| China is the largest country in sericulture industry in the world, accounted for 75% of world production. There are lots of silkworm pupa as its main by-products. Because of reasonable and balanced amino acid ratio, Pupa protein has been considered to be a high-quality protein sources with high nutritional value. As the fast developing country, the dietary imbalance has become one of the major problem which threatening the public health in China. Hypertension, obesity and other eating disorders have been increasing strikingly. Therefore, taking full advantage of silkworm protein, which is abundant, low cost and natural, for developing high purity products(silkworm pupa peptides) with antioxidant, antibacterial, antilipemic and other biologically activity, will have significant social and economic benefits.In this project, we use silkworm as raw material, investigate the extraction of silkworm protein and the biological activity of water-soluble peptides prepare from enzyme hydrolyzation. The results are as follows:1. Using dry silkworm pupa as raw material, following ultrasonic assist alkali soluble acid precipitation. The single factor and orthogonal tests to determine the best extraction parameters:liquid ratio (w/v) was 12:1, pH10, extraction temperature 60℃, extraction time 60 min, ultrasonic power 150 W. In this situation, the pupa protein extraction rate was 49.76%; and protein content was 94.56% after freeze-dried protein powder. The decoloration and deodorization study shows ethanol has a good decolouration and deodorizing effect, and the product after process will be light gray, odorless and a slightly silkwormish flavour.2. Six proteases (Alkaline proteinase, Flavourzyme, Compound proteinase, Trypsin, Papain and neutral protease) were selected for studying single enzyme or dual-enzyme preparation of water-soluble peptides process. In the single enzyme hydrolysis process, Alkaline proteinase is the best enzyme for silkworm pupa protein hydrolyzation, which the hydrolysis rate is the highest and up to 25.28%, moreover, the stability is also the best. In double enzyme hydrolysis process, I group combination (Alkaline proteinase and Flavourzyme) is the best for hydrolyzation, which the hydrolysis rate reached 28.13%. Our results also show all dual-enzyme are more stable and have higher hydrolysisrate than every single enzyme.3. Water-soluble silkworm pupa peptide prepare from of single enzyme hydrolyzation group (A, B, C, D, E, K) and Dual-enzyme hydrolyzation groups (F, G, H, I, J) have been investigated to show the ability of scavenging of DPPH·This experiment use BHT as a control, the results show that the scavenging DPPH·capacity of silkworm pupa peptide is 6 times greater than the peptide hydrolysis from BHT. Our research also shows the scavenging DPPH·capacity of peptides hydrolysis by single enzyme is better than those by Dual-enzym. And B group have the highest clearance rate which is 92.43%.4. Water-soluble silkworm pupa peptides prepared from single enzyme hydrolyzation groups and Dual-enzyme hydrolyzation groups have been investigated to show the ability of ACE inhibition. The results show that, peptides prepared from G group (Alkaline proteinase and Compound proteinase) have the best ACE inhibitory activity, which is 85.93%. Our research also shows the ACE inhibitory capacity of peptides hydrolysis by Dual-enzyme is better than those by single enzyme5. Amino acid composition analysis of different groups of silkworm pupa peptides from single or double enzyme hydrolyzation show that the amino acid composition is the highest in I group preparation peptide, which is 60.54%, G group of the second, was 60.17%; Except H group, Peptide form Dual-enzyme hydrolysis have higher total amino acid content and more each amino acid than peptide from single enzyme hydrolysis. Our results also show:peptide form Dual-enzyme (except H group) hydrolysis have higher aromatic and hydrophobic amino acid content than peptide from single enzyme hydrolysis. Dual-enzyme group in the aromatic and hydrophobic amino acid peptide content (except H group,) were higher than single enzyme group, of which, peptide prepare from I group hydrolysis is the highest, reaching 27.13%, peptide from group of the second, to 26.95%.6. With highest ACE inhibitory activity, and the best ability to scavenge DPPH·, G-group (Alkaline proteinase with Compound proteinase) has been selected for optimizing the process. using peptides production rate as the indictor, a single factor and orthogonal tests, the optimal hydrolysis process:pH is 8, the hydrolysis temperature 60℃, hydrolysis time 3.5h, in these conditions, the yield of water-soluble silkworm pupa peptides is 77.56%.7. After Ultrafiltration of peptide from G group (Alkaline proteinase with Compound proteinase), there are seven different segments of the water-soluble peptides according to molecular weight after ultrafiltration. Experiments were done to test the scavenging DPPH·activity and ACE inhibitory activity of these 7 different peptide respectively. The results show that the peptide molecular weight less than 1000Da have the strongest free radical scavenging capacity, which is 87.17%. ACE inhibitory activity results show that peptide whose molecular weight is between 1000Da and 5000Da has the strongest ACE inhibitory activity, reaching 86.53%, higher than the peptide before ultrafiltration. Amino acid analysis of 1000-5000Da peptides, which have strongest ACE inhibitory activity, shows that the amino acids content is 65.07%, aromatic and hydrophobic amino acid is 32.16%, are all higher than the peptide before ultrafiltration.8. Purification of 1000-5000 Da peptide using Sepadex G-25 gel filtration, and analyse the molecular weight and ACE inhibitory activity. The results show the peptide which molecular weight near 1560Da has high ACE inhibitory activity, which is 86.27%, close to the pre-purified 1000-5000Da peptide (86.53%).9. Peptides purification effects was analysed by RP-HPLC. The results show that after pupa SephadexG-25 gel chromatography filtration, the purity of 1000-5000Da peptide improved significantly.
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