High levels of animal proteins (meat) in the diet are linked with a greater risk of developing heart disease and other long-term health conditions. Recently there has been a shift to plant-based diets including plant proteins such as pulses, defined as beans, peas, chickpeas and lentils. Pulses are a nutritious and sustainable form of plant protein which are rich in fibre and iron. Despite this, the UK population does not consume the recommended daily amount of pulses (80g/day equivalent to a large handful). In contrast, bread is commonly consumed but very little is known about how bread enriched with pulses influences the amount of iron that is digested and absorbed by the body as well as risk factors for developing heart disease and type 2 diabetes.

The main purpose of this randomised controlled cross-over study is to determine how consuming bread enriched with pulses (in the form of faba bean flour at approximately 40% enrichment) compared with conventional white bread (100% wheat flour) influences the amount of iron absorbed in healthy males and females aged 18-50 years with low iron stores. Secondary aims are determining the effects on blood fats and sugar (glucose) and on feelings of fullness (also known as satiety) after eating the bread enriched in pulses and conventional white bread.

Participants will be required:

• To attend for seven study visits over a period of 60 days.
• Consume a breakfast meal containing either the faba bean-enriched bread or conventional bread over two study periods, each consisting of one 7 hour study visit and two consecutive visits of 2 hours in duration. After 28 days, participants will return for the second study period as above, with a final study visit conducted 28 days later.
• Give blood samples during 2 x 7 hour study visits
• Complete visual analogue scales to rate appetite after consuming the faba bean-enriched and conventional white bread.
• Record dietary intake prior to and during the study period.

Since higher dietary intakes of animal protein (meat) are linked with heart disease and other long-term health problems, there has been a shift in recent years to consuming more plant-based foods. Legumes (e.g. beans) provide a sustainable and nutritious source of protein and are higher in fibre and micronutrients (such as iron) compared with staple cereals. Higher intakes of pulses lower blood sugar and fat levels, increase feelings of fullness (satiety) and reduce blood pressure. The higher iron content in mature faba beans may also help to reduce iron-deficiency anaemia, the most common diet-related deficiency. However, studies have suggested that the absorption of iron from pulses (known as bioavailability) is lower than from meat due to the presence of compounds in plants that interfere with absorption (anti-nutrients). Processing techniques such as milling of faba beans are considered to lower or even eliminate these compounds. In the Raising the Pulse (RtP) project, flour milled from homegrown faba beans will be used to produce a pulse-enriched loaf which could be adopted in the UK diet and benefit both health and the environment. However, very little is known about how bread enriched with pulses may influence firstly, iron bioavailability and secondly, blood sugar and fat levels which are common risk factors for developing heart disease and diabetes.

This randomised controlled cross-over study will aim to recruit 16 healthy, non-anaemic males and females with low iron stores. Aside from the screening visit where participants will be assessed for eligibility and consent for participation gained, this study will involve seven study visits. The first study visit (Day 1) will be approximately 7 hours (420 minutes) in duration and participants will be randomly assigned to consume either the faba bean-enriched bread or the conventional white bread along with a drink containing iron-57, a naturally occurring non-radioactive form of iron found in very low levels in the diet (stable isotope) to measure iron absorption. Blood samples will be collected to measure blood haemoglobin, sugar and fat levels as well as hormones known to impact appetite, and participants will be asked to complete visual analogue scales to rate their appetite. At the visit, an ad-libitum (unrestricted) meal will be provided 6 hours after eating the test breakfast and the amount of food eaten will be used to assess satiety. Participants will then return on days 2 and 3 to consume the same test meal and complete visual analogue scales to rate appetite for 2 hours. At day 30, participants will return for the second 7 hour (420 minute) study visit to consume the other type of bread and return on days 31 and 32 for the shorter study visits. The day 30 fasting blood sample will be used to measure the enrichment of blood haemoglobin with iron-57 to determine the amount of iron absorbed from the test bread given on day 0. On day 60, participants will return to the clinical unit to give a final fasting blood sample to measure iron bioavailability (enrichment of blood haemoglobin with iron-57) from the test bread given on day 30. Dietary intake will also be recorded.

Inclusion Criteria:

• Non-anaemic, males and pre-menopausal females (must have regular periods)
• Aged 18-50 years old
• BMI between 19 - 30 kg/m2
• Low iron stores (serum ferritin between 13-40 µg/L for females and 30-90 µg/L for males)

Exclusion Criteria:

• Food allergies or intolerances to faba beans (favism) or gluten
• Diagnosed with anaemia (haemoglobin <115 g/L for females or <130 g/L for males) or haemochromatosis
• C-reactive protein (>3mg/L)
• Smokers
• A history of alcohol abuse (> 14 units/ week)
• CVD or medical history of MI or stroke in the past 12 months
• Diabetes (fasting glucose >7.0 mmol/L)
• Kidney, liver, pancreas or intestinal disease, gastrointestinal disorder or use of drugs likely to alter gastrointestinal function
• Pregnancy, planning a pregnancy in the next six months or breastfeeding
• Peri- and post-menopausal women or women with irregular periods
• Planning on a weight-reducing regimen (lost >3kg in last 6 months)
• Parallel participation in another intervention study
• Hypertension (blood pressure > 140/90 mmHg), cancer, taking medication for hyperlipidaemia (statins), or inflammation
• Taking vitamin or mineral supplements
• Donated blood in the last 3 months
• Any other unusual medical history or diet and lifestyle habits or practices that would preclude volunteers from participating in a dietary intervention and metabolic study (such as a pacemaker)

All anonymised IPD that underlie results in a publication and analytic code for the statistical analysis.

Anonymised data will be made available on reasonable request one year after manuscripts have been published.

For data sharing and preservation, anonymised IPD will be archived in the University of Reading Research Data Archive. Access will be overseen by the project PI. This information will be maintained under Professor Julie Lovegrove's and Dr Kim Jackson's authority and disposed of appropriately by the PI [Professor Julie Lovegrove]. If the PI should leave the University, the Personal Information will be under the authority of the Head of School.