There are two methods for analyzing the activity of feed enzymes: in vivo assay and in vitro measurement. Except for the in vivo method, it is difficult to compare the quality of different enzyme preparations using other methods. This is because different feed enzymes are produced by different microorganisms through fermentation, and the active enzyme contained in enzyme products varies greatly. Moreover, the optimal pH value, temperature, and substrate affinity of enzymes secreted by various microorganisms are different. Therefore, it is impossible to find a method that can measure the activity of enzymes of different varieties of the same type using the in vitro method. Therefore, the use of the in vitro method cannot evaluate the effect of enzymes in vivo. In order to ensure the stability of active enzyme produced and safeguard the interests of users, different manufacturers have developed their own methods for determining active enzyme. These methods are used to measure the active enzyme of feed additives, premixes, and complete feeds. Currently, the main methods for measuring active enzyme in feed are as follows.
This method uses substrates of feed enzymes that are chemically synthesized or extracted from nature. The enzyme reacts with the substrates under specific conditions. The reaction product is reducing sugar, and the amount of reducing sugar is determined by colorimetry, while a standard curve is established. The active enzyme is expressed as the amount of enzyme required to produce one unit of product per minute.
This method chemically modifies the substrate to make it have specific soluble chromogenic groups that can produce a specific color. After the enzyme and substrate react, the chromogenic group is released, and the depth of the color can be measured by a spectrophotometer, and a standard curve is established. The active enzyme is calculated by comparing it with the activity of known standard enzymes. This method cannot distinguish between endo- and exo-enzymes, but it is usually considered more suitable for determining exo-enzymes. Currently, the substrate required for this method is widely used in industry.
This method determines the activity of enzymes by the intermediate products of substrate molecules through chromogenic groups combined with enzyme action. For example, PNPG reacts with lactose to form a lactose-like substance. Under the action of enzymes, PNP is released, and its content is determined by other methods. The above method is usually only used in biochemical laboratories, and the substrate on which feed enzymes act is a high molecular weight substance, and it is not suitable for use.
This method determines the activity of enzymes by their ability to reduce the viscosity of a certain concentration of standard substrate. The substrates used are mainly chemically synthesized substrates and naturally extracted substrates. The active enzyme value measured is determined by comparing it with that of the concurrently measured standard enzyme activity. The characteristic of this method is that the activity of enzymes is reflected by reducing the concentration of the substrate, which is an important feature of the activity of enzymes in vivo. Chemically synthesized substrates are more effective than naturally extracted ones because the former are not conducive to the contact of enzymes.
Immunological methods used for active enzyme analysis include ELISA and immunodiffusion. These two methods are based on the reaction between the enzyme and antibody, followed by the determination of the active enzyme by ELISA through the second-step reaction, and by the printing process in the immunodiffusion method. These methods are very sensitive and can detect very low levels of enzyme protein. However, their disadvantages are that each product requires a special antibody for the enzyme, and as an antibody, they can react with non-enzyme proteins. Since the protein used for the antibody itself is specific, there is no cross-reactivity between the same type of enzymes produced by different producers. In addition, the sensitivity of using experimental animals is worth exploring.
This method involves mixing the substrate acted upon by the enzyme with a certain gel, pouring it into a culture dish, cutting a slot in the solidified gel, pouring in the standard enzyme and test enzyme liquids. After a certain period of cultivation, a hydrolysis area can be seen around the cut gel, and the size of the area is proportional to the enzyme content. In some cases, other reagents can be added to show the hydrolysis area. Although this method is simple, the cultivation time is long and generally requires an overnight culturing. Since this method determines the activity of the enzyme by the size of the area, its accuracy is lower than that of other non-diffusion methods. However, its advantages are simplicity, ease of operation, and no need for complex devices, which is suitable for feed mills.
No.28 Huiyuan Street, Economic Development Area, Shanghe County, Jinan, Shandong Province, China
Jinan Office: +86-531-5577-2899
Nanjing Office: +86-025-5270-3891