# Nutrition of the neonate - How processing of milk powder affects protein digestion?

Didier Dupont, Rachel Boutrou, Julien Jardin, Daniel Mollé, Gaëlle

Tanguy-Sai, Frédéric Gaucheron, Pierre Schuck, Joëlle Léonil, S. Ahmad, B.

Haab

► To cite this version:

Didier Dupont, Rachel Boutrou, Julien Jardin, Daniel Mollé, Gaëlle Tanguy-Sai, et al. Nutrition of the neonate - How processing of milk powder affects protein digestion?. International Conference on Recent Advances in Human Nutrition with Special Reference to Vulnerable Groups, Feb 2010, Faisalabad, Pakistan. hal-01482815

HAL Id: hal-01482815

https://hal.science/hal-01482815v1

Submitted on 4 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY-SA 4.0 - Attribution - ShareAlike - International
License
# Nutrition of the neonate - How processing of milk powder affects protein digestion?

Dupont D, Boutrou R, Jardin J, Molle D, Tanguy G, Gaucheron F, Schuck P, Léonil J,
**Ahmad S** & Haab B*

didier.dupont@rennes.inra.fr

INRA - Agrocampus Ouest, UMR STLO, 65 rue de St Brieuc 35042 Rennes France
* VAN ANDEL Research Institute, 333 Bostwick NE, Grand Rapids, MI 49503, USA

<figure><img src="image_2.png" /></figure>

DIET
AGRICULTURE
ENVIRONMENT
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

Interactions between proteins and other constituents

<figure><img src="image_5.png" /></figure>

Neo-formed components (protein aggregates, Maillard compounds...)

Does processing modify caseins digestibility in infant?

<figure><img src="image_6.png" /></figure>
# Objective

<figure><img src="image_7.png" /></figure>

Determine if heat treatment affects casein digestion

<figure><img src="image_8.png" /></figure>

Milk powders

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

Step 2 :
Heat
treatment

<figure><img src="image_12.png" /></figure>

Step 3 :
Spray drying

In vitro digestion

<figure><img src="image_13.png" /></figure>
## Modification of the adult gut model (Eiwegger et al. 2006)

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>
# 1. Characterisation of the rehydrated powders

<figure><img src="image_16.png" /></figure>
Particle size

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

heat-treatment at 105°C during 60s on 35% DM increases particle size
2. Characterisation of the digested samples by Western-blotting using αs₁-, αs₂-, β-, κ-casein specific antibodies

<figure><img src="image_20.png" /></figure>
<figure><img src="image_21.png" /></figure>

High Mw compounds detected by αS₂- and κ-casein specific Pabs

**Thermal-induced aggregates with β-lactoglobulin resistant to digestion ?**
(high Mw compounds observed with β-Ig specific antibodies)
3. Characterisation of the digested samples by ELISA using αs₁-, αs₂- β-, and κ-casein specific monoclonal antibodies

<figure><img src="image_22.png" /></figure>
# Identification of epitopes resistant to digestion by ELISA using specific Mabs

<figure><img src="image_23.png" /><figcaption>## β-casein</figcaption></figure>

<figure><img src="image_24.png" /><figcaption>## αS<sub>2</sub>-casein</figcaption></figure>

<figure><img src="image_25.png" /><figcaption>## αS<sub>1</sub>-casein</figcaption></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>
Residual immunoreactivity of casein fragments

<figure><img src="image_28.png" /></figure>

<table><thead><tr><td>Factor</td><td>p value</td><td>Significance</td></tr></thead><tbody><tr><td>T</td><td>0.00001</td><td>***</td></tr><tr><td>% DM</td><td>0.00555</td><td>**</td></tr><tr><td>T:%DM</td><td>0.03262</td><td>*</td></tr></tbody></table>

<figure><img src="image_29.png" /></figure>

Highest resistance at 35% DM heated at 105°C /60s
4. Kinetic of β-casein digestion using antibody microarrays

<figure><img src="image_30.png" /></figure>
Principle of the technique

<figure><img src="image_31.png" /></figure>

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>
# Antibody arrays – an example of results

<figure><img src="image_35.png" /></figure>

<figure><img src="image_36.png" /></figure>

1728 antigen-antibodies interactions studied simultaneously

<figure><img src="image_37.png" /></figure>

2-3 µl of sample

<figure><img src="image_38.png" /></figure>

Softwares available for data integration

<figure><img src="image_39.png" /></figure>
Kinetics of β-casein digestion

<figure><img src="image_40.png" /></figure>

<figure><img src="image_41.png" /></figure>

Kinetics of β-casein digestion differ according to the area studied

<figure><img src="image_42.png" /></figure>
5. Characterisation of the digested samples by
LC-MS-MS

<figure><img src="image_43.png" /></figure>
<figure><img src="image_44.png" /><figcaption>β-casein peptides identified in T, E, G digested samples</figcaption></figure>

<figure><img src="image_45.png" /></figure>
Bioactive peptides detected

<table><thead><tr><th>Peptide</th><th>Bioactivity</th><th>A</th><th>B</th><th>C</th><th>E</th><th>F</th><th>G</th><th>T</th></tr></thead><tbody><tr><td>β-CN(f108-113)</td><td>Anti-hypertensive</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>β-CN(f114-119)</td><td>Opioïd agonist</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>β-CN(f193-202)</td><td>Anti-hypertensive</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>β-CN(f193-209)</td><td>Immunomodulatory</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>αs1-CN(f1-23)</td><td>Antimicrobial</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>αs1-CN(f23-34)</td><td>Anti-hypertensive</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>αs1-CN(f91-100)</td><td>Anti-stress</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>αs1-CN(144-149)</td><td>Antioxidant</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>β-Ig(f9-14)</td><td>Anti-hypertensive</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>
Discussion / Conclusion

- Caseins partly resist to *in vitro* infant digestion model

- Resistant area have been partly identified

- Post-translational modifications and hydrophobicity could explain the resistance of these fragments to digestion

- Bioactive peptides profile can be modified by processing

- Heat treatment has a significant influence on casein immunoreactivity in digested samples (increase)
  Why?

<figure><img src="image_48.png" /></figure>
Thermal aggregation of whey proteins on the casein micelle

<figure><img src="image_49.png" /></figure>

To scale schematic representation of the major whey proteins and of
the casein micelle in unheated milk
Thermal aggregation of whey proteins on the casein micelle

<figure><img src="image_50.png" /><figcaption>a (Harwalkar et al., 1989)</figcaption></figure>

<figure><img src="image_51.png" /><figcaption>b (Rodriguez del Angel & Dalgleish, 2006)</figcaption></figure>

<figure><img src="image_52.png" /></figure>

Micelle-bound and serum aggregates are formed, containing denatured whey proteins, κ casein and possible traces of α<sub>s2</sub> casein. [a] and [b] indicate the 2 possible ways for the formation of the serum aggregates.

Guyomarc'h & Famelart

<figure><img src="image_53.png" /></figure>
Discussion / Conclusion

Caseins partly resist to in vitro infant digestion model

Resistant area have been partly identified

Post-translational modifications and hydrophobicity could explain the
resistance of these fragments to digestion

Bioactive peptides profile can be modified by processing

Heat treatment has a significant influence on casein immunoreactivity in digested samples (increase)

Why?

? Thermal aggregation of whey proteins on the casein micelle

<figure><img src="image_54.png" /></figure>
<figure><img src="image_55.png" /></figure>

Development of a more sophisticated *in vitro* digestion model including the flows and the pH variation in the stomach

<figure><img src="image_56.png" /><figcaption>Figure 2 Example of gastric pH records, six hour intervals: (A) four hourly feeds; (B) three hourly feeds; (C) two hourly feeds.</figcaption></figure>

<figure><img src="image_57.png" /></figure>

Comparison of *in vitro* data with those obtained on animal models (piglets)

<figure><img src="image_58.png" /></figure>

Validation of the gastric phase of the *in vitro* model towards human data (collection of gastric contents in pre-term neonates)

<figure><img src="image_59.png" /></figure>

Study of the influence of lipid-protein interactions on protein digestion

<figure><img src="image_60.png" /></figure>
*Improving infant formula for improving human life*

<figure><img src="image_61.png" /></figure>

*Thanks for your kind attention !!!*

DIET
AGRICULTURE
ENVIRONMENT