| Selenium is an essential trace element for human beings, and has important biological functions and has a close relationship with the illness, especially tumor and angiocardiopathy, which can severely endanger the health. Garlic (Allium Sativum L.) has been long known to be the good value of multi-medicinal plants and spices, and have a strong selenium bioaccumulation ability which can absorb 78.24% inorganic selenium into organic selenium.In this paper, selenium-enriched garlic from Enshi high-selenium areas was studied as raw material, the studies as follows:1. Study on the determination of selenium in garlicBased on previous studies , digestion conditions, fluorescent material to generate the conditions and equipment conditions of molecular fluorescence spectrometry DAN method are discussed and improved, the optimized method has high sensitivity and good accuracy, the linear range is for 0μg/mL～0.4μg/mL, correlation coefficient is 0.9999, the relative standard deviation is 3.92%, and recovery is for 91.8%～99.8%, detection limit is 1.57ng/mL.2. Extraction and separation of selenium-containing proteins fromSe-enriched garlicFirst of all, the major composition and the mineral elements of Se-enrichedand Se-anenriched garlic from Enshi and Shangdong , respectively were analyzed.The results show that two kinds of garlic powder rich in protein content, reach more than 13.00%, second only to the total soluble sugar content, and is not very different from other ingredients, but Se content of Se-enriched garlic powder from Enshi is 9.844μg /g, is about 15 times as the ordinary garlic powder from Shandong (0.6678μg / g).Second, according to different solubility of proteins, different types of proteins were extracted from selenium-enriched garlic by continuous extraction method, and determined the protein content and selenium content of four kinds of protein sub-class. The results showed that water-soluble protein is the main protein of the protein in selenium-enriched garlic, were 62.24% among total soluble protein, followed by alkali-soluble protein, the salt-soluble protein and ethanol-soluble protein. The selenium content of the four extracts decreased following order of water-soluble protein> alkali-soluble protein> salt-soluble protein> ethanol-soluble protein. The selenium content of Water-soluble protein is about two times as the total selenium contents of the other three proteins. So water-soluble protein from selenium-enriched garlic was selected as the object for the further research.The extraction technology of water-soluble protein from selenium-enriched garlic was studied. According to the single-factor tests and orthogonal experimental methodology, the results showed that the optima conditions of extraction were as follows: magnetic stirring extraction, pH value=7 and the extraction was taken at 4℃for 4h, the ratio water of to raw material was 35:1(v/w), then the extraction was repeated twice. Under these optimized conditions, the extraction rate of water-soluble protein was 24.38%, in which the content of selenium was 22.52μg/g. When the saturation of ammonium su1fate was selected as 80% for the protein depositing, the rate of depositing protein was 89.32%.Finally, water-soluble crude protein from selenium-enriched garlic and water-soluble crude protein from ordinary garlic were taken by chemical composition analysis, amino acid analysis, isoelectric point determination, SDS-PAGE denaturing gel electrophoresis and Native-PAGE electrophoresis analysis, FT-IR and DSC analysis of the preliminary identification.3. Preliminary purification of water-soluble protein from selenium-enriched garlicWater-soluble crude protein from selenium-enriched garlic were preliminary purified into three protein peaks(a, b, c) by Ion-exchange cellulose (DEAE-52) combining molecular excluding chromatography (Sephadex G-100), which peaks b has the highest protein concentration, selenium content and scavenging effects of hydroxyl radical. SDS-PAGE electrophoresis showed that peak a mainly contains a Alliinase protein subunit band which the molecular weight was 53.8kDa, Peak b contains 4 protein subunits which the molecular weight focuses on between 31.0kDa and 43.0kDa; the peak c has a molecular weight of about 30.0kDa protein subunits, and three protein peaks of the protein are more dissociation into a small molecule peptide chain. 4. Antioxidant activities in vitro of selenium-containing proteins from selenium-enriched garlicIn the different systems of antioxidant activities in vitro, four different solubility types of crude proteins from selenium-enriched garlic and ordinary garlic showed a good dose - effect relationship of scavenging effects on the hydroxyl radical, superoxide anion, hydrogen peroxide and protective effects on DNA damages, and scavenging effects on the DPPH organisms free radical is in general. The antioxidant activities from weak to strong in the following order: ethanol-soluble proteins <salt-soluble proteins < alkali-soluble proteins <water-soluble proteins. The antioxidant activities of crude protein fractions from Selenium-enriched garlic were higher than the corresponding crude protein fractions from ordinary garlic. And selenium-containing proteins from selenium-enriched garlic have a good reduction effect on the generation of MDA and peroxide content in liver of rats. Through the correlation analysis between the antioxidant activity and the selenium content, there was significant or extremely significant positive correlation.5. Studies on relationship between the stability and antioxidant activity of selenium-containing proteins from selenium-enriched garlicThe effects of temperature, pH value, sugar, neutral salt, NaCl, metal ions and urea denaturant on the stability of water-soluble selenium-containing proteins from selenium-enriched garlic and the changes of antioxidant activities were preliminarily studied.It provides a theoretical basis to use. Also for an in-depth study of more detailed protein structural information and functional activity of selenium-containing proteins from selenium-enriched garlic, it has laid a good foundation. |
