Separation And Identification And Antioxidant Function Of Anthocyanidins In Black Wheat Bran

Color wheat refers to the wheat has different color in the kernel pericarp or aleuronic layer. Color wheat consist of purple, black, blue, green and red pericarp ones of them. They are precious germplasm resources. Many cultivars color wheat have been proved to contain rich protein, amid acid and microelements or antioxidant activity material that are beneficial to humans. And there is plenty of natural pigment in the bran which has high application value and development prospects. This experiment with the bran of the Shandong Agricultural University self-cultivate black wheat for materials, optimized anthocyanins extraction technology and AB-8 type macroporous resin on the anthocyanins purification technology. Using UPLC/MS/MS separation and identification anthocyanins contained on the bran. Meanwhile to the anthocyanins stability and antioxidant activity is studied. The main research results are as following:(1) Bran pigment extraction technology research of black wheatUsing acidification ethanol solvent extraction anthocyanins of the black wheat bran, determined the best extraction process conditions for the anthocyanins of bran:the maximum absorption wavelength is 525nm, the extraction agents pH is 1.0 and ethanol volume fraction is 40 percent, the proportion of material and liquid is 1:20(g:mL), in 70℃constant temperature immersion extraction 60min, extraction twice the yield can reach 14.50%.(2) AB-8 type macroporous resin for the separation and purification of anthocyaninsThrough the AB-8 macroporous resin static adsorption and desorption experiment to anthocyanins of black wheat bran, optimize the codition of the adsorption and desorption, then preliminary analysis of purified pigments by HPLC meathod. The best conditions for it is that:Using pH is 1.0 and Abs is 0.600 pigment solution for adsorption, ambient reaction temperature, when 26h the AB-8 type macroporous resin reach to the adsorption balance, measured the maximum adsorption rate is 83.00%. Using pH is 1.0 and ethanol volume fraction is 70% solution for desorption, when 7h reach desorption balance and the rate of desorption is 85.53%.Through the AB-8 maqroporous resin column purification, the extractions of bran pigments colourity is 40.89, improved 9.127 times than crude extractings. It is preliminary separated and identified to purified pigment by HPLC, separated out four main ingredients which is identified as anthocyanins compounds.(3) Separate and identified component of anthocyanins in the branA systematic method for anthocyanins identification using HPLC-MS/MS-DAD was developed, including mass spectrometry full scan, daughter scan, parent scan, to isolation and identification of anthocyanins in black wheat bran. Black wheat bran were extracted by ultrasonically assisted extraction of anthocyanins with 50% acidification ethanol solution (alcohol:water:hydrochloric acid V/V=50:49:1), purification on SPE C18 was then performed. Identified 9 kinds of anthocyanins in black wheat bran:Cyanidin-glucoside, Cyanidin-rutinoside,Peonidin-glucoside, Cyanidin-malonyl-glucoside, Delphinidin-glucoside, Delphinidin-rutinoside, Malvidin-rutinoside, Peonidin-rutinoside, Petunidin-rutinoside. Among them, the 4 major of anthocyanins component:Delphinidin-rutinoside (26.07%), Delphinidin-glucoside (24.20%), Cyanidin-rutinoside (20.49%).(4) Study on anthocyanins stabilityThe pH values had a significant effect on anthocyanins. The anthocyan was stable below 60℃. Metal ions such as Mg2+,K+ andAl3+have good hyperchromic and stable effect on anthocyanins, Ca2+ and Fe2+ have no marked influence,Cu2+,Zn2+and Fe3+ have remarkably negative effect on it. Furthermore, this anthocyanin showed a poor oxidation and reduction resistance, but it do good resistance to sunlight. It was also found that conventional food additives such as sucros and citric acid did exert negative effects on the stability of the anthocyanins.Sodium benzoate of high concentration (>0.2%) for pigment has faded, while lower concentration (<0.1%) do not obvious effects. NaCl has certain hyperchromic effect and stable function to anthocyanin.(5) Research of the bran anthocyanin's antioxidant activityThe experiment evaluation in the bran anthocyanin's antioxidant capacity with ascorbic acid as comparison, through three kinds of in vitro antioxidant model:total antioxidant capacity, super free radical scavenging capacity and active oxygen scavenging capacity. The results show that:Compared withVc, the anthocyanin in the bran has higher activity in total antioxidant, super free radical scavenging and active oxygen scavenging. The total antioxidant activity is 83.689U/mL,2.07 times of Vc; super free radical scavenging activity is 2222.74 U/L and active oxygen scavenging activity is 4554.8U/mL.