Food and flavour companies are increasingly adopting high throughput screening approaches as used by the pharmaceutical industry to identify novel compounds, writes Cynthia Challener
It has been recognised for decades that human taste buds differentiate between the flavours in foods. Only within the last 20 years, however, has any knowledge about the chemistry of taste been elucidated, bringing opportunities for implementing new discovery methods for identifying flavour compounds. Both startup biotechs and established flavour companies have adopted techniques originally applied in the pharma industry to screen hundreds of thousands of compounds a year.
The taste experience is a complex process
Not only are there multiple receptors detecting flavour compounds, but up to 400 human odour receptors are involved as well. ‘Smell and taste are strongly connected, but many are focusing on taste receptors as a first step because the taste process is simpler,’ notes Hiroaki Matsunami, an associate professor at Duke University Medical Center, North Carolina, US. ‘It may well be that there are specific combinations of taste and odour receptors that activate at the same time. We hope to use cell-based assays to learn more about what specific agents activate a pleasant response.’
Humans can taste at least five basic flavours: sweet, salty, bitter, umami (savoury, activated by compounds such as glutamate and aspartic acid) and sour, and possibly a sixth ‘fat’ taste. Three, sweet, bitter and umami, are detected by G-proteincoupled receptors (GPCRs), typically located in the cell membrane, that interact with specific compounds outside the cell and in turn activate a signaling pathway, including the Transient Receptor Channel m5, or TRPm5 ion channel. For sweet and umami detection, dimers of two receptors (T1R3/ T1R2 and T1R3/T1R1) are involved, while up to 30 receptors have been found to detect bitter flavours in humans and test animals. Salty and sour sensing cells activate different ion channels that shuttle cations – Na+ in the case of table salt, for example – which eventually stimulate nerve fibres. Other ion channels related to TRPm5 are linked to the taste of spicy flavours.
Using traditional development approaches, where various compounds are individually synthesised and then tasted, even large flavour houses can evaluate only about 1000 compounds/year, according to Don Karanewsky, vice president of discovery at Senomyx, a US company using proprietary taste receptor technologies to develop novel flavour ingredients. With high throughput screening methods adopted from the pharma industry, Senomyx can screen up to 10,000 compounds/week.
The new approach involves using assays based on engineered cells that express a single taste modality. A cloned receptor is introduced into a mammalian cell, where its activation by a test compound results in mobilisation of intracellular calcium ions, which is detected by an increase in the fluorescence of calcium-sensitive dyes preloaded into the cells. Researchers use the assays to screen combinatorial and natural product libraries containing hundreds of thousands of low molecular weight, non-volatile, highly water soluble molecules.
There are, however, limitations to the technology. Because only one taste modality is evaluated per assay, successful compounds must undergo secondary evaluation to validate that they don’t also activate other taste receptors. Compounds must also be shown to have the necessary physical characteristics, such as stability under a wide range of temperatures and in hydrolytic conditions. They then go to taste panels for final testing.
‘The use of these technologies borrowed from the pharma industry offers potential rewards for product differentiation and allows for a high rate of evaluation. They are, in fact, most compelling for identification of disruptive technology,’ believes Mark Dewis, vice president of Flavors R&D at IFF, a US flavour and fragrance manufacturer. ‘Even so, they often come at a higher price, at least in the initial capital investment required, and at IFF we consider this approach to be just one important part of our extensive portfolio of technologies.’
Improving the appeal of more nutritious products
Senomyx, Redpoint Bio, a US company focused on discovering taste modulators that will improve the nutrition of foods, and IFF are all interested in developing taste enhancers for the sweet and salt modalities and blockers for bitter receptors. ‘Currently there is tremendous pressure on the food industry to improve the nutritional content of its products,’ Redpoint ceo Ray Salemme explains. ‘Sweetener and salt enhancers can lead to reductions in the amount of sugar and salt necessary to prepare foods that taste good. Bitter blockers can reduce the bitterness of artificial sweeteners and other compounds, improving the appeal of more nutritious products.’ The various taste receptors each possess many binding sites, which interact with different types of molecules. Flavour enhancers, called positive allosteric modulators, bind somewhere on to the receptor other than the primary binding site, but result in an overall higher binding affinity for the major tastant.
Both Senomyx and Redpoint have identified interesting compounds that act as sweetener enhancers. Senomyx, which focuses its efforts on modulating the activity of cell surface receptors, has received FEMA GRAS (Generally Recognized As Safe) designation for S2383, an enhancer of the highintensity sweetener sucralose and S6973, which halves the amount of sucrose required in foods without changing the taste. Meanwhile, Firmenich is conducting pre-commercialisation activities with S2383, which can reduce the sucralose content by up to 75%, and should make a decision this month whether or not to commercially develop S6973. The market for table sugar or sucrose is worth around $50bn/year, while artificial sweeteners command $3bn/year, according to Senomyx; sucralose alone generates $300m in product combinations and $100m in tabletop form. Senomyx is now developing second-generation sucrose enhancers with different physical characteristics to increase their usefulness in a wider range of foods. The firm is also working to identify compounds that could serve as enhancers for fructose and Rebausdioside-A, the sweetener found in herbs of the genus Stevia, a plant native to Central and South America.
Although originally focused on using receptorbased discovery technology to find modulators of the TRPm5 channels involved in taste signaling, Redpoint recently revamped its approach to better target the discovery of natural taste compounds. Using the newly developed methods, which are based on a combination of cheminformatics and automated behavioural animal models patterned on techniques originally developed for drug discovery, Redpoint has identified a natural compound initially identified as RP44 and recently revealed to be rebaudioside C (Reb-C, another component of Stevia), that acts as a sweetener enhancer for sucrose, fructose and high fructose corn syrup, effectively reducing the necessary sweetener content by 25% or more, depending on the application. ‘We have seen a real shift in consumer interest away from synthetic food additives and toward natural ingredients. Consequently, we have been focusing much of our efforts on finding naturally occurring flavour compounds,’ notes Salemme. The company is preparing an application for GRAS status for RP44.
Senomyx also has four savoury compounds with GRAS status, one being marketed by Nestlé in the Pacific Rim and Latin America, and another being commercialised for the Asian market by Japanese food and amino acid producer, Ajinomoto.
In all, Senomyx has partnerships with seven food and beverage companies. The company has completed successful initial safety studies and development activities with two bitter blockers. It has also functionalised 22 bitter receptors and has developed a toolbox of six bitter receptor antagonists that, when used in combination, can determine which of 18 different receptors are activated by bitter food components. Senomyx can use the toolbox as a starting point to develop compounds that block or attenuate the bitterness of prototype foods still under development, according to Karanewsky. The company also has programmes to develop salt enhancers and cool flavours that create the sensation associated with menthol.
Redpoint Bio, which originally began developing bitter blockers for use in oral pharmaceuticals and then turned its efforts to spice modulators, has returned to the identification of compounds with pharma applications – this time as the active ingredient. ‘Recently it has been discovered that taste signaling pathways found in the mouth are also present in the gastrointestinal tract and modulate the secretion of hormones that play important roles in the regulation of glucose uptake and the feeling of satiation,’ says Salemme. ‘We are using our knowledge of the TRPm5 signaling channel to develop small molecule modulators that have the potential to serve as a new modality for the treatment of obesity and diabetes.’ He adds that this type of drug could be safer than systemic therapies since it acts specifically on intestinal chemosensory cells.
IFF, says Dewis, will be commercialising a number of compounds in 2010 that have been identified using high throughput technology. They include natural and synthetic compounds that cover both taste and sensory activation through multiple taste and lability modalities.
‘While there has been limited success to date with the use of these new discovery techniques, the high throughput screening of engineered receptor cells is still at an introductory stage. There have been enough compounds identified, though, by ourselves and others, to convince me that the technology definitely works and is a compelling approach for finding the proverbial needle in a haystack,’ Dewis concludes.