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QLK5-1999-01442
ENHANCE: Green chemicals and biopolymers from rapeseed meal with enhanced end-performances |
| Contract No: | QLK5-1999-01442 |
| Source: | Final Report January 2003 |
| Source: | Third Annual Progress Report January 2003 |
| Source: | Second Annual Progress Report January 2002 |
| Source: | First Annual Progress Report 2001 |
Introduction
The general aim of the ENHANCE project was to develop non-food uses for oilmeal components as green chemicals and bio-polymers with enhanced end-use performances in the following markets:
The main objectives of the project have been detailed in the summary and are repeated in the abstract below that also details the corresponding workpackages that are used to describe the results.
Results
Workpackage 1: Economic and Market Evaluation
Most interesting and promising markets for rapeseed proteins are surfactant and adhesive applications
Costing models based on results and developments made through the project lifetime have been established. The models represent four scenarios, Scenarios I - III cover three evaluated and developed oil extraction methods, whereas Scenario IV is a generic method for extraction of protein from the resulting oil meals from Scenario I-Ill. The scale of production in the models are based on a estimated market of protein of 1000 tons per year, which account for 6.25-12.5% of the present estimated casein used in EU for adhesive purposes. A profit of 177 euros per ton is expected for a sales price of 3.3 euro per Kg of rapeseed protein.
Workpackage 2: New applications for industrial rapeseed meals
Processed products from AGAT and POLLEN varieties were prepared according to the methods already tested with EXPRESS. The extrusion was carried out by the use of a new screw assembly that allows a throughput of 80 kg/h of kernels. The previous positive conclusion on the feasibility of the process at industrial scale is confirmed although the blanching process needs a more accurate temperature monitoring to explain the differences observed for myrosinase inactivation between the varieties. The extrusion process resulted in a myrosinase inactivation but in a lesser extent as compared with the blanching process. Extrusion leads to a lower decrease of the glucosinolate content than blanching. Anyway, the differences observed in the composition among varieties could be also due to minor changes in the applied processes.
Extrusion of low fibre AGAT rapeseeds resulted in pellets with a very poor structure, with a very high fine content that made direct percolation extraction impossible due to a too low percolation rate. A re-pelletisation overcome this problem without decreasing the general quality parameters of the rapeseed pellets (NSI, milling defect, water content). Desolventisation with only superheated hexane is the best manner to keep the solubility of proteins but at the expense of a high content of residual hexane in the meal (> 5000 ppm). An additional desolventisation with superheated steam removes the hexane but leads to an almost immediate and significant protein denaturation because of the high temperatures reached during the process. Desolventisation with superheated ethanol also decreased the protein solubility and, in addition, the meals showed a high residual content of ethanol.
Results from the work carried out in 2002 showed that there were promising fungicidal and insecticidal activity in the nine new meals tested. Growth of Rhizoctonia solani, the cause of stem canker and black scurf of potatoes, was reduced in vitro and there was also good activity against Gaeurnannomyces graminis (Take-all in cereals) and Phoma exigua (gangrene in potatoes).
If the activity of the rapeseed meal against fungal pathogens can be transferred to a field situation this natural fungicide would benefit organic as well as conventional growers. Nevertheless, the amount of meal calculated (77 t/ha) as required to give control of Rhizoctonia similar to that of standard fungicide may be a hindrance in developing the meal as a soil fumigant.
The meals also showed good insecticidal activity against Tipula and Deroceras species, both of which are extremely important within the UK and other parts of Europe. In general, the variety Express showed the most promise as a soil fumigant against Rhizoctonia and Gaeurnannomyces, whereas the variety Agat was the most effective as an insecticide against Tipula and Deroceras species. Results have also shown that the inactivation of myrosinase during blanching does not destroy the activity of meal as biocide, which rebuts the initial assumption that biocidal activity of the rapemeal is exclusively due to the breakdown products released by the action of myrosinase on the glucosinolates. It is suggested that another biochemical process, one not destroyed by heat treatment, is also active within the rapemeal.
Workpackage 3: Protein product and processing basics
The genetic finger-printing of OSR varieties by AFLP analysis produced entirely different results than those previously obtained by micro-satellite analysis. A comparison between the two methods showed that micro-satellite analysis produced the most reliable estimation of genetic variability, and that further work was needed to improve the results obtained by AFLP. Efforts are being made to combine the DNA fingerprinting data obtained by micro-satellite analysis with the protein profiling data already obtained by INRA for the OSR varieties, to identify the markers that are linked to major protein types in OSR.
The contents of dietary fibres (DF), ash, oil, protein, reducing and non-reducing sugars and phenolics have been investigated in Brassica campestris L. cv. Agat, Brassica napus L. cv. Pollen, and germinated seeds and press cake from B. napus. No significant differences were found between Brassica species.
Part of the dietary fibre was found associated with proteins. As a consequence of germination a decrease in the content of non-reducing oligosaccharides and an increase of reducing carbohydrates was observed in the seeds. Most abundant phenolics in rapeseed are sinapoyl derivatives and 4-hydroxyindol-3-y-methylglucosinolate, which remain in the cake giving unwanted dark colours when oxidized.
Two steps of the analytical scheme were modified to scale up the LIT purification. The first step of depigmentation by gel filtration was successfully replaced by nanofiltration, and the ionic chromatography under acidic conditions was performed on a Source 30S instead of a Source l5S. These modifications permitted processing of 3.5 kg of rapeseed meal in one step of ultrafiltration providing 0.5 g of LTP enriched fraction per kg of rapeseed meal.
The antifungal proteins previously purified by HPLC have now been further purified. Isolated fractions are currently being sequenced for N-terminal amino acids. A simple method for obtaining proteinase inhibitor rich extracts from rape meal has been successfully tested, and further purification and characterisation work is currently underway.
The solid absorption of the 2 S and 12 S proteins from rapeseed and the C8 and C10 acylated peptides onto stainless steel plates has been investigated by the contact angle technique. Conditioning with the 2 S protein had a little influence on the contact angles and the surface characteristics of the plates, whereas the effect of 12 S protein largely depended on the pH and ionic strength at which the adsorption was achieved.
The adsccption of C8 and C10 acylated peptides was less influenced by the pH or ionic strength in comparison with the proteins. The C8 generally decreased the contact angles with water or formamide increasing the hydrophilicity of the plates. in contrast, the C10 acylated peptides largely increased all the contact angles and drastically decreased the total surface free energy of plates.
Workpackage 4: Enhancement of end-use performance through chemical and a physical/enzymatic modification
Cationisation of proteins by using DCC in methanol failed because this solvent seems to react with DCC. A further attempt was made replacing methanol by pyridine, but currently more data are necessary to prove the efficiency of the reaction. Another approach was to use EDC in water instead of DCC, which resulted in the cationisation of the protein on the basis of the IR spectrum. This reaction has the advantage that can be performed in one step and 15% of lysines were modified, but EDC is an expensive reagent.
Rapeseed proteins have been modified by use of electrophilic products produced from glucosinolates, which resulted in modified products having an increased hydrophobicity and reduced solubility in water. The best glycosylation yield was obtained by the chemical method but using a very toxic compound, NaBH3CN. The use of the chemo-enzymatic method avoided the use of this compound but the degree of modification was not enough to modify the functional properties of proteins.
The attempt of glycosylation of peptides did not success because of the low solubility of peptides at basic pH. New assays are planned on hydrolysates more soluble at basic pH. In conclusion, none of the methods proposed for the glycosylation of protein or peptides fulfilled the requirements of the project: non-toxic reagents, high yields and introduction of 'real' sugars instead of polyol chains.
New enzymatic hydrolysis of protein isolates from IVV has been carried out during this year in order to test the functional properties of the resulting hydrolysates. Special attention has been paid to the preparation of hydrolysates from the precipitated isolate because of its poor functional properties. Using acidic proteases at enzyme/substrate of 1/5,000 and 1/1,000 it was possible to generate hydrolysates mostly composed of 'long' peptides. Nevertheless, the 2S protein present in the precipitated isolate was hardly hydrolysed and it was necessary to carry out a previous reduction of the isolate to achieve an effective digestion of this protein.
Workpackage 5: Pilot-scale protein processing
The comparison of meal qualities resulting from the various processes developed in the ENHANCE project with respect to a standard mild process suggests that the following is effective. The chosen method consists of partial de-oiling by cooking extrusion, mild hexane extraction and de-solventisation with superheated hexane. This method results in meals with a good protein solubility and a low fat content. This procedure maintains a protein solubility between 52 and 59 %, and an oil content of 3 to 4 %, depending on the variety. Preparation of protein isolates from this meal resulted in a lower yield as compared with the reference meal, mainly attributed to a minor recovery of the 12 S protein. In contrast, the protein content of isolates was similar in both trials.
Concerning the protein quality of the BDF samples, it seams that the aqueous salt extraction method used at the end of 2001 gave a very good isolate S enriched in the 2 S protein with a protein solubility of 95,7 % at pt-I 7 and a protein content of 81,2 %. However this was at the expense of Protein P with a solubility of 48% at pH 7 and a protein content of 62,4 %. The simplification of the Bioraf process (samples protein S/P and protein S/P iso, 2002) reduced the protein content and solubility of both isolates S and P.
Workpackage 6: Adhesives, coatings and films from the protein enriched fraction and derivatives
The main shortcomings of the adhesives based on rapeseed proteins as the smell, the flexibility. The tack and the adhesion stability can be reduced or overcome. The smell could be significantly reduced by decreasing the pH of the formulations. The flexibility and tack could be improved by adding plasticisers and gelatinised starch or extra protein. Finally, the adhesion stability (higher viscosity) improves when fillers like silica or chalk are added. Pilot scale experiments have been carried out using rapeseed protein in the formulation of carpet adhesives. Despite of the improvements of the new glue formulations as compared with the former ones, the initial tack was still not sufficient for application as carpet glue.
Concerning storage behaviour, there was neither significant loss of adhesion nor microbial destruction recognisable. According to a preliminary evaluation of prices the costs for the additives of the ENHANCE formulations will range from 3.0 to 3.2 euros/kg for a "ready to use" adhesive. Taking into account some processing costs and the profit margin, the ENHANCE formulations will be competitive with the available natural adhesives but not, of course, with synthetic adhesives.
Rapeseed protein isolates showed good interaction with paper fibres. The effect of rapeseed proteins was investigated on the mechanical properties, SCT (stiffness) and Burst (strength), of recycled paper. Cationisation of the protein, which increases the positive charge of the protein and therefore the adhesion with the paper fibres, had no positive effect on the paper properties. Probably the cationised samples were less homogeneously distributed and less penetrated in the paper due to a lower water solubility of the samples in comparison with the native filtered isolate, which had the best effect on the mechanical properties of recycled paper among the samples tested.
Insulation plates of three different sizes, mini, medium and pilot size, were evaluated on the basis of their length at break and the tear and compression tests. Tests showed that preparation of plates by using rapeseed protein isolates as binder is feasible, the best results being those obtained from the succinylated precipitated isolate. Protein S (Bioraf) and precipitated rapeseed isolate (IVV) gave similar characteristics to the plates. However, further trials are needed to optimise the handling of the plates in the machine, the raw density and probably also the adhesive content. The surface amages when taking the plate out of the frame were responsible for the diminution of the tear strength and an accurate control of the raw density is essential for the a good binding of the fibres. A preliminary evaluation shows that the additional step of succinylation could be easily integrated in the protein extraction process, and based on the market prices, the precipitated could be competitive as binder in the preparation of insulation plates. Anyway, the determination of the degree of the succinylation required for such application is necessary for a more accurate evaluation of the costs.
Workpackage 7 : End-use performances: surfactants, detergents and wetting agents
Protein isolates, succinylated protein isolates, protein hydrolysates and acylated protein hydrolysates have been evaluated concerning their foaming and emulsifying properties and compared with commercial surface-active proteins. Large differences were observed among batches of the same product, which suggests differences in their composition although, in general, precipitated isolates showed rather poor foaming and emulsifying properties as compared with the filtrated isolates. Among the different batches prepared by BDF, only 002 protein S generated emulsions stable against creaming.
Protein hydrolysates showed foaming capacity and stability similar or even superior in some cases to those of commercial proteins at pH 3, 7 or 8.5 but they did not improve the emulsifying properties of the isolates.
Foams generated with acylated hydrolysate were very stable, the C10 derivative generating 'more hydrated' foams as compared with the C14C or C18. The C18 acylated peptides generated stable emulsions with a good rate of creaming, especialy at basic pH and when the concentration is increased.
Glycosylation of the 2S protein, either by the chemical method using NaBH3CN or by the chemical/enzymatic one, resulted in emulsions with a slower rate of creaming as compared with the native protein. The improvement in the chemical/enzymatic method appears due to the chemical step rather than to the subsequent enzymatic glycosylation.
Discussion
In general, the programme has followed the time schedule planned in the technical annex. Despite the great number of tasks and partners, the co-ordination of the experiments in the different workpackages is satisfying.
During the last year the research has been suplemented with data on new varieties of rape, Agat and Pollen. The results obtained showed only minor differences among varieties and confirmed the feasibility of some strategies previously developed for the Express variety. Based on the developments made through the project lifetime it has been possible to establish costing models for most of the ENHANCE products that will be very helpful to the objective of a technology transfer.
Partial de-oiling on a extruder cooker followed by mild extraction with hexane seems to be the more appropriate way to prepared de-oiled rapeseed meal from the point of view of the costs and the preservation of the solubility of proteins. In this sense, desolventisation with only hexane is preferred to other desolventisation methods although the drawback of the high content of residual hexane in the meal remains unsolved since the use of an additional desolventisation step is very detrimental for protein solubility.
Knowledge of rapeseed proteins has been widened by the purification and characterisation of minor proteins including LTP, anti-fungal proteins and protease inhibitors. The data on the genetic variability of rape will be compared with the protein data profiling to identify the markers linked to major protein types.
The diversification of the ENHANCE products has facilitated the take up of numerous applications. For example, meals show a clear potential as soil fumigant although the influence that rapeseed meal could have on the soil needs to be evaluated. Further investigations are also necessary to establish the components of the meal responsible for the biocide activity.
Protein isolates with a high protein content and solubility have been prepared. These isolates have shown a good potential in some applications as paper coatings, in adhesive formulations, as surface-active compounds for the generation of emulsions and foams, and in the preparation of insulation plates. Moreover, these isolates have been used as raw material to develop new derivatives with enhanced performances via chemical and/or enzymatic modifications. Enzymatic hydrolysates showed foaming properties superior to some commercial proteins and succinylated isolate was very effective as adhesive in insulation plates. Acylated peptides generate very stable foams and adsorb to stainless steel changing its surface characteristics. Grafting of rapeseed proteins with glucosinolates is a innovative reaction that results in an increase of the hydrophobicity of proteins. In other cases, efforts have been made to overcome the limitations of some chemical modifications to reach the objectives of the project. For example, the cationisation of proteins is now simplified by caning out the reaction in a single step although the reagent used is more expensive. In the case of glycosylation, the work has been focused on the optimisation of the chemical/enzymatic glycosylation. This method avoid the use of toxic compounds but unfortunately the yields obtained were not high enough to improve the functiona] properties of proteins consequently, at present, none of the methods investigated fulfilled the requirements of the project.
Conclusions
In conclusion, the results obtained confirm expectations on the viability of the processes developed throughout the project. Different varieties of rape have been studied and a wide range of products (from meal to modified proteins) has been produced from these processes. Nevertheless some aspects need further research to improve the products.
Original applications for the ENHANCE products have been identified and their performance and cost evaluation are very encouraging, in some cases, in terms of further industrial development. Adhesives, biocides, paper coatings and surfactants seem to be the most interesting end-uses for rape products.
1. Objectives
The main objectives of the project were:
2. Activities
These objectives correspond to the seven work packages of the project:
3. Progress
The most interesting and promising markets for protein are adhesive and surfactant applications.
Costing models based on four scenarios for oil extraction (I, II, HI and 'Traditional') and one scenario for protein extraction (scenario IV) have been established. The protein products resulted from the combination of the different oil extraction scenarios with the protein extraction scenario were compared to commercial protein products such as casein, fish meal, soy protein, etc. on the basis of their characteristics. From these data, an economic analysis of scenarios I and IV was made. Scenario 1 requires a total investment of 2.3 million euros for a processing of 2.4 ton/h and a profit of 25.3 euros/ton. Scenario IV needs an investment of 5 millions euros for a processing of 1 ton oil meal per hour an a profit of 176 euros/tons.
A new screw assembly for extrusion of seeds has been used. The feasability of industrial processing developed for the Express variety has been confirmed for the varieties Agat and Pollen although the blanching step needs more accurate temperature monitoring to explain the differences observed among varieties. Desolventisation with only hexane denatured the proteins to a lesser degree as compared with other desolventisation methods tested and consequently, the protein solubility is higher although the content of hexane in the meal is too high.
The use of rape meal as fumigant is limited because of the large amounts that are necessary to obtain a biocidal activity equivalent to that obtained with standard chemical biocides. In addition, the nutrients present in the meal favour in some cases the growth of the pathogen and offset the biocidal activity. A possible application of rape meal could be as renewable peat in horticulture, or as active material for moulded flower pots (technological feasibility demonstrated in this project)
The genetic analysis of OSR varieties has been supplemented by AFLP analysis. This technique showed results different to those obtained by microsatellite analyses and failed to group together varieties that are known to be related. Further work is necessary to improve the results of AFPL.
Purification of thionins, protease inhibitors and LTP from rape has largely progressed this last year. A thionin rich fraction and a fraction inhibiting trypsin are currently undergoing N-terminal amino acid sequencing at Leeds University. The analytical purification of LW has been scaled up during this period. The protein extract is now depigmented by nanofiltration and then purified by anion exchange chromatography plus cation exchange chromatography. Two isoforms of LTP of 9424.0 ± 1.0 and 9438.9 ± 1.0 Da have been identified by mass spectrometry.
Minor components of rape have an influence on the processing of rapeseed products. Thus, dietary fibre associated to proteins could hinder the enzymatic proteolysis, and phenolics, which are present both in lipid and protein fractions, give dark colours to the products when oxidized.
Numerous efforts have been made to solve the limitations of some chemical modifications. A new attempt of cationisation of proteins was performed by replacing methanol by pyridine to avoid the reaction of DCC with methanol but currently more data is necessary to prove the efficiency of the reaction. Another approach using EDC in water instead of DCC resulted in the cationisation of the protein on the basis of the IR spectrum. However, EDC is an expensive reagent.
The glucosinolate modification increases the hydrophobicity of proteins and therefore, reduces their solubility in water. Best yield of glycosylation is obtained by the chemical method but it uses a very toxic compound, NaBH3CN. The use of a chemo-enzymatic method avoid the use of this compound but when applied to proteins the degree of modification is not enough to change the functional properties. An assay of glycosylation of peptides is under way.
Using pilot scale methods it is possible to obtain protein concentrates from cold pressed oilseed rape with a protein content of 80 % and a solubility of more than 95 %, which could be sold at 3.3euros/Kg.
It has been demonstrated that rapeseed products are of interest in different applications. In the adhesive formulations based on rapeseed isolates, some initial weak points like the initial low tack, flexibility, viscosity or odour have been improved by adding other components and by decreasing the pH of the formulation. In certain cases, the rapeseed-based formulations were superior to a reference glue (Leinos 720).
Tests showed that preparation of plates by thermal insulation using rapeseed protein isolates as adhesives is feasible, the best results being those obtained from succinylated isolates. Rapeseed protein isolates showed good interaction with paper fibres and could be used to improve the mechanical properties of recycled paper. Surface properties of isolates and proteins with or without chemical modification were also very promising.
Emulsifying and foaming properties of some protein isolates and protein hydrolysates were in some cases comparable or even superior to commercial surface-active proteins. Emulsions generated with glycosylated 2 S protein from rapeseed showed a better rate of creaming as compared with the native protein. Foams generated with acylated hydrolysates were very stable, the C10 derivative generating 'more hydrated' foams as compared with the C14 or C18. The C10 acylated peptides and the 12 S protein are also able to adsorb to stainless steel and modify the surface characteristics of this material. 2S protein exhibited interesting properties as tensor for cosmetic applications.
1. Achievements
Activities
In the second year, aspects of the economic of the process (WPI) were evaluated for some niche markets for rapeseed proteins some costing models were developed for the various processes that have been developed. On the basis of the results obtained in the project, some of the technology was transferred to an industrial partner. The oil extraction processes were optimised to improve the quality of meal and extractability of proteins. New potential uses for experimental and industrial meals (WP2) were studied. Pilot extraction processing for oil and proteins were developed through innovative procedures (WP5). As far as more basic research is concerned, in addition to purification of some new proteins, DNA fingerprinting of varieties based on micro satellite analysis was carried out. Complementary information was obtained, relating to the size of various alleles of several rapeseed varieties that were tested (WP3). Chemical and enzymatic modification of the proteins was carried out at laboratory and pilot scales (WP4). Protein materials were prepared in amounts sufficient to start studies of end use (WP6, WP7).
Progress
As during the first year, the activities during the second year followed the time schedule of the technical annex. Despite the large number of tasks and partners, the co-ordination of the experiments in the various work packages was satisfactory and in general delays have been limited. Only a few deliverables have not been achieved. These delays mainly concerned the use of products in cosmetics, which needed rather large amounts of modified proteins. Some samples have been prepared at pilot scale for trials.
The results of the market evaluation confirmed the clear interest of industry in renewable biological materials, especially in the area of glues and adhesives. From the cost models it was concluded that the suggested protein extraction process could lead to a positive and profitable activity, generating protein enriched products at competitive market prices (compared to other industrial proteins) for use in the non-food sector.
As far as the oil extraction process was concerned, it was clear that the first step of oil removal using an extruder cooker appeared very promising when used prior to hexane extraction as the protein solubility of the meal was increased. In addition, trials with superheated hexane using a pilot solvent removal unit also gave satisfactory results, representative of what can be obtained with a pneumatic flash solvent removal process.
These results represent the most important outcome of the project since both processes could be developed as industrial processes for providing high quality meal for protein extraction. Production of 3 dimensional objects using rapeseed meal with a combination of extrusion cooking and moulding was shown to be feasible at pilot scale (20 kg). However, the resulting products showed medium or low mechanical properties as compared to gluten mixtures or legume seed concentrates tested previously by some of the research partners. It was found possible to produce plates for insulation tests but their insulating properties were found to be rather poor. Hence, it was decided not to proceed further with this objective. Depending on the results of investigations into the biocidal effects of meals, the preparation of "biologically active" flowerpots could be a more interesting objective.
Further investigations of the biocidal effects of the meals also gave positive results that suggested that non blanched rapeseed meal has potential as a biocide against the fungal pathogens Rhizoctonia solani and Gaeumannomyces graminis. The meal would be of particular benefit for control of Take-all in organic cereal production. The blanched and non-blanched meal also controlled the larval stage of Tipula oleracea, Deroceras reticulatum and Lumbricus terrestris. Tipula (leather-jackets) and Deroceras (slugs) cause major damage to cereal and brassica crops. The only drawback is that earthworms are also killed. Therefore this side effect may prevent the use of these meals as a biocide. Studies have to be transferred to a soil based system to determine if these negative effects occur in a field situation.
In further studies, the DNA fingerprinting of rapeseed varieties, based on micro-satellite analysis, has produced useful information regarding the sizes of alleles in the different varieties tested. A dendogram based on these results has been established for the different varieties studied. From biochemical analysis, the large variability in seed composition, observed for protein in the previous studies presented in the first annual report, was confirmed for the commercial varieties tested for the other major components (lipids: 39.3 to 48.4%, dietary fibres: 31.5 to 37.1%) and, also more surprisingly, for minor components such as glucosinolates (13 to 62 micromol per g dm). Moreover, unexpectedly, only three varieties among the sixteen studied had a total glucosinolate level below 20 micromol per g dm, although they are classified as double zero varieties.
During this second year, the major storage proteins were prepared in rather large amount for modification studies and original procedures were developed to prepared small metabolic and defence proteins, such as lipid transfer proteins and thionins.
Chemical and enzymatic hydrolysis of rapeseed proteins was successfully investigated as a means of producing rapeseed surfactants. A limited peptic hydrolysis of the filtrated isolate considerably improved foaming properties. Acylation of small peptides was also very efficient in generating surface active molecules. The reactions between glucosinolate products, derived from various amino acids, peptides, reference proteins and rapeseed protein concentrates led to highly hydrophobic grafted derivatives. The basic data, which has been obtained for the first time, was of great interest not only for modifying peptides and proteins but also for controlling the protein quality during oil and protein extraction processes.
As far as glycosylation is concerned, lactose could be used as a less expensive donor for transglycosylation and was used in the preparation of galactosylated samples for further studies.
Aspects of protein extraction, based on meals produced using various oil removal processes, were studied. It was found that the solubility of the rapeseed protein in the meal could be significantly increased so that satisfactory yields for protein extraction could be reached. The preparation for hexane extraction by partial oil removal through extrusion cooking seemed promising as the protein solubility of the resulting meal was acceptable for isolate manufacture. Using partially oil removal and similar techniques, without live steam, a protein of comparable quality could be produced with respect to oil content. These results are of major importance for further industrial development of rapeseed proteins.
The extraction procedure at the pilot plant scale on mildly extracted meal gave an overall protein recovery of 69%, including precipitated and filtered fractions. With respect to protein concentrates both an aqueous salt protein extraction and an aqueous alkaline protein extraction were investigated. Using either aqueous alkaline or salt protein extraction 70% and 53% respectively, of the oilseed proteins were recovered from the meal. From these results, it could be concluded that at least three different protein extraction processes giving satisfying yield could be developed using mild extracted meal. Chemical and enzymatic modifications of these isolates (acylation, glycosylation, and proteolysis) were successfully performed at pilot scale.
Among the various functional properties tested, the most interesting results were those relating to the adhesive properties of rapeseed protein isolated by filtration. This showed a good tack and flow behaviour. In general the gluing properties of the standard rapeseed protein suspensions were nearly the same as for the casein adhesive, or even better if some additives are added. This encouraged plans for application trials.
Film properties of rapeseed proteins for use in thermo-moulded materials was found to be in the same range for stress and strain values as other globular plant proteins (pea, soybean). As far as emulsifying and foaming properties are concerned, filtered isolates gave emulsions characterised by an excellent stability. Controlled hydrolysis, either enzymatic with pepsin or chemical with sodium hydroxide, appeared to be an efficient way to prepare foaming derivatives.
Conclusions
On the basis of the second year's results, it can be concluded that a complete procedure from seed to functional protein products has been developed. This includes original mild de-oiling processes and efficient protein extraction and modification techniques. The economic evaluation of these processes, using pilot scale data, appeared very encouraging as far as further industrial development is concerned. The functionality of the protein products, as non-modified or modified proteins, exhibit original behaviours among which adhesive, emulsifying and foaming properties seemed the most interesting for end-uses. The great genetic variability of the protein composition related to the different functional properties of the constitutive major proteins should be exploited for optimising both these processes and the product functionality.
Achievements
Future actions
WPI
WP2
WP3
WP4
WP5
WP6
WP7
Objectives
The main objectives of the project are:
Activities
The work is divided into seven work packages corresponding to the above objectives .In the first year, substantial efforts were directed towards:
Less work was done on the end-uses (WP6, WP7), the trials on protein materials being planned for the second and third years of the progreamme.
Progress
The economic analysis gave the surprising result that as far as industrial applications for rapemeal products is concerned, soymeal was not the principle competitor. Depending on the application, a very wide range of materials competed with potential rapemeal products. It became clear research should focus on the identification of "Entry Points" for rapemeal and its constituent parts, within targeted industrial sectors. Initial results indicated rapemeal bio-polymers have application in adhesive and surfactant markets. They appear unlikely to enter coating markets on grounds of cost and technical performance. They also appear to have potential use in bio-composites. Bio-cidal properties need to be more clearly identified before any analysis of market potential can progress.
A great part of the efforts during the first year were devoted to the influence of de-oiling conditions on the protein quality in the meal. Some technical adaptations were performed on the oil extraction pilot plan. From these experiments, it could be clearly shown that NSI can be greatly restored during flash desolventisation. Hence, deoiling technology seemed seems very interesting as a means to preserve protein quality in the meal. High NSI could also be obtained by cold-pressing.
Some preliminary results were obtained on the biocidal properties of rapeseed meal on Rhizoctonia and Fusarium, which will be confirmed by further studies
Concerning the protein composition of seeds, its variability was studied on thirty five varieties of rapeseed experimentally grown in controlled conditions. A great variability of the cruciferin/napin ratio was observed, lying in the range 0.6-2, with in most cases cruciferin as the major protein. The percentage of lipid transfer protein was also determined and ranged from 1.6 to 7.3 % of total protein. A complementary microsatellite approach has also been started.
The major storage proteins of the seeds were purified from Express variety by a preparative chromatography procedure. Dietary fibers (DF) were determined on a Danish grown double low spring rape variety (Maribo Seed) and various pilot protein products from BDF. The isolation of rapeseed protein results in a reduction of DF content compared to the content obtained in the intact rapeseeds, but some strong interactions between dietary fibers and proteins were demonstrated.
Analysis of phenolics was also performed in rapeseed showing the presence of major components like sinapine, sinapoylglucose, sinapic acid and sinapoylmalate. The emulsifying properties of the purified 2S and 12S fractions was studied, depending on concentrations and pH. In general, it was concluded that cruciferin emulsions were more stable. Moreover, the results obtained with the 2S/12S mixture were rather similar to those relative to with the pilot protein isolate.
Chemical modifications by acetic and succinic anhydrides was performed at 60ºC to enhance the functional properties of the rapeseed proteins. The succinylated sample were characterized by a lower solubility than the native protein but by more constant value depending on pH. Acetylated rape seed protein is comparable to native protein
The glucosinolates-protein interactions were modelled. Several compounds formed during reaction with either Cys, Trp, alpha-Glu-Trp, y-Glu-Trp, His, Ser or Asn have been characterised and structurally identified.
Enzymatic transglycosylation was performed using beta-galactosidase from A. oryzae on myoglobin as reference protein but also on purified rapeseed protein (napin), but the assays were totally unsuccessfull. A new strategy is under development. On the other hand, chemical glycosylation performed with rapeseed protein (napin) gave satisfying results.
Enzymatic hydrolysis of purified protein (napin and cruciferin) as well as protein isolate was performed in various conditions of pH, with or without thermal and reductive pre-treatment of the protein substrates. Using protein isolate, neutrase, alcalase and Amano A were the more efficient enzymes. The solubility of the hydrolysates measured at pH 3,5 and 8 was rather high. It could be be concluded that many basic informations have been collected to generate modified proteins and peptides for functional tests.
To study the extractability of the protein from the rapeseed meals, prepared by the various de-oiling processes a lab-scale process was developed using the same separation technology as in pilot-scale by means of a discontinuous centrifuge. A systematic study of the extraction parameters was performed, especially focused on the influence of retention time on composition , yields and solubility of the resulting protein fractions. In the resulting optimized conditions, an overall protein recovery yield (protein isolate I plus protein isolate II) of 59% was reached on mild deoiled meal. .
The precipitated protein fraction (isolate I) was less soluble (25.6%) compared to the filtrered one (isolate II) (93%). Using the aqueous BDF process, protein concentrates were also prepared. The process led to a rather high protein recovery yield (72.6% ) and a protein content of the concentrates in the range 48-52%.
Concerning end-uses potential, although the studies were limited during the first year, it has already been demonstrated that hydrolysed rapeseed proteins are well soluble in 20% and 40% ethanol water mixtures, which makes their use in cosmetic products possible. Rapeseed isolate could be also compression moulded into test bars with low strength but high strain. Unfortunately, It was not yet possible to process native rapeseed isolate into paper coatings.
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