植物乳杆菌LP69胞壁蛋白酶的制备及其应用研究
发布时间：2019-06-10 07:33
【摘要】：植物乳杆菌(Lactobacillus Plantarum)是常用的益生菌,课题组前期研究表明,利用植物乳杆菌LP69发酵的羊乳制品能抑制血管紧张素转换酶(Angiotensin Convening Enzyme,ACE)的活性且具有抗氧化性,主要是羊乳中的蛋白被水解产生了ACE抑制肽和抗氧化肽的缘故,水解蛋白源于植物乳杆菌LP69的胞壁蛋白酶(cell-envelope proteinase,CEP)的作用。本课题采用单因素及响应面法确定了植物乳杆菌LP69产胞壁蛋白酶的最适培养基,单因素及正交设计优化了发酵条件,分别采用钙离子法和溶菌酶法提取了胞壁蛋白酶,研究了其酶学性质。应用该酶水解脱脂羊乳制备功能性羊乳饮品,研究了功能性羊乳饮品的稳定性,获得以下结论:1.植物乳杆菌LP69产胞壁蛋白酶的最适培养基为:葡萄糖20g/L、酵母浸粉20g/L、硫酸锰0.04g/L、吐温80 1g/L,菊糖2g/L、酪蛋白胨4.78g/L、磷酸氢二钠5.27g/L、亮氨酸16.36mg/L,该条件下胞壁蛋白酶的酶活为(21.46±0.81)U/mL,比活力为(1.01±0.46)U/mg。较优化前酶活(16.39±0.81)U/mL和比活力(0.64±0.71)U/mg提高了30.9%和57.8%;植物乳杆菌LP69产胞壁蛋白酶最佳发酵条件为:培养基pH 6.0,接种量为5%,39℃下恒温培养22h,LP69胞壁蛋白酶活力达到(22.31±0.82)U/mL,比活力(1.17±0.06)U/mg,较优化培养基后的酶活及比活力又提高了3.8%和15.8%。2.钙离子法提取植物乳杆菌LP69胞壁蛋白酶的最佳条件为:缓冲液比例1:20、EDTA-Na2浓度60mmol/L、39℃下保温70min、缓冲液pH6.5,其酶活和比活力分别为(26.94±0.86)U/mL和(1.33±0.03)U/mg。溶菌酶法提取植物乳杆菌LP69胞壁蛋白酶的最佳条件:裂解液比例1:41.4、溶菌酶含量0.75mg/mL、裂解液p H 8.65、37℃下保温3h,其酶活和比活力分别为(24.03±0.39)U/mL、(1.27±0.02)U/mg。Ca~(2+)-free法提取胞壁蛋白酶的酶活比溶菌酶法提取的酶活高12.1%,比活力高4.7%,Ca~(2+)-free法用时短,该方法较溶菌酶法更为理想。3.试验研究植物乳杆菌LP69胞壁蛋白酶的酶学性质表明:最适反应pH为8,最适温度41℃,可耐受80℃高温。Ca~(2+)-free法提取的酶活相对稳定,钙离子对CEP有激活作用,钠离子、钾离子对CEP活性影响不显著,锌离子、EDTA对CEP活性表现出明显地抑制作用。采用Lineweaver-Burk法求得胞壁蛋白酶水解乳清蛋白、乳球蛋白和酪蛋白的反应动力学参数Km分别为0.745 mg/mL、0.332 mg/mL和1.176 mg/mL,Vmax分别为0.479 mg/mL·min、0.228 mg/mL·min和0.798 mg/mL·min。由Km值可知,CEP与乳球蛋白的结合能力最强,不易分离,其次依次是乳清蛋白和酪蛋白;由Vmax可知,酪蛋白的最大反应速率大,其次为乳清蛋白和乳球蛋白。4.胞壁蛋白酶酶解脱脂羊乳的最适工艺条件为:酶加量12%,酶解温度41℃,初始pH值8.5,时间4.5h,此条件下水解度(Degree of hydrolysis,DH)为(15.68±0.74)%,ACE抑制率为(83.25±1.05)%,DPPH自由基清除率为(64.91±1.27)%,羟自由基清除率为(89.17±1.13)%,抑制率和抗氧化性明显。5.β-环糊精能有效改善脱脂羊乳酶解液的苦味,其最适添加量为0.8%,卡拉胶、结冷胶和蔗糖酯为影响脱脂羊乳酶解液稳定性的3个主因子,其最适添加量分别为0.05%、0.15%和0.15%;贮存结果表明,常温放置时,其稳定系数、ACE抑制率和抗氧化性下降快,4℃下冷藏性能稳定。本研究表明植物乳杆菌LP69胞壁蛋白酶的制备技术,用于开发具有抑制ACE活性和抗氧化性的功能性羊乳是可行的,为后续功能性羊乳产品的开发提供了理论依据和技术参考。
[Abstract]:Lactobacillus Plantarum is a common probiotics, and the earlier studies of the research group show that the sheep milk product fermented by the plant lactobacillus LP69 can inhibit the activity of the angiotensin converting enzyme (ACE) and has the oxidation resistance, The protein in the goat milk is mainly hydrolyzed to produce the ACE inhibitory peptide and the antioxidant peptide, and the hydrolyzed protein is derived from the cell-enenvelope protein (CEP) of the plant lactobacillus LP69. The optimum culture medium, single factor and orthogonal design of the cell wall protease of Lactobacillus plantarum LP69 were determined by single factor and response surface method. The conditions of fermentation were optimized by single factor and orthogonal design. The cell wall protease was extracted by calcium ion method and lysozyme method, and the enzymatic properties were studied. The functional sheep milk beverage was prepared by using the enzymatic hydrolysis and defatting goat milk, and the stability of the functional goat milk beverage was studied, and the following conclusions were obtained:1. The optimum culture medium for the cell wall protease of Lactobacillus plantarum LP69 is:20 g/ L of glucose,20 g/ L of yeast immersion powder, 0.04 g/ L of manganese sulfate,80 1 g/ L of Tween 80,2 g/ L of inulin, 4.78 g/ L of casein, 5.27 g/ L of disodium hydrogen phosphate and 16.36 mg/ L of leucine, and the enzyme activity of the cell wall protease under this condition is (21.46-0.81) U/ mL, The specific activity was 1.01 (0.46) U/ mg. The optimum fermentation conditions of the cell wall protease of Lactobacillus plantarum LP69 were: medium pH 6.0, inoculum size 5%, constant temperature culture at 39 鈩，
本文编号：2496287
[Abstract]:Lactobacillus Plantarum is a common probiotics, and the earlier studies of the research group show that the sheep milk product fermented by the plant lactobacillus LP69 can inhibit the activity of the angiotensin converting enzyme (ACE) and has the oxidation resistance, The protein in the goat milk is mainly hydrolyzed to produce the ACE inhibitory peptide and the antioxidant peptide, and the hydrolyzed protein is derived from the cell-enenvelope protein (CEP) of the plant lactobacillus LP69. The optimum culture medium, single factor and orthogonal design of the cell wall protease of Lactobacillus plantarum LP69 were determined by single factor and response surface method. The conditions of fermentation were optimized by single factor and orthogonal design. The cell wall protease was extracted by calcium ion method and lysozyme method, and the enzymatic properties were studied. The functional sheep milk beverage was prepared by using the enzymatic hydrolysis and defatting goat milk, and the stability of the functional goat milk beverage was studied, and the following conclusions were obtained:1. The optimum culture medium for the cell wall protease of Lactobacillus plantarum LP69 is:20 g/ L of glucose,20 g/ L of yeast immersion powder, 0.04 g/ L of manganese sulfate,80 1 g/ L of Tween 80,2 g/ L of inulin, 4.78 g/ L of casein, 5.27 g/ L of disodium hydrogen phosphate and 16.36 mg/ L of leucine, and the enzyme activity of the cell wall protease under this condition is (21.46-0.81) U/ mL, The specific activity was 1.01 (0.46) U/ mg. The optimum fermentation conditions of the cell wall protease of Lactobacillus plantarum LP69 were: medium pH 6.0, inoculum size 5%, constant temperature culture at 39 鈩，
本文编号：2496287
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