Why do we use pH-stat to measure protein kinetics?

We have decided to use the pH-stat method in collaboration with Aarhus University as this is a real kinetic technique allowing you to measure progress of protein hydrolysis instead of only focusing on the extent of protein hydrolysis. It is a scientific well established and recognized method and we use the method as essentially described and published by scientists from Wageningen University.

The method has been well described and used in literature to evaluate protein hydrolysis for different raw materials and product types. It is a reliable and simple method, and results obtained by pH-stat are found to correlate well with other in-vitro methods.

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How does the method work – what is the principle?

How to measure protein kinetics?

There are several methods to measure protein kinetics. Most of them are based on measuring the degree of hydrolysis, which is defined as the percentage of hydrolyzed peptide bonds.

Methods to measure degree of hydrolysis assume that a free amino group and a free carboxyl group are released every time a peptide bond is hydrolyzed. Common for all these methods is that they quantify the increase in concentration of such released groups, and they use the quantification as a variable to evaluate the amount of hydrolyzed peptide bonds. The difference between the methods really lies in how they quantify the increase in the given concentration.

Why do we use pH-stat to measure protein kinetics?

We have decided to use the pH-stat method in collaboration with Aarhus University as this is a real kinetic technique allowing you to measure progress of protein hydrolysis instead of only focusing on the extent of protein hydrolysis. It is a scientific well established and recognized method and we use the method as essentially described and published by scientists from Wageningen University.

The method has been well described and used in literature to evaluate protein hydrolysis for different raw materials and product types. It is a reliable and simple method, and results obtained by pH-stat are found to correlate well with other in-vitro methods.

How does the method work – what is the principle?

As you can hear by the name, the pH-stat technique has something to do with pH.

The technique utilizes that an acid is being released every time a peptide bond is broken, and that a subsequent auto-titration will add the required amount of alkaline solution to keep the pH at 8.

Step by step:

First you mix the sample with water and force the pH to 8. Then you start the reaction, here a protein hydrolysis by adding an enzyme cocktail of three pancreatic enzymes to mimic the enzymes present in the intestine of the young animals.

The enzymes will start to hydrolyze peptide bonds, and for every peptide bond being hydrolyzed, an acid will be released, and the pH of the sample solution will drop accordingly. When the pH value drops, the pH stat automatically adds alkaline solution, in our case sodium hydroxide, to increase pH again and keep it constant at 8.

The volume of sodium hydroxide added is recorded for all timepoints and converted into degree of hydrolysis creating a curve representing the degree of hydrolysis.

A huge advantage of this method is that it provides us with a rate constant. It means that the method is not only able to tell how much of the peptide bonds that have been hydrolyzed, it also tells you how fast they have been hydrolyzed.
The faster the peptide bonds have been hydrolyzed, the more alkaline solution has been added in the beginning of the reaction time.

Why pH at 8?

The pH is forced to 8 as this is the optimum for the pancreatic enzymes used during hydrolysis. If we lower the pH to represent conditions closer to the stomach, the enzymes will start to denature which will affect the enzyme activity negatively. We cannot ensure consistent denaturing of enzymes when we force them outside their optimum, which will affect the stability and control of experimental conditions. Therefore, to secure consistent and reproducible results, the pH-stat runs at 8, which is also commonly used and described in literature.

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