Measuring the activity of Natto NKCP
The effectiveness of Natto Extract products is often difficult to compare because there is no standard test for products that help to optimize blood viscosity. Many companies marketing nattokinase compounds measure the enzymatic activity of their product in Fibrinolytic Units (FU), making the assumption that the effectiveness of these enzyme supplements lies solely in the degree with which they can break up fibrin. This is misleading, however, because:
- Fibrinolysis is just one of the in vitro activities of nattokinase. In vivo, it can have differing activities and functions.
- Fibrin is an insoluble protein and does not allow formation of uniform suspensions or plates. This makes it unsuitable for a substrate for quantifying enzymatic activity. It should therefore only be used as a substrate for qualitative analysis.
- Fibrin suspensions or plates have a network structure, and so it is unclear whether the enzymatic activity is associated with the degradation of the network structure or that of fibrin monomers.
- When fibrin is used as the substrate, the reaction proceeds in a logarithmic manner against the enzyme concentration. Thus, a linear standard curve is not obtained, resulting in an accuracy problem.
- When fibrin is used as the substrate, reactivity is likely to vary according to the enzymes used. Because there are multiple types of nattokinase, standards for all of these types is necessary. (Current analysis is based on a certain standard product of unknown origin.)
For these reasons, instead of giving the activity of NKCP in FU, Daiwa uses two other methods to determine activity, which results in a much more accurate assessment of their product:
- Method for determining peptidase activity
The sample is incubated at 37°C for 5 minutes with the synthetic chromogenic substrate S-2251 (H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride) as substrate and the PNA, which is freed by hydrolysis, is determined by measuring the absorbance at 405 mm. Peptidase activity (unit/gm) is defined 1 unit when 1nmol of pNA per minute is freed from the substrate of 2mM S-2251 at 37°C by 1gm NKCP.
- Method for determining functional ingredients (antigen measurement method)
The natto bacillus-produced protein responsible for NKCP's peptidase activity is purified to prepare antibodies specific for rabbits and mice. The amount of antigen reacting with the specific NKCP antibodies is determined using the sandwich ELISA method. The functional ingredients are represented as μg/g or mg/kg.