Study On Antihypertensive Peptides From Tenebrio Molitor (L.) Larva Protein Hydrolysate

Tenebrio molitor (L.) larva is a kind of important and high quality insect resources containing anabundant amount of protein and fat. It has known as “the king of animal protein content” because theprotein content in the body even reached about50%on a dry matter basis. Although the resource ofTenebrio molitor (L.) larva in our country was abundant because of its easily breeding, the lack ofsystematic and reasonable research has limited its utilization. The study of antihypertensive peptides fromTenebrio molitor (L.) larva has not been reported nowadays. Antihypertensive peptides from Tenebriomolitor (L.) larva protein were obtained successfully in the paper. The research can not only broadapplication areas, but also develop new ways for further utilization of Tenebrio molitor (L.) larva protein.The fat was extracted with supercritical carbon dioxide from Tenebrio molitor (L.) larva. And theextraction conditions were as follows: pressure35MPa, temperature35℃, extracting time200min. Theprotein content then reached60.5%after the fat was removed.The content of albumin, globulin, alcohol soluble protein and glutenin isolated from defatted Tenebriomolitor (L.) larva was9.0%，1.5%，2.3%and23.7%, respectively. There was still63.5%of proteinremaining in the residue. The amino acids composition of protein fractions indicated that non polar aminoacids and aromatic amino acid was abundant in glutenin and residual protein. In order to improve theutilization efficiency of protein, defatted Tenebrio molitor (L.) larva was used directly as the raw materialsin the experiments.Alcalase was chosen to prepare antihypertensive peptides from defatted Tenebrio molitor (L.) larvaprotein. The optimum enzymatic conditions were determinated as follows: substrate concentration3%,Alcalase dosage4000U/g substrate, temperature50℃, pH value8.5. The ACE inhibitory activity was thestrongest when hydrolytic degree reached20%. And the IC50value of the hydrolysates was determined at0.39mg/mL.Ultrasound (350W/g substrate) was used to treat defatted Tenebrio molitor (L.) larva protein beforeenzymatic hydrolysis in the experiment. The hydrolysis time (DH reached20%) was shorted by20%whenpretreated for10min. The effects of ultrasonic treatment on conformation of Tenebrio molitor (L.) larvaprotein were studied by means of ultraviolet absorption spectra, fluorescence emission spectra, surfacehydrophobicity, free sulfhydryl content, particle size distribution, circular dichroism, and sodium dodecylsulphate-polyacrylamide gel electrophoresis. The results indicated that ultrasonic treatment changed theconformation of albumin and glutenin isolated from defatted Tenebrio molitor (L.) larva. But the primarystructure of protein was not affected by ultrasound.The effects of ultrasound on the enzymatic hydrolysis of defatted Tenebrio molitor (L.) larva proteinwith Alcalase were also investigated. The results indicated that ultrasonic treatment (2.5and5min) canaccelerate the enzymatic hydrolysis procedures of samples. The influence of ultrasound on activity andconformation of Alcalase was also measured. The result revealed enzyme activity was significantlyimproved as being treated for5min, while extensive processing on the contrary decreased the activity ofAlcalase. The results of ultraviolet absorption and fluorescence emission showed that ultrasonic treatmentled to a change on molecular structure of Alcalase. Amino acid composition in hydrolysates (DH8%) ofTenebrio molitor (L.) larva protein was almost the same whether enzymatic hydrolysis was treated withultrasound or not.Tenebrio molitor (L.) larva protein hydrolysates was fractionated using Sephadex G-15columnchromatography after decolored by activated carbon, four peaks (Peak1，Peak2，Peak3and Peak4) wereobtained. The results of amino acids composition and relative molecular weight distribution indicated that Peak2was suitable for production of ACE inhibitory peptides. The IC50value of four fractions isolatedusing gel filtration was determined with high performance liquid chromatography. The value was0.46,0.23,0.83and2.81mg/mL, respectively.The active fractions in Peak2based on the ACE inhibiting activity were isolated and purified byHPLC. P2-F6and P2-F9among20fractions isolated from Peak2were further purified because of theirstrong ACE inhibitory activity. The amino acid sequences of ACE inhibitory peptides were Ser-Met andTyr-Ala-Asn from P2-F6and Asn-Tyr from P2-F9determined by Matrix-Assisted LaserDesorption/Ionization Time of Flight Mass Spectrometry. The IC50value of them was0.026，0.017and0.011mg/mL, respectively. Ser-Met and Tyr-Ala-Asn have not been reported as antihypertensive peptidesyet.Blood pressure of spontaneously hypertensive rats (SHR) decreased obviously after fed with thefraction of Peak2, in a single oral administration in100,200and400mg/kg.bw dosage respectively. It isalso discovered that defatted Tenebrio molitor (L.) larva protein hydrolysates has some antioxidant effect.