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

Area of Science:

• Food Science
• Analytical Chemistry
• Biochemistry

Background:

• The INFOGEST protocol is standard for in-vitro digestion and bioaccessibility assessment.
• Enzyme standardization, typically via UV spectrophotometry, is critical but time-consuming and resource-intensive.
• Current methods lack analytical validation and require large sample volumes.

Purpose of the Study:

• To miniaturize and validate a colorimetric method for enzyme standardization in the INFOGEST protocol.
• To offer a faster, automated, and more efficient alternative to UV spectrophotometry.
• To assess the reproducibility and comparability of the new method.

Main Methods:

• Adapted a colorimetric method using Folin-Ciocalteu reagent in microplates.
• Miniaturized the assay for reduced sample and reagent volumes.
• Compared the novel method against the traditional UV spectrophotometric method for pepsin standardization.

Main Results:

• The miniaturized colorimetric method demonstrated high reproducibility (8% inter-day CV).
• No statistically significant differences were observed between the new method and the traditional UV method (p > 0.05).
• The adapted method offers automation, ease of use, and reduced sample/reagent requirements.

Conclusions:

• The miniaturized colorimetric method is a validated and efficient alternative for enzyme standardization in in-vitro digestion studies.
• This approach enhances the practicality and speed of bioaccessibility assessments.
• The method's advantages include automation, reduced costs, and minimal sample/reagent usage.