CRC Sugar Industry innovation through biotechnology : final report 2B8

Abstract

Sugarcane has a highly effective carbohydrate biosynthetic and storage metabolism that has facilitated its use for the production of sucrose. Sugars are increasingly seen as low-cost, renewable organic resources which can be modified to produce food ingredients and industrial raw materials. For the sugar industry, alternative sugars offer a means of diversification in an area close to the existing core business. However a major restriction to development of alternative products has been ownership of enabling intellectual property by third parties. This project aimed to identify alternative sugars with desirable commercial properties and capture the IP to enable their production.

The initial phase of the project was a scoping study to identify novel, naturally occurring sugars and enzyme systems that may be involved in their manufacture by collating information from the literature and patent databases. Sugars that occur naturally in sugarcane and closely related species were also examined for potential as higher value products. Preferred candidates were simple modifications of sucrose where the gene sequences encoding the enzymes were available and no prior IP existed. Four sugars with potential applications as alternative sweeteners or chemical feedstocks were identified.

Two of the candidate sugars could be either purchased directly or made by chemical synthesis from a purchased precursor. The remaining two candidate sugars were not available commercially and could not be synthesised easily. We proposed to make these sugars by cloning and expressing the genes that encode the enzymes from their native sources and then using the enzymes to synthesise the novel sugars in vitro. Two enzymes were expressed and characterised. Although neither of these enzymes carried out the predicted reactions, both enzymes were new; one is a dehydrogenase and the other is a glucosidase acting on gluco-oligosaccharides.

The potential value of any novel sugar depends on its physical and sensory properties. For application as an alternative sweetener, a novel sugar ideally needs to be as sweet as sucrose but offer health benefits, particularly low cariogenicity (tooth decay) and low calorie-yield. We developed methods that can be used in the laboratory to test industry-relevant properties of sugars, specifically sweetness, cariogenicity and digestibility. A set of commercially available sugars, including several alternative sweeteners, was used to test the assays and provide a comparison with the novel sugars.

Sweetness relative to sucrose and glucose was determined by a two-way preference ingestion assay with Drosophila melanogaster (fruit flies). Production of acid by the oral bacterium Streptococcus mutans was used as an assay to detect potentially cariogenic sugars. Calorie yield of sugars was measured by assays for digestibility by yeast invertase and rat α-glucosidase/sucrase. We also tested whether the sugars were able to inhibit the digestion of sucrose, and whether the sugars could promote the growth of ‘healthy’ bacteria in the gut.

The results showed that two alternative sugars derived from sucrose have the properties required for an alternative sweetener. We also identified a disaccharide which is sweet-tasting and able to inhibit the digestion of sucrose. Further research will be required to develop an economic production system for these candidate sugars. The tests developed in this project also identified some interesting relationships between sugar structure and sensory or nutritive properties. Further analysis of these relationships may allow design of new sweeteners with optimal properties. The outcome of this work is an improved ability to develop new sugar derivatives as alternative sweeteners. The information and tools developed by the project will assist future efforts to exploit new options for diversification in the sugar industry.