Non starch polysaccharide hydrolyzing enzymes as feed additives: detection of enzyme activities and problems encountered with quantitative determination in complex samples.
Chromogenic substrates, an agar diffusion assay and viscosity reduction were used to estimate beta-glucanase and xylanase activities in water soluble extracts of different feedstuffs and digesta supernatants. The dinitrosalicylic acid reducing sugar method was employed to calibrate results from different methods based on international units (IU, glucose equivalents). The detection of dye release from chromogenic substrates was a suitable method, allowing the detection of 0.05 IU of enzyme activity per ml of extract, although measurements in digesta supernatants were limited in linearity (0.1-0.5 IU/ml supernatant). With the agar diffusion assay the detection of enzyme activity was possible over a wider concentration range (extracts: 0.05-1 IU/ml, digesta supernatants: 0.1-1 IU/ml), but visual evaluation led to inaccurate measurement. Accuracy can be improved by computer based evaluation of digital images. The use of viscosity reduction produced linear standard curves from 0.01 to 0.5 IU/ml in feed extracts, but reliability of measurements depended on modification of substrates. Quantification of enzyme activities was influenced by matrix effects of complex samples. Cereal dependant differences were found in various extracts of feed mixtures and cereal extracts. Digesta supernatants partly inhibited enzyme activity, depending on the origin of the sample. Interaction of substrates with digesta components varied between methods. The sensitivity of the methods is comparable, however, all methods require specific calibrations to account for matrix- and enzyme specific effects.
Vahjen
W
W
Institute of Animal Nutrition, Free University of Berlin, Germany.
Gläser
K
K
Froeck
M
M
Simon
O
O
D000821:Animal Feed / Q000032:analysis*; D000818:Animals; D002863:Chromogenic Compounds / Q000378:metabolism; D004064:Digestive System / Q000201:enzymology*; D002523:Edible Grain / Q000737:chemistry / Q000201:enzymology; D005503:Food Additives / Q000032:analysis*; D005504:Food Analysis / Q000379:methods*; D005947:Glucose / Q000032:analysis; D006868:Hydrolysis; D005779:Immunodiffusion; D011134:Polysaccharides / Q000378:metabolism; D015203:Reproducibility of Results; D014783:Viscosity; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000032:analysis*; D001617:beta-Glucosidase / Q000032:analysis*
D002863:Chromogenic Compounds; D005503:Food Additives; D011134:Polysaccharides; D014995:Xylosidases; D001617:beta-Glucosidase; D043325:Xylan Endo-1,3-beta-Xylosidase; D005947:Glucose