Study On The Alfalfa Leaf Protein And Preparation Of Antioxidant Peptides By Enzymic Hydrolyzing

Alfalfa leaf protein concentration (ALPC) extracted from alfalfa leaves by extracting, separating, concentrating and drying. ALPC as a sort of function protein mainly consists of matrix protein and cytoplasmic proteins with good solubility. It is considered by FAO (Food Agriculture Organization) as a new protein source valuable for further utilization.Researches and explorations of alfalfa leaf protein globally at present are still on the stage of extracting and application, without sufficient study on function, hydrolysates, and amino acid components. Bioactivities of hydrolyzed peptides are rarely reported. Therefore researches and development of bioactive peptides using alfalfa leaf protein is of theoretical and practical significance.Firstly, optimal process and parameters for extraction of soluble alfalfa leaf protein concentration (SALPC) from the compacted alfalfa leaves was studied by adding water, adjusting pH and marinating time, with a prerequisite of non-denaturalization of protein. The optimal condition for SALPC were a ratio 1:7 for material to liquid, an alkali pH9.0, a time of 50 minutes for marinating and an acidic pH4.0. The order of factors influencing protein extraction yield were the acidic pH > the alkali pH > marinating time > ratio of material to liquid. The extraction yield and protein content was 57.87% and 87.38% respectively at the optimal conditions. The SALPC nitrogen soluble index (NSI) was up to 77.32% at pH6.0～10.0Secondly, this project studied the enzymatic hydrolysis techniques. Five types of proteases were selected by comparing antioxidant activity and hydrolysis degree (DH) in vitro. The optimal protease is Alcalase FG 2.4L. The optimal hydrolytic conditions for SALPC was temperature 60℃, pH8.0, concentration of SALPC [S] 5% (w/v), [E]/[S] 4.0% by single-factor tests. The relative molecular weight of alfalfa leaf protein hydrolysates (ALPHs) was mostly under 5000, of which 93.03% under 3000 and 67.86%under 1000. ALPHs had good scavenging activity against DPPH radical.ALPHs were ultrafiltrated due to the multi-components in the SALPC produced with the optimal enzymatic process. The processing of hydrolysis inevitably brought in some salts that may influence the quality of the finished products and the analysis of the bioactivity of ALPHs. The effects of concentrations of ALPHs, pressure and temperature on ultrafiltration permeation were investigated. The results showed that the optimal conditions for ultrafiltration were pressure of 0.35MPa, temperature of 25℃, concentrations of ALPHs 35g/L . The relative molecular weight distribution of ALPHs in 1000～500,500～130 and under 300 increased respectively from 21.85%, 21.64% and 4.91% to 27.91%, 30.83% and 7.61%, after ultrafiltration using polysulfone membrane with 3000 relative molecule weight. The scavenging activity of ultrafiltered. ALPHs against DPPH radicals was increased because its hydrophobic amino acid content increased from 32.00% to 33.98%. The types of macroporous resin were selected by comparing their performances in adsorbing and desorbing, and the optimum absorbent material for ALPHs was DA201-C. Dynamical adsorption-desorption trials showed that desalt ratio of ALPHs reached 92.21% at 0.5 BV/h flow injecting with a 50 mg/mL concentration, 1 BV/h flow water washing and 1.5 mL/min of 75% ethanol desorbing. The hydrophobic amino acid content in ALPHs increased from 33.98% to 36.69% and the scavenging activity of ALPHs against DPPH radical was enhanced.The antioxidant activities of ALPHs evaluated using six different antioxidant tests in vitro had stronger and significant linear relationship with concentration. So ALPHs was a good electron and hydrogen donator. It can clearly inhibit peroxidation in linoleic acid system, with a strong scavenging activity against O2-·,·OH and DPPH·radicals. It can chelate well the Fe2+. ALPHs can get the same antioxidant effect when the concentration is as 3 to 5 time as glutathione.Good antioxidant effects of ALPHs in mice were proved sufficiently by trials on mice in vivo. that ALPHs was able to decrease significantly the content of MDA in mice (P<0.01), increase the activity of GSH-Px and SOD (P<0.01) in mice serum, heart and especially liver.ALPHs was divided into four fractions (A, B, C and D) by gel filtration on Sephadex G-15. The antioxidant and free radical-scavenging activities of the four fractions were measured using reducing power and DPPH/superoxide/hydroxyl radical scavenging assay. The antioxidant activity of ALPHs-D was the strongest and the other three were listed as B>C>A. The ALPHs-D was separated into 10 components in which the ALPHs-D-6 was a purified compound that had stronger antioxidant activity than others. The ALPHs-D-6 purified by using consecutive chromatographic methods had a molecular mass 521.2 Da and the amino acid sequence was identified as Glu-Tyr-Asp-Pro by amino acid analysis and MALDI-TOF-TOF MS/MS.