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AIR1-CT92-0026
New Processes for the Biological Transformation of Agricultural Residues for the Production of High Added Value Flavours |
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Proposal No: | AIR1-CT92-0026 |
| Date Prepared: | September 1999 | |
| Source: |
Final synthesis report 1997 Progress Report Summary Update to Progress Report Summary |
Summary
Introduction The aim of this project was to use enzymes to obtain ferulic acid and other precursors (rhamnose, arabinose, xylose), from agricultural by-products such as cereal brans and beet pulp, to purify these and then transform the precursors into aromas, such as vanillin, furanone and pyranone-type derivatives using selected microorganisms. Sugar beet pulp and cereal brans were chosen as raw material since they are rich sources of ferulic acid (1-4%), ester-linked mainly to heteroxylans and pectins, respectively. Because of the botanical origin (including plants showing C3 and C4 pathway of photosynthesis), variations in the isotopic ratio (C12/C13) of the ferulic acid could also be obtained.
Objectives The objectives of the project were to evaluate the potential for the development of natural flavours by microbial and enzymic bioconversion of specific components (ferulic acid, rhamnose, arabinose, xylose) of plant cell-wall residues including brans derived from wheat or maize and sugar beet pulp. The biological transformation included the enzymic treatment of these materials in order to obtain the flavour precursors, and the bioconversion of these precursors by selected microorganisms; with ferulic acid converted into vanillin and rhamnose, arabinose and xylose converted into pyranone or furanone derivatives.
The programme was divided into two main phases.
Phase 1 activities Production of flavour precursors by enzymic means, in which the precursors (rhamnose, arabinose and ferulic acid from beet pulp, ferulic acid arabinose and xylose from cereal brans) were obtained on a laboratory scale by extensive degradation/solubilisation of the cell walls containing these precursors by enzyme mixtures, and by more selective actions of some highly purified enzymes (ferulic acid esterase, arabinanases, polygalacturonanases, xylanases). The isolation of the precursors involved mainly chromatographic systems, while production of suitable enzymes and isolation procedures was scaled up in order to obtain appreciable quantities of precursors.
Phase 2 activities Conversion of the precursors to flavour compounds by selected microorganisms The microbial bioconversion of the precursors to vanillin and furanone or pyranone derivatives was investigated. The production of vanillin especially involved the basidiomycete Pycnoporus cinnabarinus , and a programme of strain improvement was developed on the basis of knowledge of the vanillin biosynthesis pathway . For the production of pyranone or furanone derivatives, a cumulative screening of the yeasts that are able to metabolize rhamnose, arabinose and xylose by the pentose phosphate pathway was carried out.
The selection of highly productive strains was achieved through the analysis of the whole flavour compound by high performance liquid chromatography and gas chromatography-mass pectrometry. The final process of flavour production was carried out in bioreactors first at the laboratory scale and then at the pilot-plant scale. The flavours were extracted according to EU legislation.
Results The work, focussed on sugar-beet pulp, and wheat bran and maize bran as potential sources of ferulic acid, arabinose and xylose, investigated the structure of the polysaccharides (arabinoxylans, pectins) in the cell walls. Special attention was paid to the location of the ferulic acid residues. The association between ferulic acid, sugar beet pulp, maize bran and wheat bran has been established. In sugar-beet pulp, ferulic acid is linked to 0-2 of arabinose residues and to 0-6 of galactose residues. In cereal brans, ferulic acid is linked to 0-5 of arabinose residues.
Studies on enzymes and enzymic degradation of the substrates were concentrated on ferulic acid esterases that were extensively studied. It has been shown that there is not only one enzyme but rather a range of enzymes, the activities of which depend on the nature of the sugar and the linkage with ferulic acid, as well as the length of the oligosaccharide moiety. These enzymes are only very slightly active when used directly on the raw substrates. The polysaccharides have to be pre-treated, resulting in some degradation.
The production of following cloned polysaccharidases has been up scaled:
With a combination of these enzymes, almost 100% of the ferulic acid can be released from wheat bran and sugar beet pulp. Maize bran is more resistant to the enzymic degradation and only 30% of ferulic acid could be released. Special attention was paid to the recovery of ferulic acid. Activated charcoal and resins were used for this purpose. Despite very promising initial results, no significant production of furanone and pyranone derivatives could be obtained. The use of laccase-deficient strains of Pycnoporus cinnabarinus leads to the production of vanillin up to 500 mg/l.
Another, two-step, strategy was developed using two microorganisms with complementary activities. The first stage, using Aspergillus niger, was studied for the production of vanillic acid from ferulic acid. In a secod step, the production of vanillin from vanillic acid was achieved using Pycnoporus cinnabarinus, with good yields. When using ferulic acid released enzymatically from the raw materials, yields of natural vanillin of nearly one g/l were obtained. At the beginning of the project, the maximal yield (from synthetic ferulic acid) was 60 mg/l.
In particular in a laboratory scale bioreactor, the continuous addition of ferulic acid, in the presence of plant cell wall fractions as fermentation feedstock (xylose as carbon source and cellobiose as activator of the vanillin pathway), increased vanillin production up to 700 mg per litre (patent no. 94 10889). This was a 15-fold increase over that claimed in patent 0453368A1.
The one-step strategy was studied further using strains of P. cinnabarinus and natural ferulic acid. Best results (300 mg/l of vanillin) were obtained with beet ferulic acid. It was also found possible to use ferulic acid bound to the granulated activated charcoal, although the vanillin yield was lower (150 mg/1).
It was also shown that Xanthomanas campestris could be used to produce vanillic acid from ferulic acid. High yields of natural vanillic acid can be easily obtained from beet ferulic acid (1 g/1); this vanillic acid could be subsequently biotransformed vanillin by Pycnoporus cinnabarinus.
An isotopic analysis was investigated in order to distinguish betwen natural and synthetic vanillin and it was shown possible to prove the origin of the natural vanillin, each source (beet, maize) having a different isotopic ratio.
An organoleptic evaluation of the natural vanillins was carried out. Although vanilla is always the major sensation, differences appeared between the various broths, depending on the strain and the phenolic acid used as substrate. This point is of importance since more complex "aroma" than that of synthetic vanilin can be obtained.
Conclusions New enzymes were identified for the liberation of ferulic acid and the required preparations have been selected for the production of other precursors (arabinose, xylose, rhamnose) while the location of ferulic acid on polysaccharides from cereal and beet has been demonstrated.
Attempts to produce furanone and pyranone derivatives gave disappointing results. In contrast, the work on production of natural vanillin was successful. Improved strains of microoganisms have been obtained and new strategies for the production of vanillin have been developed. At the beginning of the project, the maximum production of vanillin from synthetic ferulic acid was 60 mg/l; at the end of the project, a much higher value (about one g/l) had been reached.
ACTIVITIES
Raw materials: Sugar beet pulp and cereal brans (wheat and maize) were chosen as raw materials since they were known to contain significant levels of ferulic acid (1 4%), ester linked mainly to heteroxylans and pectins. Variation in the isotopic ratio C12/C13 of the ferulic acids could also be obtained. This reflected the fact that beet and wheat have the conventional (C3) photosynthetic pathway, but maize is a C4 plant with a modified photosynthetic pathway that discriminates less against heavier isotopes, thus changing the ratio.
Location of feruloyl groups: Using a combination of enzymatic hydrolysis and NMR it was established that in sugar beet pulp, approximately 50 % of the ferulic acid is linked to O-2 of arabinose residues and 50 % to O-6 of galactose residues; in cereal bran ferulic acid is (mainly) linked to O-5 of arabinose residues. This meant that ferulic acid esterases could be used to selectively release the natural ferulic acid.
Ferulic acid esterases: It was shown that there are a number of different esterases, the activities of which depend on the nature of the sugar and the linkage with ferulic acid, as well as the length of the oligosaccharide moiety. These enzymes, which were obtained mainly from strains of Aspergillus niger, could be classified on the basis of their preferred substrates, which included O-2 feruloylated arabinose, O-6 feruloylated galactose, O-5 feruloylated arabinose and O-6 feruloylated galactose.
Enzymatic production of ferulic acid: It was found that pure ferulic acid esterases are essentially inactive if used directly on the raw materials. An extensive degradation of the cell wall polysaccharide has to be achieved first. A thorough screening of commercial enzymatic preparations was carried out and preparations containing adequate pectinase and xylanase activities have been found. However, the amount of ferulic acid esterases in these were always limiting and the preparations had to be complemented by added esterases. The raw materials used as a source of phenolic compounds appeared to have different degradability and hence variable yield. With sugar beet pulp, commercial preparations were able to release about 60 % of the total amount of ferulic acid. Addition of ferulic esterase increased this yield to 95 %. On a laboratory scale, 95 % of the total ferulic acid was released from wheat bran using a combination of enzymes including an endoxylanase from Trichoderma viride. So far, no enzyme mixtures have been identified which can extensively hydrolyse the maize bran. This is due to its very complex structure which means that no more than 30 % of the ferulic acid could be released.
Separation of ferulic acid: It has been demonstrated that at the laboratory scale some adsorbents (resins, activated charcoal) have a very high specificity for ferulic acid and can be used for the efficient production of pure ferulic acid after desorption with ethanol.
Production of furanone and pyranone derivatives: By screening of yeasts able to metabolise rhamnose, xylose and arabinose were investigated. Strains of Pichia capsulata and Pichia angusta were selected for their ability to produce 4 hydroxy 2,5 dimethyl 3(2H) furanone in concentrations of 60 and 30 ppm. However, results were inconsistent.
Filamentous fungi as producers of vanillin: It was found that Pycnoporus cinnabarinus I 937 was able to transform ferulic acid to vanillin, the concentration of vanillin reaching 45 mg/l. This has been patented (Patent N° 0453368A1). In order to develop a process for the production of natural vanillin, the pathways of the biotransformation of ferulic acid by strain I 937 have been identified and investigated. Three divergent routes resulting in lowering the yield in vanillin, were identified. These either formed polymers (due to laccase) or degraded the vanillin to vanillic acid, vanillyl alcohol or methoxyhydroquinone. To overcome some of these problems, laccase deficient strains were obtained by conventional selection, resulting in a ten-fold increase in production, with the concentration of vanillin obtained increasing to 450 mg/l with pure ferulic acid as precursor.
Two filamentous fungi improve vanillin production: In biotransformation of ferulic acid to vanillin, the major limiting step was degradation of the ferulic acid propenoic chain into vanillic acid. To improve vanillin production, a new two step strategy was developed combining two filamentous fungi exhibiting complementary capabilities of bioconversion (Patent N° 94 10889). The first step involved Aspergillus niger I 1472 for the biotransformation of ferulic acid to vanillic acid. The second step involved laccase deficient Pycnoporus cinnabarinus 30555.ss23 or Phanerochaete chrysosporium which used vanillic acid as precursor, instead of ferulic acid.
Improved fermentation processes: Xylose and cellobiose, which are derived from the raw materials described above during the same process, were used as fermentation feedstocks. Xylose was utilised as carbon source for the mycelium-producing phase, while cellobiose was added for the vanillin-producing phase to channel the flow of vanillic acid through the reductive pathway leading to vanillin. Under the best conditions, yields were increased to several g/l.
EXPLOITATION
This work is being continued (scaled-up) under support from the FAIR programme ( FAIR2-CT95-1099).
PARTICIPANTS
The work was coordinated by INRA Nantes (F), with the following partners: INRA Marseille (F), IFR Norwich (UK), ARD Pomacle (F), University College Galway (EIR), Novo Nordisk (DK), INETI Queluz (P), Pernod-Ricard Creteil (F) and UCL Louvain-la-Neuve (B).
Activities
The work programme was divided into phases as follows:
Phase 1: Production of Flavour Precursors by Enzymic Means. The precursors (rhamnose, arabinose and ferulic acid from beet pulp, ferulic acid arabinose and xylose from cereal brans) were obtained on a laboratory scale by extensive degradation/solubilisation of the cell walls containing these precursors by enzyme mixtures, and by more selective actions of some highly purified enzymes (ferulic acid esterase, arabinanases, polygalacturonanases, xylanases). The isolation of the precursors mainly involved the use of chromatography. Production of suitable enzymes and isolation procedures was scaled up in order to obtain appreciable quantities of precursors.
Phase 2: Conversion of the Precursors to Flavour Compounds by Selected Micro-organisms. The microbial bioconversion of the precursors to vanillin and furanone or pyranone derivatives was investigated. The production of vanillin involved the basidiomycete Pycnoporus cinnabarinus. A programme of strain improvement was developed based on knowledge of the pathway of vanillin biosynthesis. For the production of pyranone or furanone derivatives, screening of yeasts known to yeasts that are able to metabolise rhamnose arabinose and xylose was carried out. Attempts were made to select highly productive strains will by analysis of flavour compounds by high performance liquid chromatography and gas chromatography mass spectrometry. The final process of flavour production was carried out in bioreactors first at the laboratory scale and then at the pilot plant scale, with the flavours extracted using methods that met EU regulations.
Results
The work in phase 1 focused on sugar beet pulp, wheat bran and maize bran as potential sources of ferulic acid, arabinose and xylose. The structure of the cell wall polysaccharides (arabinoxylans, pectins) bearing ferulic acids has been investigated with specific attention to the location of the ferulic acid. In sugar beet pulp, ferulic acid is linked to 0-2 of arabinose residues and to 0-6 of galactose residues. In cereal brans, ferulic acid is linked to 0-5 of arabinose residues. Extensive studies of the ferulic acid esterases indicated a number of enzymes, the activities of which depend on the nature of the sugar and the linkage with ferulic acid, as well as the length of the oligosaccharide moiety. These enzymes are only very slightly active when used directly on the raw substrates, but higher activities are seen when partially degraded polysaccharides are used. The production of following cloned polysaccharidases was scaled-up: polygalacturonases, pectin lyase, pectin-methylesterase, endoarabinanase, endogalactanase, rhamno galacturonases, rhamno galacturonan acetyl esterase, and xylanases. With a combination of these enzymes, almost 100% of the ferulic acid can be released from wheat bran and sugar beet pulp. Maize bran is more resistant to the enzymic degradation and only 30% of free ferulic acid could be released. The recovery of ferulic acid was achieved using activated charcoal and resins. Despite very promising initial results, no significant production of furanone and pyranone derivatives could be obtained. However, good results were obtained for vanillin with the use of laccase deficient strains of Pycnoporus cinnabarinus giving good yields. An alternative two step process was developed using two micro-organisms with complementary activities. The first step used Aspergillus niger to form vanillic acid from ferulic acid. In the second step, the productions of vanillin from vanillic acid was achieved using Pycnoporus cinnabarinus. An organoleptic evaluation of the natural vanillin was carried out. Although vanilla is always the major sensation, differences appeared between different broths enabling more complex "aroma" than just synthetic vanillin to be obtained.
Conclusions
Each participant was able to complete the schedule they were involved in. New enzymes have been identified which liberate ferulic acid from hemicelluloses while adequate preparations have been selected for the production of other precursors (arabinose, xylose, rhamnose). The position of ferulic acid on plant cell wall polysaccharides from cereal and beet have been identified. Although, attempts to produce furanone and pyranone derivatives were disappointing the production of natural vanillin was very successful. Improved strains of micro-organisms were obtained and new strategies for the production of vanillin have been elucidated. At the beginning of the project, the maximum production of vanillin from synthetic ferulic acid was 60 mg/l. By the end of the project this had been raised to around one gram per litre.
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