AGRE-0011 Production of a Precursor (Rhamnose) for the Synthesis of Flavouring by Using a Starch Derivative

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Biological Conversion : ECLAIR Cluster III - Carbohydrates : Flavours/Fragrances : Starch

Structure of the rhamnose-rich polysaccharide.

SUMMARY

L-rhamnose is a natural sugar found in several animal, plant and bacterial polysaccharides. Commercially available rhamnose is produced by chemical hydrolysis of arabic and karaya gums, or from rutin or citrus fruits which contain by weight 10-30% rhamnose. Rhamnose is a raw material for the chemical synthesis of furaneol, a strawberry flavour. At present this is dependent on chemical synthesis, whilst direct extraction from fruits is costly and subject to seasonal variations in supply volume and quality. As a result of this project, worldwide patents have been deposited by BioEurope covering a strain of the bacteria Klebsiella terrigena and the production process for obtaining an exopolysaccharide containing 50% rhamnose. This polysaccharide can be used as is or, after hydrolysis, as a source of rhamnose for the synthesis of natural aroma compounds such as furaneol.

INTRODUCTION

Rhamnose is a derivative of the 6 carbon sugar mannose. It can also be described as 6-deoxy-L-mannose and occurs in a combined form in the glycosides of many plants. It is traditionally extracted from specific higher plants or algae which may have a rhamnose content ranging from 10-30% by weight. In particular, commercially available rhamnose is currently produced by hydrolysis of arabic and Karaya gum or rutin. However, such supply is affected by seasonal variations. The current market for rhamnose lies between 5 and 10 tons/year, being sold for around 500 ecu/kg, giving a market volume of several million ecu per annum. At present, most of the rhamnose used in Europe is imported from some 7 rhamnose producers based in America and from 2 based in Japan. Production in Europe would limit importation needs, whilst the development of a reliable technology to provide a constant and uniform supply of rhamnose within Europe could lead to an increase in market size. The most significant use of rhamnose is for the synthesis of furaneol to produce fruity aromas, resembling those of strawberry and raspberry. This market is expandable to other aroma products including meat flavourings.

OBJECTIVES

The main objective of this project was to produce rhamnose as a precursor for the synthesis of flavourings by fermentation. In addition to being found in plants, it is present in some bacterial hetero-polysaccharides. In fact, a very diverse range of microorganisms (including Gram-positive and Gram-negative, aerobic and anaerobic bacteria, as well as unicellular algae and fungi) produce such polysaccharides, although the specific composition and properties of these vary from species to species. BioEurope has screened and selected microorganisms for such purposes. This has resulted in the identification of an organism suitable for the biotechnological production of a polysaccharide containing 50% rhamnose. As a result, worldwide patents were taken out relating to a novel production process (EP 88 403131.1; F 2 624 522; SP 63-311047/88; US 281.542). The main objective of the ECLAIR funded work was the optimisation of this process. This required improvements in yield (an increase in the concentration of rhamnose-containing polysaccharide to at least 10 g/l) by use of a mutation programme and by improved culture conditions. Other aspects of importance were methods of harvesting, extraction and purification, under conditions which would meet legislative guidelines defining natural aromas. The project also supplied polysaccharide and polysaccharide hydrolysate to a number of partners for evaluation.

APPROACH

The work programme covered productivity, enzymatic hydrolysis and rhamnose purification, as well as use of the raw material as substrate for the synthesis of natural flavourings of the polysaccharide from Klebsiella terrigena, strain BEC 441. This has a rhamnose content of 50% when produced by fermentation of starch during 2-3 days fermentation. The polysaccharide is recovered by alcohol precipitation, whilst rhamnose is recovered by acid hydrolysis of the polysaccharide, followed by neutralisation and separation of the sugars by ion-exchange chromatography.

RESULTS

The original productivity of 7g rhamnose/litre of culture medium was increased to 17g rhamnose/litre under optimised conditions at the 200 litre scale. Increased yields in terms of substrate utilisation and cost are currently under investigation. Some problems arise from the presence of proteins in the polysaccharide powder. The possibility of more specific hydrolysis of the polysaccharide fraction in order to liberate readily purifiable rhamnose was investigated by screening commercial enzymes. Of these, two (buckwheat extract and naringinase) have the potential to cleave rhamnose from the polysaccharide. The screening of methods to remove protein while leaving the polysaccharide intact continues. Purification of the rhamnose contained in the polysaccharide was accomplished using ion exchange columns.

COMMERCIALISATION

The complete process is currently being optimised at the industrial scale.

COORDINATION

The project is coordinated by BioEurope, Toulouse, France.