Research on donkey milk · Study 18

Donkey milk, lysozyme fraction & gut microbiota

In 2022, Yvon et al. examined in a preclinical stress model how complete donkey milk and a lysozyme-containing protein fraction relate to the intestinal barrier, visceral sensitivity, Paneth cell function and the gut microbiota.

Scientific context: These are preclinical mouse data from a conference abstract. The LYZ fraction was a lysozyme-containing protein fraction and must not be equated with completely pure lysozyme.
View results View microbiome
Biological focus Stress × gut × lysozyme Paneth cells · barrier · microbiota
Study 18
PublicationYvon et al.
2022
EvidencePreclinical
mouse model
Stress modelWater avoidance
9 days
FocusIntestinal barrier · Paneth cells
microbiota

The research question

What role could the lysozyme-containing fraction play?

The research group examined whether complete donkey milk or a lysozyme-containing whey protein fraction obtained from it can influence stress-related changes in the gut.

Among other things, they examined intestinal permeability, visceral sensitivity, lysozyme expression by Paneth cells and the faecal microbiota.

The crucial point: Similar findings with complete donkey milk and a lysozyme-containing fraction make lysozyme scientifically interesting. However, they do not prove that lysozyme alone was responsible.

Experimental setup

From the stress model to the microbiome

01 · Stress

9 days WAS

C57BL/6 mice were exposed to a repeated water avoidance stress condition or a control.

02 · Intervention

Milk & fraction

Control conditions, complete donkey milk and a lysozyme-containing protein fraction were compared.

03 · Gut function

Barrier & sensitivity

Intestinal permeability and visceral sensitivity were among the endpoints examined.

04 · Biology

Cells & microbiota

Paneth cell lysozyme, inflammatory parameters and the faecal microbiota were also examined.

The animal model

Water avoidance stress

The mice were repeatedly exposed to a controlled stress situation. The model serves to examine stress-related changes in gut function experimentally.

In the model, stress conditions were associated with changes in intestinal permeability and visceral sensitivity.

The animation on the right therefore links the actual stress model directly to the underlying intestinal barrier.

Water avoidance stress · schematic illustration
9 days
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Intestinal barrier · schematic
Illustration of the experimental stress model and an intestinal barrier. Not an image from the animal experiment; the representation serves as a biological visualisation.
Milk → protein fraction · illustration
LYZ
PRO
PROTEIN FRACTION · 10–30 kDa
LYZ
PRO
Schematic representation of the lysozyme-containing protein fraction. LYZ marks lysozyme as a component; the fraction was not a demonstrably completely pure lysozyme preparation.

The LYZ fraction

An interesting candidate – but not single-protein proof

In addition to complete donkey milk, a lysozyme-containing whey protein fraction was examined. The fraction contained proteins in the range of 10 to 30 kDa.

This creates an interesting research logic: if complete milk and a fraction with high lysozyme relevance show similar findings, lysozyme can be investigated more specifically as a possible biological factor.

But: a protein fraction contains more than one possible influencing factor. The study therefore does not prove a sole causal lysozyme effect.

Paneth cells

A biological line of defence in the small intestine

Paneth cells are located deep in the crypts of the small intestine and produce components of the innate antimicrobial defence. These include the body’s own lysozyme.

This endogenous lysozyme is not the same as the lysozyme taken in with donkey milk.

This very distinction is important for Study 18: it examines a biological system in which milk components, the gut environment, microorganisms and the body’s own defence mechanisms could be interconnected.

Intestinal crypt · Paneth cells · illustration
endogenous lysozyme
Schematic representation of Paneth cells and the release of antimicrobial molecules. Not a microscope image and no claim of a mechanism directly observed in this form.

Key results

What was observed in the preclinical model

01 · Stress

Intestinal permeability

The stress condition was associated with increased intestinal permeability.

02 · Sensitivity

Visceral sensitivity

The visceral response also changed under the chronic stress model.

03 · Paneth cells

Endogenous defence

Paneth cell function and its lysozyme expression were among the biological relationships examined.

04 · Intervention

Donkey milk

With complete donkey milk, more favourable findings were reported for several stress-associated parameters.

05 · Protein fraction

LYZ fraction

The lysozyme-containing protein fraction also showed changes in a comparable direction for several endpoints.

06 · Microbiota

Bifidobacterium

Under the interventions, the relative abundance of Bifidobacterium spp. moved towards that of the non-stressed control.

Gut microbiota

A dynamic microbial ecosystem

The researchers also examined changes in the faecal microbial community.

Of particular interest was Bifidobacterium spp.: with complete donkey milk and the lysozyme-containing fraction, its relative abundance moved back towards the non-stressed control conditions.

Relative abundance does not automatically mean that there were more live bifidobacteria in absolute terms. The data must therefore not be interpreted wholesale as an “improvement of the gut flora”.

Microbiota · schematic illustration
Bifidobacterium relative abundance in the microbiome examined
Abstract visualisation of microbial dynamics. Not a microscope image. The animation does not represent an absolute number of bacteria.

Biological working hypothesis

How the observed levels could be connected

01 · Stress Chronic stress Experimentally induced stress.
02 · Gut Barrier & Paneth cells Changes in intestinal functions.
03 · Ecosystem Microbiota Shifts in relative microbial abundances.
04 · Intervention Milk / LYZ fraction Several parameters moved in more favourable directions.
Important: This chain is a biological interpretation and research hypothesis. The study does not prove that a change in the microbiota caused the other findings or that lysozyme alone was the responsible factor.

Critical assessment

What Study 18 shows – and what it does not

What was observed

✓Stress-related changes in several intestinal parameters in the mouse model.
✓Paneth cell function and endogenous lysozyme were part of the biological relationships examined.
✓Complete donkey milk and the lysozyme-containing fraction showed interesting findings for several endpoints.
✓Changes in the relative abundance of Bifidobacterium spp. were reported.

What was not proven

×No clinical efficacy in humans.
×No proof that lysozyme alone was responsible.
×No therapeutic effect against irritable bowel syndrome, “leaky gut” or chronic intestinal diseases.
×No human dose or drinking amount recommendation.
×DON DONKEY’S® products were not examined in this study.

Research at DON DONKEY’S®

Making the hypothesis testable on our own product

The scientifically sensible next step is not to transfer the animal data to our own product, but first to characterise our own donkey milk analytically.

Phase A Analyse lysozyme Determine protein amount and enzymatic activity in several batches of fresh milk.
Phase B Compare powder Compare fresh milk and freeze-dried powder from corresponding batches.
Phase C Simulate digestion Examine which amount of protein and enzymatic activity remain after simulated digestion.
Phase D Test biologically Only then examine defined endpoints in suitable preclinical models.
DON DONKEY’S® 100 % pure donkey milk

DON DONKEY’S®

DON DONKEY’S® donkey milk

Study 18 makes lysozyme in particular, protein fractions and their possible interactions with intestinal systems interesting research questions.

Transparency: DON DONKEY’S® was not tested in this study. The results presented come from a preclinical mouse model and do not demonstrate any health effect of our donkey milk in humans.
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Scientific source

Lysozyme fraction of donkey milk alleviates gut hyperpermeability and visceral hypersensitivity in a mouse model of chronic stress

Yvon S., Bézirard V., Lévêque M., Froment C., Marcoux J., Burlet-Schiltz O., Ali Hamoud-Lekhal D., Olier M., Theodorou V., Peter M., Eutamène H.

Gastroenterology · 2022 · Volume 162(7) · S-997
Conference abstract Tu1523 · Digestive Disease Week 2022

DOI: 10.1016/S0016-5085(22)62361-8

Evidence status: Preclinical mouse data from a scientific conference abstract. The results are not equivalent to a clinical human study.