Respected Madam,

                          Kindly go through this methodology.

Extraction and Estimation of Trypsin Inhibitor (TI) Activity

Extraction (Sadasivam and Manickam, 1996)

Five hundred milligram of powdered sample was transferred into a pre-chilled mortar and pestle and ground with 25 ml of distilled water. The ground sample was kept in a refrigerator for 3h. with frequent shaking for the complete extraction of TI. The homogenate was centrifuged in a refrigerated centrifuge (temperature 4 ± 2°C) [REMI Cooling Centrifuge, Model C-24, Mumbai, India] at 15,000 x g for 20 min. After centrifugation, the supernatant was collected. One millilitre of the clear supernatant was diluted to 10ml with distilled water and used as TI source.

Estimation

Trypsin inhibitor activity was assessed by the method of Kakade et al. (1974). 0 to 2 millilitre (i.e., 0, 0.5, 1.0, 1.5 and 2 ml) of extract was transferred into duplicate sets of test tubes and each tube extract was made upto 2ml with distilled water. After that, 2 ml of trypsin solution [4 mg trypsin (Sigma Chemical) was dissolved in 200 ml 0.001 M HCl] was added to each test tube and kept in water bath at 37°C. To each tube, 5ml BAPNA solution [40mg N-a-Benzoyl-DL-Arginine p-nitroanilide hydrochloride (Sigma Chemical)] in 1 ml of dimethyl sulfoxide and diluted to 100 ml with tris-buffer (6.05g hydroxyl-methylaminomethane and 2.94 g CaCl2 : H2O were dissolved in 900 ml of water. The pH was adjusted to 8.2 and the volume was brought to 1000 ml with distilled water) and pre-warmed to 37°C] was added; exactly after 10 min. the reaction was terminated by adding 1 ml of 30% (v/v) acetic acid. The contents of the tubes were mixed thoroughly and the absorbance was measured at 410 nm with a help of spectrophotometer [Elico UV-VIS Spectrophotometer SL 150, ELICO - LTD] against a reagent blank [1 ml of 30% (v/v) acetic acid + 2 ml of each trypsin and distilled water +5 ml of BAPNA solution]. Protein content in the extract was determined by the method of Lowry et al. (1951) and expressed the trypsin inhibitor activity in Trypsin inhibitor Units or TIU/mg protein. One trypsin unit was expressed as an increase of 0.01 absorbance unit per 10 ml of reaction mixture at 410 nm.

• Sadasivam, S. and Manickam, A. 1996. Phenolics. In: Biochemical methods for agricultural sciences. Wiley Eastern Ltd., New Delhi, India. pp. 187-188.

• Lowry, O.H., Rorebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with folin phenol reagent. J. Bio. Chem.193: 265 – 275.

• Kakade, M.L., Rackis, J.J., McGhce, J.E. and Puski, G. 1974. Determination of trypsin inhibitor activity of soy products: a collaborative analysis of an improved procedure. Cereal Chem. 51: 376 -382.

Good Luck

Dear Livia,

You can use these methods as well:

1) Quantitative Determination of Trypsin Inhibitory Activity in Complex

Matrices

Robin E.J. Spelbrink*, Pieter Jan Gerrits, Carina Mooij and Marco L.F. Giuseppin

2)The determination of trypsin inhibitor levels in foodstuffs

Clifford Smith, Wim van Megen, Leendert Twaalfhoven and Christopher Hitchcock

DOI: 10.1002/jsfa.2740310403

As mentioned above for a well vadlidated assay in various food matrices a very recent study

Quantitative Determination of Trypsin Inhibitory Activity in Complex Matrices

Robin E J Spelbrink Pieter Jan Gerrits Carina Mooij Carina Mooij Marco L F Giuseppin

in : The Open Food Science Journal 01/2011; 511:42-46.

ABSTRACT A quantitative assay using azocasein was developed to measure trypsin inhibitory activity in emulsions and other complex systems that are refractory to analysis. The method was tested for reproducibility on pure protein solutions as well as protein-containing material rich in fats and sugars, with special attention to emulsions. In the clean situation, the overall relative standard deviation was less than 6% while for the more complex systems it was less than 16%. The procedure proved robust against deliberate variations of temperature, incubation time and substrate concentration.

thank you all

Read the attached paper

good luck

Article Measuring Trypsin Inhibitor in Soy Meal: Suggested Improveme...

This is what I did to analyse the TIA in sweet potatoes. You can try this out without a standard curve.

Please have a look at this reference ma'am. It provides an alternative method for determination of trypsin inhibitors using the casein hydrolysis method.

https://www.aaccnet.org/publications/cc/backissues/1969/Documents/chem46_518.pdf

Dear all,

Can someone tell me a method to eliminate the colur intesity of the samples? At which point Can I take a preplate?

@Harsha Bharwani , can You please help me?

It would be helpful if you can tell me what color are your samples.