Assessment of Protein Digestibility by In Vitro Enzymatic Hydrolysis with Simultaneous Dialysis

Abstract

An in vitro digestion method to assess the quality of proteins based on enzymatic hydrolysis is presented. The method consists of a two-step proteolysis at 37 degrees, a 30-minute incubation of the protein with pepsin at pH 1.9, followed by pancreatin digestion at pH 8 for 24 hours in a dialysis bag with a 1000 molecular weight cutoff for the continuous elimination of the digested products into a replaceable buffer. Three types of buffer replacement were tried. These were, in increasing order of efficiency: intermittent with either six changes (infrequent buffer replacement) or eleven changes (frequent buffer replacement) or continuously circulated at the rate of 212 ml/hour (continuous buffer replacement). By using three protein sources, i.e., casein, soybean and rapeseed proteins, it was found that the degree of digestion (dialyzed N) as well as the regularity of the process were markedly improved by increasing the frequency of buffer replacement. In spite of different pepsin digestibilities, as determined from the production of trichloroacetic acid-soluble N, the digestion of the three proteins gave equivalent results when the best procedure of buffer replacement was used (continuous buffer replacement). The deleterious effect of heat and alkali treatment on protein digestion was readily shown by this procedure and indicated casein to be a very heat-sensitive protein as compared to the others.

Critical Evaluation of in Vitro Methods for Estimating Digestibility in Simple-Stomach Animals

“…The collected products are analysed for nitrogen and for amino acids . This method has been developed from previously described methods (Mauron et al 1955;Steinhardt & Kirchgessner, 1973;Gauthier et al 1982). et al (1982) observed that the degree of digestion of casein, soya-bean and rapeseed protein was markedly increased by buffer replacements, indicating inhibition by end-products as suggested by Robbins (1978).…”

Suitable condition of enzymatic reactivity for determining the digestibility of feedstuffs phosphorus in vitro by dialysis tube method

“…As for temperature of enzymatic reactivity, the parameter of P in the in vitro dialysis method was 35uC, the same to that of CP in the experiment by (Huang et al, 1999), but lower than 39uC of Liu et al (1997) and 40uC of Zyla et al (1999). In the in vitro dialysis tube method established by Mauron et al (1955) and successively modified by Steinhart and Kirchgessner (1973), and Gauthier et al (1982), the volume of dialyzing solution was very important. The dialyzability of CP in feedstuff showed an increase with the change of dialyzing solution volume in many reports (Liu et al, 1997;Xi et al, 2003), however our dialyzability of P as well as that of Liu et al (1997) and Zyla et al (1999) were all just reverse, and the suitable volume of dialyzing solution was 100 mL among these tests.…”

Alternative prediction methods of protein and energy evaluation of pig feeds

“…The first in vitro dialysis method included pre-incubation of the sample with pepsin for 30 min at 39 °C, followed by incubation with pancreatin with continuous stirring in the dialysis tube for 6 h [63]. The dialysis method and its modifications have been used to study the kinetics of enzymatic digestion of proteins and other nutrients [64–66].…”