BioMatNet Item: AIR3-CT94-2060 - New Strategies for the Development of Integrated Processes for the Generation of Compounds with Flavouring and/or Other Functional Properties from Natural Material

AIR3-CT94-2060
New Strategies for the Development of Integrated Processes for the Generation of Compounds with Flavouring and/or Other Functional Properties from Natural Material

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Summary Information

	Proposal No:	AIR3-CT94-2060
	Date Prepared:	September 1999
	Source:	Final Consolidated Report April 1998

Final Consolidated Report April 1998

Summary

Studies on the generation of compounds with flavouring and/or other functional properties have been performed. Efforts were focused on processes using natural starting material and process conditions leading to products that can be declared as natural. Studies on the enzymatic production of 2,5-di-methyl-4-hydroxy-3[2H]-furanone (known under the name Furaneol, registered to Firmenich SA, Switzerland), the strawberry flavour were carried out. Alternative routes to Furaneol were investigated and compared. Special emphasis was given to natural production and development of economic downstream processes.

A bio-process has been developed for generating 2(E)-hexenal, a flavour compound with fresh, green odour, involving several enzymatic reactions. A thorough investigation of all by-products and their possible exploitation has been carried out. With regard to an industrial process immobilisation techniques have been applied. The enzymatic formation of fatty acid esters was carried out in anionic, cationic and non-ionic microemulsion systems using lipolase, Rhizopus delemar and Candida cylindracea lipase using butanol, hexanol and octanol as well as caprylic, lauric and oleic acid as substrates. Both the anionic and the non-ionic systems were found to be appropriate for these types of reactions. The same methodology was transferred to the esterification studies of buthanethiol, hexanethiol and octanethiol with various fatty acids using the same lipase. The production of catalytic antibodies with aldolase-like activity was performed in order to provide an alternative route to the aldolase reaction, a key step in the enzymatic production of valuable compounds including Furaneol.

Detailed studies on the thioesterification catalysed by an immobilised lipase (Lipozyme) with various thiols and acids have been performed. Optimisation of the reaction parameters has been carried out on a model reaction and has been applied in a continuous process. The thioesters with an onion-like odour were characterised.

Synthesis of Maltol, Furaneol and 2-acetoxytetrahydrofurane has been studied from an industrial point of view. Special consideration was given to the application of cheap natural starting material. Technical feasibility and safety aspects have been considered,

Introduction

Biotechnology offers the possibility of producing high value products from common natural Sources. Agricultural products can thus be utilised, not only for nutrient products but also for value added fine chemicals. The naturally manufactured products have a higher value than comparable products synthesised in classical chemical processes and would have five to ten higher value than the chemosynthesised flavour, if available. Additional positive effects will arise from the fact, that chemicals produced in a biotechnological process (natural production) will increase the consumers' acceptance.

Within the project the biotechnological synthesis of aroma compounds, such as Furaneol, hexanals/hexanals, and fattyacid-thioester derivatives was investigated. These compounds possess physiological properties and olfactory features that are of interest to the food, feed, and cosmetic industries as well as in pest and insect control. It is desirable to use starting material from natural sources. For example, Furaneol can be produced from sucrose and hexanal/hexenal can be obtained from plants.

The results from the research work performed demonstrate that biocatalytic and microbial production processes easily fulfil demands for product quality, functional and molecular properties. Biotechnological processes give the opportunity, even on an industrial scale, to produce flavouring compounds with high quality, even in high quantities. In the following summary, the compounds investigated are presented.

Activities

A major activity was an investigation of the enzymatic synthesis of Furaneol. The results were used to obtain a more detailed insight of the enzymatic engineering. Each step was characterised and process step combinations of the single enzymatic steps were performed. In addition, experiments concerning alternative routes to Furaneol were performed and compared with the enzymatic route. The optimisation of the established process was investigated. Analysis of reaction mixtures was carried out and the separation of Furaneol in an economic down stream process was investigated. Since product purification appeared as the bottleneck in the process, studies were focused on the separation problems. Product recovery via selective extraction and super-critical CO₂-extraction was carried out. A validation of the process investigated was carried out and data regarding process documentation and safety aspects have been collected.

The bio-process developed for generating 2(E)-hexenal involved several enzymatic reactions. In order to recognise rate-limiting steps of the entire bio-process, each enzymatic reaction was optimised separately with respect to pH-optimum, temperature, incubation time, and substrate concentration. 13-HPOT, the key intermediate in the bio-process is a very reactive compound, which can be metabolised in addition to its cleavage into hexenal by further enzymatic and non- enzymatic reactions. Therefore, the main emphasis was laid on the analysis of possible by-products. Further experiments focused on the isolation of flavour compounds from the reaction mixture. For this purpose the recovery of 2(E)-hexenal by solid phase extraction and gas stripping was investigated in comparison with classical solvent extraction. Since industrial application of soluble enzymes is often hampered by their instability and by a lack of reusability of the bio-catalysts, work was focused on enzyme immobilisation, which is a common method for stabilisation. In particular, this was required for the hydroperoxide lyase, the most critical enzyme in the entire bio-process. All investigations were performed with regard to the possibility of an industrial application.

The reaction of mono- and di-esters of hydrophilic diols with fatty acids in a microemulsion system using lipase was performed. Reaction parameters were investigated. Mono- and di-esters were selectively synthesised with high reaction rates. Reaction parameters were investigated. Utilisation for thioester production has been tested. In addition, the production of catalytic antibodies with aldolase-like activity was performed in order to provide an alternative route to the aldolase reaction, a key step in the enzymatic production of valuable compounds including Furaneol. The development of aldolase-like catalytic antibodies capable of using non-phosphorylated substrates was also considered as a challenging future target. Therefore mice were immunised with transition state analogues to produce anti-hapten antibodies.

The application of lipase (triacyl gycerol hydrolase E.C. 3.1.1.3.) for the thioesterification of short chain flavour-thioesters has been investigated. Detailed studies on the thioesterification catalysed by an immobilised lipase (Lipozyme™) with various thiols and acids have been performed. The role of water on the equilibrium towards esterification was investigated from a kinetic (reaction rate) and thermodynamic (equilibrium yield) point of view. The effects of different parameters such as temperature, substrate (both acid and thiol) concentrations and enzyme concentration were investigated. The relative efficiency of the trans-thioesterification and thioesterification were also compared. Optimisation of the reaction parameters was carried out using a model reaction: the synthesis in a batch reactor of the thiobutyl pentanoate from pentanoic acid and butanethiol. A purification scheme was studied, resulting in a proposal for a two step purification. The thioesters synthesised have been characterised from a structural point of view using mass spectrometry and from an olfactory point of view. Finally, a process has been developed to produce the thiobutyl pentanoate continuously.

Various methods for the synthesis of Maltol, Furaneol and 2-acetoxytetrahydrofurane were screened. Focusing on process economy, special consideration and emphasis was given to the application of cheap natural starting material. The reaction steps were studied and elaborated in detail, with Furaneol and 2-acetoxytetrahydrofurane obtained in high yields. Methods included development of a process for the synthesis of 2-acetoxy-tetrahydrofuran, a precursor used for natural compounds, from 2,3-dihydrofuran, that was proved to be technically feasible in pilot plant experiments.