Study On Extraction,purification,stability And Interaction With Protains Of Anthocyanins From Blueberry Wine Lees

As natural products,anthocyanins have many physiological functions such as scavenging free radicals,anti-oxidation,anti-tumor,anti-inflammatory,improve memory and so on.Which were the hot spots of research in recent years.Blueberry wine lees,which is the byproduct of winemaking,rich in anthocyanins.Taking advantage of blueberry wine lees and extracts the active ingredient in it,Which has a role of turning waste into treasure,and has certain economic benefits.In this paper,we took blueberry wine lees as our research target,extracted and purified the anthocyanins of it,studied the stability of the anthocyninis as well as the interaction with 3 kinds of proteins.In order to provide some theoretical basis of the applications in industry for anthocyanins in blueberry wine lees.Specific contents and results are as follows:1.The optimum extraction conditions of anthocyanins from blueberry wine lees.Selection of solvent extraction,single factor experiments such as concentration of ethanol,pH,time,temperture and solid to liquid ratio and orthogonal test were studied to obtain the best extraction process.Concents of anthocyanins were the evaluation basis.Then macroporous resins were used to purify the extract of blueberry wine lees.Through the single factor experiments such as the selection of column materials,static adsorption time,static desorption time,pH of adsorption and desorption,concentration of ethanol to determin the best purification process.Then the concents of total phenol,anthocyanins,flavonoids in the extract were determined and compared with the extracts of blueberry and blueberry wine.The results show that:The best extraction process was 50?,pH3.0,120 min,solid-liquid ratio l:20?m/v?and ethanol concentration of ethanol was 70%.The influence of extraction effects were The ethanol concentration>solid-liquid ratio>extraction time>extraction temperature>pH.The best purification process was optimum column materials for XAD-7HP,static adsorption time was 240min,desorption time was 60min,ethanol concentration 80%,adsorption and desorption pH were all 2.5.The concent of anthocyanin in extract of blueberry wine lees was 151.12±0.88 mg/g,total phenol was 436.67 ±6.80 mg/g,flavonoid was 253.03±12.13 mg/g.2.The stability of extracts from blueberry wine lees.Stability and copigmentation effect of the anthocyanins from blueberry wine lees after extraction and purifition were studied,including temperature,pH,metal ion,oxidant H₂O₂ and reducing agent Vc,LSPC and LSOPC.Putting the absorbance as evaluation basis.The results show that anthocyanins of blueberry wine lees were much more stable under 40?,the optimum pH was 1.0,H₂O₂ had a bad influence on the stability but Vc could improved the color and stability of anthocyanins.Al³⁺ could significantly enhanced the stability while LSOPC had no influence on it;The combined effects of Al³⁺ and LSOPC could obviously improved the stability during a short period of time?8d?and the effect was better than Al³⁺ alone,but it was not as good as Al³⁺alone during a long time?30d?.In terms of copigmentation,high concentrations of Al³⁺?10mol/L?and Sn²⁺?l?5mmol/L?had a good influence on the copigmentation,Mn²⁺ and K⁺ had a bad influence on the copigmentation and it was proportional to the concentration.Mg²⁺ had no influence on the copigmentation.LSPC also had a good influence on the copigmentation.The combined effects of Al³⁺ and LSPC had a obviously increaset on the copigmentation and effect was better than the two separate role.3.The identification,isolation and antioxidant activity of anthocyanins from blueberry wine lees,blueberry and blueberry wine.HPLC-ESI-MS was used to identify and compare the composition of extracts from blueberry wine lees,blueberry and blueberry wine.And the antioxidants such as Scavenging DPPH and ABTS radical,reduction ability of extracts from blueberry wine lees and blueberry were also evaluated.carboxymethyl dextran resin Sephadex LH-20,HSCCC and semi-preparation HPLC were used as a further purification for the extract from blueberry wine lees.The eluent of Sephadex LH-20 method was 10%methanol contain 2%formic acid,the solvent system of HSCCC was water/n-butanol/methyl tert-butyl ether/acetonitrile/trifluoroacetic acid?v/v,6/3/1/1/0.001?,the eluent of semi-preparation HPLC was formic acid/acetonitrile/water.The results showed that 11 kinds of anthocyanins of the extracts from blueberries were identified,there were no acidylated anthocyanins.12 kinds of anthocyanins of the extracts from blueberry wine lees were identified,containing 3 kinds of anthocyanins which were acidylated?cyanidin-3-acetyl-glucoside,cyanidin-3-acetyl-rhamnoside,delphinidin-3-acetyl-arabinoside?.9 kinds of anthocyanins of the extracts from blueberry wine were identified,containing one kinds of anthocyanins which were acidylated?petunidin-3-acetyl-rhamnoside?,and the highest content of anthocyanins from the extracts of blueberries,blueberry wine lees and blueberry wine was all malvidin-3-hexose.The IC50 values of scavenging effects on ABTS radical of anthocyanidins from blueberries and blueberry wine lees were?1.43±0.02?and?1.19±0.03??g/mL respectively.The IC50 values of scavenging effects on DPPH radical were?3.28±0.03?and?2.41±0.01??g/mL respectively;Scavenging effects on radical and reduction ability of the anthocyanins from blueberris and blueberry wine lees were much more better than Ve.The results indicated that the anti-oxidation activity of anthocyanidins in blueberry wine lees was much more better than blueberries.The method of Sephadex LH-20 could obtained the monomer of malvidin-3-hexose?containing malvidin-3-galactoside and malvidin,3-glucoside?,and the purity was 92%approximately.82%purity of malvidin-3-arabinoside was obtained by HSCCC.Through semi-preparation HPLC,4 kinds of monomers of anthocyanins were obtained,and the purity were all above 90%,containing delphini din-3-glucoside,petunidin-3-glucoside,malvidin-3-arabinoside and malvidin-3-acetyl-hexose?containing malvidin-3-acetyl-galactoside and malvidin-3-acetyl-glucoside?.However,the three methods are all unable to separate the isomers of malvidin-3-galactoside with malvidin-3-glucoside and malvidin-3-acetyl-galactoside with malvidin-3-acetyl-glucoside.4.The interaction of malvidin-3-hexose and malvidin-3-acetyl-hexose with proteins.Fluorescence spectroscopy and circular dichroism were used to studied the interaction of malvidin-3-hexose and malvidin-3-acetyl-hexose with BSA,trypsin and cellulase.Comparing the different interaction between acidylated and unacidylated malvidin.It turmed out that the interaction between the 2 kinds of anthocyanins and the 3 kinds of proteins all came into being by the style of static quenching,and there were little or no effect on the conformation of these protains.The forces of acidylated and unacidylated malvidin with BSA were all hydrophobic effects.The binding site of malvidin-3-hexose with BSA was site I and both site I and II were the binding sites of unacidylated malvidin with BSA.While the Ka of malvidin-3-acetyl-hexose with BSA was much more bigger than malvidin-3-hexose,showing that the scope and degree of interaction between malvidin-3-acetyl-hexose and BSA were deeper.Both malvidin-3-hexose and malvidin-3-acetyl-hexose had no influence on the secondary structure of BSA.The forces of acidylated and unacidylated malvidin with trypsin were not the same,which were electrostatic force and hydrophobic effects,respectively.While the Ka of acidylated malvidin with trypsin was much more bigger than unacidylated malvidin,and the acidylated malvidin had greater influence on the secondary structure of trypsin.The forces of acidylated and imacidylated malvidin with celluase were all electrostatic force.While the Ka of unacidylated malvidin with cellulase was much more bigger than acidylated malvidin,but the influences on secondary structure of cellulase for acidylated malvidin were greater than unacidylated malvidin.