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FAIR-CT96-1625
Valorisation of rapeseed/sunflower lecithins, a by-product of seed oil, in cosmetics and fermentation industries: use of natural carriers of phospholipids overproduced by filamentous fungi growing on lecithin substrates |
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Proposal No: | FAIR-CT96-1625 |
| Date Prepared: | July 2001, September 1999 | |
| Source: |
Final Report Progress Report December 1997 |
Source: Final Report 2000
Consortium: The project was co-ordinated by the Laboratoire de Biochemie et Technologie des Proteines, INRA Nantes (France), Gist-brocades BV, Delft (The Netherlands), Lucus-Meyer, Hamburg (Germany), Institue of Biomedicine, University of Helsinki, Siltavurenpenger (Finland), Unite de Chimie des Interfaces, Universite Catholique de Louvain, Louvain la Neuve (Belgium).
Introduction
The non-food valorisation of rapeseed and sunflower lecithins, a by-product of the vegetable oil industry, was carried out using an integrated approach by a complementary partnership including: a lecithin producer (Lucas-Meyer, P2) for preparation of lecithin mixtures from rapeseed and sunflower as fermentation substrate as well as liposome technology a fermentation company (Gist-Brocades, P3) using lecithins as the sole carbon source in fermentation medium for the overproduction by the fungus Aspergillus oryzae of highly active carriers of phospholipids (PhosphoLipid Transfer Proteins, PLTP) and a cosmetic company (Sanofi Beauté, P4) combining this highly active fungal PLTP with new liposome formulations to develop , "natural" and "efficient" cosmetic applications.
This project was completed in close association with a number of research or academic instittues (INRA PI, IFR P5, Institute of Biomedicine P6, Universite de Louvain APl) to study production processes mechanisms of PLTP in fungal cells and structure-function studies of PLTP in relation with membrane biogenesis.
This project aimed to answers a number of important scientific questions:
As far as technical applications are concerned this project aimed to will provide some definitive facts concerning: the efficiency of use of rapeseed/sunflower crude and lecithin fractions in industrial fermentation processes and for PLTP (and other secreted enzymes) in respect of Soya lecithins. the efficiency of the new liposome-PLTP technology in cosmetic, and as a drug delivery system.
It should be noted that in this project the industrial companies have a key role, and without the work of these industrial partners, the academic research would not be possible.
Activities
This project is divided in four main tasks: Task 1: Preparation of rapeseed and sunflower lecithins and phospholipids Task 2: Overproduction of fungal PLTP Task 3: Structure-function studies on fungal PLTP Task 4: Cosmetic applications
Task 1 has to provide the phospholipid substrates for fermentation studies (Task 2) and cosmetic applications (Task 4). The soya lecithin fractions routinely produced by partner P2 were used to perform fermentation trials (7L fermentation) necessary to optimise PLTP production. Soya lecithin was also used for large scale fermentation studies (300 L). Sunflower and rapeseed lecithins were prepared by P2 during the second year and were used for fermentation trials at laboratory scale. Phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine enriched fraction were prepared for fermentation trials and structure-function studies by partner PI and P2. Throughout the project, partner P6 has prepared the fluorescent phospholipids needed for performing some studies in Task 2 and in Task 3.
Task 2 is a key task of the project since it determines the optimal conditions for PLTP production needed for cosmetic applications and academic research. It was first shown that among the industrial strains from partner P3 tested, the reference strain provided by partner PI (Aspergillus oryzae LMTC 2.14) was the more efficient. Since numerous data have been previously obtained by partner PI on this latter strain, it was decided to use this strain throughout the project. A source of phospholipid for PLTP overproduction was defined. The different phospholipid sources prepared by partner P2 were assayed as fermentation substrates. It was shown that the use of these lecithins play a major role in the stimulation of phospholipid transfer activity in A. oryzae as well as on fungal growth. This increase varied with the phospholipid composition of the lecithin fractions used.
The most effective lecithin source was Epikuron 110 from sunflower which exhibited the highest PI content. Experiments for growth scale-up from flasks to 7L bioreactors have been performed by partner P3 in collaboration with partner PI in order to determine the optimal fermentation conditions: preculture, lecithin concentration, anti-foaming agents, fermentation time, etc.
Fermentation experiments were performed, at the 300 L scale, in order to provide sufficient amounts of mycelium for the purification of the PLTP needed for the other tasks. Unfortunately while the biomass production was high, a significnat decrease was noted in the PLTP activity. Hence, further fermentation studies at a 7L-bioreactor scale were performed in order to determine precisely various limiting factors such as oxygen availability, mixing speed, pH, phospholipid content on fungal growth and PLTP production.
The results obtained enabled a second 300L fermentation to be carried out, which was successful. New conditions were defined, leading to high cell density cultures, with increased PLTP activity. A pilot scale bioreactor was successfully validated under these new conditions.
A large scale filtration harvest method was developed for the separation of mycelia from the mycelial fermentation broth. The selected technology was a membrane press filter. Mycelia disruption was achieved by mechanical breakage of the cell wall and membranes using a high pressure homogeniser. Crude extract was obtained after removal of the cell debris using two serial filter presses with the addition of filter aid.
Purification of the protein was developed at lab scale by partner PI. A purification procedure that could be easily scaled up was developed, based on treatment of the PLTP extract by ion exchange chromatography (IEC). This purification was performed using extracts from fungal cells obtained by pilot scale fermentation (partner P3).
A problem encountered during the purification process related to the high lipid content (about 40%) of the fungal cells produced at the pilot scale (300L). Hecne, the fungal cells were first defatted using n-pentane. This defatting procedure improved PLTP yield (x4) but did not produce the highly purifed protein required for structure-function studies.
Various possible methods were investigated to improve purity. These included reversed phase HPLC, FPLC and size exclusion chromatography. However PLTP yields remained quite low (<5%).
In addition to the high lipid content of the fungal cells, another major problem was encountered due to the accumulation of phenolic compounds from the lecithin fractions into the fungal cells. These polyphenols probably interacted and denatured the fungal PLTP, as has been observed for many other proteins. Treatments of protein extracts with polyvinylpyrrolidone (PVP) were tried, but not found very effective as a means of removing the phenolic compounds, while this treatment also increased the loss of PLTP. Therefore the whole extraction-purification procedure was changed to one based on phase partitioning in PEG (polyethyleneglycol)-salt systems.
Various PEG grades were tested (PEG2000, PEG4000, PEG6000 and PEG8000). It was found that a procedure combining extraction in potassium salt-PEG2000 resulted in the elimination of phenolic compounds (that accumulated in the PEG upper phase) while PLTP was recovered in the lower aqueous salt phase. When this procedure was was applied to non defatted cells, both phenolic compounds and lipids were recovered in the PEG upper phase. Such a phase partition procedure appeared of interest and possible value, since it is frequently used for the purification of industrial proteins, although on an industrial scale PEG is expensive but it is possible to recycle it.
Unfortunately, this treatment resulted in an instability of PLTP activity. New physicochemical conditions for PEG partitioning were explored. Finally, using a hexane treatment of aqueous extract, followed by an ion exchange chromatography, the problem of PLTP loss due to lipids was partially solved leading to a pre-purified PLTP with a reasonable yield.
From a laboratory batch of pure PLTP, polyclonal antibodies were prepared and the localisation of A. oryzae PLTP was achieved using immuno-cytochemistry (electron microscopy) and subcellular fractionation by isopicnic ultracentrifugation. It was shown that the protein is mainly associated with the Golgi apparatus.
A cDNA encoding the 19kDa PLTP was isolated and the protein sequence was deduced from the sequence of this cDNA. In addition, the cDNA corresponding to the mature protein was cloned in E. coli by partner PI in order to provide significant amount of protein for structure-function studies. The protein was recovered in inclusion bodies that were slightly solubilised, even in strong denaturing media containing concentrated urea and reducing agent.
This suggests that this fungal protein should display particular structural and folding properties, probably related to its high proline content and high thermal stability while it has few disulfide bonds in comparison to plant PLTPS.
A problem encountered in heterologous expression could also be related to the low yield obtained when the protein is purified from fungal biomass. For this reason a new eukaryotic expression is being developed to produce fungal protein in A. oryzae.
Northern blot analysis has been used to investigate the 19kDa PLTP gene expression product in comparison with the transfer activities of PLTP extracts. An inhibition of transcription and PLTP activities was observed when the lecithin content was increased in the culture medium, indicating the importance of an optimal concentration.
Effect of lecithins on sporulation and pellet morphology was studied. While lecithins improve dispersion of dormant spores, they resulted in the formation of large aggregates a few hours after inoculation. Although the pellet size is larger in glucose medium than in lecithin medium after one day of fermentation, this difference was not observed in the following days of fermentation.
Task 3 is dependent of the production of PLTP in task 2. Most of the structure-function studies planned in the project require significant quantities of PLTP. Since highly purified fungal PLTP could not be provided to the academic partners, preliminary studies concerning the physicochemical basis of lipid transfer were performed using other PLTP sources.
On the basis of the scientific expertise of partner PI, PLTP isolated from plant sources were chosen. These proteins are less specific than the fungal PLTP and their specific activity is lower than the fungal protein. Nevertheless, structure prediction and sequence alignment revealed some interesting structural homologies between fungal PLTP and related plant proteins. These differences in the lipid transfer properties mean that a comparison study can provide some interesting insight to an understand of the physicochemical basis of lipid transfer.
While studying the lipid specificity of this plant proteins in the transfer process, partner P6 showed that fungal and plant PLTPs displayed similar behaviour. This led to the suggestion that, in contrast to previous conclusions, fungal and plant PLTP are both non-specific lipid transfer proteins. This new and unexpected conclusion also strengthens previous results, suggested from sequence alignments and secondary structure predictions, that plant and fungal PLTP are structurally related.
This task aimed to determine the structural and physico-chemical basis of lipid transfer by the corresponding proteins in order to improve their uses in cosmetic applications. interestingly, partner P6 showed also that some cyclodextrins are capable of enhancing lipid transfer with a specificity quite similar to what it was observed for plant and fungal PLTPS. Since this carbohydrate do not interact with lipid bilayers, it is suggested that cyclodextrins and therefore PLTPs bind and transfer monomeric lipids leading to a shift in the liposome-monomer equilibrium. The absence of interaction between PLTP and membrane interface was also highlighted by partner P5. Therefore it seems that the specificity of PLTPs towards different lipid molecules is related to difference in the liposome to lipid monomer equilibrium. Finally, the high specific transfer activity observed for fungal PLTPs is probably related to the ability of protein to adsorb at membrane interface and to shift the equilibrium towards the formation of lipid monomers.
A topological study performed by partner PI from the structural data available in the data bank have shown that, if the volume of the lipid binding cavity differs between different plant proteins, its high plasticity enables it to increase in volume yet keep similar binding properties. Interestingly this high plasticity is related to the ability to bind other hydrophobic molecules. Partner P5 confirmed that the lipid transfer by PLTPs is due to a real transfer and not to a fusion mechanism. It was also shown that PLTPs are capable of enhancing the transfer of lecithins from the bulk aqueous phase to the air-water and oil-water interfaces. This property could have interesting applications for improving the foaming and emulsifying properties of lecithins in some cosmetic products.
Task 4 concern the cosmetic applications of fungal PLTP in association with liposome formulation. As discussed above, since the highly active fungal PLTP or enriched extracts could not be prepared, this task focused on the development of methodology to follow the transfer of phospholipids into living cells. Partner P6 developed a new, efficient procedure to follow the transfer of phospholipids from liposomes to cells by fluorescence imaging. This new fluorescence imaging method prevents photobleaching of the pyrenyl fluorescent lipid probes by an enzymatic deoxygenation of the medium and eliminate background fluorescences by a simple substraction procedure. It was successfully applied to the transfer of phospholipids in different animal cells.
This efficient new technique was also used by partner PI to study the transfer of lecithins from growth media to fungal cells (Task 2), it was also sued to follow the transfer of phospholipids by cyclodextrins in various cellular models. It was found that cyclodextrins were also capable of enhancing intermembrane transfer of phospholipids with a specificity close to that displayed by plant and fungal proteins. This study demonstrated that cyclodextrin can greatly enhance the transfer of phospholipids from liposomes to cells. However, only monomeric phospholipids were transferred and due to the low shift of the liposome to monomer equilibrium, a large amount of cyclodextrin is needed. To reduce this, strategies can be developed to improve the surface hydrophobicity of cyclodextrin in combination with the association of PLTP and cyclodextrins.
Summary
Introduction This project that concerns the non food valorisation of rapeseed and sunflower lecithins, a by-product of oil industry, is being carried out adopting an integrated approach between:
These activities are being carried out in closed association with academic organisations that are studying production processes mechanisms of PLTP in fungal cells and structure-function studies of PLTP in relation with membrane biogenesis.
Objectives
This project will provide answers to some important scientific questions.
For technological applications this project will provide some definitive response to questions concerning the following points:
Activities In the first year the work has focused mainly on the production of lecithin substrates, on the fermentation studies, on the production and purification of fungal PLTPS. The localisation of the fungal PLTP have been investigated. The isolation and sequencing of a cDNA coding for the fungal PLTP have been performed.
Results These are presented in relation to the four main tasks of the project:
Task 1 is a key task since its objective is to provide the phospholipid substrates for fermentation studies (Task 2) and cosmetic applications (Task 4). Since harvest of the required European seeds was not available at the beginning of the project, the soya lecithin fractions routinely produced by Lucas-Meyer were used to perform the first fermentation on PLTP production. It was also decided to use soya lecithin for large scale fermentation studies (300 l to 3m3) since the required amount of sunflower and rapeseed lecithin could not be provided at the required time on an industrial scale. The European lecithin fractions will be prepared, in the first part of the project, only for laboratory fermentations and cosmetic applications. These slight deviations from the initial programme was adopted in order to limit delays. Pure phospholipids will be prepared from these fractions for some academic studies.
By the end of the first year the various rapeseed and sunflower lecithin fractions had been prepared by Lucas-Meyer and were available for fermentation and cosmetic applications. Fluorescent phospholipids required for some studies were prepared.
Task 2 concerns the production of PLTP needed for cosmetic applications and academic research. A collection of Aspergillus oryzae strains used in industrial fermentations was tested and compared with the strain selected previously for use in the laboratory studies. From this preliminary study it appears that none of the industrial strains from partner were more efficient than the reference strain. Since a large amount of background data had been previously obtained concerning this strain, it was decided to use this in the future studies. These included the definition of the optimal fermentation conditions (pre-culture, concentration of lecithins, anti-foaming agents, time of fermentation) and enabled scale-up from flasks to 10l and then 300l, using an ethanol insoluble fraction from soya lecithin.
The scaling-up indicated that the selected strain is efficient and can be used under industrial conditions. From the 10 l fermentation studies it was concluded that 5 days and 50g/l of lecithins were required to give high biomass yields without change in PLTP activity. A 300l fermentation experiment was performed in order to purify the PLTP needed for the other tasks. Unfortunately while the biomass production was high, a significant decreased in PLTP activity was observed. Under the conditions of scale-up, the lipid content of fungal cells after 5 days was about 40% of dry weight. These lipids are probably intracellular, since they are mainly composed of non polar triglycerides, storage lipids, and are un-extractable by washing the cells on filters.
The production of fatty fungal cells in large scale fermentation could be a problem for the purification of PLTP since these proteins are known to adsorb at oil-water interfaces. Various defatting procedures were tested prior to PLTP extraction but did no lead to a significant increase in yield of PLTP. These results suggested that the intracellular metabolism of lipids was affected in the high density fermentation used at a pilot scale. Therefore further experiments will be performed to optimise PLTP production. If this is successful it will be scaled-up further to 3m3.
During this first year partner the purification procedure was investigated in detail in order to find a new protocols that can be easily scaled up. In the new procedure, the PLTP extract was first purified by ion exchange chromatography (IEC) and then by size exclusion chromatography (SE), using extracts from fungi grown in laboratory fermentation. PLTP enriched fractions from this procedure will be used for cosmetic applications. For some academic studies, it is necessary to work with highly purified proteins. A reversed phase HPLC procedure, that has been shown to be efficient in the final purification of plant PLTPS, is being investigated.
In order to avoid long delays that could arise following the restructuring of the Sanofi group, the responsibility for purification of the first batch of PLTP produced by the 300 L fermentation, has been re-assigned.
The localisation of PLTP in A. oryzae was investigated using immuno-cytochemistry (electron microscopy) and subcellular fractionation by isopicnic ultracentrifugation. The results obtained indicated that the fungal PLTP is localised within the cytoplasm of cells and partly associated with the Golgi apparatus. To obtained more information on the intracellular routing of the protein, a cDNA corresponding to the PLTP was isolated. A 700 bp cDNA was obtained by using a new technique, the RACE-PCR (rapid amplification of cDNA ends polymerase chain reaction). The protein sequence was deduced from the sequence of this cDNA. In comparison with the purified protein the deduced sequence revealed an N-terminal extra-peptide sequence in the mature protein. Therefore, it appears that PLTP is synthesised as a pre-proprotein suggesting a specific intracellular routing. This cDNA will be helpful in the production of homologous integrative or autonomous vector for the over-expression of the protein in A. oryzae. An investigation of the efficiency of sunflower and rapeseed lecithins as a carbon source for fungal growth and production of PLTP was initiated at the beginning of 1998.
Task 3 is dependent of the production of PLTP in task 2. Most of the structure- function studies planned in the project require large quantities of PLTP. A 300 l fermentation is sufficient to provide the protein material necessary for such studies. Unfortunately, as discussed above, the first fermentation was unsuccessful. So, preliminary academic studies concerning the physicochemical basis of lipid transfer were performed on other PLTP sources (isolated from wheat and maize kernels). These proteins are less specific and their specific activity is lower than the fungal protein. In respect of these differences in the lipid transfer properties, a comparative study could be of interest in establishing the physicochemical basis of lipid transfer. Furthermore the use of these plant proteins should save substantial quantities of the fungal proteins required to develop the new methodologies used in the project.
Task 4 concerning the cosmetic application were initiated 1998.
Discussion During the first year, in spite of some adjustment to the time table, significant progress was achieved, with the production of rapeseed and sunflower lecithins, the selection of an efficient A. oryzae strain, the isolation and sequencing of a cDNA coding for the fungal PLTP, and preliminary studies on the intracellular localisation of this protein. Some difficulties have been, encountered in the scaling up of the fermentation process which has delayed the purification of PLTP by around six months. However significant data are now available concerning fermentation process to allow initiation of determination of the best conditions for future research tasks.
To avoid delays it was decided to introduce plant PLTP into this project. These proteins have been routinely purified and are available in non limiting mass quantities for academic research activities. First, these proteins will allow the development of methodology by the academic partners without the need to use the more limited amount of fungal material. Secondly, these proteins are a good reference material for use in the project. A lot of information is available on these proteins and preliminary results suggested structural analogies between plant and fungal PLTP.
The restructuring of the Sanofi group has led to important geographic and personnel changes during the first year of this project. Both the cosmetic and chemistry branches of Sanofi involved in the cosmetic applications and large scale purification of PLTP, respectively, were affected by these changes.
Future activities For the beginning of 1998, the priority will be given to the purification of fungal PLTP.
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