Methods for determination of bioactivity, quantity, removal, or inactivation of cereal amylase trypsin inhibitors in cereals, flours, plants, and complex foods
AssigneeBeth Israel Deaconess Medical Center, Inc.
InventorsDetlef SCHUPPAN, Victor ZEVALLOS
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Abstract
The present invention features methods of extracting amylase trypsin inhibitors (ATIs) from processed and unprocessed foodstuff, determining bioactivity of ATIs, quantifying the amount of ATIs in a foodstuff, and reducing the content of ATIs in a foodstuff.
Claims42
A method of reducing amylase trypsin inhibitor (ATI) content or reactivity of a foodstuff, said method comprising:
- (i) incubating said foodstuff in an extraction buffer for a duration sufficient to reduce the ATI content of said foodstuff by at least 50% followed by removing said extraction buffer from said foodstuff;
- (ii) (a) incubating said foodstuff with an extraction buffer in a mixture;
- (b) centrifuging said mixture to form a pellet and supernatant;
- (c) drying said supernatant;
- and
- (d) reconstituting the dried supernatant in a reconstitution buffer, wherein extracted ATIs are present in said reconstitution buffer;
- (iii) (a) determining the bioactivity of and/or quantifying the amount of the ATI in the foodstuff;
- and
- (b) reducing the amount of the ATI in the foodstuff by extracting and degrading the ATI using one or more reducing agents, proteases, and/or synthetic enzymes engineered to reduce and/or degrade the ATI in the food stuff;
- (iv) cultivating a cereal with oxidized glutathione (GSSG) and/or protein bound glutathione (PSSG) and forming the foodstuff with the cereal;
- (v) cultivating a cereal with sulfur rich or sulfur depleted fertilizers to reduce the reactivity of ATI and toll-like receptor 4 (TLR4) in the foodstuff;
- or
- (vi) fermenting said foodstuff with a bacteria with high disulfide reducing and/or proteolytic ability.
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A method of determining the quantity and/or bioactivity of ATIs in a sample, said method comprising:
- (i) incubating ATI-reactive cells with an extract suspected of comprising ATIs and determining an ATI-response in said ATI-reactive cells;
- (ii) incubating an immobilized TLR4, TLR4-MD2, or TLR4-MD2-CD14 with an extract suspected of comprising ATIs, wherein said immobilized TLR4, TLR4-MD2, or TLR4-MD2-CD14 is immobilized to a solid phase and bound to a labeled ATI, removing the extract, and determining the amount of labeled ATI in the extract or bound to said immobilized TLR4, TLR4-MD2, or TLR4-MD2-CD14 that is immobilized to the solid phase;
- (iii) incubating an immobilized TLR4, TLR4-MD2, or TLR4-MD2-CD14 with an extract suspected of comprising ATIs, removing the extract suspected of comprising ATIs, and determining the amount of ATIs that are bound to said immobilized TLR4 using an antibody in an ELISA;
- and/or
- (iv) incubating said foodstuff in an extraction buffer thereby forming an extract;
- optionally incubating said extract with an alkylating agent, optionally separating the ATIs from said extract, quantifying said ATIs, and optionally adding at least one protease and/or a synthetic enzyme engineered to reduce and/or degrade ATIs to said extract.
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Dependencies are inferred from the claim text.