Preparation, Purification And Identification Of ACE Inhibitory Peptides Derived From Goat Milk

Angiotensin converting enzyme(ACE) is one of the main reasons which elevate the blood pressure in hypertensive patients, so inhibition of ACE activity becomes an important way to adjust blood pressure by taking drugs. In the clinical, however, long-term use of synthetic drugs causes several side effects. Previous research proved that ACE inhibitory peptides from dairy proteins, which are capable of lowering blood pressure and are safe and non-toxic side effects. Shaanxi Province is rich in goat milk, but the main products are bulk goat milk powder and goat infant formulas, homogeneity is serious. In this study, goat milk was used as raw milk, the probiotic Lactobacillus with ability for producing ACE inhibitory peptides were firstly screened and identified, and then the preparation processes of fermentation by two strains and enzymatic hydrolysis by commercial proteinases were optimized, the ACE inhibitory peptides were isolated, purified and identified from fermented goat milk and casein hydrolyzate by ultrafiltration, gel filtration chromatography, RP-HPLC methods and MALDI/TOF-TOF-MS/MS. The main results are as following:(1) The probiotic Lactobacillus producing ACE inhibitory peptides by fermenting goat milk were screened and identified. Among the 28 selected strains probiotic Lactobacillus, there are 19 strains having ACE inhibitory activity and the inhibition ratio between 2.27±0.09% and 85.52%±2.59%. The ACE inhibition ratio in fermented goat milk from Lactobacillus LB6, LR22, LT33 and LP69 are more than 60%. The identification results of 16 S r DNA showed that LB6 is L. delbrueckii subsp. bulgaricus, LR22 is L. rhamnosus, LT33 is L. reuteri and LP69 is L. plantarum.(2) The conditions for producing ACE inhibitory peptides for L. bulgaricus LB6 and L. plantarum LP69 were optimized by single factor, Plackett-Burman test and response surface methodology. The optimal peptides-producing conditions for L. bulgaricus LB6 were determined as follows: the pasteurization time of 15 min, inoculum size of 5.0%, whey powder of 0.8% and calcium lactate of 0.5% at 35 ?for 12 h. Under the conditions, the ACE inhibitory activities in fermented goat milk achieved 85.88±0.63%. The optimal peptides-producing conditions for L. plantarum LP69 were as follows: the addition of calcium chloride and Tween-80 are 0.07% and 0.04%, respectively, 35 for 14 h. Under ?the optimal conditions, ACE inhibitory activities in fermented goat milk achieved 88.91±0.52%, which was 19.48% higher than that of the control.(3) The peptides-producing media for ACE inhibitory peptides from goat milk fermented by L. bulgaricus LB6 and L. plantarum LP69 were optimized. The soy peptone, glucose and casein are main factors of fermentation medium for L. bulgaricus LB6 according to the Plackett-Burman test. The composition(g/L) of optimal peptides-producing medium for L. bulgaricus LB6 are as follows: goat milk powder 110 g, soy peptone 3.0g, glucose 1.15 g and casein 1.50 g, respectively, the ACE inhibitory activities in fermented goat milk achieved 92.31±0.56%. The results of Plackett-Burman experiments showed that the main elements of peptides-producing medium for L. plantarum LP69 were lactose and casein. The composition(g/L) of optimal peptides-producing medium for L. plantarum LP69 are as follows: goat milk powder 140 g, lactose 4.9g and casein 2.3g, respectively, the ACE inhibitory activities in fermented goat milk achieved 89.16±0.54%.(4) The preparation of ACE inhibitory peptides from casein of goat milk was optimized by enzymatic hydrolysis and kinetic parameters of Alcalase and two enzymes(Alcalase and Trypsin) were obtained. The ACE inhibition rate order in casein hydrolysates of goat milk is as follows: Alcalase>Trypsin> Proteinase K>Papain>Neutral protease. The optimal enzymatic hydrolysis conditions by Alcalase and Trypsin are as follows: substrate concentration 10%, E/S 8.25%, the ratio of Alcalase to trypsin 1:1, p H 8.4, 69?for 90 min. The ACE inhibitory activities achieved 91.99±1.18% in the conditions, the kinetic parameter Km and Vmax for Alcalase and Trypsin are 0.127mg/m L and 0.154mg/m L·min, respectively, which indicate that the preparation of ACE inhibitory peptides from goat milk casein by Alcalase and Trypsin is betterr than that by Alcalase.(5) The ACE inhibitory peptides derived from goat milk were separated,purified and identified. The IC50 values of crude ACE inhibitory peptides from fermented goat milk by L. bulgaricus LB6(LB6FM), L. plantarum LP69(LP69FM) and casein hydrolysates of goat milk by Alcalase and Trypsin(GCH) are 0.677mg/m L, 0.502mg/m L and 0.729mg/m L, respectively. After ultrafiltration, ACE inhibitory peptides are all mainly concentrated in MW<1k Da components, the IC50 values of LB6 FM, LP69 FM and GCH decreased to 0.348mg/m L, 0.312mg/m L and 0.284mg/m L, respectively. The fragments F2 from LB6 FM, G2 from LP69 FM and H2-2 from GCH were purified by Sephadex G-15 and had highest inhibitory activity. The order for ACE inhibitory activity of purified fractions from strong to weak is as follows: G2(IC50 0.125mg/m L)>H2-2(IC50 0.134mg/m L)>F2(IC50 0.150mg/m L). Furthermore, the nine ACE inhibitory peptides were identified from F2, G2 and H2-2 by RP-HPLC and MALDI/TOF-TOF-MS/MS. By inquiring relevant database of bioactive peptides and references including Mascot?AHTPDB?Swe Pep?Pep Bankand EROP-Moscow, Four peptides including SKK, RHPHPH, YFYPQ and LSQPKVLP in the present study, which corresponds to the fragment of ?s2-casein f(151-153), ?-casein f(118-123), ?s1-casein f(159-163) and f(180-187), respectively, have not been reported in milk fermented with probiotics or casein hydrolysates and are novel ACE inhibitory peptides from goat milk.Studies have shown that preparation of ACE inhibitory peptides through fermenting goat milk by probiotics or by hydrolyzing casein from goat milk by enzymes is feasible, the isolation and identification of 9 ACE inhibitory peptides provide theoretical basis and technical support for development of functional goat dairy products in the future.