Preparation Of Antihypertensive Hydrolysate From Yeast With Bacillus Enzyme

Hypertension is a chronic disease that affects the human health over the world.Hypotensive peptides are natural angiotensin-converting enzyme?ACE?inhibitors.Polypeptides from yeast,a nutrient-rich edible fungus,which was hydrolyted by proteases have a high ACE inhibitory activity.Non-drug treatment is an important way for hypertension.Yeast-based hypotensive polypeptides are expected to be a safe and green functional food for non-drug treatment of hypertension.In this paper,Bacillus enzyme was used to prepare the functional polypeptides by hydrolyzing the yeast,after the fermentation process for producing protease by Bacillus were optimized.The in vivo and in vitro antihypertensive effects of the enzymatic hydrolysate from yeast?EHY?were investigated.The main research content is listed as follows:?1?The fermentation conditions of Bacillus HU528 were optimized by choosing the protease activity of the supernate as response value.The culture medium?w/v?was composed of 1%Tryptone,0.5%glucose,0.08%CaCl₂ and 0.04%NaCl.The Bacillus HU528 was incubated for 48 h at 37°C with the liquid volume 30%and pH 7.0.Under these fermentation conditions,the crude enzyme was achieved with the protease activity of 1652.75±4.15 U/m L by removing cell from broth.In addition,the optimum pH and temperature of the crude enzyme for casein hydrolysis are 7.0 and 50°C,respectively.Furthermore,the crude enzyme exhibits a?-glucanase activity.?2?The protease produced by Bacillus was isolated and purified by ammonium sulfate precipitation,followed by cation exchange chromatography.The results from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry indicated that the protease had a highest matching score with an extracellular neutral metalloproteinase from Bacillus subtilis.The purified protease had an optimum pH 8.0,optimum temperature 55°C to hydrolate casein,with a superior stability at pH 7.5-8.5 and 20-40°C.Mn²⁺and SDS significantly promoted the enzyme activity,while Fe³⁺,EDTA,DTT and PMSF inhibited the enzyme activity.?3?The crude enzyme was used to hydrolyze the edible yeast,and its hydrolysis conditions was investigated.The results showed that the optimum conditions for the hydrolysis were solid to liquid ratio of 1:10?w/v?,enzyme dosage of 7000 U/g dry yeast,temperature of 55°C,initial pH of 9.0,hydrolysis for 5 h.After solid-liquid separation and spray drying,the polypeptide content of EHY was 67.32%?w/w?.The resultant polypeptide presented a good solubility under neutral and alkaline conditions?pH 7.0-10.0?with a nitrogen solubility index of?96%.Moreover,the molecular weight of most polypeptides ranged 300Da-3kDa in EHY.?4?The in vitro activity of EYH on inhibiting ACE,as well as the in vivo activity on reducing blood pressure of spontaneously hypertensive rats?SHR?,was investigated.The 50%inhibitory concentration of EHY on ACE activity was 123.66?g/m L,and polypeptides should be the major active inhibitors.The systolic blood pressure of SHR was significantly reduced when the gavage dose was 133.3 mg/kg·bw.Furthermore,the blood pressure of SHR was similar to that of normal rats?WKY?at the gavage dose of 1200 mg/kg·bw,and the antihypertensive effect was stable over time.