The use of enzymes in the animal nutrition industry has gained widespread use, says Keith Filer with Alltech, Inc., in Thailand. “Enzymes provide an additional tool for improving animal performance by releasing more nutrients to the animal,” he says. “A wide variety of carbohydrase, protease, phytase and lipase enzymes have proven to be useful. Analytical techniques have been developed to allow enzyme producers, regulatory agencies and enzyme users to determine the enzyme activity of these products.”
Enzyme assays can be used to detect enzymes in finished products as well as after they’ve been added to feeds, says Filer. However, each sample provides challenges that should be understood to ensure the results are interpreted properly.
Enzyme samples can be divided into two general categories, explains Filer. “The sample is either a concentrated sample or it is one that has been added to feed. Enzyme assays in their most simple form can be explained as an incubation of the enzyme with substrate for a given time period. The reaction is stopped and either an end product is detected or the disappearance of the substrate in determined.
“The substrate and incubation conditions such as temperature, pH and time vary with the specific enzyme,” he continues. “Each must be optimized for the enzyme.”
Filer notes that the most common assays can be divided into categories depending on detection methods. End-product formation assays include reducing sugar assays, dye-labeled substrate and color metric assays. Types of enzymes detected by these methods include protease, cellulose, xylanase and phytase. Assays that detect substrate changes include the color metric and viscometric processes. Enzymes detected with this type of assay include amylase, xylanase and cellulose.
“The most common enzyme analyzed in feed is phytase, which utilizes a color metric end-product formation assay, although a number of labeled substrates are produced commercially that can be used for other enzymes in feed samples,” explains Filer.
Common issues for both concentrate and feed samples include sample size, utilization of a representative sample, and extraction of the enzyme into a liquid environment.
Feed samples provide a number of additional challenges. The enzyme concentration is greatly reduced when an enzyme is added to feed. The sensitivity of the assay must be such that this effect can be overcome. Using a feed sample also introduces potential enzyme inhibitors, says Filer. “Sampling techniques have been devised to ensure representative samples are taken,” he notes. “Extraction of the enzyme into a buffer can be difficult but with a small amount of research, time and temperature conditions can be developed to address this issue.”
Simple filtration techniques are available to clarify samples and remove inhibitory compounds. “Under most instances, these challenges have been addressed and the activity of an enzyme can be accurately detected regardless of sample type,” concludes Filer.
Editor’s Note: This commentary is sponsored by Alltech. Through 29 years of research-driven product development, Alltech has created a range of natural solutions for the feed and food industries. For more information, please visit the Web sites at www.alltech.com.
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