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22 Enzymes Methods​

22-02.01 Measurement of alpha-Amylase in Plant and Microbial Materials Using the Ceralpha Method

Microbial alpha-amylases find widespread application in the modification of starch in cereal products and in cereal processing. The level of endogenous alpha-amylase in cereal grains and products significantly affects the industrial exploitation of these commodities. In breadmaking, the level of alpha-amylase must be sufficient to produce saccharides that can be absorbed and utilized by yeast but not so high as to cause excessive starch dextrinization, which can lead to sticky crumb and problems in processing. In the brewing industry, the level of malt alpha-amylase is a key quality parameter. alpha-Amylase also finds application as a silage additive, to assist in the degradation of starch and thus to provide fermentable sugars for bacterial growth. The procedure described here (the Ceralpha method) can be used to measure cereal flour, malt, and fungal and bacterial alpha-amylases. Samples are extracted with appropriate buffer and, following filtration or centrifugation (if required), are suitably diluted. Aliquots of diluted extract are incubated with substrate solution under defined conditions of temperature, time, and pH, and the reaction is stopped with an alkaline solution that also develops the color.

The assay is absolutely specific for alpha-amylase. The substrate mixture contains the defined oligosaccharide “nonreducing end-blocked p-nitrophenyl malto­heptaoside” (BPNPG7) in the presence of excess levels of a thermostable alpha-glucosidase (which has no action on the native substrate due to the presence of the “blocking group”). On hydrolysis of the oligosaccharide by endo-acting alpha-amylase, the excess quantities of alpha-glucosidase in the mixture give instantaneous and quantitative hydrolysis of the p-nitrophenyl maltosaccharide fragment to glucose and free p-nitrophenol. The absorbance at 400 nm is measured, and this is a direct measure of the level of alpha-amylase in the sample analyzed. The assay can be used over the pH range 5.2-7.0 and at temperatures of up to 60°. The optimal pH for cereal and fungal alpha-amylases is 5.4 and for bacterial alpha-amylase is 6.5.

22-05.01 Measurement of alpha-Amylase in Cereal Grains and Flours—Amylazyme Method

This method determines the amount of alpha-amylase in malted cereal grains and cereal flours from sound and weather-damaged grains. Because the levels of alpha-amylase in flours from weather-damaged and sound grain are about 0.1% of that in malted grains, two assay formats are employed. For malted cereals, the enzyme is extracted with buffer and suitably diluted before assay. For flours from sound and weather-damaged grains, the assay is performed directly on flour slurries. In both formats, the activity is converted to Ceralpha units. This method is the only alpha-amylase procedure that employs a defined substrate (i.e., end-blocked p-nitrophenyl maltoheptaoside; in the presence of excess quantities of amyloglucosidase and alpha-glucosidase).The substrate employed is dyed and cross-linked amylose in tablet form. As the particles of substrate are hydrolyzed by alpha-amylase, soluble dyed fragments are released into solution, and the color released is directly related to the level of alpha-amylase in the assay mixture.

22-08.02 Measurement of alpha-Amylase Activity with the Rapid Visco Analyser

This method is based on the ability of alpha-amylase to liquefy a starch gel. The enzyme activity is measured as the stirring number (SN), defined as the apparent viscosity in rapid visco units at the 180th sec of stirring a hot aqueous flour suspension undergoing liquefaction. By the action of the hydrolytic enzyme alpha-amylase, viscosity decreases and SN increases. Enzyme activity is also an indication of sprouting of grains. This method is applicable to both meal and flour of all small grains.

22-10.01 Measurement of alpha-Amylase Activity with the Amylograph

This method uses the amylograph to estimate alpha-amylase activity (diastatic activity) in an aqueous suspension of flour as it gelatinizes during heating. The high viscosity of the starch gel is counteracted by the action of alpha-amylase, which liquefies starch granules as the slurry is heated. The amylograph value, or peak viscosity, also called malt index, is therefore inversely correlated with alpha-amylase activity. The method measures alpha-amylase that naturally occurs in flour or is added as malt; it does not respond to fungal alpha-amylase.

22-11.01 Measurement of Gassing Power by the Pressuremeter Method

This method estimates gassing power or gas production of dough, which is highly correlated with starch damage. In the presence of excessive alpha-amylase activity, gas production is very high, due to increased production of fermentable sugars. To measure yeast activity, refer to Method 89-01.01.

22-12.01 Measurement of alpha-Amylase Activity in Flour Supplemented with Fungal alpha-Amylase—Modified Amylograph Method

This is a modification of Method 22-10 that allows the amylograph to be used on flour supplemented with alphaase of fungal (Aspergillus oryzae) origin. The initial high viscosity of a pregelatinized wheat starch-flour slurry is decreased by the hydrolytic action of alpha-amylase. Viscosity continues to drop until the gelatinizing starch from wheat flour causes a buildup in viscosity, forming a dip, or minimum viscosity (MV). The MV, like the peak viscosity (PV) of the regular amylograph procedure, is inversely proportional to the amount of alpha-amylase present and can be used as an index of diastatic activity in flour. This method is applicable to fungal alpha-amylase-treated flour, malted flour, or untreated flour.

22-14.01 Measurement of Gassing Power by Volumetric Method

This method provides an estimate of gassing power or gas production of fermenting dough. Gas production is much higher if there is excessive alpha-amylase activity due to increased production of fermentable sugars.

22-15.01 Measurement of Diastatic Activity of Flour or Semolina

Processing characteristics of flour are influenced by increased levels of hydrolytic enzymes, primarily alpha-amylase. This method estimates diastatic activity in flour or semolina by measuring the amount of maltose produced through hydrolysis of starch. Maltose can be measured by ferricyanide procedure with or without titration with thiosulfate.

22-40.01 Measurement of Trypsin Inhibitor Activity of Soy Products—Spectrophotometric Method

This method determines total and residual trypsin inhibitors in soy products, including raw and toasted soybean meals and flours, soy protein concentrates and isolates, and corn-soy mixtures. Trypsin inhibitor activity is determined by incubating the sample with a known substrate (BAPA) and trypsin. Trypsin activity is indicated by an increase in absorbance at 410 nm. Inhibition of trypsin, by the inhibitor present in the sample, decreases the absorbance increase. The method can be modified slightly to determine trypsin inhibitor content in other food and feed products and plant materials.

22-62.01 Measurement of Proteolytic Activity—Spectrophotometric Method

This method estimates proteolytic enzyme activity in active proteinase preparations. The method is based on enzymatic hydrolysis of denatured hemoglobin at pH 4.7, followed by precipitation of unhydrolyzed substrate with trichloroacetic acid. Solubilized hemoglobin is then measured spectrophotometrically. Ratio of enzyme concentration to absorbance is linear within range of assay.

22-80.01 Qualitative Test for Peroxidase in Oat Products

In the processing of oat products, the inactivation of lipase enzyme is of particular importance for maintenance of adequate flavor in the stored product. Since peroxidase has greater thermal stability than lipase, it may be used as an indicator of lipase inactivation. This method is applicable to oat groats, steel cut oat groats, rolled oats, oat flour, and oat bran.

22-85.01 Measurement of Polyphenol Oxidase in Wheat Kernels (L-DOPA Substrate)

Discoloration in dough is an end-use quality defect. A bright, clear color that remains stable over time is desired. Discoloration, in the form of grey, brown, or green shades, or general “dullness,” has long been attributed to polyphenol oxidase (PPO). PPO is located predominately in the bran coat of wheat kernels. Problems with enzymatic discoloration occur in Asian noodles (especially alkaline noodles) and in any doughs, including refrigerated doughs, that are held for periods of time before baking or boiling. This method is a relatively rapid, small-scale, nondestructive test for PPO; applicable to both breeding and commercial applications. It utilizes L-dihydroxyphenylalanine (L-DOPA) as a substrate, and meets the criteria for a practical test for breeding programs. The L-DOPA method here is specific for whole wheat kernels, but it can be adapted for use on flour or ground wheat samples.

22-90.01 Measurement of Urease Activity

The method is a modified Caskey-Knapp method that yields residual urease present in soybean products under conditions of the test. It is applicable to soybean meals, soy flour, and millfeeds.