Protein Quality of Fava Bean and Honey Chlorella in Humans

The objective is to determine the nutritional quality of protein from honey chlorella and faba bean meat analogue in healthy humans, especially in terms of protein and amino acid digestibility as well as nitrogen retention. Ileal digestibility is determined in ileal digesta collected postprandially with a naso-ileal tube. The protein sources are intrinsically labelled in 15N in order to track dietary nitrogen and amino acids in the samples.

Ethical and regulatory aspects:

The protocol has been approved to the Ethical Committee and authorized the French Agency of Drugs and Health. The personal data management will be in accordance with the regulation on personal data protection ( " regulation n° 2018-493 du 20 juin 2018").

Meal:

The meal will consist in either a drink containing 40 g of honey chlorella or a mixed meal containiong 80 g of meat analogue, to provide 20 g protein. Sensory tests will be realized to optimize the organoleptic properties of the meal.

Volunteers:

Sixteen subjects will be included in the study. The investigators plan to recruit around 40 volunteers to accommodate for the usual 60% dropouts.

Four days before the experiment, the volunteers will follow a standard diet adapted to their body weight to control their protein intake (1.3 g protein/kg body weight).

The volunteers will arrive at the Human Nutrition Research Centre of Avicenne Hospital on the morning before the day of the experiment. They will be equipped with a double lumen intestinal tube that will be allowed to progress through the intestinal tract for 24h. One of the lumen is radio opaque and serves to perfuse a non-absorbable maker in the intestine (slow marker method). The other lumen is dedicated to the continuous aspiration of the effluents, 15 cm below the perfusion site. The measurement of the non-absorbable marker in the effluents allows the determination of the effluent flow rate.

On the evening before the experimental day, they will ingest 3 oral doses of deuterated water 99 % to reach 5g/kg total body water, to label the intestinal endogenous proteins.

On the day of the experiment, the position of the tube will be checked by radiography to verify its location at the terminal ileum. A catheter will be inserted in the forearm vein for blood sampling. The perfusion of the non-absorbable marker, PEG-4000 (20g/l), will start at a flow rate of 1ml/min. The intestinal flow and the basal ileal sample will be collected during 30 min. Basal plasma sample will be sampled.

Then, at t=0, the volunteers will drink the test-meal. Until t=8h, intestinal content will be continuously collected by aspiration and pooled every 30 minutes. Blood will be sampled every 30 minutes during 4h and hourly thereafter. The urine will be collected every 2 h for 8 h.

Measurements:

In the ilel digesta:

PEG-4000 by turbidimetric method, total N and 15N enrichment by EA-IRMS, amino acid concentrations by UPLC (after acid hydrolysis HCl 6N at 110°C for 24h), 15N amino acid enrichment by GC-c-IRMS (after purification on cation exchange resin and derivatization), 2H amino acid enrichment by GC-c-IRMS, microbiome analysis (DNA sequencing), peptide analysis (LC-MS).

In the plasma:

Glucose, urea (colorimetric methods), insulin (ELISA), amino acid concentrations (UPLC), 15N enrichment in plasma protein, urea and free amino acids (EA-IRMS, after purification on cation exchange resin and derivatization).

In the urine:

Urea (colorimetric method) and 15N urea (EA-IRMS, after purification on cation exchange resin and derivatization).

Main outcomes are:

Real ileal digestibility of protein and amino acids (digestive losses), Postprandial deamination (metabolic losses), Net Postprandial Protein Utilization

• Nitrogen metabolites in blood in response to the ingestion of 15N fava bean [Срок оценки: 0 to 8 hours after the test meal]
• Plasma glucose in blood in response to the ingestion of 15N fava bean [Срок оценки: 0 to 8 hours after the test meal]
• Plasma insulin in blood in response to the ingestion of 15N fava bean [Срок оценки: 0 to 8 hours after the test meal]
• Ileal microbiota [Срок оценки: -0.5 h before the meal]
• Peptide composition of ileal digesta [Срок оценки: 0-8 h after the test meal]