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NMR has been used to gain a breakthrough in the understanding of a novel low-calorie sweetener that is thousands of times sweeter than sucrose and has none of the bitter after-taste of other sweeteners. Food manufacturers are ever on the look out for new sweet-tasting compounds that are easy to produce or extract, have the same effect on the palate as sugar but with close to zero food calories. There are several such artificial sweeteners already in widespread use, including saccharin, aspartame, sucralose, neotame, acesulfame. Some of these compounds are said to produce a bitter aftertaste or have other problems, such as perceived health risks. Moreover, in an open market there is always room for rival products. Market researchers estimate that demand for artificial sweeteners, natural or otherwise, will be over $1 billion (about £500m) by 2010. The main use will be in the manufacture of low-calories soft drinks and as a substitute for regular sugar on the kitchen shelf. One such sweetener is derived from an extract of the fruit of the tropical plant Pentadiplandra brazzeana Baillon, which has been consumed by people in West Africa for thousands of years. Intriguingly, the extract known as brazzein tastes sweet only to old world monkeys and humans. It is, one of a handful of sweet-tasting proteins, and was first identified in the early 1990s by researchers at the University of Wisconsin-Madison. Brazzein is 2000 times sweeter than sucrose when compared to a 2% sucrose solution in water and 500 times sweeter compared to 10% sugar solution. However, it has a taste closer to sucrose itself than other sweet proteins, such as thaumatin, is soluble in water and its sweetness is maintained even at 80 Celsius, which is important for foods that must be pasteurised, for instance. Wisconsin's Fariba Assadi-Porter uses NMR to study the structure-function relationships in brazzein and its analogues in order to understand precisely why this compound so stimulates our sweet receptors. Stable-isotope-assisted multinuclear NMR is the key that unlocks detailed chemical and structural information at the atomic level when the compound is present in living cells and interacting with the receptor. The team's current model of the binding of brazzein to the sweet receptor, suggests that the 54-amino acid protein chain binds at several points to the receptor. The sweet proteins do not share the same chemical properties as sugars like sucrose. In fact, these proteins do not interact with the same binding sites as sugars. "What makes these proteins sweet is far more complex than small sweeteners like sucrose, aspartame, etc," Assadi-Porter told SpectroscopyNOW, "There are many factors that dictate sweetness, such as the three-dimensional fold and large positive charges." She and her colleagues are now attempting to understand the detailed structural and chemical properties that are important for the sweetness of brazzein and its interaction with the sweet receptor. Now, Californian startup company Natur Research Ingredients of Los Angeles has obtained an exclusive licence from Wisconsin Alumni Research Foundation (WARF), the technology transfer wing of the University, to manufacture and distribute the compound under the cunning brand name Cweet. Natur CEO Loren Miles, explains that while the sweetener community has known about brazzein's potential for many years, there was not until now, a commercially viable manufacturing process. Assadi-Porter, however, has developed an expression and purification protocol that is perfectly suited to mass production by the food industry. In earlier work, Assadi-Porter explains that she and her colleagues synthesized a gene that codes for the brazzein protein and then transformed this gene into a laboratory bacterium that could easily be fermented to make large quantities of brazzein. The protein can then be extracted using protein folding techniques and oxidation of the disulfides to yield a synthesised form of the natural protein, which is identical to the original extract according to the NMR data. Taste tests also reveal it to have full activity, Assadi-Porter says. She points out that the same approach using mutants of the original gene can be used to express experimental analogues of brazzein, several of which are sweeter still. This technology for making brazzein mutant proteins is based on our original technology which needed refolding and oxidation protocols," Assadi-Porter told SpectroscopyNOW, "We have a couple of new technologies for larger protein production," she adds, "The first technology paper has now been accepted for publication in Protein Expression and Purification Journal." "Cweet will represent one of the first natural intense protein sweeteners to be available as an alternative to sucralose and aspartame," adds Miles. She points out that demand from the food industry and health-conscious consumers for a natural, yet intense, sweetener is growing fast. Cweet is yet to go through the US Food & Drug Administration (FDA) approval process. Having appropriated a natural ingredient from a native species of West Africa, Natur is planning to establish a foundation there that will contribute to humanitarian causes once production and distribution has started. Related links: The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.
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 Assadi-Porter, understanding sweet solutions
 Brazzein, a thousand times sweeter than sucrose |
