EFFECTS OF ANTINUTRIENTS ON THE NUTRITIONAL VALUE OF LEGUME DIET

Obiettivo

A. INTRODUCTION

Why a COST Action for this topic

Soya and other legume products are used extensively in human food and animal feedstuffs but they are not utilized efficiently mainly because of their content of different antinutrients which interfere with the digestion and absorption of nutrients from the digestive tract. Recent studies have revealed that lectins (agglutinins), trypsin inhibitors and non-starch polysaccharides are major antinutrients in legume-based diets, and when present, they severely restrict nutrient utilization.

Agglutinins, through their recognition and specific binding to brush border epithelial membrane receptor glycans, interfere with the digestion and absorption of proteins, modify the renewal and structure of brush border cells and change the microbial ecology of the gut. Due to the accumulation of cellular debris and undigested food in the lumen of the damaged gut, toxic lectins can promote the proliferation of opportunistic commensal and food-borne bacteria, including pathogens. This occurs widely in practice in both monogastric and ruminant nutrition and exacerbates the direct antinutrient effects of toxic lectins. Trypsin inhibitors, by reducing the digestibility of dietary proteins aggravate these antinutritive effects. Dietary

agglutinins and trypsin inhibitors can interfere with the endocrine and immune functions of the body and some of these harmful effects may be mediated or reinforced by a reduction in the bioavailability of minerals, such as iron, zinc and copper. Although it is often suggested than the poor nutritional value is due to the presence of the seed agglutinin and/or the trypsin inhibitors, the mechanism of the antinutritive effects is largely unknown. At present, to reduce this constraint, most legume products are heat-treated. As this is expensive and can lead to the production of toxic by-products, it is clear that there is an urgent need to find more science-based and cost-effective methods of detoxification. It is hoped that the results of this work will lead to an understanding of the toxicity and provide methods for the production of safer and more nutritious dietary products.

The programme brings together research groups both within the EC (FLAIR Concerted Action No 9) and those outside (COST 909) and would be a natural continuation of COST 909 Action. However, this new proposal takes into account the achievements of the previous actions, builds on and extends them to achieve new and advanced objectives. By increased regular contacts, exchanges and meetings of members of the proposed action, the scope and the outcome of the collaboration will be enhanced. This will be further facilitated by the setting up of a common data base and computerized communication between the groups. The results will be published in scientific journals and given at workshops and major conferences.

It is envisaged that increased collaboration between the various individual research centres will promote efficiency and increase scientific output. The participation of Hungary as a COST country is particularly important because the proposed partners from the Central Food Research Institute (Budapest) have unique immunological input, facilities and expertise with blood serum proteins that are not available in the other centres of the proposed project. These resources are absolutely essential for a successful outcome of the project. In addition, the primary rumen epithelial tissue culture which had been set up by the proposed members from the Veterinary University (Budapest) will be a priceless asset with which we can extend these studies to ruminant nutrition. This primary culture is not available in other laboratories. It is hoped that not only the groups' results will be published as a book after the conclusion of the COST Programme and as Proceedings of some of the earlier workshops but also that as the Rowett has strong links with European food and feed manufacturers, the results of our pre-competitive research findings and the new technologies resulting from them will be transferred to industry. A close collaboration with other European, national and bilateral projects is expected and that would enhance the scientific value of the outcome.

B. ACTION OBJECTIVES:

The main objective of the proposed COST Action is to combine pertinent European expertise for the identification of factors which limit the nutritional utilization of soya and other legume proteins and from the understanding of their mechanism of action to find and test new methods for the production of safer and more nutritious legume food- and feedingstuffs.

Secondary objectives:

It is intended that with the facilities available to the group for technology transfer to use the results of the Action to improve both human and animal nutrition and health. The considerable savings of energy, manpower and land-use due to the more efficient use of soya (and other legume seed) products will have a major impact on our environment and public health.

C. THE SCIENTIFIC PROGRAMME OF THE PROPOSED COST ACTION:

Although the poor nutritional value of soya and other legume proteins is often ascribed to antinutritive components of the seed, mainly the agglutinins and trypsin inhibitors, direct evidence for the involvement of these antinutrients in toxicity or how they exert their effects, is not readily available. Thus, detoxification is usually achieved by empirical, expensive and potentially harmful procedures of heat-treatment. Clearly, there is a need for finding new methods which are firmly based on the principles of the mechanism of toxicity.

First a systematic and detailed search for lectins and trypsin inhibitors in different plant families will be made, particularly in those which are presently used as food or feedstuffs or which may be used as such in future. There will be a major effort to isolate and clone the genes of those lectins which are likely to be beneficial for gene transfer in transgenic plants.

As toxicity may be in part the result of the deleterious effects exerted by dietary lectins after their absorption via the gut wall into the blood circulation, in the proposed work, toxicity will, in the first instance, be assessed from the binding, endocytosis and transcytosis of the FITC-labelled agglutinins with human Caco-2 cells in culture, followed by measuring the inhibitory potency of suitable glycans and/or metabolic inhibitors in the same system. These studies will also be extended to include rumen epithelial cells in primary culture to assess the effects on dietary lectins on rumen metabolism and digestive efficiency.

The antinutritive effects of soya and other legume seed fractions or the purified agglutinins and/or the trypsin inhibitors will be tested by feeding experiments with rats. The extent of the depression of the digestion/absorption of the nutrients of the diet, damage of the brush border structure and the increase of epithelial cell turnover and renewal induced by these partially purified or pure legume seed components will be used as a measure of their antinutritive activity. In similar studies, the effects of

the legume components on the dietary bioavailability, absorption and excretion of iron, zinc and copper and how these affect the concentration of minerals in blood and various tissues of the body will be investigated and quantified. The potential for detoxification will be explored by testing in vivo the effects of those specific inhibitors, which were effective in reducing the binding, endocytosis and transcytosis of the agglutinins in vitro with Caco-2 cells. Protease supplements which can reverse the trypsin inhibitory activity of legume meals will also be tested by similar techniques in rat feeding experiments.

Humoral or gut-associated immune responses to dietary antigens including antinutrients can either reinforce or suppress antinutritive effects. Thus, although it is known that systemic allergic reactions to soya and other legume components occur, their effects on the gut immune system are virtually unknown. In the proposed work there will be a concerted effort to measure the changes induced in the secretion of IgA and IgM antibodies in the gut to legume seed antinutrients together with monitoring the changes in the concentration of serum proteins and the level, type and antigen-specificity of humoral antibodies in rats during feeding.

The effects of the combined administration of antinutrients and the most powerful inhibitors of binding of the agglutinins and/or proteases on immune responses will also be measured.

Methods for the reversal of harmful immune reactions will also be explored. The effects on immune responses of the administration of antinutrients in combination with glycans which are potent inhibitors of lectin-binding or with protease supplements which effectively neutralize trypsin inhibitor activity, will be determined. Based on these results, new methods for the detoxification of legume meals will be devised and tested by animal experiments.

Lectins will also be tested to see whether their antinutrient properties were due to their indirect promotion of bacterial proliferation in the gut. The reversal of this antinutrient activity will be attempted by the inclusion in the diet of glycans which are potent inhibitors of lectin-binding to the gut. Lectins will also be tested for their potential activity as blockers of harmful bacterial overgrowth in the gastro-intestinal tract.

Accordingly and on the basis of the specific interests, resources, expertise and goals of the participating groups, the work in the proposed COST Action is subdivided into appropriate research objectives as follows:

1. To investigate the lectin specificity and content of plants which are part of our diet and to isolate lectins in sufficient amounts for biochemical and nutritional investigations.

2. To study the genes of lectins which are not deleterious for consumers but confer nutritionally advantageous properties.

3. To find specific plant lectins and/or polysaccharides which as natural dietary supplements will abolish or minimize the harmful bacterial colonization of the small intestine and other parts of the gastrointestinal tract.

4. To assess the toxicity of soya and other agglutinins in vitro, the interaction with and binding to undifferentiated and differentiated human Caco-2 cells of the FITC-labelled agglutinins will be studied by fluorescence microscopy and by ELISA method and to study their potential metabolic effects, the endocytosis and transcytosis of the lectins will be measured in vitro with intact monolayers of Caco-2 cells grown on filters in the presence or absence of various metabolic and other inhibitors.

5. Similar binding studies of FITC-labelled lectins will be carried out with rumen epithelial tissue and primary rumen epithelial tissue culture cells to assess their metabolic effects on rumen tissue and bacteria.

6. To study the antinutritive effects of purified soya and other legume agglutinins and trypsin inhibitors in rat feeding experiments by measuring the digestion and absorption of the proteins, and other nutrients of the diet, assessing the damage to brush border structure and by testing for changes in cell turnover and renewal.

7. To select and test in rats (in pigs) food lectins and saccharides as blockers of the binding and proliferation of harmful and/or pathogenic bacteria in the small intestine.

8. To compare the effects of protein fractions containing trypsin inhibitors and agglutinins on the bioavailability, excretion and tissue levels of minerals, iron, zinc and copper.

9. To develop polyclonal antibodies to the major antinutrients of legume seeds and work out immunological methods, such as direct ELISA and indirect competitive ELISA, for monitoring the survival in and progress through the gut of antinutrients.

10. To measure the secretion of IgA and IgM antibodies in the gut and changes in the concentration of serum proteins and the level, type and antigen-specificity of humoral antibodies in response to antigenic and/or allergenic antinutrients in the legume diets.

11. To extend the studies to products which have been heat-treated or supplemented with sugars specific for the lectins or with proteases to reduce the harmful effects of inhibitors.

It is expected that as a result of these studies new and cost-effective methods of detoxification of legume seed meals will be developed without expensive heat-treatment. There is increasing evidence of a major influence of the diet on the health of the population in Europe. Improvements to the diet could lead to major savings in the cost of the health service and reduction in the work days lost through ill-health. Policies designed to satisfy the nutritional requirements of consumers need to consider food toxicology, hygiene and safety and should be consistent with reduced input of unnatural food substances such as antibacterial agents, antibiotics and other additives. The proposed research will develop novel scientific methods for the prevention and control of bacterial infection in the digestive tract of higher animals, including humans, by natural dietary means. It could lead to the introduction of alternative crops, enriched in lectins by genetic selection or by the transgenic technique, which would be amenable to small-scale farming methods. Processing methods for the production of food supplements from suitable crop or transgenic plants containing these lectins in high concentrations could in future be developed by small and medium-sized enterprises or large food companies within European States, increasing their international competitivity.

The proposed research will make a substantial contribution to our understanding of digestive and cell biology and reveal new aspects of food-gut interactions. Although some aspects of humoral responses to various plant antigens have been studied, the effects of the highly antigenic and potentially allergenic antinutritive components of soya bean on the gut associated lymphoid system and the secretion of s-IgA and IgM antibodies are virtually unknown. Thus, the proposed research is expected to open new avenues in the research of immune responses to dietary components. The evaluation of the overall nutritional value of soya and other important legume products will also be looked at in the context of the effects of the antinutrients on mineral balance and absorption of particularly important metal ions, such as zinc, iron and copper. One of the main attractions of the proposal is that, for the first time, a comprehensive study will look at all these aspects in a concerted fashion and this will allow us to develop an overview of the problem rather than to tackle it piecemeal.

In addition, as the importance of soya and other legume products in both human and animal nutrition is likely to increase even further, elimination of the major constraint which limits the efficiency of their nutritional utilization will have major impact on economics and health. It is envisaged that from the understanding of the mechanism of interaction between antinutrients and the gut, new safe and cost-effective methods of processing will merge eliminating the need for costly and potentially damaging heat-treatment. With this, after full development of the programme and when eventually applied, the results will give a major edge to European industry.

D. TIME-TABLE

The proposed Action is planned to last for five years and the various research objectives will be carried out in parallel in the participating laboratories. Detailed time-table and lists of tasks will be worked out by the Management Committee at its first meeting.

E. ORGANIZATION, MANAGEMENT AND RESPONSIBILITIES

A Management Committee will be established after the Memorandum of Understanding is signed by the parties included in the COST Action and will meet twice per year. This Committee will establish the time-table, tasks and rules of the Action at its first meeting.

F. ECONOMIC DIMENSION OF THE COST ACTION

It is estimated that to carry out the objectives of this COST Action will require about 20 man-years with a total estimated cost of about ECU 1 000 000 per year to be covered from national resources in the participating countries.

Current status
There are several Working Groups : One is concerned with the dietary use of lectins to slow down/stop the progress of tumour development and a second one concerned with the effects of structure on biological activity of antinutrient lectins. A third group is composed of MC members whose main interests are the low molecular weight antinutrients.
A special network of information exchange and collaborative programmes between Spanish and Scottish scientists participating in COST 98 Action was set up during the Short Term Scientific Mission visit of Drs Pusztai and Bardocz to Madrid in 1995. This network is now being extended to include more British participants who are not members of COST 98 MC.
During 1995 COST 98 Action financed 8 Short Term Scientific Missions while in 1996 the number of missions was ten.
There were three WG meetings held in the last two years.
A first WG Meeting of WG No. 1 on "Lectins - Modulation of Immune Function was held in Budapest (H),1/4 Feb 1996.
WG No. 2 held its first WG Meeting in Toulouse (F) on "Three-dimensional Protein Structure and Molecular Modelling Techniques for Lectins" on 21/24 Mar 1996.
A third WG Meeting of WG No. 1 with major participation (over 50 scientists) of scientists outside COST 98 Action took place in Witten (D) on "Immunomodulating and Antitumour Effects of Lectins of Mistletoe and Kidney Bean" on 07/10 Oct 1996.
COST 98 Action was represented at the COST Interaction Conference in Basel (CH) in October 1995 by Professor W.H. Holzapfel and at the 2nd NordFood Conference and EUREKA Food Brokerage Event held in Reykjavik (IS) on 17/21 Aug 1996 by Professors Uwe Pfüller and Pierre Rougé.

Programma(i)

IC-COST - European cooperation in the field of scientific and technical research (COST), 1971-

Argomento(i)

5 - Food Technology

Invito a presentare proposte

Data not available

Meccanismo di finanziamento

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Coordinatore

CEC

Contributo UE

Nessun dato

Indirizzo

Rue de la Loi, 200
1049 Brussels
Belgio

Costo totale