Preparation Of Angiotensin-converting Enzyme Inhibitory Peptides From Tea And Research On Its Antihypertensive Activity

Hypertension is one of the most common cardiovascular diseases,which can cause damage to the brain,cardiovascular system and kidney.Hypertension is an important factor leading to diseases such as stroke,heart failure and coronary heart disease.The use of natural foods to prepare antihypertensive peptides has become an ideal choice for non-drug control of hypertension.There is a high production of low-value tea and the utilization rate of it is low in China.Whatsmore the added value of the formed products is not high.Tea protein is a high-quality protein resource.However,its development and utilization is very low.In this study,the angiotensin-converting enzyme inhibitory peptides were prepared by enzymatic hydrolysis method from tea protein,and the antihypertensive activity in vivo was studied.The research contents and results were as follows:(1)A higher extraction rate of tea protein was obtained used by a complex enzyme alkaline extraction method.First,the tea residue was treated with a compound plant hydrolase(Viscozyme)at a concentration of 2.5%for 3 h,and then treated with Na OH solution.The conditions were as follows:the Na OH concentration was 0.12 mol/L,the solid-liquid ratio was 1:50,the extraction time was 2 h,and the extraction temperature was90°C.The protein extraction rate of tea was up to 74.27%.Tea protein was further purified by precipitated(p H 3.0),desalted and decolorized.The purity of the tea protein obtained after lyophilization was 75.36%.(2)A fast HPLC detection method of ACE inhibition rate in vitro was established.The conditions were as follows:The separation was performed on an Agilent Eclipse Plus C18column(4.6×150 mm,5?m);the column temperature was 35°C;the mobile phase of acetonitrile/water(v/v)was 30:70(containing 0.05%formic acid);the flow rate was 0.8m L/min;the detection wavelength was 228 nm;isocratic elution,and the injection volume was 10?L.Under this chromatographic condition,the marcumyl histidyl leucine and hippuric acid were separated.The retention time was within 3 min.The accuracy,reproducibility,stability and recovery experiments proved that this method was fast,accurate and reproducible.(3)Tea protein was hydrolyzed by acid protease,alkaline protease,neutral protease,pepsin and trypsin respectively.The results showed that the ACE inhibitory activities of the tea polypeptides obtained by enzymatic hydrolysis were from strong to weak,followed by acidic protease>trypsin>pepsin>alkaline protease>neutral protease.By orthogonal test,the conditions of enzymatic hydrolysis of tea protein were obtained as followings:the enzyme concentration was 2%,the reaction system p H was 4.0,the hydrolysis temperature was 40°C,and the hydrolysis was 2 h.Under this condition,the ACE inhibitory rate of tea polypeptide was 77.0%.(4)The nutritional value and functional properties of tea protein were analyzed and evaluated.The analysis of amino acid composition of tea protein showed that there were 17kinds of amino acids,and the contents of glutamic acid,leucine and aspartic acid were the highest in tea protein.The nutritional value of tea protein was 72.22,which was significantly higher than that of soy protein.The tea protein had the lowest solubility near the isoelectric point(p H 4.0).And the solubility of the tea protein rose fast under strong alkali conditions.The water absorption,oil absorption,foaming and the foam stability of tea protein were good.Although the emulsifying property was low,the emulsion stability was high.The good functional properties of tea protein showed that it has high utilization value in food processing.(5)The stabilities of tea ACE inhibitory peptides were analyzed.The temperature had little effect on hypotensive activity of tea ACE inhibitory peptides.The tea ACE inhibitory peptides had high activity under acidic conditions and low activity under alkaline conditions.Different metal ions had different effects on the activity of tea ACE inhibitory peptides.The activities of tea ACE inhibitory peptides were effected significantly by Ca²⁺,Mg²⁺,Cu²⁺and Zn²⁺,whereas were less influenced by K⁺(ACE inhibition rate was62.68%?64.72%).The tea ACE inhibitory peptides could maintain high ACE inhibitory activity within 1 h after pepsin treatment.The ACE inhibitory rate showed a slow upward trend after treated by trypsin.Tea ACE inhibitory peptides had high anti-digestive function,which was beneficial to maintain high ACE inhibitory activity in vivo.(6)After ultrafiltration(3000 Da),tea ACE inhibitory peptides were analysised by Nano-LC-MS/MS.Three 7-peptide molecules LAEQAER,VECTIPK,MASLALK and one11-peptide molecule DAYVGDEAQSK were identified.the hydrophobic amino acid ratio of MASLALK reached 71.4%.The molecular weight range of 4 peptides was732.4302?1181.5233 Da.(7)The effect and mechanism of tea ACE inhibitory peptides on blood pressure in SHR rats were studied.The blood pressure of rats in one-time administration began to decrease at 1 h,and decreased to the lowest value at 2 h,which had achieved remarkable or the extremely remarkable difference level compared with the model control group.Then the blood pressure of the rats gradually increased,and after 6 h the blood pressure level was basically restored to the level of blood pressure before administration.The tea ACE inhibitory peptides had no effect on the heart rate of the rats.In the long-term administration experiment,the administration of tea ACE inhibitory peptides had a significant antihypertensive effect at the first week,and the blood pressure continued to decrease steadily at the second week,and did not rebound back to the original high blood pressure level,indicating that the antihypertensive ability of tea ACE inhibitory peptides was stable and persistent.The tea ACE inhibitory peptides had significant inhibitory effect on renin,ACE,Ang II and ALD in rat plasma.The tea ACE inhibitory peptides could improve the panic and irritability of hypertensive rats.Anatomical experiments also confirmed that tea ACE inhibitory peptides had no obvious damage to rat internal organs.In the 800 mg/kg·BW dose range,tea ACE inhibitory peptides had no liver toxicity,which indicated that the tea ACE inhibitory peptides were probably an natural and safe plant-derived blood.antihypertensive peptide.