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Functional Additives
Friday, July 13, 2007 12:47:08 PM
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Measuring protein quality in vitro
Dr Daniel Lemos

Marine Aquaculture Laboratory (LAM)

Oceanographic Institute,

University of Sao Paulo, Brazil


Aqua Feeds: Formulation & Beyond, an electronic magazine produced by Feedware.com in association with Aquafeed.com



Determination of protein digestibility in ingredients is needed for several purposes: (1) selection of suitable ingredients; (2) quality control in purchasing; and (3) precise formulation of feeds.


In finished feeds, protein digestibility is an indicator of feed quality and a predictor of animal performance. The direct way to assess digestibility is to feed the animals with the ingredient or feed, and then collect the feces and perform analyses necessary to estimate digestibility.


However, this in vivo approach is laborious, time consuming and subject to several methodological limitations. In vitro methods to assess digestibility are relatively more convenient, widely adaptable, and easier to replicate.


All in vitro methods use enzymes to digest ingredients. Variations in methods include the source and number of enzymes, use of chemical buffers and the degree to which digestion is controlled by factors such as pH and temperature.


As opposed to the single-enzyme method, multi-enzyme methods use analytical grade proteases from different sources (bovine, porcine, microorganisms). In these methods, pH of the reaction may be controlled or uncontrolled.


Based on how pH is managed, the methods are divided into two types: one is called pH shift or drop and the other is called pH stat. In the pH shift/drop method, the degree of protein digestion is considered to be proportional to the reduction in reaction pH because it is due to the release of H+ ions after breakage of peptide bonds, and the pH drops.


In the pH-stat system, pH is maintained constant throughout hydrolysis by automated addition of an alkali such as sodium hydroxide (NaOH). The volume of NaOH added in the reaction is proportional to the degree of protein hydrolysis. The advantage of this method is that at constant pH, the enzyme remains stable and close to optimal functioning.


Validation and standardisation are extremely important in the practical use of in vitro methods. Methods that work well in terrestrial animals may not be appropriate in aquatic species. The number of enzymes and catalytic properties of the enzymes in shrimp, for example, are different from those of other species.


Using enzymes from the shrimp digestive system in a pH-stat method, a more reliable in vitro digestibility system has been developed in the author's Marine Aquaculture Laboratory. Significant correlation of data from this method with data from in vivo methods and growth performance of shrimp indicate great promise for the method.


Enzymes for the pH-stat method are obtained from hepatopancreas excised after sacrificing healthy shrimp. Age of shrimp selected for enzyme extraction is preferably in the size range of the shrimp to which the test ingredient or feed would be offered.


The hepatopancreas must be immediately stored at - 20 deg C or below. At this temperature, proteases are stable for months.


When the laboratory is ready for the assay, the hepatopancreas is homogenised with chilled distilled water and centrifuged at 4 deg C for extracting the enzymes. Since the degree of protein hydrolysis (DH percentage) depends on the enzyme extract's activity, the extract must be standardised according to total protease activity for comparison of DH percentage of different feedstuffs for a given species.


For more of the article, please click here.


Article made possible through the contribution of Feedware.com and Aquafeed.com.

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