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Content, composition and functional properties of proteins from seeds of pea mutants. - AR0106

Description
Project Code: AR0106

Funded By Department for Environment, Food and Rural Affairs

Lead Research Centre
John Innes Centre
Norwich Research Park
Colney
Norwich

Abstract of Research Proposal
Most of the carbon entering the pea seed during development is partitioned either into protein or starch. There are, therefore, two methods for increasing the proportion of protein in the dry seed. Firstly, to manipulate those genes which directly affect the protein content and secondly, to shift the partitioning of carbon towards protein by reducing the synthesis of starch. This proposal will study the effect on the content and composition of protein following the reduction of starch content. We will use mutants developed previously which are affected in starch synthesis. It has been shown that the content and composition of pea seed proteins is greatly affected by mutations at two loci, r and rb. We have identified a range of mutant alleles at both of these loci plus mutations at a further four loci affecting starch, including lines which have little or no starch in the dry seed. The protein content and composition of these lines will be determined and related to the nutritional value of the meal before and after processing. The proposal is a response to the problem of improving protein crops for use in animal feedstuff production within the UK as defined in the ROAME A of Programme 8. Specifically, the project will provide information and material for improving the content of protein within the pea crop which will make it more attractive to compounds for the production of high protein pea meal, which will be more competitive with imported commodities such as Soya, as a raw material for animal feed. The project will also improve the selection and development of material by plant breeders, which conforms to industry requirements for the animal feedstuff market as requested in A3 bii of the forward objectives of the amended ROAME A policy document.

Summary of Objectives
1) Validate the chemical and physical methods for estimating total protein content in pea meal and use three methods to determine the protein content in the seeds of mutant lines (alleles at the r, rb, rug3, rug4, rug5 and lam loci).
2) Determine the protein composition (albuminins, globulins, glutelins and prolamins) in the seeds of representative mutant pea lines analysed in Objective 1.
3) Using mutant lines selected following Objectives 1 and 2, assess the effect of changes in protein content and composition on those functional properties of proteins which relate to the processing of animal feedstuff including the development of methods for air classification of pea meal.
4) In collaboration with a commercial processor, determine the properties of proteins from meal and protein concentrates before and after processing using selected mutant lines.
5) Determine the effects of protein content, composition and functional properties on the nutritional value of feedstuff before and after processing.



Executive Summary of Final Report

The first objective of the project was to develop a rigorous chemical method for calculating the true protein content of pea seeds, based on amino acid analysis of pea flour from whole seeds. Also, it was necessary to determine the total nitrogen content and nitrogen-to-protein conversion factors for calculating the true protein contents of wild-type and mutant peas. In order to determine the effects of the pea seed mutations on carbon partitioning in the embryo for starch and protein and to understand the relationship between protein content and crop yield, we analysed the effects of seed size and the contribution made by the testa on protein content. This analytical part of the project was carried out in collaboration with Pedigree Masterfoods and with the University of Nottingham. From the results of these chemical analyses it was possible to identify pea seed mutants with higher total protein and sulphur amino acid contents, which were likely to be nutritionally more valuable for animal feedstuff.

In addition to determining variation between the pea seed mutants for total protein content, we also carried out a comparative analysis of the protein composition of wild-type and mutant pea seeds. We were able to demonstrate differences in the proportion of the major seed proteins, legumin (11S) and vicilin (7S) using entirely different extraction (no salt) and detection (analytical ultracentrifugation) techniques. We were able to show that a reduction in legumin (11S) was accompanied by a corresponding increase in albumin (2S-4S) content. We also carried out more detailed work on the hydrdynamic properties of the purified legumin and vicilin from seed of the wild type and from the r and rug3 mutants. This study confirmed that the mutations did not affect the structure of the proteins directly; legumin and vicilin from the mutants have the same molar mass as wild type. This work was carried out in collaboration with the University of Nottingham (National Centre for Macromolecular Hydrodynamics). The studies on the composition of the globulin fraction from wild-type and mutant peas confirmed similar variation in vicilin:legumin ratios to previously published data. Moreover, no changes in the molar mass of the purified vicilin and legumin from wild-type and mutant peas was observed.

Another aspect of the protein characterisation part of the project was to determine the proportion of all of the different protein groups within the seed of the different mutants. We developed a method, based on successive extraction of pea meal with aqueous and organic solvents at different pH. In general, the results of this study confirmed the data from analytical ultracentrifugation. In addition, we were able to demonstrate that there was a wide variation in proportion of proteins present in the seed of different mutants that cannot be classified as albumins and globulins. These included the prolamins (ethanol soluble), glutelins (alkali soluble) and cell wall/membrane associated proteins (insoluble).

The albumin fraction is the most diverse group of proteins within the seed. We compared the albumins from the wild type and the various mutants using rocket immunoelectrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The methods used for this part of the project had been developed within an allied JIC MAFF project (AR0105). Using this method we were able to demonstrate that there were no quantitative changes to PA2 (a major albumin protein), but there were changes in the relative amount of another polypeptide of approximate molecular mass 45kDa for some mutants. The overall appearance of the albumin protein profiles on SDS-PAGE gels suggested that the lam mutant had a considerably larger number of qualitative changes than the other mutants, whose protein profiles, apart from the 45kDa polypeptide, were almost identical.

On the basis of the chemical compositional information the wild-type and two of the mutants (r and rug3) were chosen for further work leading to digestibility trials. Seed from one of these mutant lines (rug3) lacked starch, the remaining storage products being protein and lipid. In collaboration with the Institute of Food Research, air-classification was used to produce high protein fractions from these selected peas for use in a poultry digestibility trail and for further analysis. It was decided that the poultry trial would be carried out using the native pea meal and meal that had been processed through an extruder. Before extrusion, the effect of temperature on the samples was studied using differential scanning calorimetry. It was not possible to determine glass transition temperatures by DSC analysis, however, it was possible to identify the peaks in the DSC profiles that were due to vicilin (7S) and legumin (11S) in the flours and, in particular, in the air-classified fractions. This enable us to compare the melting behaviour of these proteins and to estimate their denaturation temperatures as a function of flour moisture content, an important factor in determining the conditions for extrusion processing. This was carried out for wild-type and for the r and rug3 mutant material. The renaturation behaviour of the protein in the samples was determined from the DSC profiles for previously heated or denatured samples. This provided information on the conditions required to produce extruded materials from the different peas in a form which could be fed to poultry.
The extrusion work was carried out in collaboration with the University of Nottingham. Despite the fact that the rug3 seed lacked starch, we were able to produce an extruded product using this material. The visible appearance of this product was considered unusual for peas and more closely resembled that which would have been obtained from soybeans. Mechanical properties of the extruded products were determined in collaboration with the Institute of Food Research using INSTRON. This work indicated that there were significant differences in the hardness of the different materials and that materials were harder than expected.
The poultry digestibility trials were conducted at the University of Nottingham using semi-synthetic poultry diets. All amino acids were evaluated, but the trends for lysine, methionine and threonine were examined in detail since these are nutritionally essential for poultry and are also usually considered in assessing the nutritional quality of any raw material. Differences were found between the digestibility of the protein from wild-type and from the two mutants, both in terms of the content of digestible amino acids and the coefficient of digestibility. As well as this effect of genotype there were also some significant effects of processing and processing x genotype interactions. There was some evidence air-classification of meal from the r mutant produced a protein fraction that was more digestible. The limited fund available did not allow us to undertake a growth trail using poultry, which would have demonstrated more clearly potential differences between the various lines for use in animal feedstuff.
The other problem with using an animal based system for trialling the different pea lines was the requirement for relatively large amounts of material to make up the poultry diets. We decided, therefore, to explore the possibility of using an in vitro system for assessing digestibility of raw and processed pea meals, and to compare the result of this with the in vivo digestibility trial using poultry. The system used had been developed at the IFR in Norwich from a previously published method. The basis of the technique is to determine the rate of production of amino acids and short chain polypeptides following digestion of the protein by porcine pancreatin extract. The procedure is carried out in dialysis tubing, the small molecular weight molecules produced during the digestion diffusing through the membrane and are measure using a spectrophotometer. This method uses very small amounts of material and large numbers of samples can be analysed in a relatively short time.
Small differences in digestibilities were observed when in vitro analysis of raw meals, from the various pea lines, was performed. A trend of digestibility, similar to that seen in the in vivo study, was shown to exist, wild-type meal having the greatest, and the rug3 mutant the lowest digestibility. No significant differences were found between the pea lines when meal, processed by extrusion prior to digestion was measured in vitro. However, extrusion was shown to improve the digestibilities of all the pea meals. Following extrusion, digestibility of meal from the rug3 mutant was significantly improved its digestibility in comparison to that of the raw meal. Air classifying of wild-type meal had little effect on its digestibility. It was concluded that the in vitro technique provided an accurate and highly reproducible method of determining digestibility of protein-rich feedstuffs.
Overall we have shown that the protein produced by the pea seed mutants differs for content, composition and properties. We have also shown variation in digestibility of the proteins measured using either in vivo or in vitro methods. There is undoubtedly scope for increasing the use of pea seed protein in animal feedstuff and for improving the quality of the protein for this purpose. It would be useful in the future to study further the interaction between the digestibility of the proteins as affected by the starch fraction since this will almost certainly be present, even if air classified, protein-rich fractions are used. This could most easily be carried out using the in vitro system developed within the current project. It would also be important to link these studies to growth rate studies using animal systems such as poultry.

Funding
£223,200

Start date: 01/04/98
Completion date: 31/03/01



Project Documents
• Final Report : Content, composition and functional properties of proteins from seeds of pea mutants.   (2568k)
Time-Scale and Cost
From: 1998

To: 2002

Cost: £310,635
Contractor / Funded Organisations
John Innes Centre (BBSRC)
Keywords
Arable Farming              
Biotechnology              
Crop Improvement              
Crops              
Farming              
Fields of Study
Arable Crops