Lab 10 Browning Reactions

Question 1

Describe the Maillard reaction.

Answer 1

• Non-enzymatic browning that begins with a reaction between a reducing sugar and an amino acid.
• Carbonyl group of sugar reacts with amino group of amino acid
• Creates a glycosylamine/Schiff’s base that is unstable and undergoes an Amadori rearrangement, and then continues to react
• Dark brown compounds called melanoidins are formed as end products

Question 2

Describe the Maillard reaction in milk.

Answer 2

• Lactose is the reducing sugar
• Milk does not contain many free amino acids, leaving the epsilon-amino group of the side chain of lysine in casein proteins to predominantly react with lactose
• The free amino group of the N-terminus of peptides can also react with reducing sugars

Question 3

What factors influence the Maillard reaction?

Answer 3

• Increases in (1) temperature, (2) pH, and (3) water activity are all known to increase the rate of formation of Maillard reaction products
  
  • (4) Unprotonated amino acids (basic conditions) facilitate Maillard browning
  • Maillard reaction does not require as high of temperatures as caramelization for colour changes to occur

Question 4

What is o-phthaldialdehyde?

Answer 4

• o stands for ortho which refers to a benzene ring structure with substituents on adjacent carbons
• OPA reacts with amino groups in the presence of 2-mercaptoethanol and yields a blue fluorescent product with excitation and emission wavelengths of 340 and 455 nm, respectively
• Colour can be detected using spectrophotometry at 340 nm
• Is used to measure lysine levels in milk samples to determine degree of Maillard browning

Question 5

Discuss OPA concentration and pH dependence.

Answer 5

Question 6

What is the main amino acid degraded during the Maillard reaction in milk?

Answer 6

Lysine (specifically the epsilon amino acid)

Question 7

Explain the purpose of each of the components of the OPA reagent in a spectrophotometric assay for lysine determination in milk. [4]

Answer 7

• OPA: This is the primary reagent that reacts with the primary amino group of lysine to form a highly fluorescent isoindole derivative. The OPA is dissolved in ethanol to help it dissolve and disperse evenly in the solution.
• Sodium tetraborate buffer: This buffer helps to maintain a stable pH during the reaction. The pH of the solution affects the reactivity of the OPA and the formation of the fluorescent product. A pH of 9.7-10.0 is optimal for this reaction.
• SDS (sodium dodecyl sulfate): This is a detergent that helps to solubilize and denature the proteins in the sample, exposing their lysine residues for reaction with the OPA.
• 2-mercaptoethanol: This is a reducing agent that helps to reduce the OPA molecule, allowing it to react with the primary amino group of lysine to form the fluorescent product.

Question 8

What is the purpose of diluting the milk samples in 10% SDS prior to spectrophotometric analysis?

Answer 8

The purpose of the SDS is to denature the protein so that the lysine residues are more readily accessible for detection.

Question 9

How is a HunterLab colorimeter standardized?

Answer 9

With black and white tiles