In this article we will look at the method used to determine enzymatic activity as determined by the Falling Number Apparatus.

The Falling Number method was first described by Hagberg and Perten in the early 1960’s for the purpose of providing a rapid means of determining the extent of sprout damage in wheat or rye (Doty, 1980). It has found widespread acceptance because of its rapid analysis time, simple operation, and high degree of reproducibility (Pyler, 1986). Sprout damage in wheat is of critical concern if the end-use for the flour being milled is bread production. As little as 5% heavily sprouted wheat in a mill mix of otherwise sound grain can make the mixture unacceptable for bread production (Perten, 1985). Sprouting in wheat results in a higher than normal level of alpha-amylase in the flour. Wheat that has been harvested before sprouting has occurred contains low levels of alpha-amylase (Posner, 1997).

α-amylase is of greater concern in bread production than β-amylase for four reasons: (1) it is able to hydrolyze damaged raw starch; (2) it has a higher thermal stability allowing it to act on gelatinizing starch for 3-4 minutes when the interior of the bread loaf is 140-150F, (3) it is stable at the common pH of bread dough: 5.0 - 5.6, and (4) it is activated by calcium ions that inactivate β- amylase (Pyler, 1986). In addition to this, β-amylase is only able to act upon the non-reducing ends of starch chains from which it splits off maltose, and it is unable to act upon intact raw starch (Doty, 1980). The activity of β-amylase is also dependent upon the level of starch damage in the flour as damaged starch has more sites at which β-amylase can act. Starch is damaged in the milling process and typically accounts for around 8% of the total starch in hard wheat flours (Pyler, 1986).

α-amylase is far more able to reduce the long starch molecules into smaller pieces upon which the β-amylase can act. α-amylase is able to act upon interior portions of the starch molecules. The result of this is that the β- amylase now has more open sites upon which is can act and produce more maltose molecules: a source of energy for yeast involved in fermentation. It is the combined action of these two molecules that can convert nearly the entire starch molecule into fermentable sugar (Doty, 1980)

This amylolytic action in dough occurs once the dough ingredients are combined and mixed. The conversion of starch to maltose and other yeast fermentable sugars is critical to the bread baking process. This conversion results in several changes in dough properties including: a decrease in absorption capacity, a slackening of dough consistency, and the development of a stickier dough. The rate at which these changes occur is directly proportional to the amount of starch damage and α-amylase level of the flour. As we noted above, flour milled from sound, un-sprouted wheat has a very low amylase content and requires supplemental α-amylase to have the required functional properties for bread production. Hard wheat flours typically have a total sugar content of 1.5%. This level is initially boosted to 2.0 to 2.5% during mixing by the rapid action of α-amylase upon the damaged starch (Pyler, 1986).

Typically, wheat is harvested once the grain has dried to an appropriate moisture level that takes into consideration both optimality for harvest and suitability for prolonged storage. In a dry, normal growing season this is done before the grain has had a chance to germinate and sprout. The direct implication of this is that alpha-amylase levels are typically quite low and supplementation of the milled flour must occur. The Falling Number Method is used to measure the level of alpha-amylase activity in newly harvested wheat as a means of detecting sprout damage and as a method for determining the proper supplementation rates of barley malt, or other alpha-amylase enrichment (Doty, 1980).

Bread flours with normal diastatic activity (milled from sound, un- sprouted wheat and supplemented with alpha-amylase through the addition of barley malt, or fungal amylase) typically having falling number values in the range of 220 to 250 seconds. Flours deficient in diastatic activity will typically have values in excess of 400 seconds and over supplemented flours or flour milled from sprout damaged wheat can have the minimum value of 60 seconds.

The Falling Number Method is based on the starch liquefying action of alpha- amylase and expresses this as the time in seconds required to stir and allow the stirrer to fall a measured distance through a heated aqueous flour gel that is undergoing liquefaction (Pyler, 1980). The instrument consists of a boiling water bath, a stirring head, and a timer. Also needed are, a test tube and a stirring rod. The stirring head consists of motors and gears which allows for precise and uniform stirring insuring reproducibility of the results (Doty, 1980).

The procedure for flour involves mixing 7 g of flour with 25 mL of distilled water in a test tube. The tube is shaken and the stirring rod is inserted and then the whole assembly is placed in the boiling water bath. The timer is automatically started and a stirring process is activated and continues for 55 seconds and a rate of 2 strokes per second. At the end of 60 seconds the stirring rod is released from the up position and allowed to fall through the heated flour-water slurry. Upon completion of the vertical fall the timer stops and displays the elapsed time in seconds (Pyler, 1986). The descent of the stirring rod through the slurry is related to the amylase activity of the sample. Upon completion of the stirring action the amylase present in the flour starts to break down the gelatinized starch reducing the viscosity of the slurry (Doty, 1980).

If analysis of a wheat sample is required then a 300 g sample is ground in a hammer mill to obtain a flour sample. From here the procedure is the same as above for the flour sample (Doty, 1980).

There are several factors that have the potential to affect the results of the Falling Number Method. These include the sampling method, the preparation of the samples, moisture content of the samples, boiling temperature (affected by altitude), heat treatment of the grain, and the stirring procedure (Perten, 1967). This last issue is not of particular importance today as the Falling Number Instruments most commonly found are fully automated and require little operator input aside from the initial shaking of the sample.

The falling number method as described is an absolutely essential analysis technique in both the milling and baking industries. As alpha-amylase plays such a critical role in baking, the development of this rapid, simple, and highly reproducible technique has proven to be invaluable.