Research On Antioxidant Activity Of Silkworm Pupa Peptides And Purification Of Antioxidant Peptides

Many chronic diseases and human aging phenomenon have something to do with the imbalance of free radicals inside human body. External antioxidants can help people to maintain the balance of free radicals inside body. It's possible to develop effective antioxidant drugs and health foods on the base of the research, screening and modification of natural foods with antioxidant activity. Silkworm pupa is a high quality protein resources, containing 18 amino acids, including 8 essential amino acids, the content of which is more than 40%。The protein content of pupa is about 50%.It is more nutritional than soybean protein whit high quality protein resources.In this paper, the detection methods of antioxidant activity of silkworm pupa protein peptides, the enzymatic hydrolysis conditions of silkworm pupa protein, and the purification of peptides with DPPH radicals scavenging activity of silkworm pupa protein peptides were investigated.1. Degrease of silkworm protein powder and pretreatment: Selecing the conventional solvent n-hexane, acetone, ethyl acetate, and ethanol as a single degreasing solvent to get a optimized degreasing rate, the best degreasing solvent aceton is acetone. The protein powder was crushing by three metod of ultrasound crushing, microwave crushing and soaking deionized water. With the crushing of protein content in the supernatant as an index, the best conditions for ultrasonic crushing is 100HZ, interval 2sec, 10min.2. Detection methods of vitro antioxidant activity of silkworm pupa protein powder peptides were established. Antioxidant capacity of silkworm protein powder fowl protein were detected, including scavenging activity of super oxide anion, hydroxyl radicals, and DPPH free radicals, reductive ability and ability to prevent lipid peroxidation. Experimental results showed that silkworm protein powder peptides showed some antioxidant activity in different detection systems. Scavenging activity of DPPH free radicals was the highest. It showed good prospects in development.3. Further enzymatic hydrolysis technics of silkworm pupa protein was determined. After single enzyme hydrolysis experiment as DPPH free radicals scavenging for target, the best enzyme was pepsin. After L16(45) orthogonal test, the optimum conditions of pepsin digestion were temperature 50℃, initial pH1.5, 8000U/g of E/S, 6h of enzymatic hydrolysis time, 4% of substrate concentration.4. Antioxidant peptides were purified as hydroxyl radical scavenging ability for target. The best Sephadex G-25 gel chromatography separation conditions of silkworm pupa protein peptides were determined: eluted with deionized water, 0.2mL/min elution speed, 10mg/mL of sample concentration, and 0.50mL sample. Silkworm pupa protein peptides could be separated into three peaks, among which the three peak had the highest hydroxyl radical scavenging activity. Use Deae filler for ion exchange, the third peak is mainly a peak A. Peak A in HPLC analysis further showed that it is a single substance. It is a molecular weight of 892 Da tyr-containing peptides by mass spectrometry.