FAIR-CT96-1979 Wheat Gluten as Biopolymer for the Production of Renewable and Biodegradable Materials

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Biopolymers/Gums : FAIR Area 1.2 - Green Chemicals and Polymers Chain : Packaging : Paints/Coatings/Plastics : Protein/Amino Acid

	Proposal No:	FAIR-CT96-1979
	Date Prepared:	December 2000, May 1999, April 1998, January 1997
	Source:	Final Report Abstract Second Annual Progress Report First Annual Progress Report Proposal Abstract

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Final Report Abstract

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Proposal Abstract

SUMMARY

This project will be looking at the development of plant constituents as a source of renewable materials for industry. It starts with an established agro-industrial product (protein known as gluten derived from wheat) which is mainly used in the food industry as an additive to improve bread made from soft wheat. The objective of this project is to investigate non-food use of films made from gluten, for example as biodegradable packaging material.

OBJECTIVES

The increased demand for environmentally friendly, renewable polymers can be met using plant macro-molecules as substitutes for synthetic, petroleum based, products. In addition to starch and cellulose which have been extensively studied, proteins exhibit potential as film forming agents for many applications. The project will focus on:

• food packaging: including biodegradable films with selective permeability for gases (oxygen and carbon dioxide) water and aroma
• plastic films for agricultural use
• coatings for various substrates.

The use of wheat gluten as a substitute for synthetic polymers appears as a very interesting option for many reasons:

• Wheat is a major crop in Europe, well adapted to the lands and climates, but new uses are needed to ensure continual development
• Gluten is available in large amounts at a reasonable cost
• The physicochemical properties of wheat gluten make it suitable for many non food uses, including plastic materials. Properties of interest are mechanical, film forming, adhesive, which can be modified by physical, chemical and enzymatic treatments.

Previous studies have shown that it is possible to make strong transparent films from gluten. For large scale applications, knowledge is needed on the relationship between protein composition and structure on the one hand and the processability and properties of gluten products on the other. In this project, the suitability of various gluten proteins to produce plastic materials, especially films, through their physical, chemical and enzymic processing, will be investigated with the aim of relating protein structure and properties at the molecular level (protein interactions, formation of covalently linked networks) to the macroscopic properties of the protein network and the final product (mechanical properties, barrier properties). This will enable processing conditions to be adopted to obtain specific bulk and film properties, optimised for various applications.

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First Annual Progress Report

Objectives
The combined project objective is to produce biodegradable materials from wheat gluten by evaluating the potentialities of the different protein fractions, as native or after modification, as well as their processabilities. The project aims to give answer about the potentialities of gluten for providing films for food packaging or agricultural uses, coatings for different substrates (paper, cardboard, paints...) or thermoplastic materials.

For utilisation of gluten as biopolymer more knowledge is needed on the interdependence between protein composition and structure on the one hand, and processability and the final properties of gluten products on the other hand. For this purpose the main scientific objectives of this program are summarised by the following tasks:

• To exploit through fractionation procedure the great differences in the physicochemical and functional properties between monomeric gliadins and polymeric glutenins.
• To adapt through physical, chemical, enzymatic modifications or even mutagenesis wheat proteins to the preparation of biomaterials
• To get more knowledge about mechanism of protein network formation in biomaterial films
• To develop processes to prepare biomaterials from wheat proteins. The influence of protein processing both under aqueous and low-moisture conditions on the final properties will be investigated.
• To characterise wheat proteins materials and model their behaviour to predict their properties

Activities
During this first year, the work was mainly devoted to task 1, 2 and 3 whereas most of the studies relative to task 4 and 5 are delayed to the next three years. First, the protein material was prepared and provided to all the partners for further studies. Second, the conditions for modifying wheat proteins by chemical reagents (anhydrides, aldehydes), enzymes (transglutaminase) or for expressing model periodic polypeptidic polymers were studied. The third main task was dealing with the formation of the films and protein thermoplastic materials. Properties of the protein dispersions added with various plasticisers, the conditions of films preparation, the thermoplastic properties of gluten depending on water content were studied in detail. On this basis some assays of processability by wet processing were carried out. Moreover some data, especially about thermoplastic properties and mechanical properties of films could be modelled.

Progress Industrial gluten was selected for all the programme among four batches on the basis of biochemical and physicochemical analysis. Experimental gluten from specific varieties and enriched gliadin and glutenin fractions were prepared at the pilot scale, characterised and sent for further assays to the partners. To improve water resistance properties of gluten materials, hydrophobisation by chemical reagents were performed and surface hydrophobicity of the proteins followed by fluorescence. By studying the ability of transglutaminase to modify wheat proteins, a very high insolubilisation of gluten was observed, due to cross-linking whereas treated gliadins became more soluble in the neutral pH region. Finally, homoblock polypeptides could be expressed in E. Coli as model of true peptidic polymers. Using industrial gluten (native or deamidated) or gliadins enriched fractions, films could be prepared by casting in aqueous, alkaline or acidic/ethanolic conditions. From rheological properties of the film forming dispersions it could be assumed that the various polyol type plasticisers interact in different manner with the proteins. Tetraethylene glycol and glycerol appeared to be the more attractive. Depending on plasticiser and protein/plasticiser ratio, the stress of gliadin films varied in the range 1 to 3.5 Mpa and the strain 100 to 600%. For whole gluten, these values were comprised between I and 1.5 Mpa and 375 to 450% respectively. The glass transition temperature (Tg) for gluten was shown by modulated DSC to be dramatically dependent of the water content. It dropped from approximately 110 degrees C to 10 degrees C when the water mass fraction increased from 2 to 20%. The delta Cp was confirmed to be 0.4J/g*per degree C. Using the model of Gordon-Taylor, it was shown that the fit was satisfactory in the range of 7 to 15% for water mass fraction. The feasibility of wet processing was also investigated by casting, spraying and brushing of aqueous dispersion of proteins onto a solid substrate. Even if it was not possible to use brushing, continuous films could be prepared by casting or spraying, using native or deamidated glutens.

Achievements
These were as follows:

• I Industrial glutens was chosen among four batches and provided to the partners. Seven wheat varieties and lines have been produced for further experimental gluten extraction
• The pilot scale preparation of experimental gluten and enriched gliadin and glutenin fractions
• The conditions for hydrophilisation of gluten proteins are well defined and the methods for surface hydrophobicity measurements have been developed. Effects of oil additives was not successful and confirm the interest of chemical modifications.
• Gluten became insoluble after transglutaminase reactions whereas gliadins solubility curve was drastically shifted towards acidic pH.
• Homoblock polypeptides composed by the repetition of a gliadin consensus repetitive sequence could be expressed in E. Coli.
• Conditions of films preparation are defined. Glycerol and tetraethylene glycol appeared as efficient platicisers. The methodology for studying the rheology of the dispersions has been set-up.
• Thermoplastic properties of gluten are drastically influenced by water content.
• Continuous films could be processed by casting and spraying but not by brushing.

Future activities
Some complementary experiments to prepare gliadins and glutenins purified fractions will be performed. The physicochemical studies will be focused on the understanding of the protein-protein and protein-plasticisers interactions not only in the protein dispersions but also in the biomaterials, prepared either by wet or low moisture processing. The influence of modifications on these interactions and more generally on the characteristics of these biomaterials have to be examined in detail. Moreover, complementary experiments are needed to study the thermoplastic behaviours of gluten in presence of various plasticiser like polyols or amine plasticisers. Some efforts will be done to improve the methodology for evaluating the properties of the protein films, especially for developing miniaturised tests for studying the film ability of purified samples available in small amounts. Next year is also be concerned by studies on processability of gluten. Especially, first experiments using low moisture processing will be undertaken.

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Second Annual Progress Report

Description of work

During this first year, work was mainly devoted to task 1, 2, and 3 whereas most of the studies relative to task 4 and 5 were delayed to the next three years. In the second year again substantial effort was put in the first three tasks. Additionally, research was performed on processing and modelling, which are the subjects of task 4 and 5, respectively.

Regarding fractionation, experiments were initiated to prepare large amounts of gluten sub-fractions differing by their prolamin composition. These experiments are a follow-up of the preliminary experiments on preparation, characterisation, and processing of these sub-fractions that were carried out in the first one and a half year of the project. Furthermore, grain and flour were prepared of different wheat lines that were selected for their protein and gluten characteristics. This work will be followed by gluten extraction.

Wheat proteins were modified in different ways. Research was performed on genetic modification (recombinant repetitive polypeptides), enzymatic modification (transglutaminase and peroxidase), physical modification (additives and heat and pressure treatment), and chemical modification (mainly anhydrides).

One of the main research subjects in the previous year was the physico-chemical properties of wheat gluten in relation to films prepared from the protein. Besides films prepared from native wheat gluten, which was selected in year l (FAIR 2 gluten), films from gliadin and glutenin enriched fractions together with films prepared from different wheat varieties were prepared and analysed. Amongst others, the film forming solutions, process conditions, effect of plasticizer and storage conditions, post treatment of protein films, and barrier properties of the films were subject of investigation.

Furthermore, it was demonstrated that wheat gluten could be processed by different techniques into films and coatings on substrates as glass, poly(ethylene) and poly(styrene). Additionally, the effect of additives on processing ability was investigated.

On several of the above mentioned research subjects modelling of the results was applied.

State of progress

Additional to previous experiments, four wheat varieties were selected from which well identified gluten will be extracted. The varieties showed large genetic variability for high molecular and low molecular weight glutenin, and gliadin content. Furthermore, fractionation methods to prepare gliadin and glutenin sub-fractions and to purify low and high molecular weight glutenin sub-units were scaled up.

In the framework of genetic modification conditions for high level expression and purification of peptides (PQQPY)₈, (PQQPY)₁₆, and (PQQPY)₃₂, in E. coli were optimised. The peptides were analysed by CD and FTIR spectroscopy. Physical modification (heat, pressure) did not affect macroscopic protein properties. However, addition of especially stearic acid substantially decreased the water vapour permeability of wheat gluten films. The mechanical properties of (deamidated) wheat gluten films could be modified by enzymatic treatments using transglutaminase and peroxidase. Hydrophobisation of wheat gluten with caproic acid anhydrides was optimised and scaled up. Additionally, experiments with hydrophobisation reagents with long alkyl chains were carried out.

Films could be prepared from industrial FAlR 2 gluten, from gluten from different wheat varieties, and from gliadin and glutenin enriched fractions. Each partner prepared the films according their own procedures. Both mechanical and barrier properties were determined to characterise the different films. It appeared that the influence of preparation method was larger than the variation in gluten source or prolamin composition. Rheological measurements, FTIR spectroscopy, DSC measurements, and solubility experiments were carried out to obtain more knowledge about protein-protein interactions. Following the experiments of the first year, wet processing techniques such as brushing, casting, and spraying were applied using gluten dispersions differing in solid content, pH, or additives. Although viscosity varied, all techniques could be used to prepare coatings. The adhesion of the coatings to different substrates was also assessed.

Finally, statistical experimental set-ups and modelling procedures were used to study the effect of processing conditions and residual plasticizer content on the mechanical properties of wheat gluten films.

Achievements

1. Gluten was extracted from different wheat varieties. Additional varieties have been produced from which gluten also will be extracted;
2. A method of purification of low and high molecular weight glutenin sub-units was scaled up;
3. Films were prepared from gliadin and glutenin enriched fractions. The method of preparation determined the film properties to a large extent;
4. Heat and pressure treatment of wheat gluten before film preparation did not affect the film properties. However, post-treatment of gluten films with heat increased the strength of the films;
5. The water vapour permeability could be decreased by adding stearic acid to wheat gluten films;
6. Homoblock polypeptides composed by the repetition of a gliadin consensus repetitive sequence was organised in b-turns and was furthermore able to form a network maintained by hydrogen bonds;
7. Transglutaminase can be added to a film forming solution in which it cross-links deamidated gluten. Both stress and strain of the modified films was increased;
8. Hydrophobisation reactions can be performed at 100 g scale. Furthermore, reaction conditions and the type of reagent influence modification efficiencies;
9. FTIR methodology was developed to study protein conformation in solution, doughy state, and films;
10. The large influence of plasticizer (including water) on the mechanical properties of films was stressed;
11. Rheological behaviour of gluten dispersions can be adjusted by changing solid content, pH, or by adding additives without affecting the ability to form films and coatings using different application techniques.

Future actions

The future actions more or less originate from the initial project planning and time table. The influence of gliadin and glutenin sub-fractions in gluten on film properties will be an ongoing research subject. One of the important items in the next year will be setting up a ring test on the properties of protein films prepared by the different partners. Furthermore, modifications will focus on hydrophobisation of wheat gluten and on processing these modified samples either by casting procedures or low moisture techniques such as thermomoulding. Cross-linking reactions will be performed enzymatically and chemically and the results will be compared to the results of the post-treatments performed the previous year. Network formation studies will be performed with the developed methodology.

Finally, attention will be given to wet processing and to processing of (modified) gluten by thermomoulding and extrusion.