NMR-milk metabolites and extraction methods

Purpose

This is an example study that showed how NMR can be used to study the component of milk and the influence of milk extraction methods

Materials

  • Micropipette

  • Eppendorf vials with lids

  • NMR tubes (5 mm)

Chemicals

Software

  • Topspin 4.0, Amix 4.0, Matlab and Chenomx 8.7

Methods:

Two extraction approaches for milk samples

Method #1: Extraction with Methanol

  1. Add 700 μL 2% milk into a Eppendorf vial

  2. Add 700 μL methanol into the same Eppendorf vial

  3. Close the lid of the vial

  4. Vortex for 5 minutes

  5. Centrifuge for 10 minutes (settings: 14.8 RPM Speed, temperature 5°C)

  6. Collect aqueous layer and transfer into a new PCR vial (do not disturb non-aqueous layer)

  7. Close the lid of vial

  8. Centrifuge for 10 minutes

  9. Open the lid of vial and place into the SpeedVac Instrument

  10. Dry for 7 hours at room temperature

  11. Add 700 μL of buffer into vial

  12. Vortex for 5 minutes

  13. Centrifuge for 10 minutes

  14. Transfer 650 μL of sample into NMR tube

  15. Put a lid/stopper on the NMR tube

  16. Analyze sample on the NMR instrument

Method #2: Extraction with Chloroform

  1. Add 700 μL 2% milk into a Eppendorf vial

  2. Add 700 μL chloroform into the same Eppendorf vial

  3. Close the lid of the vial

  4. Vortex for 5 minutes

  5. Centrifuge for 10 minutes (settings: 14.8 RPM Speed, temperature 5°C)

  6. Collect aqueous layer and transfer into a new PCR vial (do not disturb non-aqueous layer)

  7. Close the lid of vial

  8. Centrifuge for 10 minutes

  9. Open the lid of vial and place into the SpeedVac Instrument

  10. Dry for 7 hours at room temperature

  11. Add 700 μL of buffer into vial

  12. Vortex for 5 minutes

  13. If it is not dissolved, sonicate for 1 minute

  14. Vortex for 10 seconds

  15. Centrifuge for 10 minutes

  16. Transfer 650 μL of sample into NMR tube

  17. Put a lid/stopper on the NMR tube

  18. Analyze sample on the NMR instrument

Analyze using the Bruker software:

Topspin simple analysis


Initial data analysis



This section was created by Ngan Nguyen, Undergraduate student, Department of Chemistry, NC A&T State University

Data were collected and processed by Ngan Nguyen and Kamirra Pearson, Undergraduate students, Department of Chemistry, NC A&T State University