RIBOFLAVINASE ENZYMES AND THEIR USE TO PREVENT OFF FLAVOR IN BREWING
The present disclosure provides methods, compositions, apparatuses and kits comprising several enzymes involved in the hydrolysis or breakdown of riboflavin beer or a malt-based substrate for beverage production. The present invention relates to improvements in the production of beer and similar light-sensitive beverages, in particular improvement of the flavor stability of such light-sensitive beverages by enzymatic hydrolysis or degradation of riboflavin during beer production.
1 . A method for the inhibition of formation of 3MBT (3-methylbut-ene-thiol) in a malt beverage comprising adding to the malt beverage an effective amount of a riboflavinase.
2 . A method according to claim 1 wherein the riboflavinase is a riboflavin hydrolase.
3 . A method according to claim 2 wherein the riboflavin hydrolase comprises an enzyme having at least 80% sequence identity to MOXRcaE1 (SEQ ID NO:8) or an active fragment thereof or MOXRcaE2 (SEQ ID NO:12) or an active fragment thereof.
4 . A method according to claim 3 wherein the riboflavin hydrolase comprises an enzyme having at least 80% sequence identity to MOXRcaE1 or an active fragment thereof.
5 . A method according to claim 4 wherein the riboflavin hydrolase comprises an enzyme having at least 90% sequence identity to MOXRcaE1 or an active fragment thereof.
6 . A method according to claim 5 wherein the riboflavin hydrolase comprises an enzyme having at least 95% sequence identity to MOXRcaE1 or an active fragment thereof.
7 . A method according to claim 6 wherein the riboflavin hydrolase comprises an enzyme having at least 99% sequence identity to MOXRcaE1 or an active fragment thereof.
8 . A method according to claim 7 wherein said riboflavin hydrolase comprises MOXRcaE1 or an active fragment thereof.
9 . A method according to claim 3 wherein said riboflavin hydrolase comprises an enzyme having at least 80% sequence identity to MOXRcaE2 or an active fragment thereof.
10 . A method according to claim 9 wherein said riboflavin hydrolase comprises an enzyme having at least 90% sequence identity to MOXRcaE2 or an active fragment thereof.
11 . A method according to claim 10 wherein said riboflavin hydrolase comprises an enzyme having at least 95% sequence identity to MOXRcaE2 or an active fragment thereof.
12 . A method according to claim 11 wherein said riboflavin hydrolase comprises an enzyme having at least 99% sequence identity to MOXRcaE2 or an active fragment thereof.
13 . A method according to claim 12 wherein said riboflavin hydrolase comprises MOXRcaE2 or an active fragment thereof.
14 . A method according to claim 2 further comprising adding an effective amount of a second riboflavinase enzyme.
15 . A method according to claim 14 wherein said second riboflavinase comprises a riboflavin reductase.
16 . A method according to claim 15 wherein the riboflavin reductase is selected from the group consisting of an enzyme having at least 80% sequence identity with MOXRcaB1 (SEQ ID NO:6) or an active fragment thereof or MOXRcaB2 (SEQ ID NO:10) or an active fragment thereof.
17 . A method according to claim 16 wherein the riboflavin reductase comprises an enzyme having at least 80% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
18 . A method according to claim 17 wherein said riboflavin reductase comprises an enzyme having at least 90% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
19 . A method according to claim 18 wherein said riboflavin reductase comprises an enzyme having at least 95% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
20 . A method according to claim 19 wherein said riboflavin reductase comprises an enzyme having at least 99% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
21 . A method according to claim 20 wherein said riboflavin reductase comprises MOXRcaB1 or an active fragment thereof.
22 . A method according to claim 16 wherein the riboflavin reductase comprises an enzyme having at least 80% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
23 . A method according to claim 17 wherein said riboflavin reductase comprises an enzyme having at least 90% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
24 . A method according to claim 18 wherein said riboflavin reductase comprises an enzyme having at least 95% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
25 . A method according to claim 19 wherein said riboflavin reductase comprises an enzyme having at least 99% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
26 . A method according to claim 20 wherein said riboflavin reductase is MOXRcaB2 or an active fragment thereof.
27 . A method according to any of the preceding claims in which the malt beverage is selected from the group consisting of a beer, ale, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, and non-alcoholic malt liquor.
28 . A method according to claim 27 in which the malt beverage comprises a beer.
29 . A malt beverage comprising an effective amount of a riboflavinase.
30 . A malt beverage according to claim 29 wherein the riboflavinase comprises a riboflavin hydrolase.
31 . A malt beverage according to claim 30 wherein the riboflavin hydrolase is selected from the group consisting of an enzyme having at least 80% amino acid sequence identity to MOXRcaE1 (SEQ ID NO:8) or an active fragment thereof or MOXRcaE2 (SEQ ID NO:12) or an active fragment thereof.
32 . A malt beverage according to claim 31 wherein said riboflavin hydrolase an enzyme having at least 80% amino acid sequence identity to MOXRcaE1 or an active fragment thereof.
33 . A malt beverage according to claim 32 wherein said riboflavin hydrolase comprises an enzyme having at least 90% amino acid sequence identity to MOXRcaE1 or an active fragment thereof.
34 . A malt beverage according to claim 33 wherein said riboflavin hydrolase comprises an enzyme having at least 95% amino acid sequence identity to MOXRcaE1 or an active fragment thereof.
35 . A malt beverage according to claim 34 wherein said riboflavin hydrolase comprises an enzyme having at least 99% amino acid sequence identity to MOXRcaE1 or an active fragment thereof.
36 . A malt beverage according to claim 35 wherein said riboflavin hydrolase comprises MOXRcaE1 or an active fragment thereof.
37 . A malt beverage according to claim 31 wherein said riboflavin hydrolase comprises an enzyme having at least 80% sequence identity to MOXRcaE2 or an active fragment thereof.
38 . A malt beverage according to claim 37 wherein said riboflavin hydrolase comprises an enzyme having at least 90% sequence identity to MOXRcaE2 or an active fragment thereof.
39 . A malt beverage according to claim 38 wherein said riboflavin hydrolase comprises an enzyme having at least 95% sequence identity to MOXRcaE2 or an active fragment thereof.
40 . A malt beverage according to claim 39 wherein said riboflavin hydrolase comprises an enzyme having at least 99% amino acid sequence identity to MOXRcaE2 or an active fragment thereof.
41 . A malt beverage according to claim 40 wherein said riboflavin hydrolase comprises MOXRcaE2 or an active fragment thereof.
42 . A malt beverage according to claim 29 further comprising adding an effective amount of a second riboflavinase enzyme.
43 . A malt beverage according to claim 42 wherein said second riboflavinase comprises a riboflavin reductase.
44 . A malt beverage according to claim 43 wherein the riboflavin reductase comprises an enzyme having at least 80% amino acid sequence identity to MOXRcaB1 (SEQ ID NO:6) or an active fragment thereof or MOXRcaB2 (SEQ ID NO:10) or an active fragment thereof.
45 . A malt beverage according to claim 44 wherein the riboflavin reductase comprises an enzyme having at least 80% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
46 . A malt beverage according to claim 45 wherein said riboflavin reductase comprises an enzyme having at least 90% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
47 . A malt beverage according to claim 46 wherein said riboflavin reductase comprises an enzyme having at least 95% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
48 . A malt beverage according to claim 47 wherein said riboflavin reductase comprises an enzyme having at least 99% amino acid sequence identity to MOXRcaB1 or an active fragment thereof.
49 . A malt beverage according to claim 48 wherein said riboflavin reductase comprises MOXRcaB1 or an active fragment thereof.
50 . A malt beverage according to claim 44 wherein the riboflavin reductase comprises an enzyme having at least 80% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
51 . A malt beverage according to claim 50 wherein said riboflavin reductase comprises an enzyme having at least 90% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
52 . A malt beverage according to claim 51 wherein said riboflavin reductase comprises an enzyme having at least 95% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
53 . A malt beverage according to claim 52 wherein said riboflavin reductase comprises an enzyme having at least 99% amino acid sequence identity to MOXRcaB2 or an active fragment thereof.
54 . A malt beverage according to claim 53 wherein said riboflavin reductase comprises MOXRcaB2 or an active fragment thereof.
55 . A malt beverage according to any of the preceding claims in which the malt beverage is selected from the group consisting of a beer, ale, dry beer, near beer, light beer, low alcohol beer, low calorie beer, porter, bock beer, stout, malt liquor, and non-alcoholic malt liquor.
56 . A malt beverage according to claim 27 in which the malt beverage is a beer.
57 . A method according to claim 1 wherein the riboflavinase is a riboflavin destructase.
58 . A method according to claim 57 wherein the riboflavin destructase comprises an enzyme having at least 80% identity to SmeBluB1 (SEQ ID NO:2) or an active fragment thereof or PspBluB1 (SEQ ID NO:4) or an active fragment thereof.
59 . A method according to claim 58 wherein the riboflavin destructase comprises an enzyme having at least 80% sequence identity to SmeBluB1 or an active fragment thereof.
60 . A method according to claim 59 wherein the riboflavin destructase comprises an enzyme having at least 90% sequence identity to SmeBluB1 or an active fragment thereof.
61 . A method according to claim 60 wherein the riboflavin destructase comprises an enzyme having at least 95% sequence identity to SmeBluB1 or an active fragment thereof.
62 . A method according to claim 61 wherein the riboflavin destructase comprises an enzyme having at least 99% sequence identity to SmeBluB1 or an active fragment thereof.
63 . A method according to claim 62 wherein the riboflavin destructase comprises SmeBluB1 or an active fragment thereof.
64 . A method according to claim 58 wherein the riboflavin destructase comprises an enzyme having at least 80% sequence identity to PspBluB1 or an active fragment thereof.
65 . A method according to claim 64 wherein the riboflavin destructase comprises an enzyme having at least 90% sequence identity to PspBluB1 or an active fragment thereof.
66 . A method according to claim 65 wherein the riboflavin destructase comprises an enzyme having at least 95% sequence identity to PspBluB1 or an active fragment thereof.
67 . A method according to claim 66 wherein the riboflavin destructase comprises an enzyme having at least 99% sequence identity to PspBluB1 or an active fragment thereof.
68 . A method according to claim 67 wherein the riboflavin destructase comprises PspBluB1 or an active fragment thereof.
69 . A method for the inhibition of formation of 3MBT (3-methylbut-ene-thiol) in a food comprising adding to the food an effective amount of a riboflavinase.
70 . A method according to claim 69 wherein the food is selected from the group consisting of milk, milk-based dairy product, fermented milk products, ice-cream, vegetable oil, olive oil, soy milk, soy bean oil and oil containing salad dressing.
71 . A method according to claim 69 wherein said riboflavinase is selected from the group consisting of a riboflavin hydrolase, a riboflavin reductase and a riboflavin destructase.