Preparation And Application Of Cell-Envelope-Proteinase From Lactobacillus Plantarum LP69

Lactobacillus plantarum is a kind of common probiotics.The previous study of our group showed that the goat milk fermented by Lactobacillus plantarum LP69 have the ability of reducing the Angiotensin I-Converting Enzyme?ACE?activity and antioxidant.The main reason is that the protein in goat milk were hydrolyzed to produce polypeptides.Some of the polypeptides have the ACE inhibitory activity,there are also some polypeptides have antioxidant activity.The cell-envelope proteinase?CEP?is the core enzyme in the process of hydrolyzing goat milk protein to produce polypeptides.In this study,The single factor experiment and Box-Behnken Design?BBD?response surface method were employed to optimize the culture medium composition,the single factor experiment and orthogonal design were used to optimize the fermentation conditions.Using Ca²⁺-free method and lysozyme method to extract the CEP,the enzymatic properties of CEP were also studied.Finally,CEP was used to hydrolyze skim milk to prepare the functional goat milk beverage and it's stability was also studied.The main results are as follows:1.The optimal culture medium composition of Lactobacillus plantarum LP69 producing CEP were: glucose 20g/L,yeast extract 20g/L,manganese sulfate 0.04g/L,Tween 80 1g/L,inulin 2g/L,casin peptone 4.78g/L,disodium hydrogen phosphate 5.27g/L,leucine 16.36mg/L.The enzyme activity and specific activity reached?21.26±0.56?U/mL and?1.11±0.05?U/mg in the optimal culture medium.Compared to the enzyme activity and specific activity before,?16.39 ± 0.81?U/mL and?0.64 ± 0.71?U/mg,the values improved 30.9% and57.8%.The optimal fermentation conditions of Lactobacillus plantarum LP69were: initial pH value 6,inoculum size 5%,time 22 h,fermentation temperature37 oC.The enzyme activity and specific activity reached?22.31±0.82?U/mL,?1.17±0.06?U/mg under the optimal conditions,which improved 3.8% and15.8% compared with the results after optimization of culture medium composition.2.The optimal extraction conditions of CEP in Lactobacillus plantarum LP69 for Ca²⁺-free method are: buffer ratio 1:20,EDTA-Na2 concentration 60mmol/L,incubation 70 min,incubation temperature 39 o C,buffer pH 6.5.The enzyme activity and specific activity of CEP extracted in these conditions were?26.94±0.86?U/mL and?1.33±0.03?U/mg.The optimum condition of lysozyme method are: lysate ratio 1:41.4,lysozyme contentration 0.75mg/mL,lysate buffer pH 8.65,incubation temperature 37 oC and incubation time 3h.The enzyme activity and specific activity of CEP were?24.03±0.39?U/mL and?1.27±0.02?U/mg.The enzyme activity and specific activity of CEP extracted by Ca²⁺-free method are higher than those of lysozyme method?12.1% and 4.7%?,and Ca²⁺-free method is more time-saving and effective.3.The study of enzymatic properties of CEP in Lactobacillus plantarum LP69 showed that the optimal reaction pH of CEP is 8,the optimal temperature of CEP is 41 oC and CEP could withstand 80 oC.The CEP extracted from Ca²⁺-free method is more stable.Calcium ions could promote the activity of CEP,sodium ions and potassium ions have no obvious effect on CEP,zinc ions and EDTA have a strong inhibitory effect on CEP activity.According to Lineweaver-Burk method,the kinetic parameters of CEP on whey protein,lactoglobulin and casin are Km = 0.745 mg/mL,0.332 mg/mL,1.176 mg/mL,Vmax = 0.479mg/mL·min,0.228 mg/mL·min,0.798 mg/mL·min.From Km value,the binding capacity between CEP and lactoglobulin is the strongest,and then whey protein,while the binding ability of casin and CEP is the the weakest.The Vmax value showed that the maximum reaction rate of casin is the biggest,and then whey protein and lactoglobulin.5.The optimal hydrolysis conditions of CEP hydrolyzing skim milk are:enzyme amount 12%,temperature 41 oC,pH 8.5,hydrolysis time 4.5h.Under the conditions,the degree of hydrolysis?DH?was?15.68±0.74?%,ACE inhibitory activity was?83.25±1.05?%,DPPH radical scavenging activity was?64.91±1.27?%,and hydroxyl radical scavenging activity was?89.17±1.13?%.The ACE inhibitory activity and antioxidant activity are obvious.6.?-cyclodextrin could debitter the goat milk enzymatic hydrolysate,the beverage tastes best when adding 0.8% ?-cyclodextrin.the stability results show that carrageenan,gellan gum and sucrose ester are best stabilizer for goat milkbeverage,and the orthogonal design indicated that the optimal combine conditions are carrageenan 0.05%,gellan gum 0.15% and sucrose ester 0.15%.In addition,the storage experiment shows that the stablity,ACE inhibitory activity and antioxidant activity all decrease faster in room temperature,while4? is more stable and it's the better condition for storage the beverage.This study showed that the preparation technique of CEP from Lactobacillus plantarum LP69 could be used to develop the functional goat milk?ACE inhibitory activity and antioxidant activity?,which provided theory evidence and technical reference for the development of functional goat milk.