Improvement Of Determination Of Carminic Acid And Cochineal Dye's Extraction And Quality

Cochineal (Dactylopius coccus Costa) dye can be used in food, medicine and cosmetics in the United States Food and Drug Administration. There are little research on the pigment in our country. Experiment parameters of the extraction of cochineal dye were optimized. For purification method, the method of ultrafiltration membrane and silica gel column chromatograph were used, and determine the main ingredient. The factors affecting pigment stability are discussed.The main results were as follows:(1) when mobile phase was consist of acetonitrile, methanol and phosphoric acid (0.01%) (volume ratio 10:15: 75), flow rate was 0.8 mL/min, column temperature at 20℃, peak shape was the best. This mothed used for determined car- minic acid both at 280 nm and 494 nm. Carminic acid(purity:100%) was utilized as standard sample, made standard curve, obtain regression equation Y=8.6048X, R²=0.9990, and Y=60.7360X,R²=0.9999, respectively. Both equa-tion's linear relationship were fine. when determine cochineal red pigment, the result showed that in this method, sam- ple had good stability(determined at 280 nm, RSD=0.16%; at 494 nm, RSD= 0.32%), equipment had high precision(determined at 280 nm, RSD=0.42%; at 494 nm,RSD=0.54%) and sample had high recovery rate(determined at 280 nm, recovery rate was 96.8, at 494 nm was 94.3).compare high-performance liquid chromatography (HPLC) and spectrophotometry to determine the purity of carminic acid samples, these two methods make quantitative analysis of 10 kinds of samples, the result of spectrophotometry measurements is slightly higher than high-performance liquid chromatography, but the results of the two methods no insignificant difference.Significant differences were exist between cochineal cultivated in middle area of Yunnan province and in Peru, except bulk density. But the carminic acid contents is high. The conclusion is that Cochineal cultivated in middle area of Yunnan province can also used for making carminic acid. Through compare cochineal of three generations, the results show that even there some difference existi in cochineal of three generations, but the difference of carminic acid contents are not significant, so cochineal of three generations can all used for make cochineal dye.(2) In order to develop a method for microwave-assisted extract cochineal dye, single-factor test was carried out. The results show that cochineals should soak in water for 40min before microwave treatment, and extract with water for 40min after microwave treatment. On the basis of single-factor test, the microwave-assisted extraction process parameters about extraction of cochineal dye were optimized with response surface method. The results suggest that the optimal parameters should as follows: ratios of liquid to material 7.5, microwave power 714W, microwave treatment time 73s. Under this conditon, the best extracting times is 12.In order to develop a method for microwave - ultrasonic synergistic extract cochineal dye, On the basis of single-factor test (included microwave power, treatment time, ratios of liquid to material and ultrasonic power), optimized extraction process parameters with response surface method, best condition should as follows: microwave power 464W, treatment time is 18.48min, ratios of liquid to material 8.23, ultrasonic power 627W. Under this conditions, the suitable extraction times is 4.Compare the microwave-assisted extract mathod and microwave-ultrasonic extract mathod, we can find out that use microwave-assisted extract mathod, energy consumption are lower than microwave-ultrasonic extract mathod. But time consumption is longer, and extraction times is more than microwave-ultrasonic extract mathod.(3) Utilize the papayin hydrolisis cochineal dye, then penetrates the cochineal dye through the 100KDa, 30KDa, 10KDa, 3KDa and the 1Kda ultra filter. Determines the volume of different solution, and determines the carminic acid concentration, the azotic material concentration; And compare with cochineal dye that has not treated with the papayin, the results show that after the papayin hydrolisis, in the cochineal dye, macro-molecule protein degenerated in some extent, because in the cochineal dye, much of sensitization material are the macro-molecule protein, therefore, the papayin hydrolisis can eliminates the sensitization protein of cochineal dye.(4) To assess the free radical scavenging capacity of carminic acid, the research was carried out to investigate the free radical (including DPPH·, ABTS+·and O₂ˉ·) scavenging activity of it, make comparison of free radical scavenging activity between carminic acid and ascorbic acid. The result showed thatBoth carminic acid and ascorbic acid have free radical scavenging activity for DPPH·, ABTS+·and O₂ˉ·. Carminic acid's can clean away 83% DPPH·in solution, 85% ABTS+·in solution, 46% of O₂ˉ·in solution. It's show that carminic acid is a food addictive which has free radical scavenging activity.