The person-based approach to enhancing the acceptability and feasibility of interventions

Lucy Yardley
Ben Ainsworth
Emily Arden-Close
Ingrid Muller
The person-based approach to enhancing the acceptability and feasibility of interventions
10.1186/s40814-015-0033-zM E T H O D O L O G Y Open Access
Background: This paper provides three illustrations of how the "person-based approach" can be used to assess and enhance the acceptability and feasibility of an intervention during the early stages of development and evaluation. The person-based approach involves using mixed methods research to systematically investigate the beliefs, attitudes, needs and situation of the people who will be using the intervention. The in-depth understanding of users' perspectives derived from this research then enables intervention developers to design or modify the intervention to make it more relevant, persuasive, accessible and engaging. Methods: The first illustration describes how relevant beliefs and attitudes of people with asthma were identified from the existing qualitative and quantitative literature and then used to create guiding principles to inform the design of a web-based intervention to improve quality of life. The second illustration describes how qualitative "think-aloud" interviews and patient and public involvement (PPI) input are used to improve the acceptability of a booklet for people with asthma. In the third illustration, iterative think-aloud methods are used to create a more accurate and accessible activity planner for people with diabetes. Results: In the first illustration of the person-based approach, we present the guiding principles we developed to summarise key design issues/objectives and key intervention features to address them. The second illustration provides evidence from interviews that positive, non-medical messages and images were preferred in booklet materials for people with asthma. The third illustration demonstrates that people with diabetes found it difficult to complete an online activity planner accurately, resulting in incorrect personalised advice being given prior to appropriate modification of the planner. Conclusions: The person-based approach to intervention development can complement theory-and evidence-based development and participant input into intervention design, offering a systematic process for systematically investigating and incorporating the views of a wide range of users.

Background

This paper illustrates how the "person-based approach" [1] can be used to assess and enhance the acceptability and feasibility of an intervention during the earliest stages of development and evaluation, in order to maximise the likelihood of a successful outcome when the intervention is subsequently evaluated in a full feasibility study. The aim of the person-based approach is to ground the development of behaviour change interventions in a sensitive awareness of the perspective and lives of the people who will use them, obtained through mixed methods research and particularly iterative qualitative studies. The person-based approach enables the intervention developer to understand how different people in different situations may view and engage with the intervention, which elements may seem particularly relevant and useful to them and which may be rejected-and thus how the intervention can be made more attractive, persuasive and feasible to implement. The first section of this paper briefly introduces the key elements of the person-based approach, and the following sections then illustrate its application in a range of intervention contexts, highlighting methodological issues and demonstrating how crucial insights can be gained from using this approach.

Core elements of the person-based approach

In-depth qualitative research is a core feature of the person-based approach at the intervention planning and development stage. Theory and evidence from trials of similar interventions can suggest intervention components with the potential to be effective but seldom provide guidance as to which are most important or how best to implement them. Expert and participant involvement can help to provide the patient perspective but is unlikely to be able to represent the views of the entire target user population. Table 1 outlines activities (column 2) suggested as part of the person-based approach to complement activities usually undertaken for intervention planning and development (column 3). At the planning stage, the person-based approach involves carrying out mixed methods research (e.g. interviews, focus groups, observation, questionnaire studies) to systematically investigate the beliefs, attitudes, needs and situation of the people who will be using the intervention in order to identify intervention components that are likely to be necessary, feasible and salient [2,3]. During development, further mixed methods research (e.g. thinkaloud interviews, diary studies, usage analyses) is needed to elicit views of every element of the prototype intervention and allow the researcher to understand the range of ways in which it may be used [4][5][6]. The intervention is modified on the basis of this research, and then further evaluation is carried out to check whether the changes made have succeeded in making the intervention acceptable, interesting, and easy to use and adhere to.

During the intervention planning phase, we have found it useful to produce guiding principles consisting of two elements: intervention design objectives, and key features of the intervention that can achieve these objectives. The intervention design objectives are based on the key context-specific behavioural needs, issues or challenges that have been identified during the planning stage.

The key features of the intervention consist of characteristics of the intervention which should address these objectives. The guiding principles are not exhaustive, and do not replace detailed planning of all the behaviour change elements in an intervention, but can help developers to easily recall and refer to the principal and distinctive features of the intervention which seem particularly important to achieving the intervention objectives.

Method

In this paper, we provide three brief, previously unpublished, illustrations of how we used the person-based approach for planning and developing interventions for people with asthma and diabetes. These examples were selected to illustrate three different ways in which the person-based approach can contribute to the earliest stages of intervention planning: by providing a process for identifying the key required elements and characteristics of the intervention design, by eliciting crucial early feedback about the acceptability of intervention elements, and by helping to detect and remedy barriers to effective usage of the intervention. Note that these activities may be carried out iteratively, concurrently or in a different order, not all these activities will be necessary or possible to undertake for every intervention, and this is not intended as an exhaustive list of the types of mixed methods research that could be useful

Results and discussion

Illustration 1: creating guiding principles to guide intervention development

Creating guiding principles for an intervention is necessarily an iterative process. It is preferable to begin to formulate them at an early stage in order to inform intervention planning-but in the initial stages of intervention planning, it is necessary to base them on incomplete evidence from literature review and primary research. Consequently, the guiding principles must be progressively refined as intervention planning and development proceeds. This case study provides a transparent account of how guiding principles can be formulated from incomplete evidence in the early stages of intervention planning.

There are a number of steps that must first be undertaken to provide the context for developing the guiding principles [1]. The initial stage of planning the development of a digital intervention for the self-management of asthma (My Breathing Matters: "MBM") was to describe the key objective of the intervention (taken from the proposal): the improvement of patients' quality of life (QoL) by supporting illness self-management by pharmacological and non-pharmacological means. The next step was to identify the key issues, needs and challenges the intervention must address in order to achieve this objective. For this purpose, we synthesised evidence from both qualitative [Morton K et al: What makes chronic illness self-management interventions acceptable and feasible: a thematic synthesis of users' experiences, in prep] and quantitative [Mclean G et al: Digital interventions to promote self-management in adults with asthma: systematic review and meta-analysis] systematic reviews of relevant literature, as well as information from mixed methods research undertaken for the development and feasibility testing of relevant previous interventions (RAISIN, an online intervention designed to improve medication self-management for patients with asthma [7], and BREATHE, a digital video-based intervention designed to help patients with asthma practice breathing retraining [8]). Our working summary of evidence used to identify the behavioural needs and issues relevant to developing these particular guiding principles is presented in Additional file 1, which illustrates the range of sources on which the person-based approach can draw in the initial stages-maximising available resources to start to design an effective intervention before later refining acceptability and feasibility using mixed methods research. Qualitative research makes a crucial contribution to this process of identifying user needs and issues relating to the acceptability and feasibility of interventions, as these can generally be elicited best by open-ended, in-depth exploration of the user perspective and the implementation context.

Drawing on these resources, we identified important characteristics of the population (patients with asthma who would be using MBM). With this specific population in mind, we could identify essential relevant issues that the design of the intervention would need to address-for example, particular psychosocial aspects of the population or certain beliefs the users would be likely to hold. For users of MBM, we identified these as the following:

1. Most people with non-optimal asthma control nevertheless do not consider themselves as patients with active asthma [9][10][11]. 2. Therefore, users are not likely to adhere to medication, nor to use an asthma management plan, and may be sceptical of necessity and efficacy of both [9,12]. 3. Other factors contributing to increased symptoms (and reduced QoL) are often not known or acknowledged, particularly (i) anxiety and stress [13] and (ii) lifestyle (e.g. smoking/obesity/avoidance of physical activity [10]).

Having described the intervention objective, the psychosocial characteristics of the target population, and the specific needs, issues and challenges relevant to this population, we were then able to develop the guiding principles themselves (see Table 2). The process of developing guiding principles involves first formulating key intervention design objectives to target each key issue. The next stage is to identify key intervention features that will address each design objective. These can include features of the intervention that may have been present in other interventions, as well as features unique to MBM.

The key intervention features are not a comprehensive list of each core ingredient to the intervention; they should be the overarching features around which the bulk of the intervention is developed. The key features can prove particularly useful for planning detailed intervention development (i.e. does a particular intervention component have the key elements necessary to achieve a design objective?) as well as for implementing user feedback (i.e. are changes suggested by a user in line with the guiding principles?). Creating the guiding principles required several meetings of the research team, which were valuable at this early stage of planning in terms of allowing team members with different expertise and priorities to highlight aspects of the intervention (and the population that it aims to help) that they considered crucial.

Illustration 2: using qualitative research to improve the acceptability of an intervention

Qualitative research is invaluable in enabling researchers to improve the acceptability of an intervention. We elicit and observe reactions to every element of the intervention, using think-aloud techniques [14], which enable researchers to observe people using the intervention while saying their thoughts out loud. This has provided valuable insight into people's views and experiences of an intervention. We then iteratively modify the intervention to improve acceptability.

In this section, we highlight four points (presenting breathing training as something for everyone, the design of the front cover, the importance of continuing with medication and use of personal stories) that arose during the development of the Breathing Freely booklet, which was used in BREATHE, a trial of breathing training for asthma [15]. Semi-structured think-aloud interviews were carried out with 29 individuals with asthma to explore their views of breathing training in general and reactions to the booklet (see [8]).

Before the booklet was pilot-tested using think-aloud interviews, we had several meetings with a patient representative. He said he felt that breathing training should be presented as something that is helpful for everyone, rather than as treatment for an illness, as people do not like being labelled. We therefore amended the original booklet to provide the following information on page 5: "Breathing training can be carried out by almost anyone and can help people to feel more comfortable with carrying out their daily activities." Based on feedback from participants that it would be more helpful to spell out the benefits of breathing training, this was later amended to: " Q: How will doing the breathing retraining benefit me? A: Breathing retraining can be carried out by almost anyone. It is good for everyone, not just for people with asthma, as you learn to breathe more efficiently. Carrying out the breathing retraining may help you to: Feel less wheezy or short of breath, Use your short term 'reliever' puffer less often, Do more or walk further before you feel out of breath, Feel more relaxed and Feel more in control of your asthma."

We changed the front cover image on the booklet several times. Our early images included one of a woman doing yoga, based on feedback from our PPI representative that it would be appropriate to give the booklet a non-medical focus. However, given comments we received from participants that the title did not seem directly related to asthma, we decided to change the picture to something more asthma related. We felt that as we were developing a medical information booklet, a picture of lungs would be appropriate. However, a comment we received from one participant was that: "Yeah I don't like the picture of the lungs but others might feel differently. But like a friendlier looking booklet, it doesn't really matter but if you want people to pick it up…" (participant 8). We therefore changed the image to our final image, one of someone blowing a dandelion, which people liked, because they felt it indicated being able to breathe and blow, and therefore demonstrated that breathing training would teach them skills. For example, one participant felt that "…because the picture is really good but I think it gets a good message across that you will have the breath to, you know, to kind of blow on a dandelion…" (participant 16). Similarly, participant 18 said "I think it's quite good because it indicated being able to breathe and blow and breathing out is quite a lot more difficult for asthmatics than breathing in."

One other important change we made was to more explicitly highlight the importance of continuing with regular asthma medication. Although our original booklet stated that "… you should continue to take your medications as usual as well as doing the breathing training. The breathing training is not an alternative to your preventer medication", one participant said that "I think it's likely that some people, even though it states not to in the booklet, that some people might neglect, if they feel like they've increased their breathing capacity, they might neglect to actually use their inhaler … it probably should be stressed a bit more that you really shouldn't uhh use this as a different option from medication, it's just an additional thing to help, it's not a cure or it's not gonna make it completely better. It's just gonna relieve some symptoms." (participant 3). In later versions, we therefore bolded the sentence "You should continue to take your preventer medication as usual", so participants were in no doubt about this. This was picked up by a later participant who said "You should still take your medication. So it's not like a kind of alternative to taking medication. You should do it as well."(participant 6). Qualitative interviews therefore demonstrated the importance of the change we had made.

Another motivational tool included in the booklet was the use of personal stories. We included a story about "Tom", who practised his breathing exercises while commuting to work on the train, and "Sue", who used to get out of breath from carrying heavy shopping bags. These stories were well received by the majority of participants, as shown by the following quote: "having the little story in the bubble is nice because, like people can relate to it sort of thing, so they will be like 'oh yeah I get that' so it can help me sort of thing, they will think if it can help them it can help me as well." (participant 4). In line with this quote, participants liked the stories because they could relate to them: "it says Sue's story about getting out of breath like when shopping in town and carrying heavy bags and like I've thought of times when I've gone to West Quay shops and have lots of bags and I'm with sort of my housemate or something and she wants to keep going round places and I just get really like: 'uhh I can't carry these bags anymore, I can't walk anymore' , and getting really tired and I sort of…it sort of makes me feel like why can I not keep just walking around but sometimes it just gets umm a little bit stressful … I like her story cause I can kind of understand." Participants also liked the stories because they showed how much progress the individuals in the stories had made and, by extension, how much it was possible for them to make, as shown here "…Tom's story, like, shows how much of a benefit [breathing training] is to him. Like going from five seconds to twenty seconds is like a significant amount." (participant 6).

This example illustrates how in-depth qualitative research provides a unique and crucial opportunity for a diverse sample of users to raise issues that are salient to them and have not been previously considered by researchers. One of our earlier front covers may even have dissuaded participants from using the booklet, and the earlier versions may have inadvertently led participants to stop taking their medication, leading to worsening asthma, but all participants were satisfied with and appropriately motivated by the final version. Qualitative research is thus an essential tool to improve as well as to demonstrate the acceptability of an intervention.

Illustration 3: using qualitative research to improve the feasibility of an intervention

Qualitative research during intervention development can provide insights beyond assessing the acceptability of an intervention. This section will illustrate how qualitative research methods have been used to improve the feasibility of an intervention.

We developed a web-based intervention (Healthy Living with Diabetes, ISRCTN43587048) to encourage physical activity in people with type 2 diabetes and lower levels of health literacy. A key feature of the intervention was an interactive physical activity planner, designed to help people form concrete and achievable plans for increasing physical activity by building on their current activities. Users were presented with a variety of physical activities and asked on how many days they did each activity in a typical week and for how many minutes each time. The intervention used an algorithm to calculate total minutes of physical activity per week, and users were then presented with tailored feedback informing them whether they need to increase their physical activity levels.

Think-aloud interviews [14] were carried out with a range of people from the target population to assess the acceptability, usability and feasibility of the intervention. Observing participants using the intervention while explaining their thought processes reveals how users enable the researcher to understand and interpret information and instructions. After only a few interviews, it became apparent that users were struggling to complete the physical activity planner accurately; often the context of what they said made it clear that they were misinterpreting instructions or misrepresenting their physical activities. As a result, people with sedentary lifestyles were being presented with intervention pages congratulating them on already being active enough. As we gained insights into how and why people were unable to complete the planner appropriately, we iteratively modified the planner, carrying out further think-aloud interviews repeatedly (with novice users) to check whether the modifications had succeeded in correcting the problems identified.

The original physical activity planner was presented as a single table that required users to enter the number of days and minutes they spent on each activity. Participants were struggling to complete this accurately, and early modifications were made to separate the planner onto two web pages so users could first indicate how many days per week they did certain activities before being asked on a second web page to specify how many minutes they spent on the activity. The original planner also included "climbing stairs" as one of the activities, but early interviews indicated that users were not able to confidently or accurately estimate how much time was spent on this activity. Consequently, the decision was made to remove climbing stairs from the physical activity planner.

Subsequent think-aloud interviews indicated that the usability of the planner had been improved but that users were still vastly overestimating their current levels of physical activity. Further changes were made to the planner, and think-aloud interviews were conducted after each change to assess the impact it was having on people's ability to complete the planner accurately (see Fig. 1 for an illustration of changes to the planner). One modification was to add general guidance notes, such as "only include activities that make you feel a bit warmer and breathe a bit harder than normal". Another change was to add activity-specific guidance notes, such as "only count fast walking for at least 10 minutes non-stop". Finally, we added a large, red speech bubble with the text "very gentle activities such as walking slowly or washing the dishes do not count!".

The feasibility of the planner was hugely improved by implementing these changes, and further think-aloud interviews carried out after the final changes had been made indicated that most participants were now able to accurately provide their current physical activity levels. There was still concern that people tended to overestimate their activity levels and hence receive incorrect feedback that they were already sufficiently active. Our final modification was therefore to adapt the algorithm to increase the minimum total amount of physical activity that would lead to this feedback message. Thinkaloud interviews were carried out on the final version of the intervention before we were confident that the physical activity planner was feasible and could be implemented. In total, we conducted 35 think-aloud interviews with a range of users from our target population and observed further 4 people completing the physical activity planner.

Conclusions

The aim of this paper was to illustrate how applying the person-based approach to intervention development can improve the acceptability of interventions and the feasibility of implementing them, from the target users' perspective. The first two examples highlight the importance of matching the fundamental design of the intervention to the identity and goals of users. Qualitative research and our PPI feedback helped us to understand that most people with asthma like to see themselves as fundamentally healthy, despite intermittent minor symptoms [16], and may therefore be reluctant to engage with an intervention that appears too medical or onerous and hence irrelevant to their situation and needs. Consequently, to increase the acceptability of our asthma interventions we presented them as providing convenient, "light touch" methods to promote and maintain healthy breathing rather than manage long-term illness. Our third illustration demonstrated the necessity of using iterative qualitative research to explore and improve the feasibility of interventions. If we had not done this, then we would not have discovered users' difficulties reporting their current activity levels correctly, and our intervention would have provided inactive users with tailored advice indicating that they did not need to increase their physical activity levels-the exact opposite of the study aim. These illustrations also highlight the potentially difficult decisions research teams have to make when modifying interventions. Sometimes, decisions can be made on the basis of intervention planning and the guiding principles. Another way we address this is by making iterative changes and assessing the impact of those changes with further qualitative interviews. This paper provided just three examples of the value of applying the person-based approach, which we have used very extensively [17] and have found essential and effective for ensuring that our interventions are acceptable and feasible. The approach is not intended to replace but to complement and integrate with theory-and evidence-based intervention development-as illustrated by our development of person-based guiding principles for intervention development, which drew on literature review and expert opinion and was carried out alongside a detailed mapping of the complete set of intervention ingredients and associated behaviour change techniques. The second illustration in this paper showed how the person-based approach can also be used to complement participant input into the design of interventions, by specifying a systematic, rigorous process for eliciting and analysing the views of a wide range of users. By using these methods in the earliest stages of intervention development, we are able to ensure that by the time our interventions are evaluated in formal feasibility studies, we have maximised the likelihood that they will be found to be not only acceptable but engaging, feasible to implement and effective.

Fig. 1
1Healthy living with diabetes physical activity planner

Table 1
1Activities relevant to applying the person-based approach at each stage of intervention development and early evaluation Stage of intervention development and evaluation Specific person-based approach activities useful at each stage aOther activities relevant to person-based approach normally undertaken as part of intervention development1. Intervention planning
• Synthesise previous qualitative studies of user
experiences of similar interventions
• Carry out qualitative research to elicit user views of the
planned behaviour changes and intervention (including
relevant previous experience, barriers and facilitators)

• Consult experts and stakeholders (e.g. members of
user groups, practitioners, purchasers of healthcare
services)
• Examine relevant theory and evidence from previous
trials
• Observe real-life context of intended intervention

2. Intervention design
• Use themes arising from the intervention planning stage
to identify key issues, needs and challenges the
intervention must address
• Create guiding principles, comprising:
a) Key intervention design objectives
b) Key distinctive features of the intervention needed
to achieve objectives

• Carry out intervention mapping of behavioural
determinants and behaviour change techniques
• Create logic model describing hypothesised
mechanisms of action of intervention

3. Intervention development
and evaluation of acceptability
and feasibility

• Elicit and observe user reactions to every intervention
element (e.g. using think-aloud techniques), iteratively
modifying intervention to optimise acceptability and
feasibility
• Carry out detailed longitudinal mixed methods case
studies of independent intervention usage

• Develop detailed procedures for intervention plus
information/advice, manuals, scripts, training, etc. for
patients and/or health professionals
• Pilot intervention using mixed methods to evaluate
acceptability and feasibility

a

Table 2
2Guiding principles for My Breathing Matters-an intervention to improve functional quality of life of primary care patients with asthma, by helping them to control their condition using pharmacological and non-pharmacological methods Design objectives that address each key issue Key intervention features relevant to each design objective i) To engage people who do not view themselves as having active asthma • Maintain positive illness context throughout (i.e. promote health rather than manage illness) • Simple, unobtrusive interface to provide optional (and flexible) support only when needed ii) To persuade and educate users to implement appropriate pharmacological management • Persuade and educate users regarding the necessity, efficacy and safety of preventative asthma medication • Facilitate easy completion of an action plan with primary care support iii) To encourage users to employ non-pharmacological methods of improving QoL • Educate users on benefits and offer psychological methods to improve quality of life (e.g. cognitive behavioural techniques for symptom management)• Provide tailored access and address patient concerns about relevant positive lifestyle
changes, such as weight loss if overweight, smoking cessation if current smoker,
physical activity if inactive

AcknowledgementsWe would like to thank Emma Teasdale, Sarah Tonkin-Crine and Natasha Pitre for their contributions to the BREATHE study; Ali Rowsell and the Diabetes Literacy Study Group for their contributions to the Diabetes Literacy project; and Mike ThomasAdditional fileAdditional file 1: Evidence for key behavioural issues that the intervention is trying to address. (DOCX 51 kb)Competing interestsThe authors declare that they have no competing interests.Authors' contributionsAll authors contributed to the writing of this manuscript. All authors read and approved the final manuscript.Submit your next manuscript to BioMed Central and take full advantage of:
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The effect of wheat prebiotics on the gut bacterial population and iron status of iron deficient broiler chickens

Elad Tako
Raymond P Glahn
Marija Knez
James Cr Stangoulis
The effect of wheat prebiotics on the gut bacterial population and iron status of iron deficient broiler chickens
R E S E A R C H
Background: Currently, there is a lot of interest in improving gut health, and consequently increasing Fe absorption, by managing the colonic microbial population. This is traditionally done by the consumption of probiotics, live microbial food supplements. However, an alternative, and often very effective approach, is the consumption of food ingredients known as prebiotics. Fructans and arabinoxylans are naturally occurring non-digestible oligosaccharides in wheat that exhibit prebiotic properties and may enhance intestinal iron (Fe) absorption. The aim of this study was to assess the effect of prebiotics from wheat on Fe bioavailability in vitro (Caco-2 cells) and in vivo (broiler chickens, Gallus gallus).Methods: In the current study, the effect of intra-amniotic administration of wheat samples extracts at 17 d of embryonic incubation on the Fe status and possible changes in the bacterial population in intestinal content of broiler hatchlings were investigated. A group of 144 eggs were injected with the specified solution (1 ml per egg) into the amniotic fluid. Immediately after hatch (21 d) and from each treatment group, 10 chicks were euthanized and their small intestine, liver and cecum were removed for relative mRNA abundance of intestinal Fe related transporters, relative liver ferritin amounts and bacterial analysis of cecal content, respectively.Results:The in vivo results are in agreement with the in vitro observations, showing no differences in the hatchling Fe status between the treatment groups, as Fe bioavailability was not increased in vitro and no significant differences were measured in the intestinal expression of DMT1, Ferroportin and DcytB in vivo. However, there was significant variation in relative amounts of bifidobacteria and lactobacilli in the intestinal content between the treatments groups, with generally more bifidobacteria being produced with increased prebiotic content.Conclusions: In this study we showed that prebiotics naturally found in wheat grains/bread products significantly increased intestinal beneficial bacterial population in Fe deficient broiler chickens. With this short-term feeding trial we were not able to show differences in the Fe-status of broilers. Nevertheless, the increase in relative amounts of bifidobacteria and lactobacilli in the presence of wheat prebiotics is an important finding as these bacterial populations may affect Fe bioavailability in long-term studies.

Introduction

Micronutrient malnutrition, often called 'hidden hunger', is a serious health problem worldwide. The most prevalent micronutrient deficiency is iron (Fe) deficiency, affecting about 40% of the world's population, particularly women and children in developing countries [1,2]. Cereal crops are an important source of minerals and other nutrients for humans. Wheat is currently the primary staple food for nearly one-third of the world's population, providing >50% of the total daily energy intake [3]. Wheat grains contain significant amounts of dietary fiber and phytate.

It is believed that diets high in phytate contribute to Fe deficiency [4,5]. In contrast, some naturally occurring non-digestible oligosaccharides in wheat, known as prebiotics, have been suggested to have an enhancing effect on Fe absorption [6,7].

Prebiotics have the ability to support the growth of probiotics [8,9]. The fermentation of prebiotics by colonic bacteria gives rise to production of unbranched SCFA such as acetic, propionic, butyric, and lactic acids, thereby lowering intestinal pH [10,11], inhibiting the growth of potentially harmful bacteria [11] and improving mineral absorption [12][13][14][15]. Next to inulin and fructo-oligosaccharides (FOS), the two most extensively studied prebiotics, arabinoxylans (AXs) are also considered as a potential new class of prebiotic components [16]. AXs are present mainly in the bran portion, for example in wheat bran (6.7%). However, the thin aleurone layer surrounding wheat endosperm contains 60-70% AX [17]. Overall, they constitute 60-69% of non-starch polysacharides in wheat bran [18] and 88% in wheat endosperm [19].

Inulin and FOS have been shown several times to selectively stimulate bifidobacteria [20][21][22]7]. Regarding the absorption of Fe, it has recently been shown that intra-amniotic administration and dietary inulin improved the Fe status of Fe deficient broilers [9]. mRNA abundance of DMT1 and ferroportin in addition to liver ferritin were higher in the inulin group compared to control (p < 0.05) [9]. Similarly, Yasuda et al. [15] demonstrated improvement in hemoglobin repletion efficiency in young anemic pigs fed corn soybean meal supplemented with 4% dietary inulin. Recently, Freitas et al. [23] showed the benefit of inulin and oligofructose prebiotics on the regeneration of haemoglobinic mass and increased intestinal Fe absorption in anaemic rats. However, to date, the comparison of the effect of prebiotics (with different fructan and arabinoxylan content) both from raw and processed wheat on Fe absorption has not been investigated.

Therefore, the primary aim of this study was to assess the effect of prebiotics from wheat on short-term Fe bioavailability in-vitro (in Caco-2 cells) and in-vivo (broiler chickens). Within this aim there were several objectives: (A) to compare the effect of wheat treatments with varied arabinoxylan and fructan content on Fe availability and (B) to evaluate the differences in the efficacy of prebiotics from processed and unprocessed wheat grain flour. Finally, in view of the fact that people do not consume raw cereal grains and that processing and baking are common practices worldwide, our last objective was (C) to examine the effect of prebiotics from baked wheat products (breads baked for 45 min and 5 min) on Fe status and gut microbiata of iron deficient broilers hatchlings.

Materials and methods

Wheat and bread samples

The four samples of double haploid lines included in this study were from the Berkut × Krichauff doubled haploid (DH) population, previously used to map quantitative trait loci (QTL) for high AX in this population [24]. The population was grown in 2009 at Roseworthy in South Australia, at an adequate-rainfall site. The lines were chosen based on their fructan and arabinoxylan content (the lines with the highest and the lowest fructan and arabinoxylan amounts were included). These samples were compared with commercially bought white and wholemeal flour mixes and their baked products (Laucke Flour Mills; www.laucke. com.au).

Bread and chapatti sample preparation

Bread loaves (1 kg, standard size) were baked using an automatic bread maker Breville BBM100 (Sydney, Australia). An identical baking program was established for the preparation of all breads.

The endpoint of baking was set as "Medium Crust." The total baking time was 3 h and 5 min, including 25 min kneading, 1 h and 40 min fermentation and 1 h baking at 155°C.

The average amount of the flour mix used for preparation of a loaf of bread was 600 g.

Weight of other ingredients (yeast and water) was 420 g for white and 550 g for wholemeal bread. Once the baking was completed bread loaves were removed from the oven, left to cool on a rack and subsequently frozen to −25°C.

Chapatti breads were prepared using 120 g of the flour mix and 80 ml of water. Dough was kneaded by hand for 20 min than left to rest for a further 30 min (room temperature) before cooking. The dough was shaped into a flat 6-inch disc and cooked on a hot skillet (Sunbeam Classic Skillet SK 4200P 25 cm; 210°C) for 1-2 min until brown spots formed on both sides of the dough. Cooked chapatti was left to cool to room temperature and put at −25°C freezer.

The samples (both yeast leavened and chapatti breads) were freeze-dried for 48 h (Dynavac freeze drying unit) to remove all moisture before grinding (Retch 2M1000b;

Retch, Ochten, The Netherlands). All milled samples were stored in plastic containers at room temperature until analysis.

Sugar, phytate and micronutrient analyses

Sugar and phytate analyses were performed using High Performance Liquid Chromatography (HPLC) on a Dionex ICS-3000 unit as described by Huynh et al. [25] for fructans, Nguyen et al. [24] for AXs and by Lehrfeld et al. [5] for phytate. The micronutrient concentration of samples was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) [26] using an ARL 35B ICP analyser.

Extraction of the wheat samples

Procedure was conducted as previously described by Vidanarachchi et al. [27]. Briefly, the wheat exudate was dissolved in distilled water (50 g/L) and the solution was filtered through a 600 μm screen to remove particulate matter and then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was collected and dialysed (MWCO 12-14 kDa, Medicell International Ltd., London, UK) exhaustively against distilled water (48 h). The dialysate was collected and then lyophilized to yield a fine offwhite powder (wheat extract). In addition, in order to avoid embryonic dehydration, the samples were diluted prior to in-ovo injections (solution concentration was not higher than 320 OSM).

In vitro iron bioavailability assessment

An in vitro digestion/Caco-2 cell culture model [28] was used to assess in vitro Fe bioavailability. With this method, bread/flour samples (0.5 g) were subjected to simulated gastric and intestinal digestion.

Briefly, the intestinal digestion is carried out in cylindrical inserts closed on the bottom by a semipermeable membrane and placed in wells containing Caco-2 cell monolayers bathed in culture medium. The upper chamber was formed by fitting the bottom of Transwell insert ring (Corning) with a 15000 Da molecular weight cut off (MWCO) membrane (Spectra/Por 2.1, Spectrum Medical, Gardena, CA). The dialysis membrane was held in place using a silicone ring (Web Seal, Rochester, NY).

Iron uptake by the cell monolayers was assessed by measuring ferritin concentrations in the cells. Three replicates of each Fe bioavailability measurement were performed, each utilizing a separate sample of the wheat extract.

In terms of materials for the study, Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD) at passage 17 and used in experiments at passage 29. Cells were seeded at densities of 50,000 cells/ cm2 in collagen-treated 6 well plates (Costar Corp., Cambridge, MA). The integrity of the monolayer was verified by optical microscopy. The cells were cultured at 37°C in an incubator with 5% CO2 and 95% air atmosphere at constant humidity, and the medium was changed every 48 h.

The cells were maintained in Dulbecco's modified Eagle medium plus, 25 mmol/L HEPES, and 10% fetal bovine serum. 48 h prior the experiment, the growth medium was removed from culture wells, the cell layer was washed, and the growth medium was replaced with minimum essential media (MEM) at pH 7.0.

The MEM was supplemented with 10 mmol/L PIPES, 1% antibiotic/antimycotic solution, 4 mg/L hydrocortisone, 5 mg/L insulin, 5 μg/L selenium, 34 μg/L triiodothyronine, and 20 μg/L epidermal growth factor. This enriched MEM contained less than 80 μg Fe/L. All ingredients and supplements for cell culture media were obtained from Gibco (Rockville, MD). The cells were used in the Fe uptake experiment at 13 days post seeding. In these conditions, the amount of cell protein measured in each well was highly consistent between wells. In experiment day, 1.5 mL of the digested sample was added to the insert's upper chamber and incubated for 2 h. Then, inserts were removed and 1 mL of MEM was added. Cell cultures were incubated for 22 h at 37°C.

Harvesting of Caco-2 cells for ferritin analysis

The protocols used in the ferritin and total protein contents analyses of Caco-2 cells were similar to those previously described [8,29,9,28,30].

Briefly, growth medium was first removed from the culture well by aspiration and the cells were washed twice with a solution containing 140 mmol/L NaCl, 5 mmol/L KCl, and 10 mmol/L PIPES at pH 7.0. The cells were harvested by adding an aliquot of deionized water and placing them in a sonicator (Lab-Line instruments, Melrose Park, IL). The ferritin and total protein concentrations were determined on an aliquot of the harvested cell suspension with a one-stage sandwich immunoradiometric assay (FER-IRON II Ferritin assay, Ramco laboratories, Houston, TX) and a colorimetric assay (Bio-Rad DC Protein assay, Bio-Rad, Hercules, CA), respectively.

Caco-2 cells synthesize ferritin in response to increases in intracellular iron concentration. Therefore, we used the ratio of ferritin/total protein (expressed as ng ferritin/mg protein) as an index of the cellular Fe uptake. All glassware used in the sample preparation and analyses was acid washed.

In-vivo (intra amniotic administration procedure) bioavailability analysis Birds, diets, and study design One hundred and sixty Cornish-cross fertile broiler eggs were obtained from a commercial hatchery (Moyer's chicks, Quakertown, PA), from a maternal flock 35 weeks in lay. The eggs were incubated under optimal conditions at the Cornell University Animal Science poultry farm incubator.

Intra-amniotic administration

The intra amniotic administration procedure was previously described [9,31]. Briefly, at 17 days of embryonic incubation, eggs containing viable embryos (n = 150) were weighed and divided into 13 groups (n = 12), each with an average egg weight of 49 ± 1.15 g.

All treatment groups were assigned eggs of similar weight-frequency distribution.

A group of 144 eggs was then injected with the specified solution (1 ml per egg) with a 21-gauge needle into the amniotic fluid, which was identified by candling [31]. The intra-amniotic treatment solution included the following: 'Control 1': non injected eggs; 'Control 2': inulin solution (4%-inulin/ddH2O); 'Control 3': arabinose solution (4%-arabinose/ddH2O) and 10 samples (extracted in ddH2O).

'Control 1' (n = 12) was non injected group that paralleled routine procedures in commercial hatcheries. After all the eggs were injected, the injection holes were sealed with cellophane tape and the eggs placed in hatching baskets such that each treatment was equally represented at each incubator location.

Hatchability was similar in all treatment groups and was approximately 90%. Immediately after hatch (21 d) and from each treatment group, 10 chicks were euthanized by carbon dioxide exposure. The digestive tracts and livers were quickly removed from the carcass and separated into various sections for tissue (small intestine and liver~1-2 cm;~2-3gr, respectively) and cecum.

Samples were collected for relative mRNA abundance of intestinal Fe related transporters, relative liver ferritin amounts and bacterial analysis of cecal content, respectively. The samples were immediately frozen in liquid nitrogen, and then stored in a −80°C freezer until analysis. All animal protocols were approved by the Cornell University Institutional Animal Care and Use Committee.

Blood analysis and hemoglobin (Hb) measurements

Blood samples were collected weekly from the wing vein (n = 10,~50 μL) using micro-hematocrit heparinized capillary tubes (Fisher, Pittsburgh, PA). Samples were collected immediately after hatch. Blood Hb concentrations were determined spectrophotometrically using the cyanmethemoglobin method (H7506-STD, Pointe Scientific Inc. Canton, MI) following the kit manufacturer's instructions.

Isolation of total RNA

Total RNA was extracted from 30 mg of the duodenal tissue (tissue was harvested from the proximal duodenum, n = 10) using QiagenRNeasy Mini Kit (RNeasy Mini Kit, QiagenInc.,Valencia, CA) according to the manufacturer's protocol. Briefly, tissues were disrupted and homogenized with a rotor-stator homogenizer in buffer RLT®, containing β-mercaptoethanol. The tissue lysate was centrifuged for 3 minutes at 8,000 g in a micro centrifuge. An aliquot of the supernatant was transferred to another tube, combined with 1 volume of 70% ethanol and mixed immediately.

Each sample (700 μL) was applied to an RNeasy mini column, centrifuged for 15 s at 8000 g, and the flow through material was discarded. Next, the RN easy columns were transferred to new 2-mL collection tubes, and 500 μL of buffer RPE® was pipetted onto the RNeasy column followed by centrifugation for 15 s at 8000 g. An additional 500 μL of buffer RPE were pipetted onto the RNeasy column and centrifuged for 2 min at 8000 g.

Total RNA was eluted in 50 μL of RNase free water. All steps were carried out under RNase free conditions. RNA was quantified by absorbance at A 260/280. Integrity of the 28S and 18S ribosomal RNAs was verified by 1.5% agarose gel electrophoresis followed by ethidium bromide staining.

DNA contamination was removed using TURBO DNase treatment and removal kit from AMBION (Austin, TX, USA).

DMT1, DcytB and Ferroprtin gene expression analysis

As previously described [8,29,32,9] Determination of the linear phase of the PCR amplification was performed (Access RT-PCR system, Promega, Madison, WI) with pooled aliquots removed at 15,20,25,30,35,40,45,50, and 55 cycles.

Amplification of the chicken duodenal DMT1, DcytB and Ferroportin genes were performed for 32, 40 and 30 cycles respectively, which consisted of denaturation (95°C, 30 s), annealing (48°C, 1 min), and extension (72°C, 1 min); ribosomal 18S was amplified at 32 cycles under identical conditions in a different tube. All PCR products were separated by electrophoresis on 2% agarose gel, stained with ethidium bromide, and quantified using the Quantity One 1-D analysis software (Bio-Rad, Hercules, CA).

Ferritin and Fe in the liver

Liver samples were treated as described by Mete et al. [33]. Briefly, 1 g of sample was diluted into 1 mL of 50 mM Hepes buffer, pH 7.4, and homogenized on ice at 5000 g and for 2 min. One mL of each homogenate was subjected to heat treatment for 10 min at 75°C to aid isolation of ferritin since other proteins are not stable at that temperature [33,34,32,9]. After heat treatment the samples were immediately cooled down on ice for 30 min.

Thereafter, samples were centrifuged at 13000 g for 30 min at 4°C until a clear supernatant was obtained and the pellet containing most of the insoluble denaturated proteins was discarded. All tests were conducted in duplicates for each animal (n = 6).

Electrophoresis, staining and measurement of gels

Native polyacrylamide gel electrophoresis was conducted using a 6% separating gel and a 5% stacking gel. Samples were run at a constant voltage of 100 V. After electrophoresis, the gels were treated with either of the two stains: Coomasie blue G-250 stain, specific for proteins, or potassium ferricyanide (K3Fe(CN)6) stain, specific for iron.

The corresponding band found in the protein and iron stained gel was considered to be ferritin [32,35,33,34]. The gels were scanned with Bio-Rad densitometer. Measurements of the bands were conducted using the Quantity-One 1-D analysis program (Bio-Rad, Hercules, CA). The local background was subtracted from each sample.

Horse spleen ferritin (Sigma Aldrich Co., St. Louis, MO) was used as a standard for calibrating ferritin protein and iron concentrations of the samples. Dilutions of the horse spleen ferritin were made and treated similarly to the liver supernatant samples in order to create a reference line for both protein and iron-stained gels. Iron levels were determined using the same calibration since approximately 20% of the weight of horse spleen ferritin is iron.

Saturation levels of ferritin with iron were calculated as the percentage of the iron present in the protein to the maximum amount of iron atoms (4500 iron atoms/ ferritin molecule) ferritin can incorporate [32,35,33,34].

Collection of microbial samples and DNA isolation

The cecum was removed and treated as described previously (Zhu et al., 2002;Tako et al., 2008;Tako and Glahn, 2012). The contents of the cecum were squeezed out into a sterile 50-mL tube containing 9 mL of sterile PBS and homogenized by vortexing with glass beads (3-mm diameter) for 3 min.

Debris was removed by centrifugation at 700 × g for 1 min, and the supernatant was collected and centrifuged at 12,000 × g for 5 min. The pellet was washed twice with PBS and stored at −20°C until DNA extraction. For DNA purification, the pellet was resuspended in 50 mM EDTA and treated with lysozyme (Sigma Aldrich Co., St. Louis, MO; final concentration of 10 mg/mL) for 45 min at 37°C. The bacterial genomic DNA was isolated using a Wizard Genomic DNA purification kit (Promega Corp., Madison, WI). The DNA concentration was determined spectrophotometrically.

Primer design and PCR amplification of bacterial 16S rDNA

Primers for Lactobacillus (forward: 5'-CAT CCA GTG CAA ACC TAA GAG-3'; reverse: 5'-GAT CCG GTG CAA ACC TAA GAG-3') and Bifidobacterium (forward: 5'-GGG TGG TAA TGC CGG ATG-3'; reverse: 5'-CCA CCG TTA CAC CGG GAA-3') were designed according to previously published data [36][37][38]9,35]. Universal primers identifying all known bacteria were designed using the invariant region in the 16 s rDNA of the bacteria (forward: 5'-CGT GCC AGC CGC GGT AAT ACG -3'; reverse: 5'-CGT GCC AGC CGC GGT AAT ACG-3'). The universal primer set was used for determining the total microflora population. For PCR amplification of the bacterial targets from cecal contents, 5 μL of DNA extract was added to 45 μL of PCR mixture containing 27.5 μL of nuclease-free water, 5 μL of each primer (10 μg/mL), 1.5 μL of nucleotide (dNTP) mix, 5 μL of PCR buffer, and 1 μL of Taq polymerase (Go-Taq, Promega). The PCR thermal conditions were as follows: 1 cycle of 94°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 1 min, 68°C for 2 min, and finally 1 cycle of 68°C for 7 min. The PCR reaction was run with different numbers of cycles (25, 30, 35, 40, 45, or 50) for each primer set, and 35 cycles was in the center of the exponential increase in PCR products.

The PCR products were separated by electrophoresis on 2% agarose gel, stained with ethidium bromide, and quantified using a Gel-Pro analyzer version 3.0 (Media Cybernetics, Bethesda, MD). To evaluate the relative proportion of each examined bacteria, all products were expressed relative to the content of the universal primer product and proportions of each bacterial group are presented, where the total of the examined bacteria was set at 100%.

Statistical analysis

Results were analyzed by one way ANOVA using the SPSS statistical software (SPSS 17.0; SPSS Inc, Chicago, IL). Differences between treatments were compared by Tukey's test and significance level of p < 0.05 was used for all comparisons. Values in the text are means ± SD.

Results and discussion

The content of prebiotics and phytate and the concentration of micronutrients in wheat samples Among the DH lines there were samples with similar levels of phytate and the content of fructans plus arabinoxylans varying from 7.7% to 10.6% (Table 1).

To examine the effect of prebiotics from processed wheat (and compare them with prebiotic effects of raw material) two commercially bought flour samples were included in this study (Laucke's white and wholemeal flour). These samples contained lower amounts of fructans and arabinoxylans (3.2% and 5.8% fructan plus arabinoxylan), but also much lower levels of phytates. In order to get a better insight into the prebiotic efficacy of baked products we tested the samples of leavened white and wholemeal yeast bread, and chapatti bread.

Yeast leavened breads are most frequently consumed breads in Western countries, while chapatti is the most common type of unleavened wheat bread consumed in Asia. In Pakistan, for example, more than 70% of the total wheat produced is used to make chapattis [39]. Baked samples of yeast leavened breads contained lower levels of phytate (phytate is degraded during bread baking processes, from 50% to 90%), and lower levels of prebiotics (30%, fructan reduction, no changes in the content of arabinoxylans), when compared with their corresponding flour samples (Table 1).

There were similar amounts of arabinoxylan, fructan and phytate in the flour and their analogous chapatti breads. The concentrations of micronutrients were lowest in white flour samples and highest in DH line samples.

In vitro bioavailability results

The quality controls of in vitro study (ie. cell baseline, FeCl3, FeCl3 + AA) were all within the expected and relative ranges which indicate a valid and functional run of the bioassay. As predicted, the results showed improved Fe bioavailability in the presence of white and wholemeal flour and bread samples (Figure 1). The inhibitory effect of phytate on Fe bioavailability is well known [40,41]. Therefore, the lower amounts of phytate in white and wholemeal samples (compared to DH lines) are most likely responsible for observed enhanced Fe bioavailability.

The results of this study confirm previous research [42,30] that reduction of phytate increases iron bioavailability in Caco-2 cells and one would expect enhanced availability if tested in the animal gut. Wheat grains contain endogenous phytase, an enzyme that can release covalently bound phosphate groups from the inositol ring, thus reducing the anti-nutritional effect of phytic acid [43].

These enzymes act by hydrolysing phytic acid into lower myo-inositol phosphate esters with a lower capacity to bind minerals [44]. The fermentation process provides the optimal conditions for phytase activity.

During conversion of flour into bread, phytate content decreases as a consequence of the activity of native phytase. However, as shown in this study the phytate is not reduced to an extent as to greatly improve Fe bioavailability in whole-wheat products. Different factors determine the reduction of phytate content during baking processes such as phytase activity, dough pH, presence of calcium salts and the degree of flour extraction [45].

The essential factor that determines the bioavailability of a compound is the release and solubility of the compound from the food matrix [42]. Even so, relative to controls, bioavailability values of all wheat samples examined in this study were low. Based on previous research, we can assume that such low values (ie. close to baseline, or below) are due to the presence of inhibitors, in this case phytate. However, it should be noted that Fe availability of wheat products is not affected entirely by phytic acid concentration and that other components of Table 1 The content of prebiotics (arabinoxylans and fructans) and phytate, and the concentration of micronutrients (zinc, iron, phosphorous) in various flour and bread samples Values are means +/− SD (n = 4). AX = arabinoxylan.

wheat grains and bakery products may also have an effect on iron availability; i.e. proteins, calcium [42].

There were no significant differences in Fe bioavailability between treatment diets for hemoglobin (Table 2). Similarly, no significant variations were observed in the intestinal expression of DMT1, Ferroportin (major intestinal Fe transporters) and DcytB (Fe reductase). The relative liver ferritin amounts between the treatment groups were also not different. One possible explanation is the amount of material that was administrated in-ovo, as we were limited by the osmolality of the injected solution (not more than 320OSM, as embryonic dehydration will occur).

Another reason could be the duration of the study; it was a short term (one time administration) study, which may not be long enough time to observe the efficacy of these particular treatments on Fe bioavailability measures. Similar to previous studies, the poultry [29,9] and Caco-2 cell [28,30] models are sensitive and responsive to bioavailable Fe.

In conclusion, the in vivo results are in agreement with the in vitro observations showing no differences in the hatchling Fe status between the treatment groups.

Bacterial populations in the intestinal content of chickens

Overall, there was a significant increase in the relative amounts of bifidobacteria and lactobacilli in intestinal content between the treatments groups ( Figure 2). Considerably more bifidobacteria has been produced in the presence of most samples (exception is white flour sample where there were no statistically significant differences in the production of bifidobacteria and lactobacilli).

Making individual comparisons, there were no statistically significant differences in both lactobacilli and bifidobacteria production between DH line treatments. As expected, due to the higher amounts of prebiotics present (difference in prebiotic content up to 7.4%) there were significantly higher production of bacteria in the presence of DH line treatments compared to the  treatments with commercially produced white and wholemeal flour samples (p < 0.01). There were statistically significant difference in expansion of bacteria between the wholemeal and white flour (wholemeal more bifidobacteria, white more lactobacillus) (p = 0.02; p = 0.04, respectively).

Additionally, there were no discrepancies in lactobacillus production among the treatments of white and wholemeal flour and their corresponding baked products.

In the presence of white flour baked products, more bifidobacteria was produced in the white flour treatment (p < 0.003); while there were no changes in production of bifidobacteria between the wholemeal flour and corresponding baked products. Finally, there were no variations in bacterial production between the white and wholemeal breads.

In conclusion, prebiotic segments of wheat enhanced the growth of colon microflora, with generally more bifidobacteria being formed. This is an important finding as these bacterial populations may affect Fe bioavailability and improve gut health.

Our findings are concurring with previously published papers that demonstrate an increase in bifidobacteria population in the ceca in the presence of inulin [29,46,47]. Some recently completed human trials clearly show the association between the production of gut bacteria and Fe absorption.

In Fe-deficient young women in South India lower levels of lactobacilli were measured which suggest that bacterial flora have an important role in the Fe absorption process [48]. In addition, it has been demonstrated that bifidobacteria have a nutritional advantage compared with other intestinal microorganisms due to their β-1,2glycosidase activity [49,7].

As mentioned earlier, one of the possible reasons for increased mineral bioavailability in the presence of prebiotics is that they serve as a substrate for short chain fatty acid (SCFA) synthesis and as a consequence of SCFA production, more of the "beneficial bacterial community" is formed which further enhances the absorption of minerals (such as Ca, Zn and Fe) in the colon [12,13,50]. SCFA positively affect Fe bioavailability by releasing Fe from complexes by a lowering of the pH. The lowering of the pH enhances mineral absorption by releasing the iron from protein complexes [51]. Furthermore, one of the fermentation products, propionate, can create soluble complexes with Fe and consequently make the Fe more absorbable [51].

In addition, SCFAs can enlarge the absorption area through stimulation of the epithelial cells to proliferate. Lactobacilli and Bifidobacteria are beneficial for humans with low Fe status as bioavailability of Fe is improved as a result of the reduction in the amounts of the pathogenic becteria using dietary Fe [52].

In-vitro data show that more bioavailable Fe has been measured in the presence of samples containing lower levels of phytate. However, high amounts of phytate in samples were not a limiting factor for the stimulation of beneficial gut microbiata production. Generally speaking, the production of beneficial bacteria was higher in the presence of samples with higher amounts of prebiotics (wholemeal flour and bread samples) which clearly demonstrates that the consumption of wholemeal bread as opposed to white bread is not a contributing factor in the development of Fe deficiency. Similarly, the baking process (including the fermentation) does not seem to have a crucial role in microbial production as there were no discrepancies in bacterial production among the treatments of white and wholemeal flour and their corresponding baked products.

It has been shown that both bifidobacteria and lactobacilli can produce phytate degrading enzymes [53]. It seems that prebiotics influence the production of probiotics which degrades the phytate via the action of phytate degrading enzymes and in this way may positively influence Fe absorption. Previously, we have been able to show the change of receptors of Fe in different places of the intestine in pigs and chickens in the presence of prebiotics [8,9]. However, even with those studies, it sometimes took more than six weeks to observe the changes in all Fe related parameters (i.e. the concentration of Hb). Therefore, we believe that further improvement of Fe status in vivo (i.e. changes in Fe related proteins and enzymes) in the presence of wheat products may be seen in a longer term study.

Conclusion

Iron deficiency is the most prevalent nutritional deficiency worldwide. At present, there is more and more interest in improving gut health, and consequently increasing Fe absorption, by managing the colonic microbial population.

This study demonstrated that prebiotics naturally found in wheat grains/bread products significantly increased intestinal beneficial bacterial population in Fe deficient broiler chickens. The prebiotic segments of wheat enhanced the growth of colon microflora, with generally more bifidobacteria being created. This is an important finding as formed bacterial populations improve gut health and can consequently contribute to increased Fe absorption. With this short-term feeding trial we were not able to show differences in the Fe-status of broilers. Nevertheless, the bacterial analysis clearly suggests that we may have more significant differences (i.e. increased Fe bioavailability and improved Fe status) in a longer term feeding trial. Based on the results presented here, additional in-vitro and in-vivo trials are required to increase our knowledge on the nutritional and essential prebiotic properties of wheat grains and their baked products in improving Fe absorption.

, briefly, PCR was carried out with primers chosen from the fragment of the chicken (Gallus gallus) duodenal DMT1 gene (GeneBank database; GI 206597489) (forward: 5'-AGC CGT TCA CCA CTT ATT TCG-3'; reverse: 5'-GGT CCA AAT AGG CGA TGC TC-3'), DcytB gene (GI 20380692) (forward: 5'-GGC CGT GTT TGA GAA CCA CAA TGT T-3'; reverse: 5'-CGT TTG CAA TCA CGT TTC CAA AGA T-3') and Ferroportin gene (GI 61098365) (forward: 5'-GAT GCA TTC TGA ACA ACC AAG GA'; reverse: 5'-GGA GAC TGG GTG GAC AAG AAC TC-3'). Ribosomal 18S was used to normalize the results (GI 7262899) (forward: 5'-CGA TGC TCT TAA CTG AGT-3'; reverse: 5'-CAG CTT TGC AAC CAT ACT C-3').

Figure 1
1Summary of in vitro bioavailability results. Red columns -Ferritin concentrations in Caco-2 cells exposed to samples (ng of ferritin/ mg of protein). Values are means +/− SD (n = 6), P < 0.05. Blue columns -Sample Fe concentration (means +/− SD (n = 3), P < 0.05, ppm). WM = wholemeal.

Figure 2
2The relative amounts of Lactobacilli and Bifidobacteria generas in intestinal contents of chickens expressed in arbitrary units (AU). Values are means +/− SD (n = 10). a, b Mean values within each bacterial species tested with unlikesuperscript letters are significantly different (p < 0.05).

Table 2
2Blood hemoglobin (Hb) concentration (g/dL), liver ferritin protein amounts, and duodenal mRNA abundance of DMT1, Dcytb and ferroportin in broiler chickens fed with different flour and bread samplesSample
Blood Hb
Liver ferritin
Dcytb
DMT1
Ferroportin

DH line number 7
10.22 ± 1.40
0.56 ± 0.02
0.65 ± 0.04
0.75 ± 0.05
0.69 ± 0.02

DH line number 121
11.31 ± 1.40
0.56 ± 0.02
0.65 ± 0.04
0.75 ± 0.04
0.68 ± 0.04

DH line number 114
10.50 ± 1.65
0.55 ± 0.04
0.65 ± 0.03
0.75 ± 0.04
0.68 ± 0.03

DH line number 150
10.91 ± 2.05
0.56 ± 0.03
0.65 ± 0.02
0.76 ± 0.04
0.69 ± 0.02

Crusty white bread mix flour
10.64 ± 1.79
0.55 ± 0.03
0.65 ± 0.03
0.76 ± 0.03
0.69 ± 0.03

White yeast leavened bread
11.13 ± 1.50
0.55 ± 0.03
0.65 ± 0.04
0.76 ± 0.04
0.69 ± 0.03

White chapatti bread
11.59 ± 0.88
0.55 ± 0.04
0.66 ± 0.05
0.76 ± 0.04
0.69 ± 0.04

Wholemeal bread mix flour
11.32 ± 1.39
0.55 ± 0.03
0.66 ± 0.03
0.75 ± 0.03
0.69 ± 0.03

Wholemeal yeast leavened bread
11.59 ± 1.36
0.56 ± 0.02
0.65 ± 0.04
0.76 ± 0.03
0.69 ± 0.03

Wholemeal chapatti bread
10.61 ± 0.89
0.56 ± 0.04
0.65 ± 0.04
0.76 ± 0.02
0.69 ± 0.02

Changes in mRNA abundance are expressed relative to the expression of 18S rRNA in arbitrary units (AU). Values are means +/− SD (n = 10).

http://www.nutritionj.com/content/13/1/58
Competing interestsAll of the authors read and approved the final version of the manuscript. None of the authors had a conflict of interest.Authors' contributions ET carried out the in vivo study, analyzed and interpreted data and helped to draft the manuscript, co-authored the paper. RG was responsible for in vitro study; reviewed the final draft of the manuscript. MK carried out preparation of bread samples and completed sugar and phytate analysis, evaluated data, wrote the manuscript and prepared the manuscript for submission to the journal. JS responsible for the design of the study, organization of micronutrient analysis, reviewed the final draft of the manuscript, co-authored the paper. All authors read and approved the final manuscript.
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The effect of wheat prebiotics on the gut bacterial population and iron status of iron deficient broiler chickens. Tako, Nutrition Journal. 1358Cite this article asCite this article as: Tako et al.: The effect of wheat prebiotics on the gut bacterial population and iron status of iron deficient broiler chickens. Nutrition Journal 2014 13:58.