Purification Of CEP From Lactobacillus Acidophilus JQ-1and The Study On Hydrolyzing Of β-casein By CEP To Produce ACE Inhibitory Peptides

The β-casein hydrolysed by Lactobacillus acidophilus was found to have the AngiotensinI-Converting Enzyme (ACE) inhibitory activity to effectively reduce the blood pressure. The objectof this research was to separate and purify the cell-envelop-protease (CEP) of Lactobacillusacidophilus JQ-1, optimization of enzymatic hydrolysis of β-casein with CEP in order to producethe angiotensin-I converting enzyme (ACE) inhibitory peptides was investigated systematicallyusing response surface methodology. Once the most powerful ACE inhibitory peptides areproduced, the β-casein hydrolysates will be characterized in this research.The suitable culture conditions and the liquid fermentation conditions of Lactobacillusacidophilus in MRS medium were optimized for producing cell wall proteinase (CEP) throughorthogonal tests and single-factor test, response surface methodology. Suitable conditions to obtaincell wall proteinase (CEP) from Lactobacillus acidophilus were optimized through orthogonal testsof carbon source and nitrogen source. The optimal conditions were as following:2%glucose and1%beef peptone. The effects of original pH of MRS medium, fermentation temperature and timeon cell wall proteinase (CEP) specific activity were studied by single-factor test and responsesurface methodology. The highest cell wall proteinase (CEP) specific activity was media with theoriginal pH value of8.0, fermentation temperature of37℃and fermentation time of20h. Also,the test was proved to be feasible for condition optimization of cell wall proteinase (CEP)production.The extraction and purification process of cell-envelope proteinase (CEP) from Lactobacillusacidophilus JQ-1is determined in the present study. The lysozyme with nonionic surfactant wasused to extract the CEP, and the conditions of CEP extraction was optimized as follows: thallus inlysis buffer solution at the ratio of1:5(g/ml) and then incubating the suspension for4.0h at33℃,centrifugating the suspension and then collecting the supernatant (crude cell-envelope proteinasesolution). Using PEG-20000concentrate the crude enzyme solution, and purifying the crudeproteinase by DEAE-Sephadex A-25and Sephadex G-100column chromatography successively.The enzyme is purified by about50.951folds from the crude cell-envelope proteinase solution andthe recovery of activity is about12.569%. The purified enzyme has a monomer structure and themolecular mass of about45kD by SDS-PAGE. The angiotensi converting enzyme (ACE)inhibitory rate of β-casein hydrolyzate by the purified enzyme is48%. Optimization of enzymatic hydrolysis of β-casein with cell wall proteinase (CEP) fromLactobacillus acidophilus JQ-1in order to produce the angiotensin-I converting enzyme (ACE)inhibitory peptides was investigated systematically using response surface methodology. Underoptimal conditions (enzyme to substrate (E/S) ratio (w/w) of0.08and pH of8.00at42o C), theACE inhibitory activity of hydrolysates was72.91%and the total peptides was12.68mg/ml.Scanning electron microscopy (SEM) micrographs indicated that the tightness of the β-caseinsurface structure was gradually weakened and small holes appeared after enzymatic treatment,while Fourier transform infrared spectroscopy (FTIR) spectra indicated remarkable changes in thechemical composition and macromolecular conformation of β-casein after enzymatic hydrolysis.Differential scanning calorimetry (DSC) analysis indicated that the corresponding hydrolysates hadhigher thermal stability. The enzymatic hydrolysis also led to an increase in the free sulfydrylcontent of β-casein hydrolysates compared to raw β-casein, which led to the increase of theantioxidant activity of β-casein hydrolysates.The structural characteristic of casein hydrolyzed by cell wall proteinase (CEP) fromLactobacillus acidophilus was analysed by granularity and Fourier transform infrared spectroscopy(FTIR). The effect of different degree of hydrolysis (DH2.4%、4.5%、7.1%、8.3%) on functionalproperties of casein was determined by emulsibility, emulsion stability, solubility, antioxidant andACE inhibitory activity. The results of FTIR spectra indicated remarkable changes in the chemicalcomposition and macromolecular conformation of casein after enzymatic hydrolysis, that was tosay the secondary structure of casein was changed with the enzymatic treatment. The nanoparticlesize analysis showed that the casein particles decreased during the initial hydrolysis time(DH<4.5%), but increased for further hydrolysis, which resulted in the highest emulsion stabilityunder DH4.5%. The emulsibility of casein first increased (DH<7.1%) then decreased with thedeeping of hydrolysis, and this trend was consistent with its solubility. In addition, the caseinhydrolysates had certain ACE inhibitory activity and antioxidant properties, the DPPH removalcapacity within a certain range inceeased as DH increased, along with the increase of theconcentration. The highest DPPH removal capacity was35.00%under DH8.3%and thehydrolysates concentration of5mg/ml. Therefore, the enzymatic hydrolysis with CEP caneffectively improve the structural and functional properties of casein, which will provide powerfultheoretical basis for the production of ACE inhibitory peptides derived from casein.