Separation And Identification And Properties Study Of Anthocyanins In Color Wheat Bran

This experiment with the bran of color wheat by Henan for materials, optimized bran anthocyanin extraction condition and macroporous resin purification technology, using the high performance liquid chromatography with diode array detector and tandem mass spectrometry separation identification and quantitative analysis on the anthocyanin in bran and explored the stability of anthocyanin, the oxidation resistance and thermal degradation characteristics. The results are as follows:Firstly, the extraction condition of color wheat bran anthocyanin was optimized by ultrasonic assisted solvent to extract to determine the best extraction conditions: the pH=1.0 with the volume fraction of 80% ethanol as extraction solvent, solid-liquid ratio 1:15(W/V), extracted 80 min, and extracted three times.Secondly, using the high performance liquid chromatography with diode array detector and tandem mass spectrometry technology, through the anthocyanins liquid chromatography, mass spectrometry full scan and secondary spectrum scanning separation identification and quantitative analysis on this five kinds of color wheat bran anthocyanin. The study found that the content size of bran anthocyanin in five kinds of color wheat: LM6(85.97 mg/kg)>ZH(47.93 mg/kg)>ZL2(33.68 mg/kg)>ZL1(29.32 mg/kg)>LM1(26.15 mg/kg). 9 kinds of anthocyanin was identified in five kinds of color wheat bran, cyanidin-3-glucoside and cyanidin-3-rutinoside are the anthocyanin component which the five kinds color wheat all contain. The anthocyanin component of ZH and LM1 is similar, including cyanidin-3-glucoside, cyanidin-3-rutinoside, peonidin-3-galactoside and peonidin-3-glucoside etc, among them, the content of cyanidin-3-glucoside is highest, 44.24% of the total content of anthocyanin in ZH, 53.71% in LM1, is the first major anthocyanin for the two color wheat. The anthocyanin component of ZL1、ZL2 and LM6 is similar, mainly consist of delphinidin and cyanidin anthocyanin, including delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside, among them, the delphinidin-3-glucoside is the first major anthocyanin for ZL1 and ZL2, the proportion is respectively 36.64% and 34.27%, while in LM6, the delphinidin-3- rutinoside is the first major anthocyanin(35.75%) and delphinidin-3-glucoside is the second major anthocyanin.Thirdly, the pH,temperature, metal ion and Vc etc additive affecting on the stability of anthocyanin extracted liquid for color wheat bran were researched using UV-vis spectroscopy technology in the experiment. The results have shown that color wheat bran anthocyanin is more sensitive to the change of pH and temperature and stable under low temperature(65 ℃ or less) and acidic conditions. When the metal ion concentration is 2.0mmol/L, K+, Na+ and Ca2+ have certain stable and hyperchromic effect on anthocyanins, Zn2+ and Ba2+ have little effect on the stability of anthocyanins, and Fe3+ and Cu2+ fade the anthocyanins solution obviously. Vc has obvious fade effect on the anthocyanin, a certain concentration of citric acid(0.5% or higher) and sucrose(5% or higher) have faded effect on the anthocyanin, and the salt has certain stability hyperchromic effect.Fourthly, the anthocyanin of in color wheat bran was purified by AB-8 macroporous resin and ethyl acetate successively, optimized the purification condition of the macroporous resin: using static adsorption and desorption experiment, the best sample initial concentration of anthocyanin crude product is 75mg/mL, the best eluent solvent is 60% ethanol solution; the anthocyanin liquid of 320 mL, resin adsorption reach saturation; 180 mL elution solvent will elute anthocyanin completely in the sample flow rate of 2 mL/min and the elution flow rate of 1 mL/min. The purity of anthocyanin samples after purification is 4.23 mg/g, compared with the beginning, purity increases 9.04 times, and purification effect is better.Fifthly, the color wheat bran antioxidant capacity was evaluated by anti DPPH free radical, anti hydroxyl free radical, anti superoxide anion radical,three kinds of in vitro antioxidant model, and compared the difference of antioxidant effect of anthocyanins sample for color wheat bran before and after purification. The study found that anthocyanins purification samples effect on the DPPH? clearing is better than that of crude products, when sample concentration is 3.0 mg/mL, the clearance rate reaches 84.45%. For ?OH and O2-? scavenging effect, anthocyanins coarse sample removal effect is superior to the purification, concentration of 3.0 mg/mL, anthocyanins crude products clearance rate on ?OH and O2-? is as high as 99.44% and 99.44% respectively.At last, the thermal degradation properties of anthocyanins extacted liquid for color wheat bran were studied using UV-vis spectroscopy technology. The results have shown that anthocyanins thermal degradation in color wheat bran fits pseudo-first-order kinetics reaction model, and in different pH conditions, the required activation energy in color wheat bran anthocyanin thermal degradation from big to small orderly is pH 2.2(66.76KJ/mol)>pH 3.0(53.22 KJ/mol)>pH 3.8(46.34 KJ/mol). The rye bran anthocyanin at pH 2.2 shows higher thermal stability.