| Anthocyanins are a hot spot in the study of natural active substances and have been widely used in many fields such as food and health care products.Blueberries are a popular fruit that contain high amounts of anthocyanins in their pulp and peel.As a by-product of fruit juice and fruit wine processing,blueberry pomace were mainly composed of peel and blueberry seeds.It was usually used as animal feed or directly discarded,which wasted resources,polluted the environment and decreased the utilization rate.According to reports,blueberry pomace is rich in anthocyanins,which have high development value.In this project,blueberry pomace was used as raw material,anthocyanins was extracted from ultra-high pressure and purified by AB-8macroporous resin combined with ultrafiltration tube.The anthocyanin components were analyzed by high performance liquid chromatography(HPLC).Further research on the biological activities of blueberry pomace anthocyanins will lay a theoretical foundation for the comprehensive development and utilization of blueberry pomace.The main research contents and conclusions are as follows:1.Study on the optimization of ultra-high pressure extraction process for anthocyanins from blueberry pomace.The results show that the optimal extraction process was as follows:pressure of 400 MP,extraction time of 9 min,ethanol concentration of 60%,solid-liquid ratio of1:20(g/m L).Under these conditions,the yield of anthocyanins was 5.93±0.06 mg/g.Compared with traditional solvent extraction(CSE),the extraction effect of ultra-high pressure-assisted extraction(UPE)was better,and the yield of anthocyanins was increased by 25.11%.2.Purification and component analysis of anthocyanins from blueberry pomace.The optimum resin was screened from NDS-17,HPD-300,NKA-9,AB-8 and HPD-400.The results show that AB-8 has the highest adsorption capacity and higher desorption capacity,and the optimal purification conditions were as follows: the adsorption time was 4 h,the desorption time was 1 h,the eluent was 80% ethanol solution,and the loading/elution flow rate was 1.5 m L.Under this condition,the freeze-dried powder of anthocyanins with a purity of 22.08% was obtained,and its purity was increased by 19.71 times compared with that before purification.After preliminary purification by macroporous resin and further purification by ultrafiltration tube,blueberry pomace anthocyanins with a purity of 52.93% were obtained,and the purity was increased by 2.40 times.The components were analyzed by HPLC,and the results showed that blueberry pomace anthocyanins contained 13 different anthocyanin monomers,among which the content of malvidin-3-galactoside was the highest.3.Study on antioxidant activity and antibacterial activity of blueberry pomace anthocyanins.The in vitro antioxidant activity of blueberry pomace anthocyanins was analyzed by·DPPH scavenging rate,·OH scavenging rate,and FRAP iron reduction ability.The results showed that blueberry residue anthocyanins had the strongest scavenging ability to ·DPPH,and there was no significant difference compared with the same concentration of Vc.The scavenging rate of ·OH and FRAP iron reduction ability were significantly weaker than those of the same concentration of Vc.The antibacterial activity of blueberry anthocyanins against Escherichia coli(E.coli),Salmonella enteriditis(Se),and Listeria monocytogenes(Lm)was analyzed.The results showed that the minimum inhibitory concentrations of blueberry pomace anthocyanins on Se,E.coli and Lm were 5 mg/m L,5 mg/m L and 10 mg/m L,respectively.When the anthocyanin concentration was 10 mg/m L,the diameters of the inhibition zones were 20.07±1.10 mm,19.93±1.90 mm,and 17.47±1.50 mm,respectively.It indicated that blueberry pomace anthocyanins had certain antioxidant activity and antibacterial activity.4.Effects of blueberry pomace anthocyanins on the proliferation,apoptosis and autophagy of HSC-T6 cells.MTT assay,lactate dehydrogenase(LDH)assay,flow cytometry assay,Hoechst33258 staining,transmission electron microscopy,acridine orange staining and Western blot analysis were performed.The results showed that anthocyanins inhibited the proliferation of HSC-T6 cells,and the inhibitory effect was stronger with the increase of concentration and time.Hoechst 33258 staining showed the typical apoptosis feature of HSC-T6 cells treated with blueberry pomace anthocyanins.The apoptosis rate was detected by flow cytometry,it was found that when the concentration of blueberry pomace anthocyanins was 100 μg/m L and 200 μg/m L,the apoptosis rates were 6.56%±0.25% and 19.53%±1.96%,respectively,which were 1.55 and4.61 times higher than the control group.Observation under transmission electron microscope showed that compared with the control group,the concentration of anthocyanins in blueberry pomace increased,and the autophagic vesicles of HSC-T6 cells increased.Acridine orange staining showed that the number of lysosomes in HSC-T6 cells increased significantly with the increase of anthocyanin concentration.The results of Western blot analysis showed that when the concentration of anthocyanins in blueberry pomace reached 100 μg/m L,the protein expressions of pro-apoptotic proteins Bax and Caspase-3 increased(p < 0.05),which were 1.41 times and1.25 times higher than those of the control group,respectively;The anti-apoptotic protein Bcl-2showed a downward trend(p < 0.05),which was 1.64 times lower than that of the control group,indicating that blueberry pomace anthocyanins had significant proliferation inhibition and apoptosis induction effects on HSC-T6.Compared with the control group,after the cells were treated with 200 μg/m L blueberry pomace anthocyanins,the expression levels of autophagy-related proteins Beclin-1 and LC3-II were increased by 2.30 times and 2.53 times,respectively,and the expression of p62 protein was significantly decreased by 2.19 times compared with the control group,indicating that blueberry pomace anthocyanins significantly promote autophagy in HSC-T6 cells. |
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