A Simple Validated Method for the Estimation of Pepsin Activity in Microtiter Array for the INFOGEST Protocol.

Foods

Departamento de Análisis Instrumental, Facultad de Farmacia, Universidad de Concepción, Concepción 4030000, Chile.

Published: October 2023

AI Article Synopsis

  • The INFOGEST protocol is a method used to simulate food digestion for assessing bioactive compounds' bioaccessibility, relying on standardized enzyme activity such as pepsin measured through UV-spectrophotometry.
  • This traditional method has limitations, including the need for specific equipment, large sample sizes, and lengthy processing times.
  • The study presents a new, miniaturized colorimetric method utilizing the Folin-Ciocalteu reagent, which offers advantages like automation, reduced sample volume, and quicker results, all while demonstrating comparable accuracy and reproducibility to the standard UV method.

Article Abstract

The INFOGEST protocol has been widely used as a static simulation of gastrointestinal food digestion for bioaccessibility assessments on bioactive compounds. The standardization of the activity of several enzymes, such as pepsin, via UV-spectrophotometry of digested hemoglobin at 280 nm is a key step in the protocol. Standardization is a crucial stage since it is necessary to determine the quantity of enzyme to be added to the sample for digestion. However, this method is yet to be analytically validated; it requires quartz cuvettes and large volumes of samples and is time-consuming. Thus, we reviewed and adapted a well-known colorimetric method in microplates array by using the Folin-Ciocalteu reagent, and this study is the first to report for miniaturization of this method, the advantages of which include its automation, ease of use, the low volume of samples required, the minimal use of reagents, and speed. This method was compared to the traditional UV method, and the comparison results show no statistical difference between the inter day means for each group ( > 0.05). The proposed method was validated, showing high reproducibility (8% as inter-day CV) and statistically comparable results with the traditional UV spectrophotometric method.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC10606326PMC
http://dx.doi.org/10.3390/foods12203851DOI Listing

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