The Research On Protein Hydrolysis And The Antioxidative Peptide Derived From Hempseed (cannabis Sativa L. )

Hempseed (Cannabis sativa L.) has been used traditionally for edible and medicinal purposes for thousands of years in China. Hempseed typically contains 20%-25% protein, 25%-30% oil, 10%-15% insoluble fiber, and are rich in vitamins and minerals. Albumin, a globular protein, and edestin, are the two main proteins in hempseed. Both are rich in the essential amino acids, which are vital to human health.Also, hempseed protein are exceptional rich in such as lysine, arginine and glutamic acid. These two high quality proteins are easily digested. It can be concluded that hempseed is an excellent new plant protein source.This investigation focuses on the radical scavenging ability evalution of hydrolysates and peptides obtained from sequential enzymatic hydrolysis and liquid fermentation by Bacillus subtilis on hempseed protein. Our goals are not only to produce hydrolysates and peptides with higher antioxidative abilities by optimizing the hydrolyzing conditions, but also to discover the potential applications so as to enhance the biological and economical value of hempseed. The main results are as per following:After extracting the oil with organic solvent by ultrasound, hempseed protein was extracted by alkali from the hempseed meal. It clearly indicated that ultrasonic can be improve the yield of crude protein significantly. The acting conditions are; power 200 W, solid-to-solvent: 1:10(g/mL), on-off ratio: 20:20 s/s, time: 25 min. Using orthogonal experiment design, the conditions for best protein yield are; pH 8.5,acting temperature 45℃,solid-to-liquid: 1:12(g/mL),acting time: 60 min. The yield of crude protein was 86.1%,and the protein content of which was 86.6%. The yield was higher about 12.2% and the extraction time less 1/3 than the control.The effect of six single enzymes, multistep enzymatic hydrolysis, and Bacillus subtilis liquid fermentation hydrolysis of hempseed protein were respectively studied in this review. It was found that the hydrolysates by alcalase has better free radical scavenging ability (DPPH, HO·radical scavenging ability, reducing power, Fe2+chelating ability) than the others. In multistep enzymatic hydrolysis, the best result was hydrolysis by alcalase for 5 h, followed by Papain 3 h.This results in hydrolysates with overall superior free radical scavenging abilities relative to other combinations and/or single enzymatic hydrolysis. The production of anti-oxidative peptides by liquid fermentation of Hempseed protein by Bacillus was also evaluated in this paper. The best liquid fermented conditions, was obtained through orthogonal matrix experiment procedures, results were: initial pH 7.0, substrate concentration 5% (w/w), brown sugar power concentration 1.5% (w/w), fermentation temperature 37℃, speed of rotation 180 r/min , MnSO4. H2O 0.1% (w/w), MgSO4.7H2O 0.04%, CaCl2.2H2O 0.04% and NH4Cl 0.1%, from which peptide yield reached the highest, at 50.87%. The liquid hydrolysates with overall superior radical scavenging ability were those obtained within the 42 h-54 h fermentation timeframe. Among them, the optimum is a fermentation period of 48 h, which has the best DPPH, HO·, O2·- radical scavenging and lipid peroxidation inhibition activity.Comparing hydrolysates obtained from either enzymatic of fermentation hydrolysis, Hydrolysates at 20 mg/mL can protect against the formation of methemoglobin by free radicals for periods of about 60 min. Also, Raman spectroscopy was used to measure the effect of sodium nitrite in converting Fe2+ into Fe3+. Both enzymatic hydrolysates and fermented hydrolysates 2 % (w/w) showed remarkable inhibition of the oxidation products of chicken oil, namely peroxides and malondialdehyde. Also, the test showed that the citric acid has a synergistic effect on the aforementioned hydrolysates. It was shown hydrolysates 4 mg/mL were clearly protection DNA which demadged by radicals. It is clearly shown that the fermented hydrolysates have a remarkably high content of arginine, which account for 4.44% of the total mass.In order to evaluation the radical scavenging ability of hydrolysate and its purified peptide, IC50 value used for comparison. The purified petide has a better ability than its hydrolysate, and the fermented hydrolysate was better than enzymatic hydrolysate. IC50 value of DPPH, HO˙,O2·- ,scavenging ability和Fe2+ chelating ability of fermented petide (F2) and enzymatic peptide were 2.01 mg/mL,0.6 mg/mL, 3.21 mg/mL, 1.12 mg/mL and 1.73 mg/mL, 0.44 mg/mL, 2.89 mg/mL, 0.88 mg/mL respectively. Finally, two potent antioxidative peptide were isolated and their sequence were identified by W-TOF ESI mass spectroscopy.Purified peptide from enzymatic hydrolysates (E2):Phe-Leu-Lys-Leu-Thr-Ala-Glu-Arg (MW=997.4924 Da)Purified peptide from fermented hydrolysates (F2):Arg-Ser-Arg-Lys-Gly-Phe-Glu-Trp-Leu-Arg-Val-Lys (MW=1306.7063Da)These two peptides were structure specially and have a good inhibition of lipid peroxidation.