Reivindicaciones
CLAIMS 1. A method for the in situ production of an emulsifier in a foodstuff, wherein the method comprises the step of adding a lipid acyltransferase to the foodstuff. 2. A method according to claim 1 wherein at least 2 emulsifiers are produced. 3. A method according to claim 1 or claim 2 wherein the emulsifier is produced without increasing or substantially increasing the free fatty acids in the foodstuff. 4. A method according to any one of claims 1-3 wherein the lipid acyltransferase is one which is capable of transferring an acyl group from a lipid to one or more of the following acyl acceptors : a sterol, a stanol, a carbohydrate, a protein or a sub-unit thereof, glycerol. 5. A method according to claim 2 wherein at least one of the emulsifiers is a carbohydrate ester. 6. A method according to claim 2 wherein at least one of the emulsifiers is a protein ester. 7. A method according to any one of the preceding claims wherein one or more of a sterol ester or a stanol ester or a protein ester or a carbohydrate ester or a diglyceride or a monoglyceride is produced in situ in the foodstuff. 8. A method according to claim 7 wherein the sterol ester is one or more of alpha- sitosterol ester, beta-sitosterol ester, stigmasterol ester, ergosterol ester, campesterol ester or cholesterol ester. 9. A method according to claim 6 wherein the stanol ester is one or more beta- sitostanol or ss-sitostanol. 10. 10. A method according to any one of the preceding claims wherein the lipid acyltransferase is characterised as an enzyme which possesses acyl transferase activity and which comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S. <Desc/Clms Page number 184> 11. A method according to any one of the preceding claims wherein the lipid acyltransferase enzyme comprises H-309 or comprises a histidine residue at a position corresponding to His-309 in the amino acid sequence of the Aeromonas hydrophila lipolytic enzyme shown as SEQ ID No. 2 or SEQ ID No. 32. 12. A method according to any one of the preceding claims wherein the lipid acyltransferase is obtainable from an organism from one or more of the following genera : Aeromonas, Streptomyces, Saccharomyces, Lactococcus, Mycobacterium, Streptococcus, Lactobacillus, Desulfitobacterium, Bacillus, Campylobacte7 ; Vibrionaceaej Xylella, Sulfolobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, Mesorhizobium, Ralstonia, Xanthomonas and Candida. 13. A method according to any one of the preceding claims wherein the lipid acyltransferase comprises one or more of the following amino acid sequences: (i) the amino acid sequence shown as SEQ ID No. 2 ; (ii) the amino acid sequence shown as SEQ ID No. 3 ; (iii) the amino acid sequence shown as SEQ ID No. 4; (iv) the amino acid sequence shown as SED ID No. 5; (v) the amino acid sequence shown as SEQ ID No. 6; (vi) the amino acid sequence shown as SEQ ID No. 12, (vii) the amino acid sequence shown as SEQ ID No. 20, (viii) the amino acid sequence shown as SEQ ID No. 22, (ix) the amino acid sequence shown as SEQ ID No. 24, (x) the amino acid sequence shown as SEQ ID No. 26, (xi) the amino acid sequence shown as SEQ ID No. 28, (xii) the amino acid sequence shown as SEQ ID No. 30, (xiii) the amino acid sequence shown as SEQ ID No. 32, (xiv) the amino acid sequence shown as SEQ ID No. 34, or an amino acid sequence which has 75% or more identity with any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 32 or SEQ ID No. 34. 14. A method according to any one of the preceding claims, wherein the emulsifier is one or more of the following: a monoglyceride, a lysophosphatidylcholine, DGMG. <Desc/Clms Page number 185> 15. Use of a lipid acyltransferase to prepare from a food material a foodstuff comprising an emulsifier, wherein the emulsifier is produced without increasing or without substantially increasing the free fatty acids in the foodstuff, and wherein the emulsifier is generated from constituents of the food material by the lipid acyltransferase. 16. Use according to claim 15 wherein at least two emulsifiers are produced. 17. Use according to claim 16 wherein at least one of the emulsifiers is a carbohydrate ester. 18. Use according to claim 16 wherein at least one of the emulsifiers is a protein ester. 19. Use according to any one of claims 15-18 wherein one or more of a sterol ester or a stanol ester or a protein ester or a carbohydrate ester or a diglyceride or a monoglyceride is also produced in situ in the foodstuff. 20. Use according to claim 19 wherein the sterol ester is one or more of alpha- sitosterol ester, beta-sitosterol ester, stigmasterol ester, ergosterol ester, campesterol ester or cholesterol ester. 21. Use according to claim 20 wherein the stanol ester is one or more beta- sitostanol or ss-sitostanol. 22. Use according to any one of claims 15 to 21 wherein the lipid acyltransferase is characterised as an enzyme which possesses acyl transferase activity and which comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S. 23. Use according to any one of claims 15-22 wherein the lipid acyltransferase enzyme comprises H-309 or comprises a histidine residue at a position corresponding to His-309 in the amino acid sequence of the Aeromonas hydrophila lipolytic enzyme shown as SEQ ID No. 2 or SEQ ID No. 32. 24. Use according to any one of claims 15-23 wherein the lipid acyltransferase is obtainable from an organism from one or more of the following genera : Aeromonas, St eptomyces, Saccharomyces, Lactococcus, Mycobacteriuna, Streptococcus, Lactobacillus,. Desulfitobacterium, Bacillus, Campylobacter, Vibrionaceae, Xylella, Sulfolobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, MesorhizobSum, Ralstonia, Xanthomonas and Candida. <Desc/Clms Page number 186> 25. Use according to any one of claims 15-24 wherein the lipid acyltransferase comprises one or more of the following amino acid sequences: (i) the amino acid sequence shown as SEQ ID No. 2; (ii) the amino acid sequence shown as SEQ ID No. 3 ; (iii) the amino acid sequence shown as SEQ ID No. 4 ; (iv) the amino acid sequence shown as SED ID No. 5 ; (v) the amino acid sequence shown as SEQ ID No. 6; (vi) the amino acid sequence shown as SEQ ID No. 12, (vii) the amino acid sequence shown as SEQ ID No. 20, (viii) the amino acid sequence shown as SEQ ID No. 22, (ix) the amino acid sequence shown as SEQ ID No. 24, (x) the amino acid sequence shown as SEQ ID No. 26, (xi) the amino acid sequence shown as SEQ ID No. 28, (xii) the amino acid sequence shown as SEQ ID No. 30, (xiii) the amino acid sequence shown as SEQ ID No. 32, (xiv) the amino acid sequence shown as SEQ ID No. 34, or an amino acid sequence which has 75% or more identity with any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 32 or SEQ ID No. 34. 26. Use according to any one of claims 15-25, wherein the emulsifier is one or more of the following: a monoglyceride, a lysophosphatidylcholine, DGMG. 27. A food or feed enzyme composition which contains a lipid acyltransferase. 28. A food or feed enzyme composition according to claim 27 wherein the lipid acyltransferase is characterised as an enzyme which possesses acyl transferase activity and which comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S. 29. A food or feed enzyme composition according to claim 27 or claim 28 wherein the lipid acyltransferase enzyme comprises H-309 or comprises a histidine residue at a position corresponding to His-309 in the amino acid sequence of the Aeromonas hydrophila lipolytic enzyme shown as SEQ ID No. 2 or SEQ ID No. 32. 30. A food or feed enzyme composition according to any one of claims 27-29 wherein the lipid acyltransferase is obtainable from an organism from one or more of the following genera: Aeromonas, Streptomyces, Saccharomyces, <Desc/Clms Page number 187> Lactococcus, Mycobacterium, Streptococcus, Lactobacillus, Desulfitobacterium, Bacillus, Campylobacter, Vibrionaceae, Xylella, Sulfolobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, Mesorhizobium, Ralstonia, Xanthomonas and Candida. 31. A food or feed enzyme composition according to any one of claims 27-30 wherein the lipid acyltransferase comprises one or more of the following amino acid sequences: (i) the amino acid sequence shown as SEQ ID No. 2; (ii) the amino acid sequence shown as SEQ ID No. 3; (iii) the amino acid sequence shown as SEQ ID No. 4; (iv) the amino acid sequence shown as SED ID No. 5; (v) the amino acid sequence shown as SEQ ID No. 6 ; (vi) the amino acid sequence shown as SEQ ID No. 12, (vii) the amino acid sequence shown as SEQ ID No. 20, (viii) the amino acid sequence shown as SEQ ID No. 22, (ix) the amino acid sequence shown as SEQ ID No. 24, (x) the amino acid sequence shown as SEQ ID No. 26, (xi) the amino acid sequence shown as SEQ ID No. 28, (xii) the amino acid sequence shown as SEQ ID No. 30, (xiii) the amino acid sequence shown as SEQ ID No. 32, (xiv) the amino acid sequence shown as SEQ ID No. 34, or an amino acid sequence which has 75% or more identity with any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 32 or SEQ ID No. 34. 32. Use of a food or feed enzyme composition according to any one of claims 27- 31 in accordance with any one of claims 15-26 or in the method according to any one of claims 1-14. 33. A foodstuff obtainable by the method according to any one of claims 1-14. 34. An immobilised lipid acyltransferase enzyme. 35. An immobilised lipid acyltransferase according to claim 34 wherein the lipid acyltransferase is characterised as an enzyme which possesses acyl transferase activity and which comprises the amino acid sequence motif GDSX, wherein X is one or more of the following amino acid residues L, A, V, I, F, Y, H, Q, T, N, M or S. <Desc/Clms Page number 188> 36. An immobilised lipid acyltransferase according to claim 34 or claim 35 wherein the lipid acyltransferase enzyme comprises H-309 or comprises a histidine residue at a position corresponding to His-309 in the amino acid sequence of the Aeromonas hydrophila lipolytic enzyme shown as SEQ ID No. 2 or SEQ ID No. 32. 37. An immobilised lipid acyltransferase according to any one of claims 34-36 wherein the lipid acyltransferase is obtainable from an organism from one or more of the following genera: Aeromonas, Streptomyces, Saccharomyces, Lactococcus, Mycobacterium, Streptococcus, Lactobacillus, Desugitobacterium, Bacillus, Campylobacter, Vibrionaceae, XyZella, Sul, folobus, Aspergillus, Schizosaccharomyces, Listeria, Neisseria, Mesorhizobiuna, Ralstonia, Xanthomonas and Candida. 38. An immobilised lipid acyltransferase according to any one of claims 34-37 wherein the lipid acyltransferase comprises one or more of the following amino acid sequences: (i) the amino acid sequence shown as SEQ ID No. 2; (ii) the amino acid sequence shown as SEQ ID No. 3; (iii) the amino acid sequence shown as SEQ ID No. 4; (iv) the amino acid sequence shown as SED ID No. 5; (v) the amino acid sequence shown as SEQ ID No. 6; (vi) the amino acid sequence shown as SEQ ID No. 12, (vii) the amino acid sequence shown as SEQ ID No. 20, (viii) the amino acid sequence shown as SEQ ID No. 22, (ix) the amino acid sequence shown as SEQ ID No. 24, (x) the amino acid sequence shown as SEQ ID No. 26, (xi) the amino acid sequence shown as SEQ ID No. 28, (xii) the amino acid sequence shown as SEQ ID No. 30, (xiii) the amino acid sequence shown as SEQ ID No. 32, (xiv) the amino acid sequence shown as SEQ ID No. 34, or an amino acid sequence which has 75% or more identity with any one of the sequences shown as SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 12, SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 32 or SEQ ID No. 34. 39. A method of identifying a suitable lipid acyltransferase for use in accordance with the present invention, comprising the steps of testing an enzyme of interest using one or more of the"Transferase Assay in a Low Water <Desc/Clms Page number 189> environment", the"Transferase Assay in High Water Egg Yolk"or the "Transferase Assay in Buffered Substrate", and selecting a lipid acyltransferase if it is one which has one or more of the following characteristics: (a) when tested using the"Transferase Assay in a Low Water Environment", measured after a time period selected from 30,20 or 120 minutes, has a relative transferase activity of at least 1% ; (b) when tested using the"Transferase Assay in High Water Egg Yolk"in an egg yolk with 54% water, has up to 100% relative transferase activity; or (c) when tested using the"Transferase Assay in Buffered Substrate"has at least 2% acyltransferase activity. 40. A method according to claim 39 wherein the lipid acyltransferase is selected if it is one which has more than two of the following characteristics (a) when tested using the"Transferase Assay in a Low Water Environment", measured after a time period selected from 30,20 or 120 minutes, has a relative transferase activity of at least 1% ; (b) when tested using the"Transferase Assay in High Water Egg Yolk"in an egg yolk with 54% water, has up to 100% relative transferase activity ; or (c) when tested using the"Transferase Assay in Buffered Substrate"has at least 2% acyltransferase activity. 41. A method according to claim 39 wherein the lipid acyltransferase is selected if it is one which has more than three of the following characteristics (a) when tested using the"Transferase Assay in a Low Water Environment", measured after a time period selected from 30,20 or 120 minutes, has a relative transferase activity of at least 1% ; (b) when tested using the"Transferase Assay in High Water Egg Yolk"in an egg yolk with 54% water, has up to 100% relative transferase activity; or (c) when tested using the"Transferase Assay in Buffered Substrate"has at least 2% acyltransferase activity. 42. A method according to claim 39 wherein the lipid acyltransferase is selected if it is one which has all of the following characteristics (a) when tested using the "Transferase Assay in a Low Water Environment", measured after a time period selected from 30,20 or 120 minutes, has a relative transferase activity of at least 1% ; (b) when tested using the"Transferase Assay in High Water Egg Yolk"in an egg yolk with 54% water, has up to 100% relative transferase <Desc/Clms Page number 190> activity ; or (c) when tested using the"Transferase Assay in Buffered Substrate" has at least 2% acyltransferase activity. 43. A lipid acyltransferase identified using a method according to any one of claims 39-42.
