Raw material selection and analysis for the isolation of proteinase inhibitor II from whole potatoes
A method for assaying the proteinase inhibitor content of tissue of a plant. The proteinase inhibitor and other protein products are extracted from the plant tissue by preparing a mixture of solvent and comminuted plant tissue to form a solid fraction and a liquid fraction which contains the proteinase inhibitor and other protein products. The liquid fraction is heated to a temperature and for a time period sufficient to denature at least some of the other protein products without substantially denaturing the proteinase inhibitor. The denatured, undesired protein products are removed by centrifugation or filtering to prepare a clarified extract solution. The amount of proteinase inhibitor inhibitor in the clan ed extract is measured. If the assay is used in the selection of plant tissues for use in the extraction of proteinase inhibitor, plant tissues with a proteinase inhibitor content less than a pre-selected standard are rejected.
1. A method for assaying, without dialyzing, the proteinase inhibitor content of tissue of a plant, comprising the steps of:
(a) extracting the proteinase inhibitor and other protein products from the plant tissue by preparing a mixture of solvent and comminuted plant tissue to form a solid fraction and a liquid fraction comprising the proteinase inhibitor and other protein products;
(b) heating the liquid fraction to a temperature and for a time period sufficient to denature at least some of the other protein products without substantially denaturing the proteinase inhibitor;
(c) removing the denatured protein products to prepare a clarified extract solution; and
(d) measuring the amount of proteinase inhibitor present in the clarified extract.
2. The method of claim 1 wherein the solvent comprises formic acid and sodium chloride.
3. The method of claim 2 wherein the solvent comprises about 0.5% to about 2.5% formic acid and 0 to 3.0 N sodium chloride.
4. The method of claim 1 wherein heat treating the liquid fraction is conducted at between about 60° to about 90° C.
5. The method of claim 4 wherein heat treating the liquid fraction is conducted for between about 30 to about 180 minutes.
6. The method of claim 1 wherein the step of removing the denatured protein products is carried out by centrifugation.
7. The method of claim 1 , further comprising the step of comparing the measured proteinase inhibitor content against a standard.
8. The method of claim 1 , wherein the plant tissue is potato tubers.
9. The method of claim 1 , wherein the proteinase inhibitor is Proteinase Inhibitor II.
10. A method that does not include a dialyzing step of selecting potatoes for use as raw materials in a process for the extraction of proteinase inhibitor from the potatoes, comprising the steps of:
(a) extracting the proteinase inhibitor and other protein products from the potatoes by preparing a mixture of solvent and comminuted potato to form a solid fraction and a liquid fraction comprising the protease inhibitor and other protein products;
(b) heating the liquid fraction to a temperature and for a time period sufficient to denature at least some of the other protein products without substantially denaturing the proteinase inhibitor;
(c) removing the denatured protein products to prepare a clarified extract solution
(d) measuring the amount of proteinase inhibitor present in the clarified extract; and
(e) rejecting any potatoes that have a proteinase inhibitor content less than a selected standard.