![[BioMatNet Database - FAIR Program]](../images/Fair.gif) |
FAIR-CT97-3559
Production of bioflavours by fungal spores |
 |
Proposal No: | FAIR-CT97-3559 |
| Date Prepared: | July 2003, January 2001, November 1999 | |
| Source: |
Final Report Abstract and Executive Summary Second Annual Progress Report First Annual Progress Report |
Objectives
The final goal of the project was to develop a system in which precursors can be converted to bioflavours by fungi. The process had to be scaled up and the applicability of downstream processing had to be investigated.Activities
Within the first year of the project, useful strains were selected and a list of interesting flavour components had become available. Also the carrier material used in the solid state fermentation and the composition of the liquid medium were established. In addition, the enzymes involved in the transformation of geraniol to 6-methyl-5-hepten-2-one (MHO) were elucidated from a strain of Penicillium digitatum. In the second part of the project, the process was further optimised. A method was established to measure the viability of the spores. In addition the metabolic pathway for converting geraniol to methylheptenone by P. digitatum and the toxic effect of these compounds was established.In other work the starting compound was sciareol (2-ethylenyl decahydro-2-hydroxy-alpha-2,5,5,8a-pentamenthyl-2-naphthalenepropanol) and the target was ambroxan (dodecahydro-3a,6,6,9a-tetramethylnaphthol[2,1-b]furan).The biotransformation of selareol to ambroxan was attempt using three microorganisms: Hyphozyma roseonigra, Saccharomyces cerevisiae and Aspergillus niger. Solubility of sclareol and product isolation were the two major physical problems to resolve. Yield optimisation and biotransformation conditions were the main objectives of the work.
Results
In the first period of this research project a fast and simple screening procedure was developed based on some model biotransformations, e.g. the bioconversion of linalool to linalool oxides by Aspergillus niger and the bioconversion of geraniol, nerol and citral to 6-methyl-5-hepten-2-One by P. digitatum. This method consisted of Solid Phase Microextraction (SPMIE) of the sporulated surface cultures of the fungi in small 40-ml SPME vials. The technique is based on the partition of the compounds to be adsorbed between the aqueous phase of the sample or the solid agar medium and the fused silica fiber of the SPME device. Precursors were sprayed upon the sporulated surface cultures as solution in EtOH and the bioconversions were monitored by headspace SPME extraction at different time intervals during 2 to 30 min.Method optimisation was carried out using standard solutions containing linalool and linalool oxides for optimisation of the linalool bioconversion reaction on the one hand and a solution of alpha-pinene and its bioconversion products for the optimisation of the alpha-pinene bioconversion reaction on the other hand. It was found that the best SPME-fiber for both headspace and liquid extraction of both the linalool and alpha-pinene standard solution was divinylbenzene/carboxen on PDMS (50/30 microm).
The best headspace adsorption time for extraction of the linalool solution was 30 mm, whereas the adsorption of the hydrocarbon terpenes (alpha-pinene and its conversion products) showed a maximum after 15 min.
With this method fungi were screened for their bioconversion of citronellol. Whereas submerged liquid cultures of A. niger were not able to convert citronellol, sporulated surface cultures of this fungus converted the substrate to cis- and trans-rose oxide with small yields (up to 1%). Other hioconversion products observed were nerol oxide, linalool and alpha-terpineol (yields < 1%). The same conversions were noticed with A. tubingensis and P. roqueforti. This bioconversion was enantioselective since more cis- than trans-rose oxide was obtained.
Submerged liquid cultures of P. roqueforti yielded two unidentified metabolites after conversion of citronellol (yield up to 5%). The bioconversion of geraniol and citral by sporulated surface cultures and spore suspensions of P.digitatum was monitored by SPME. It was found that maximal conversion took place after 6-8 hours.
Using SPME 60 fungal cultures were screened for their capacity to bioconvert alpha-pinene. Both A. niger and P. chrysogenum were able to convert pinene to a number of metabolites, including camphene, terpinene, cymene, limonene, terpinene, terpinolene, fenchone. endo-fenchol, camphor, borneol, terpineol, verbenone. feuchyl acetate and some minor unidentified compounds. When sporulated surface cultures were used, the dominating bioconversion products obtained were cis-verbenol, cis-pinocamphone, myrtenol, isobomeol and verbenone. The main bioconversion product obtained from P. digitatum was cis-verbenone (yield 3-4%).
Screening of fungi for their bioconversion of limonene using SPME yielded a very wide range of metabolites, of which alpha-terpineol was the dominant conversion product. This bioconversion however was much more successful when liquid cultures were used then when sporulated surface cultures were applied. Whereas Corynespora cassiicola yielded limonene-1,2-diol as the main conversion product (yield up to 60%). Alpha-terpineol was the dominant metabolite obtained from P. digitatum (yield up to 46% after 8 h). Finally the bioconversion of citronellol was studied using SPME as the screening technique. From the headspace of A. niger, a wide range of conversion products was recovered of which citronellol was the main metabolite, Other conversion product were iso-pulegol. Iso-isopulegol, menthone and iso-menthone.
During the second period of this research project two further bioconversion processes were optimised. Namely the bioconversion of (S)-(+)-linalool to cis-furanoid arid cis-pyranoid linalool oxides by A. niger and the bioconversion of (R)-(+)-limonene to (R)-(+)-alpha-terpineol by P. digitatum. In both bioconversion reactions, liquid cultures were used.
First, the biotransformation of (S)-(+)-linalool by different A. niger strains was studied. One strain(DSM 821) was able to convert the substrate to cis- and trans-furanoid linalool oxide (yield 30% and 5%, respectively), and cis- and trans-pyranoid linalool oxide (yield 11% and 1.5%, respectively).
The main metabolites, cis-(2S,5R)-furanoid and cis-(3S,6S)-pyranoid linalool oxide, have a sweet, floral, creamy odour and are used in perfumery. The culture conditions involved, such as the composition of the broth and the type and concentration of co-solvent applied and possible adaptation to the substrate during inoculation were investigated. It was found that (S)-(+)-linalool was converted much better than (R)-(-)-linalool, and that no significant chemical conversion of the substrate occurred in controls at pH 3.5.
Three co-solvents for improving the solubility of linalool in the culture broths were compared, namely MeOH, EtOH and acetone. The highest bioconversion yields were obtained when the substrate was applied as a diluted solution in acetone. Secondly, the biotransformation of (R)-(+)- and (S)-(-)-limonene by P. digitatum was further investigated. One strain of P. digitatum was able to convert (R)-(+)-]imonene to pure (R)-(+)-a-terpineol in 8 hours with a yield of up to 93%. The culture conditions involved such as the composition of the broth, the type and concentration of co-solvent applied and the sequential addition of substrate were investigated. It was found that (R)-(+)limonene was converted much better in alpha-terpineol than (S)-(-)-limonene, and that no significant chemical conversion of the substrate occurred in control flasks at pH 3.5.
The highest bioconversion yields were obtained when the substrate was applied as a diluted solution in EtOH. Since the substrate (R)-(+)-limonene was strongly adsorbed to the glass surface of the culture flasks, the influence of silylation of the glass vessels was investigated. However, the bioconversion yields in the silane treated flasks were significantly lower than in the non-treated flasks.
Finally the biosynthesis of fungal volatiles by various sporulated surface cultures was monitored by solid phase microextraction (SPME). The de novo production of the fungal metabolite (÷)-aristolochene by sporulated surface cultures of P. roqueforti was observed for the first time with the fungus grown on solid media. The influence of culture conditions was investigated. When comparing malt extract agar with sabouraud dextrose agar, the highest yield of the fungal metabolite (0.04 mg/mL of culture) was obtained with the latter medium. The biosynthesis of (+)-aristolochene showed a maximum during the fourth day after inoculation. Headspace analysis of volatile fungal metabolites by SPME could be used for monitoring and fast detection of mycotoxin producing fungi.
Objectives
The main aim of the project was to develop a system in which precursors can be converted to bioflavours by fungi. The process had to be scaled up and the applicability of downstream processing had to be investigated. Within the first year of the project, useful strains were selected and a list of interesting flavour components established. Also the carrier material in the solid state fermentor and the medium composition had to be established In addition, the enzymes involved in the transformation of geraniol were to be elucidated for Penicillium. In the second year the process had to be further optimised at labscale. A method had to be established to measure the viability of the spores, while the metabolic pathway for converting geraniol to methylheptenone and the toxic effect of these compounds had to be established.
Activities
First year. in order to optimise growth, sporulation and conversion by the strains used, temperature and the media composition were studied in flasks and petri dishes. A requirement list was made for the carrier materials that can be used for solid state fermentation. Absorption tests and growth/sporulation tests were done on several carrier materials to find the optimal conditions. Furthermore screening, has been performed to find new strains that can produce interesting compounds.
Second year., research focussed on optimisation of spore production for those that convert geraniol to methylheptenone. A higher spore concentration appeared to be economically important. For this reason, a lot of attention was paid to identify the best fermentor, growth circumstances, carrier material and a good method to measure the viability of the spores. Considerable work was carried out to find interesting flavour products made by several strains, for the model product used in the first place was found to not be of economic interest. The enzyme system of Penicillium digitatum,converting geraniol to mnethylheptenone, has been identified. The promising enzymes within the enzyme system were investigated in more detail. Additionally research was carried out to find the optimal conditions for the fungus Hyphozymia to convert sclareol to diol and ambroxan.
Third year. work has been devoted to studies concerning technical and economically feasible process optimisations and scale up on spore recovery from solid supports, spore storage, dry spore suspensions as well as on mycelia and culture conditions. Technical aspects addressed included binding of target compounds to carrier materials, separation and toxicological properties. Fundamental studies were devoted to the purification of interesting enzymes from the bioflavour biosynthetic pathways and finding possible links between primary and secondary metabolism. Finally, in order to further improve the economical aspects a survey was conducted on the biosynthesis of sesquiterpenes, the bioconversion of citronellal to menthol/menthone and tests along with other bioconvcrsion products of potential industrial interest
A rough economic evaluation of the production of methylheptenone indicated that the product is not valuable. Therefore, the decision was made to develop the production system for the transformations of geraniol to methylheptenone as well as the biotransformation of sciareol to diol and ambroxan. This product is of interest to the fragrance industry. In addition the search for other interesting flavour compounds was continued.
During the first year of the project considerable progress was made in the development of the solid-state biotransformation process and the elucidation of the enzyme system responsible for the conversion of geraniol to MHO.The toxicity of the different products and intermediates involved in the biotransformation process were also investigated, with focus on work with Aspergillus and Penicillium. Some of the work used an agar based mineral salts medium with asparagine as nitrogen source. It was established that amino salts also catalyse the transformation of citral into MHO chemically, while it was found that acid condition and heat treatment had influence on the stability of the various precursors used.
The method to determine the viability of spores is based on measuring the activity of an enzyme, which is involved in the respiratory system. The enzyme activity can be measured by reduction of a tetrazolium salt, which gives a yellowish solution. The reliability of the method has been tested at different pH, temperature and enzyme concentration and a protocol has been developed and its reliability tested. Several strains of Aspergillus such as A. niger, A. ochraceus, A. japonicus etc, strains of Penicillium such as P. digitatum, P. lividum, P. chrysogenum etc. and various strains of fungi from the following genera: Mortiella, Rhizopus, Beauveria, Corynespora, Cuninghamella and Botryodiplodia were tested for their ability to convert linalool, citronellol, geraniol, citral and citronellal. The screening was carried out using SPME in fluid cultures and sporulated surfaces. Sporulated surface cultures gave, in general, better conversion results than submerged liquid cultures. Using A. niger the products will were more enantio selective. In the first year the key compoundswere identified and selected using a membrane selection process based on a gas phase process. During the second year the possibility of using a liquid phase process has been investigated. For both possibilities a model of the downstream processing has been made.
The fungus Hyphozyma roseonigra was tested for its ability to convert 2-ethylenyl decahydro-2-hydroxy-alpha-2.5,5,8a-pentamenthyl-2-naphthalenepropanol (sclareol) to decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphtaleneethanol(diol). The conditions for growth to optimize the conversion have been investigated. The problem of sclareol solubility was resolved by means of several approaches: by adding different emulsifiers and by chemical modification of sclareol (addition of hydrophilic groups). The conditions of microorganism culture were optimised studying the effect of pH, temperature and salt composition on growth and sporulation. Then, several parameters of the biotransformation process were optimised (time, agitation, oxygenation, and salt concentration). as well as the product extraction using different solvents. Among the substances used for the biotransformation (selareol, sclareol epoxyde and triol) sclareol and trio1 are the only substances that were transformed by Hyphozyma roseonigra.
Trials with Aspergillus niger and Saccharomyces cerevisiae showed that these strains did not transform sclareol, sclareol epoxyde or triol in the conditions under which the experiments were conducted. After optimisation a first experiment of pilot plant biotransformation was carried out with Hyphozyma roseonigra. The osmophoric properties of the product obtained were evaluated by panel test.
Finally, a feasibility study was carried for:
Achievements year 1
Achievements year 2
Achievements year 3
Objectives
The general aim of the project is to develop a system in which precursors can be converted to bioflavours by fungi. The process has to be scaled up and the applicability of downstream processing has to be researched. Within the first year of the project, useful strains should be selected and a list of interesting flavour components should be prepared. In addition the carrier material in the solid state fermenter and the composition of the culture media established. In addition, the enzymes involved in the transformation of geraniol to 6-methyl-hepten-2-one (MHO), will be elucidated using a strain of Penicillium digitatum. In the second year, the process should be further optimised at labscale. A method should be established to measure the viability of the spores. Furthermore, the metabolic pathway for converting geraniol to MHO by P. digitatum and the toxic effect of these compounds should be established.
Activities
In the first year, in order to optimise growth, sporulation and conversion by strains of Aspergillus niger and Penicillium digitatum, temperature and the media composition were studied in flasks and petri dishes. A requirement list was made for the carrier materials that can be used for solid state fermentation. Absorption tests and growth/sporulation tests were done on several carrier materials to find the optimal conditions. Further screening was performed to find new strains which can produce interesting compounds. In the second year, research has focussed on optimisation of spore production for the conversion of geraniol to methylheptenone. A higher spore concentration appeared to be economically important. For this reason, a lot of attention was paid to obtaining the best fermenter, growth conditions, carrier material and a good method to measure the viability of the spores. Considerable work was carried out to find interesting flavour products made by several strains, for the model product used now is not of economic interest.
The enzyme systems of Penicillium digitatum, which is used in converting geraniol to methylheptenone, has been clarified. The promising enzymes within the enzyme system will be investigated in more detail. Additionally research will be carried out to find the optimal conditions for the fungus Hyphozyma roseonigra to convert sclareol to diol and ambroxan.
Progress
From rough economic evaluations for the production of methylheptenone it appeared that the product is not valuable. So the decision was made to develop the production system for the transformation of geraniol to methylheptenone by Penicillium digitatum or Aspergillus niger for use as a model system. At the same time the search for interesting flavour compounds would be continued. together with partner 3. Other work carried out concerned the bio-transformation of sclareol to diol and ambroxan. This product is of interest to the fragrance industry.
During the first year of the project considerable progress has been made in the development of the solid-state biotransformation process and the elucidation of the enzyme system responsible for the conversion of geraniol to MHO. Research continued in more detail during the second year, during which the toxicity of the different products and intermediates involved in the process was also researched. Work focussed on the A. niger and P. digitatum, with some work carried out using an agar based mineral salts medium with asparagine as nitrogen source. It was established that amino salts also catalyse the transformation of citral into MHO chemically. Some research was carried out on this aspect and it was found that acid conditions and heat treatment influenced the stability of several precursors used.
A method to determine the viability of spores was based on measuring the activity of an enzyme which is involved in the respiratory system. The enzyme activity can be measured by reduction of a tetrazolium salt, which gives a yellowish solution. The reliability of the method has been tested at different pH, temperature and enzyme concentration and a protocol has been developed.
Several strains of Aspergillus such as A. niger, A. ochraceus, A. japonicus etc, strains of Penicillium, for instant P. digitatum, P. lividum, P. chrysogenum etc. and strains of fungi Mortiella, Rhizopus, Beauvelia, Corynespora, Cuninghamella and Botryodiplodia were tested on their ability to convert linalool, citronellol, geraniol, citral and citronellal. The screening was carried out using SPMEE, in fluid cultures and sporulated surfaces. Sporulated surface cultures gave, in general, better conversion results than submerged liquid cultures. Using A. niger as for bioconversion the products will be more enantio selective.
The fungus Hyphozyma roseonigra was investigated for its ability to convert 2-ethylenyl decahydro-2-hydroxy-alpha-2,5,5,8a-pentamenthyl-2naphthalenepropanol(sclareol) to decahydro-2-hydroxy-2,5,5,8a-tetramethylnaphthaleneethanol(diol). The conditions for growth to optimize the conversion have been researched.
During the first year key compounds were identified and separated by membrane selection based on a gas phase process. During the second year the possibility of a liquid phase process was researched. For both the possibilities a model for the downstream processing is made.
Achievements
Work during year one resulted in:
During the second year:
Future actions
Based on discussions during the second annual progress meeting the conclusion was that there is an option of dividing the process in two parts if the conversion level cannot be optimised. For the next reporting period the following actions are planned:
Summary
The main aim of the project is to develop a system in which precursors can be converted to bioflavours by fungi. The process has to be scaled up and the applicability of downstream processing has to be researched. Within the first year of the project, useful strains should be selected and a list of interesting flavour components should be known. Also the carrier material in the solid state fermentor and the impregnation liquid composition should be established. In addition, the enzymes involved in the transformation of geraniol to 6-methyl- 5-hepten-2-one (MHO), will be elucidated out of the Penicillium digitatum strain.
Activities
In order to optimise growth, sporulation and conversion by the strains Aspergillus niger and Penicillium digitatum, temperature and the media composition have been studied in flasks and petri dishes. In relation with the development of spores and the conversion of geraniol to MHO by spores a method will be developed to determine the viability. The enzyme system responsible for the conversion will be clarified by using spore solutions and extracts. A requirement list is made for the carrier materials that can be used for solid state fermentation. Absorption tests and growth/sporulation tests are done on several carrier materials to find the optimal conditions. Furthermore screening has been performed to find new strains which can convert interesting compounds.
Progress
Within the first year of the project considerable progress has been made in the development of the solid-state biotransformation process and the elucidation of the enzyme system responsible for the conversion of geraniol to MHO. For the strain Aspergillus niger glucose (35 g/1) as carbon and ammonium sulphate as nitrogen source give optimal results with a ratio of 20/1. Penicillium digitatum was grown in solid state fermentation with sucrose (35 g/1) and urea as respectively carbon and nitrogen source. The ratio 2.5/1 was used for the mixture carbon/nitrogen source. Carrier materials are tested for their ability of water absorption. For each group of carrier materials, one is taken and tested if growth and sporulation of the strains Aspergillus niger and Penicillium digitatum occurs. Wheat bran only needed to be impregnated with water; hemp and polyurethane foam (PUR) were impregnated with the optimised media. Aspergillus niger grew best on wheat bran and hemp and gave on these materials the best conversion from geraniol to MHO. Penicillium digitatum did grow and sporulated best on hemp and wheat bran. This fungi also gave the best conversion rate from geraniol to MHO on PUR.
An agar based mineral salts medium with asparagine as nitrogen source was also investigated. It was established that amino salts also catalyse the transformation of citral into MHO chemically. The method to determine the viability of spores is based on measuring the activity of an enzyme, which, is involved in the respiratory system. The enzyme activity can be measured by reduction of a tetrazolium salt, which gives a yellowish solution. The method will be optimised further.
Several strains of Aspergillus niger and Penicillium were tested on their ability to convert citronellol, citranellal, linalool, geraniol, nerol and citral. The conversion of these substrates was done on submerged liquid cultures and sporulated surface cultures. Sporulated surface cultures gave in general better conversion results than submerged liquid cultures. From the two methods used for measuring the conversion of the terpenoids, solid phase microextraction (SPME) was more sensitive and had the advantage of being fast and solvent free.
Some Aspergillus strains were tested for their ability to convert 2- ethylenyl decahydro- 2-hydroxy-alpha-2,5,5,8a-pentamenthyl-2naphthalenepropanol (sclareol) to decahydro-2- hydroxy-2,5,5 8a-tetramethylnaphtaleneethanol (diol). The strains were grown on several media, to optimise the conversion results.
Results
The main achievements to date are as follows:
Future actions
Based on discussions during the annual progress meeting it was concluded that there is an option of dividing the process into two parts if the conversion level can not be optimised. So it was suggested that the spores should be harvested and concentrated, after which the conversion process can be performed. Hence, for the next reporting period the following actions are planned:
© Copyright 2006 Policy Statements
Updated
by CPL Press:
03/07/2007
- biomatnet@biomatnet.org
 |
 |
 |
News |
 |
Events |
|
|