The Research Of Tenebrio Molitor’s ACE Inhibitory Peptides Enzymolyzed By Papain

Tenebrio molitor, a.k.a. bread worm, contains all kinds of protein and amino acid. In addition, it also has rich microelements, vitamins, dietary fiber, chitin, chitosan, natural hormones, polysaccharide, etc. Recently, with further understanding of tenebrio molitor, the number of raisers is growing. It is a important issue that how to make full use the resource of Tenebrio molitor. In this study, the degreased tenebrio molitor protein was hydrolysed by papain to prepare ACE inhibitory peptides, which broadens the range of tenebrio molitor application and introduces a new way to the development of tenebrio molitor protein resources. The main results are as follows:(1) By using single factor test and response surface methodology to optimize the preparation process of ACE inhibitory peptides, the optimal conditions are:the substrate concentration is 7 g/100 mL; the amount of enzyme is 1%; pH is 6.5; temperature is 55℃; hydrolysis time is 7 hours. Under these conditions, the polypeptide concentration is 7.63 mg/mL, the theoretical ACE inhibitory rate reaches 59.48% and the practical ACE inhibition rate is 58.86%, and the half inhibition concentration is 1.77 mg/mL.(2) Ultrafiltration and Sephadex G-15 gel were used to separate and purify the enzymatic hydrolysates. After ultrafiltration, enzyme hydrolyzation are divided into five groups based on the molecular weight:>30 kDal,10 kDal-30 kDal,5 kDal-10 kDal,3 kDal-5 kDal and <3 kDal. The last group has the highest ACE inhibitory rate and reaches up to 65.43%, with the half inhibition concentration 1.16 mg/mL. Ultra pure water was used as the eluent of Sephadex G-15 gel chromatography. With sample concentration 100mg/mL, flow rate 0.3 mL/min and the sample volume 3 mL, three peaks were observed, and the highest ACE inhibition rate is M2,72.11%, with the half inhibition concentration 0.605 mg/mL. The highest content of amino acid is proline (Pro), followed by glutamate (Glu) and Valine (Val).(3) Enzymolysis solution, ultrafiltered groups and parts of chromatography were checked on the fungi (Alternaria), bacteria(Escherichia coli, Staphylococcus aureus) inhibition. The enzymolysis liquid and the enzyme fractions with molecular weight >30 kDal have the inhibitory effects on three kinds of bacteria. The enzyme fractions with molecular weight from 10 kDal to 30 kDal have the highest inhibitory effects on Alternaria, with the diameter of inhibition zone up to 5、15 mm. The enzyme fractions with molecular weight>30 kDal have the highest inhibitory effects on Escherichia coli and Staphylococcus aureus, with the diameter of inhibition zone up to 7.37 mm and 7.33 mm.(4) The structure and physicochemical properties of M2 was analyzed. M2 is a hybrid peptide, whose molecular weight of main part is 378 Dal and possible amino acid sequence is Gly-Arg-Phe. ACE inhibitory rate of M2 showed slight changes after simulated human gastrointestinal digestion in vitro.0.2-1 mol/L NaCl had few effects on the ACE activity of M2, but high temperature (>60℃) weakened its activity. M2 demonstrated good stability in neutrual and acid conditions while declined dramatically under alkaline environment.