First of all, I would like to thank you for the possible effective answers. I am currently working on the method AACC 22-40.01 (Measurement of Trypsin Inhibitor Activity of Soy-Products- Spectrophotometric Method). I have questions regarding to this method and it would be a great pleasure to have your support.

First, I would tell you about the used measurement standard conditions : Reagents:

Trypsin: Trypsin from bovine pancreas ≥ 7,500 BAEE units/mg (Sigma)

Substrate: BAPA N-α-benzoyl-DL-arginine-p-nitroanilide hydrochloride

Extracted samples: 1g soy feed product for 3 hr with 0,01 N NaOH

Analytic measurement: Spectrophotometric at 410 nm

- Is the mentioned Trypsin from bovine the correct choice or preferable?

- Which kind of Trypsin standard you are used in your work to determine the TI (mg/g)?

- Could you please give me detailed Information (Manufacture, BAEE- Value, Kind of Trypsin)?

- In many publications the absorbance of trypsin standard is defined with an absorbance

of 0,38. Is 0,38 a fixed value for this method or it is variable? Is there a defined specification or range?

- I considerate five sample extracts in ml. For these extracts (0; 0.6; 1.0; 1.4; 1.8 ml )

I have measured for each the absorbance at 410 nm.

The general formula for calculating TI (mg/g) of sample is:

TI (mg/g) of sample= ((ABSstandard - ABSsample)/(19))*dilution factor

sample extract ml Absorbance differential Abs. dilution factor

0,6 ----------------------- 0,1121-------- 0,2679 --------------2777,78

1,0 ---------------------- 0,1968 --------- 0,1832 ---------------1666,67

1,4 ----------------------- 0,2464 --------- 0,1336 -------------- 1190,48

1,8 ----------------------- 0,3514 --------- 0,0286 -------------- 925,93

standard -------------- 0,380

Should I calculate each sample individually? In AACC method is described to calculate the average of these samples? Which parameter should be averaged? Or should I take one absorbance value of extract samples? Can you please give me a detailed explanation? In lectures often talk about 60% - 40% Inhibition. What is it about 40-60% range?

I would be very pleased to receive your answer, opinions and concrete proposals to this case.

Best regards