate) dissolved in 100mL redistilled water.

【Sample Preparation】

Sample Pretreatment

• Liquid samples with added VB12 (fruit juices, drinks)

1. Add 1mL homogeneous sample to a 50mL sterilized centrifuge tube.
2. Add 40mL sterile water or deionized water, vortex to mix well.
3. Filter it with 0.22μm sterilized filter and collect the filtrate.
4. Depends on the VB12 concentration range, dilute the filtrate further in 1.5mL or 2mL sterilized reaction tubes with sterile water from kit.

• Jelly with added VB12

1. Weigh out 20g jelly sample to a 100mL sterilized centrifuge tube.
2. Add 30mL sterile water or deionized water, vortex to mix well. Extract in the 95℃-Water Bath for 30 minutes. Mix it every 10 minutes (make sure the centrifuge tube is sealed), then cool it down to 30℃ rapidly.
3. Add sterile water or deionized water to 80mL, then pipette 4mL sample solution (equivalent to 1g sample) to a 50mL sterilized centrifuge tube. Then, add deionized water exactlyto 40mL, vortex to mix well.
4. Filter it with 0.22μm sterilized filter and collect the filtrate.
5. Depends on the VB12 concentration range, dilute the filtrate further in 1.5mL or 2mL sterilized reaction tubes with sterile water from kit.

• Milk powder, baby food, bakery food and wheat flour with added VB12

1. Weigh out1g (mL) homogeneous sample to a 50mL sterilized centrifuge tube.
2. Add 40mL sterile water or deionized water, vortex to mix well. Extract in the 95℃-Water Bath for 30 minutes. Mix it every 10 minutes (make sure the centrifuge tube is sealed), then cool it down to 30℃ rapidly.
3. Filter it with 0.22μm sterilized filter and collect the filtrate. Or centrifuge (≥8000g) it for 5 minutes, pipette the middle layer.
4. Depends on the VB12 concentration range, dilute the filtrate further in 1.5mL or 2mL sterilized reaction tubes with sterile water from kit.

• Total content of VB12 (added and native) for dairy product, cereal, and baby food

Method 1: Extraction by enzymatic treatment

When testing the content of VB12 (native) in food, samples should have enzymatic treatment with NaCN or KCN.

1. Weigh out 1g (mL) homogeneous sample to a 50mL sterilized centrifuge tube.
2. Add 20mL Acetate buffer (pH 4.0), vortex to mix well. Then, add 250μL NaCN Solution (1%, use freshly).
3. Add 300mg Taka-Diastase, and incubate at 37℃ without light for 1 hour (vortex regularly). Then, add sterile water or deionized water to 40mL.
4. Then, extractthe sample in the 95℃-Water Bath for 30 minutes. Mix it every 10 minutes (make sure the centrifuge tube is sealed), then cool it down to 30℃ rapidly.
5. Filter it with 0.22μm sterilized filterand collect the filtrate. Or centrifuge (≥8000g) it for 5 minutes, pipette the middle layer.
6. Depends on the VB12 concentration range, dilute the filtrate further in 1.5mL or 2mL sterilized reaction tubes with sterile water from kit.

Note: When enzymatic treatment is required for sample preparation, a blank control is recommended to ensure that the enzyme reagent used does not contain VB12.

Method 2: Extraction by high pressure

1. Weigh out 1g (mL) homogeneous sample to a 50mL sterilized centrifuge tube.
2. Add 1mL VB12 Extraction buffer, then add 30mL deionized water, vortex to mix well.
3. Extractthe sample at 121℃ high pressure for 15 minutes, then cool it down to 30℃ rapidly.
4. Adjust pH to 6.8 with 1M NaOH, then add sterile water or deionized water to 40mL.
5. Filter it with 0.22μm sterilized filter and collect the filtrate.
6. Depends on the VB12 concentration range, dilute the filtrate further in 1.5mL or 2mL sterilized reaction tubes with sterile water from kit.

Note: When the final concentration of sample is diluted, the mass concentration of Sodium Pyrosulfite is less than 0.03mg/mL. If the mass concentration of Sodium Pyrosulfite is higher than 0.03mg/mL, the sampling amount should be increased (that is, when the dilution factor is less than 1:8.3, the sampling amount should be increased).

Sample Dilution

1. Dilution factor calculations

Using reasonable dilution factor can ensure the accuracy of the test when the sample concentration falls within the standard curve.

For example, for sample with 1.2μg/100g of VB12, dilution factor can be calculated by taking the concentration of VB12 divide by Standard 2. Dilution factor = 1.2μg ÷ 0.06μg = 20, choose 20 dilution factor.

2. Sample Dilution Procedure
3. 1:9 (50μL sample + 450μL sterile water fromkit)
4. 1:1 (250μL of solution from a) + 250μL sterile water fromkit)

Note: Mix well before each dilution. Dilute the samples fresh before usage. The result is the most accurate for the samples when diluting between the concentration of Standard 2 and Standard 3.

Precautions during preparation

1. For samples with lower VB12concentration, weigh 5g of samples instead (it should take into consideration when calculate the result).
2. Lower concentration samples with darker color (darker color may affect the assay result) can be filtrated by Whatman 934-AH. Collect the filtratefor
3. Samples are difficult to filter should be centrifuge (≥8000g, 5 minutes) first to remove the particles. Then, filter it with 0.22μm sterilized filter.
4. If the samples needed to be diluted further for the extraction (dilution factor greater than 10), dilute them 10-fold or lower than 10-fold in gradient. At each gradient, ensure the samples are homogeneous (make sure to vortex well). If samples are not homogeneous, it will affect the result significantly. Dilution method should follow the steps of Sample Dilution.
5. Prepare Standards and samples in triplicates.
6. Unknown samples should dilute in two different gradients to make sure it falls within the standard curve.
7. If samples are not being used immediately, store them at 4℃ and avoid light exposure. Only use them within the same day.