Studies On Slenium Biofortification And Chemical Speciation In Rice Grain

Selenium (Se), a crucial trace element, is required in humans as well as animals for their growth and development. Rice is one of the leading food crops for more than half of the population in the world. In China, rice is growing in about 30 million-hektare area all year around, nearly 30% of cereal growing area, which produces 190 million ton rice grain per year, with 40% of total cereal production. More than 60% of Chinese population feed rice as staple, and nearly 180 million ton rice is consumed every year. Therefore, rice is an important source of Se for the world population, especially for the people in China who depend on the rice as staple food. However, low levels and considerable differences of Se were found in Chinese rice products, mainly depending on the Se concentration of the soil and rice genotype. It is feasible that increasing the micronutrient concentrations in these crops through biofortification could increase the dietary intake of these elements in these regions significantly. However, the beneficial nutritional value and cancer chemopreventive character of Se are based on both the concentration ingested and on its chemical forms. Thus, information on total Se content of Se-enriched products is insufficient to assure the biological activity of the products. Although the biological activities of Se-enriched rice prepared by foliar application were evaluated in our previous works, the speciation distribution, bioavailability of Se in rice samples should be clear in order to understand the mechanism of its biological activity in vitro or in vivo.In this study, the variation of Se content of rice in different cultivar collected from main growing regions in China were investigated, based on which the Se nutrition of the population living in these areas staple was studied. The inorganic Se uptake by rice via foliar application was transformed into organic Se species with high bioavailability. With the help of speciation studies, the major Se species presented in rice was determined, and the distribution, uptake and transportation of Se in rice grain were elucidated. The distribution of Se in large biomolecules and the speciation of Se in protein were investigated. Moreover, an in vitro gastrointestinal digestion was employed to evaluate the potential elemental bioaccessibility of Se-containing protein products. To well understand the mechanism of biological activity in vitro or in vivo of Se-enriched rice, the selenoproteins were identified utilizing metallomic strategy for illumination of their possible function. The primary research result involves:1. The mean content of rice products collected from main growing regions in China was 0.022 mg/kg. Moreover, the variation of Se level with different cultivars was considerable. The average daily intake of the people feed rice as staple was estimated to 8.3～22.0μg/day, which lower than 50μg/day, the level of RDA proposed by Chinese Nutrition Society. Therefore, the Se intake was obviously low for the people who depend on low-Se rice as staple food, which potentially threaten their health.2. The concentration of Se fertilizer increased from 0 g/ha to 100 g/ha (1 ha=10,000 square meters), which gave rise to the primary Se content of polished rice, significantly increasing from 0.032 mg/kg to 0.207～1.79 mg/kg. Moreover, the Se content was increased in polished rice, bran and husk with Se fertilizer spray. Se was mainly distributed in rice bran. The protein content could be significantly increased, compared with the control, without affecting grain yield and the content of starch, lipid and ash in rice. With Se fertilizer concentrations increased, the content of Cu, Hg, Pb and Cd in rice was significantly decreased, which indicated the possible metabolic antagonism between Se and heavy metals Cu, Hg, Pb and Cd. Interestingly, the certain concentrations of Se fertilizers are highly advantageous to the uptake of Mn and Fe by rice. The composition of amino acids in Se-enriched rice was similar to that of non-supplemented rice.3. The majority of Se found was organic Se, while only 2.48% of total Se was determined to be inorganic Se, in addition a minuscule amount was found in lipid fraction (0.03%). Nearly 54% of the total Se was metabolized into extracted proteins albumin-Se, globulin-Se glutelin-Se and prolamin-Se. Comparing the Se contents of the subsequent protein fractions, it was found that the glutelin portion contained the largest amount of Se with approximately 31.3% of the total Se in the rice. Therefore, dilute sodium hydroxide was applied to release the most Se-containing protein which was then analyzed with an orthogonal experiment. Based on our results, the suggested optimal conditions are a volume-weight of 20:1,0.08 M NaOH, an extraction time of 3 h and a temperature of 35℃to extract Se-containing protein 2 times, and then to precipitate protein under pH 5.4 for the highest extraction efficiency of Se-containing storage protein. A Se-containing rice protein product with 83.5% protein and 9.09μg/g Se was sequestered using the optimal extraction method. Using in vitro gastrointestinal digestion, this rice protein product with high molecular weight Se-containing protein can readily be digested to low molecular weight peptides and SeMet (52.3% of total Se in protein extract).4. A combination of a-amylase and protease XIV with ultrasonic bath was found to be optimal for the extraction of selenospecies, with an increased extraction efficiency of 92.8% and 88.7% for non-supplemented and Se-enriched rice, respectively. The duration for the enzymatic extraction is substantially shortened from 20 h to approximately 5 h, which helps prevent conversion of the Se species initially extracted, especially SeMet. Speciation and distribution of Se in Se-enriched rice and non-supplemented rice was evaluated by ion-paring reversed phase (IP-RP) and strong anion-exchange (SAX) chromatography coupled with inductively coupled plasma mass spectrometry (ICPMS) detection. The chromatograms obtained revealed that the major selenospecies found in Se-enriched rice was SeMet, which was further identified by nanoelectrospray ionization ion trap mass spectrometry (nanoESI-ITMS). Only 6.8% of inorganic Se as SeⅣand SeⅥwas found in Se-enriched rice extract with minor SeCys2 and trace SeOMet present. SeMet was increase from 26.7% of total Se in non-supplemented rice to 86.7% in Se-enriched rice, which was major metabolic Se species. SeⅣwas primary inorganic Se in Se-enriched rice, with little increase in inorganic Se compared with non-supplemented rice. In conclusion, due to the similarities between Se and S metabolic uptake, S can be replaced by Se in relation to the function of sulfate transporters and key enzymes of the S assimilatory pathway. Therefore, inorganic Se was uptake, translocated, and assimilated via rice leaf into grain, and finally storage as SeMet in rice protein.5. The recoveries of water extraction, NaCl extraction, NaOH extraction and EtOH extraction were 9.6%,16.8%,48.2% and 14.9% respectively. The purified protein binding Se was as following order:glutelin> globulin> albumin> prolamin. The molecular weights of four proteins were determined by SEC-HPLC-UV-ICPMS. The results showed that Se was mainly incorporated into glutelin and globulin higher than 7 kDa, while albumin and prolamin were not the most protein binding Se. The molecular weight of fraction F1 in glutelin was 199.8 kDa, which was collected, subject to trypsin digestion and analyzed by capHPLC-ICPMS and nanoHPLC-Chip/ITMS. Three Se-containing peptides with sequence and molecular weight were identified. Three possible rice proteins were searched by protein database according peptide fragment. Unfortunately, no SeMet fragments were evident in these proteins.