Controlling accessibility of tastant to receptors via binding to a carrier protein: Moderating the taste of rebaudioside A

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Intake of added sugars exceeds discretionary dietary allowances, regardless of energy needs of the US population and often leads to adverse health conditions. Half of the added sugars consumed in the United States are in the form of high fructose corn syrup (HFCS), a major sweetener in beverages. Therefore, the food industry has been constantly evaluating and using several alternate high intensity sweeteners (HIS) to duplicate the taste of sugar, usually with no calories. Last century led to the inventions of several artificial sweeteners which are often questioned for their safety and role in controlling energy intake and weight management. Growing demand for natural ingredients has led to a rise in the popularity of steviol glycosides, natural sweeteners extracted from the leaves of Stevia rebaudiana that have been in use for centuries in Paraguay. High purity rebaudioside A (Reb A), one of the steviol glycosides is GRAS for use as a table top sweetener. Though regarded as a promising alternate, Reb A is often associated with bitter after-taste, limiting its applications. In this study, a new approach that uses bovine serum albumin (BSA) to control the receptor-accessible part of Reb A was developed. The critical micelle concentration (CMC) of Reb A was established to be 4.5 mM and 5 mM at pH 3 and 6.7 respectively. All experiments were conducted at concentrations of Reb A below its CMC in order to have Reb A available as a monomer for interaction with BSA. The first part consisted of evaluating the stability of bitter inhibitor developed by Kurihara et al. 1998. Particle size analysis data and spectrofluorometric measurements of aqueous solutions of varying phosphatidic acid (PA) and ?-lactoglobulin (BLG) concentrations confirm that PA existed as a separate layer of vesicles at neutral pH and two different temperatures viz. 30°C and 50°C. The light scattering experiments further confirmed that it is almost impossible to have PA bind to BLG in aqueous environment leading to a conclusion that lipoprotein made up of PA and BLG is unstable under conditions pertinent to food systems. Second part of the study consisted of characterizing the binding between Reb A (1 mM) and BSA (20 ?M) via saturation transfer difference (STD) NMR. A binding epitope was established via slopes of saturation transfer to define the receptor accessible and non-accessible part of Reb A. STD NMR experiments confirm that the RPC has fast exchange of the bitterness-instigating hydrophobic diterpene of Reb A into the binding sites of BSA. This study shows that below its CMC Reb A binds weakly to BSA to generate a Reb A-protein complex (“RPC”). NMR titration studies were conducted to evaluate the stability of RPC, which shows that RPC is only modestly stable under varying conditions of pH (3.0-6.7) and temperature (4°C-40°C). The binding affinities of the Reb A-BSA complex were determined to be in the range of 5-280 mM. These results indicate that present approach can be used for Reb A and other amphiphilic tastants to alter (1) the strength of their interaction with receptors to varying degrees and (2) their accessibility to taste receptors as a result of their binding to a carrier protein.

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Rizvi, Syed S H

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Feigenson, Gerald

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Food Science and Technology

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M.S., Food Science and Technology

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Master of Science

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bibid: 10717138

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