Research On Amino Acids’ Bitterness Masking And Development Of Novel Bitterness Evaluation Methods

Bitterness is closely related to people’s daily life, and it’s one of five basic taste attributes. Bitter taste receptors (TAS2Rs) are G-protein coupled receptors, of which intracellular signal pathway is a classic calcium signal pathway-TAS2Rs (GPCRs)â†'intracellular G proteinâ†'phospholipase Câ†'inositol1,4,5-trisphosphate. For human beings and animals, bitterness is sometimes a warning of toxic food. In most cases, bitter compounds induce aversion, resulting in bad compliance of foods or drugs, especially for children and the aged. Therefor, bitterness evaluation is necessary in oral products development, and then the bitterness evaluation methods are needed.It’s reported that some tasteless small molecules can reduce bitterness of solution. Here this phenomenon is believed to be most probably due to the intermolecular forces between small molecules and bitter compounds in solution. Denatonium benzoate, acetaminophen and some kinds of amino acids were selected and series of experiments were finely designed to reveal this potential mechanism. Also we applied Raman spectroscopy and H’NMR spectroscopy to detect the potential changes Compared with their single solutions, some H shifts in H1NMR were found in tw mixed solutions, showing us that intermolecular forces possibly exist. The mixture solutions combined with one amino acid and one bitter compound. Based on these finding, their bitterness masking ability were further evaluated by human gustatory test and animal gustatory test.To date, most bitterness evaluation methods can be included in4methods as below-human gustatory test, animal gustatory test, TAS2Rs expressed cells based calcium imaging and electronic tongue. The first two methods can be conducted without high requirements of instruments; however, their results are always in large deviation. Electronic tongue is a fast and safe method to evaluate bitter solutions, and accurate data can be given by applying deliborate calibration and suitable pattern recognition methods. However, it is too expensive to be applied in our researches.In this article, we decided to develop our own cell besed calcium imaging system. Among25human bitter taste receptors (TAS2Rs), TAS2R39can specific combine with some typical bitter compounds, such as denatonium benzoate, acetaminophen and vitamine B, which could possibly facilitate our further researches. Besides TAS2R, Ga protein is also needed in intracellular calcium signal transportation. Here, vector construction, vector transfection, cell screening and calcium imaging will be reported. After finely adjustment of procedures, conditions and reagents, the efficiency of transfection is now more than70%for both two proteins. Also, preliminary calsium imaging experiments were conducted. However, two vectors expressed fluorescent protein, green fluorescent protein (GFP) and red fluorescent protein (RFP), did not compromise with Fluo4-Am, a calcium indicators.Instead of series steps of signaling in calcium imaging, a simpler method could be developed based on combination between fluorescent bitter compounds and TAS2Rs. Here, the extraction and quantitation of pig airway smooth muscles (ASM) were reported and further series of fluorescent experiments were designed to compare the selectivity between the extraction of ASM and other tissues. In this research, the coefficient correlation of protein concentration-absorption reached98.3%and that of quinine HCl concentration-absorption reached98.2%. Futher test showed that the fluorescence of quinine HCl were generally stable and no more decrease happened after6hours. In comparison between ASM and other tissues, the selectivity of ASM extraction was obviously higher than other tissues’. But also, the thermal denaturation ASM samples were of the same with normal ASM in comparison of selectivity to quinine HCl.