Tolerance to an Artisanal Bread in a Population Intolerant of the Conventional Bread and Study of Associated Markers

Abbreviations:

• GSRS-IBS: Gastrointestinal Symptom Rating Scale for Irritable Bowel Syndrome
• GIQLI: Gastrointestinal Quality of Life Index
• HADS: Hospital Anxiety and Depression Scale
• FSS: Fatigue Severity Scale
• LBP: Lipopolysaccharide-Binding Protein

Inclusion Visit (visit 0 [V0])

At the inclusion visit (V0) at Avicenne Hospital (Bobigny), participants will receive study instructions and materials. They will have up to 15 days to:

• Complete questionnaires (modified GSRS-IBS, Bristol Stool Scale, GIQLI, HADS, FSS)
• Collect a baseline stool sample, stored at -18°C at home until their first hospital visit (V1).

Wash-out phase (Day-15 [D-15] to Day 0 [D0]):

Participants will follow a gluten-free and low-fructan diet for 15 days. During this period:

• Daily food intake will be recorded in a diary, with meal photos sent to the research team via a dedicated email
• On D-8, participants will complete the modified GSRS-IBS and Bristol Stool Scale.
• On D-1, participants will perform a 24-hour urine collection after ingestion of lactulose (10g) and mannitol (10g) in 200 mL water, divided into three containers:
• First morning urine
• 0-5 h collection after sugar ingestion (fasting except water)
• 5-24 h collection Stool will also be collected on D-1 and stored at 4°C until V1.

First investigation day (Day 0 [D0] - V1):

At 9 a.m., participants arrive the Clinical Research Center (at Avicenne Hospital) with their stool and urine samples.

After completing the questionnaires (modified GSRS-IBS, GIQLI, HADS, FSS, and Bristol Stool Scale), a venous catheter will be inserted for blood sampling.

A 10 mL blood sample will be collected before the test meal. Participants will then consume the test meal (105 g of "artisanal" bread with water).

Postprandial blood samples (2 mL each) will be collected every 30 minutes for 3 hours (total blood volume: 22 mL) The modified GSRS-IBS questionnaire will be completed every hour during the postprandial period If a bowel movement occurs during the visit, the stool sample collected at that time may be used to replace the one collected the previous day.

Intervention phase (D1 to D14):

Participants will continue the same gluten-free, low-fructan diet, with the addition of the "artisanal" bread, which will serve as the sole source of gluten source and the main source of fructans.

• D1: 75 g of bread
• D2: 100 g of bread
• D3-14: 125 g of bread daily (≈ 8.5 g gluten, <1.2 g fructans) Bread consumption may be distributed throughout the day. On D8, participants will again complete the modified GSRS-IBS and Bristol Stool Scale.

Second investigation day (D15 - V2):

On D14, participants will repeat the 24-hour urine and stool collection as previously described. On D15, they will return to the research center and undergo the same procedures as V1.

Measurements:

• Questionnaires: modified GSRS-IBS (gastrointestinal symptoms), GIQLI, HADS, FSS.
• Blood: LBP (marker of inflammation), zonulin (marker of intestinal permeability), glucose, insulin.
• Urine: lactulose and mannitol, with calculation of the lactulose/mannitol ratio as a marker of intestinal permeability.
• Stool: microbial community analysis (microbiota) and fecal calprotectin (marker of inflammation). Baseline stool samples (before wash-out phase) will also be used to inoculate an artificial colon model for later investigation of the effects of bread on the microbiota.

Inclusion criteria

• Normal weight (18 < BMI < 25 kg.m-2)
• Male or female
• 18 to 65 years old
• Adults reporting intolerance to "conventional" bread
• Absence of celiac disease
• Affiliated to a social security scheme
• Free, informed and express consent

Exclusion criteria

• People under protective supervision: legal guardianship, tutorship or curatorship
• People with eating disorders (anorexia, bulimia, binge eating)
• Any known food allergy
• Women who are pregnant or potentially pregnant (based on a positive urine pregnancy test at inclusion)
• Breastfeeding women
• Alcohol abuse (>2 drinks/day). At the time of inclusion, the investigator will assess whether the participant exhibits harmful alcohol consumption
• Untreated hypertension, uncontrolled diabetes, or diseases of the digestive tract (excluding functional intestinal disorders), liver, kidneys, or severe heart disease. The investigator will assess the presence of these conditions on standard clinical criteria and the participants' self-reported medical history at the time of inclusion
• Participation in a clinical study in the three months preceding the study
• Absence of express consent