# Integrating in vitro digestion and peptidomics into novel food allergenicity assessment: A study on Clostridium tyrobutyricum biomass

## TL;DR

This study introduces a new method combining digestion experiments and computational analysis to evaluate the potential for a novel food to cause allergies.

## Contribution

A novel integrated workflow using in vitro digestion, peptidomics, and in silico tools for allergenicity assessment of complex food mixtures.

## Key findings

- Peptide persistence and abundance can be combined with in silico predictions to estimate allergenic potential.
- The workflow provides a flexible and efficient way to assess multiple allergenicity endpoints from a single dataset.
- The method offers insights into how in vitro digestion affects proteins and their allergenic potential.

## Abstract

Assessing the allergenicity of novel foods (NFs) is a complicated process that requires data on multiple product aspects. Although the link between allergenicity and protein digestibility remains debated, integrating next-generation risk assessment (NGRA) tools into this process is increasingly recognized as essential. Here, we present an integrated approach combining in vitro digestion, peptidomics, and in silico analysis to evaluate the allergenic potential of a complex NF mixture.

Clostridium tyrobutyricum biomass was digested in vitro, and samples collected at multiple digestion time points were analyzed by SDS-PAGE and LC-MS/MS-based proteomics. Generated peptides were filtered using allergenicity-relevant criteria and examined for sequence, size, persistence, and abundance. Proteins were further screened with online tools to predict cross-reactivity with known allergens, and resulting peptides were analyzed to determine whether potential epitopes persisted after digestion.

This workflow presents a flexible and efficient methodology to assess multiple allergenicity-relevant endpoints from a single dataset, offering a valuable screening step within current risk assessment frameworks. By integrating complementary experimental and computational tools, the method enables a more holistic and mechanistic understanding of allergenic potential in complex mixtures. Overall, the current work demonstrates how peptide persistence and abundance can be meaningfully integrated with in silico predictions to better estimate allergenic potential, while outlining current limitations and uncertainties in NGRA-based allergenicity assessment.

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•There is a need for the integration of advanced tools into novel food risk assessment.•Peptidomics offer great insight into the effect of in vitro digestion on proteins.•Allergenic potential can be assessed based on generated peptides.•Integration of this approach could complement the current regulatory framework.

There is a need for the integration of advanced tools into novel food risk assessment.

Peptidomics offer great insight into the effect of in vitro digestion on proteins.

Allergenic potential can be assessed based on generated peptides.

Integration of this approach could complement the current regulatory framework.