Study On Pupa ACE Inhibitory Peptides Preparation And Its ACE Inhibitory Activity

Tussah(Antherea pemyi Guerin,-Meneville)called silkworm is native to China and feed on oak leaves,which is an economic insect among Lepidoptera,Saturniidae. Silkworm pupa, also known as " Little Bee children, is a developmental stage in the life cycle of the silkworm, is the main byproduct of the silk reeling industry. China is the world’s leading silk producer, can produce annually 500,000 tons of fresh silkworm chrysalis. China State Economic and Trade Commission, released in 2001, "Silk Industry in the Tenth Five-Year Plan" is expected to 2015, silkworm production over 600,000 tons of raw silk output will reach more than 80000 t. In 2010, the silkworm production in China has more than 640,000 tons, which is a record high.Through silk reeling,lt grege silk can get It dried silkworm,the pupa production is very impressive.Due to the lack of deep-processing technology,most pupa is used as feed and fertilizer,which is low utilization and added value.Dried silkworm include 45%-50% crude protein and 20%-30% grease. In pupa grease,oleic acid,linoleic acid and linolenic acid make up unsaturated fatty acid whose content is 75%, palmitic acid and stearic acid make up saturated fatty acid whose content is 20%. Pupa also contain a variety of vitamins(vitamin A, vitamin B and vitamin D, etc), trace elements (iron, zinc, selenium, etc) and a little crude fiber, chitin, antibacterial peptides, hormone, lysozyme and polysaccharide, etc.The silkworm protein contain 18 kinds of amino acids needed by human body,common 17 kinds of amino acids in pupa account for 34.57% of total amino acids. Therefore, the comprehensive development and utilization silkworm has an important meaning and very broad prospect.This paper focuses on the research of preparation ACE inhibitory peptides from defatted pupa protein and its purification.This paper begins with the enzymation of defatted pupa protein.Firstly,selecting the appropriate protease,getting the result of single factor experiments and optimization experiment through the Box-Behnken’s response surface.Moreover,macroporous resin, glucan gel, half preparation RP-HPLC in turn purified pupa ACE inhibitory peptides,finally,obtaining the amino acid sequence,which provides experiment basis for synthesizing ACE inhibitory peptides.The main results are as follows:(1) The experiment of selecting proteaseThis experiment chooses neutrase, papain, alcalase, flavourzyme, protamex and trypsin protease to test enzyme solution effect with the indicators of DH and ACE inhibitory rate.Finally, alcalase protease has the best ACE inhibitory rate,32.46%.So, selecting alcalase protease to have the next experiments. (2) The optimizing enzymation experiment of defatted pupa proteinThis paper has the single factors experiment of substrate concentration, temperature, time, enzyme quantity and pH before the optimization and get the better values,on which the Box-Behnken’s response surface basing obtained the best technical parameters of the enzyme solution:11.88% substrate concentration, temperature of 50.22℃, pH9.46, enzyme quantity 7.03% and enzyme time of 4h.In these conditions,the IC50 of enzymic hydrolysates is 1.32mg.ml-1 the curve equation:y=-9.83747X2+40.42596 X+13.61453 (R2=0.9868).(3) The experiment of macroporous resin purification pupa ACE inhibitory peptidesThe experiment examines the adsorptionrate and desorptionrate of the D101, AB-8, DA201-C, HPD-400, HPD-826 macroporous resins.The result was the macroporous resin of DA201-C had highest adsorptionrate and desorptionrate,respectively 45.79% and 66.43%.So selecting DA201-C to have the next experiments.The experiment examine the influence of the sample concentration, flow and ethanol elution concentration on the purification of pupa ACE inhibitory peptides,and its results were as follow: 50mg/mL of sample concentration,0.5mL/min of flow and 75% of ethanol elution concentration.When the ethanol elution concentration is 75%,the eluent(a) has the highest values of hydrophobicity, aromatic amino acid content and ACE inhibition rate, respectively 5.98kJ/mol,11.13% and 57.79%.Moreover, hydrophobicity, aromatic amino acid content and ACE inhibition rate were positively correlated.In these conditions,the eluent(a)’s IC50 was 0.632mg.mL-1,the curve equation:y=-21.74174X2+76.79228X+10.1188 (R2=0.999).(4) Sephadex G-25 purification pupa ACE inhibitory peptidesIt had three peaks after Sephadex G-25 purifying eluent(a),and their ACE inhibition rate respectively were 24.82% of a-P1,34.37% of a-P2,52.64% of a-P3(0.2mg/mL of peptides concentration).So selecting a-P3 to have the next experiments. (5) The half preparation RP-HPLC purification and structure identification of pupa ACE inhibitory peptidesThrough half preparation RP-HPLC purification,it found 10 peaks,including a-P3-6 having the highest ACE inhibition rate 52.07%(0.05mg/mL of peptides concentration).After another half preparation RP-HPLC purification,it found 2 peaks,including a-P3-6-b having better ACE inhibition rate 54.27%(0.05mg/mL of peptides concentration).After the amino acid sequence analysis of a-P3-6-b,it got its sequence:Val-Glu-Ile-Ser.(6) The research on the physical and chemical properties of pupa ACE inhibitory peptidesThe solubility of pupa ACE inhibitory peptides(a-P3) were above 80% within pH 2-10.The ACE inhibition rate of pupa peptides changed a little after the impact of gastrointestinal enzyme.Its ACE inhibition rate(0.8mg/mL) was reduced by 1.90% through the impact of pepsase and trypsin.lt had little effect on ACE inhibition rate when the temperature in 20 to 50℃. pupa ACE inhibitory peptides(a-P3) was stable under the neutral and partial acidic condition. The ACE inhibition rate dramatically decreased when the NaCl concentration is above 0.8 mol/L.This paper is part of the research of national silkworm industry technology system comprehensive project(Number:CARS-22-ZJ0503).