Proteins - Characterization of hydrolysates

11 important questions on Proteins - Characterization of hydrolysates

What is the problem with studying protein hydrolysates of food products?

In vitro you can just see if the animal is gaining weight or not.
Not populair anymore since they use animals.

However in vivo is hard because there are many properties that you need so simulate. So it is hard to connect your data to the body.

What is the downside of a multifactorial design?

You can find the most optimal conditions but still it is hard to lik it to the body.

What are the main questions of protein hydrolysis?

  1. What happens during hydrolysis
  2. How do the conditions affect the outcome.
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How manny compleces can a protein have?

A protein with N cutting sites will form ((N+1)*(N+2))/2 different peptides.

For beta-casein – PrtP1 around 30 possible cutting sites
have been identified so 496possible peptides

How do you used the pH stat method to determine the degree of hydrolysation?

You see how much acid or base you need to add in order to get to pH 6


During hydrolysis:
  • Release of COOH and NH2 groups
  • Depending on pH: Release of protons (COO-) or uptake of protons (NH3+) from solution
  • pH stat: Automatic re-adjustment of pH by addition of HCl / NaOH

What are the 2 types of meganisms for protein hydrolysation?

V8 and BLP.

V8 is for globular proteins. First one protein is hydrolyzed completely and then the next. Gives a shallow slope.

BLP is for random coil proteins. More proteins are hydrolyzed at the same time. Gives a steep slope.

How can you measure the molecular weight distribution of the peptides?

  • RP-UPLC (-UV / - MS) (at alkaline or udner reduced conditions)
  • Size-exclusion chromatography (at natural conditions)

For what is knowing the molecular weight distribution of use?

If you wand to make sure your peptides are not to small for for instance foaming enz.

How do they use MALDI-TOF-MS for identification of peptides?

The MS spectra is matched with a data base. The coverage by this data base is only 50 to 65 percent. So with this you can identify the parental protein, it is not sufficient for a complete quantitative description of the hydrolysate in mechanism studies.

How do they use LC-MS for identification of peptides?

For complete analysis of hydrolysate. 85% coverage

How does the quantification of peptides go?

By MS however UV signals are way better.

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