發(fā)布時(shí)間：2019-06-10 07:33

【摘要】：植物乳桿菌(Lactobacillus Plantarum)是常用的益生菌,課題組前期研究表明,利用植物乳桿菌LP69發(fā)酵的羊乳制品能抑制血管緊張素轉(zhuǎn)換酶(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產(chǎn)生了ACE抑制肽和抗氧化肽的緣故,水解蛋白源于植物乳桿菌LP69的胞壁蛋白酶(cell-envelope proteinase,CEP)的作用。本課題采用單因素及響應(yīng)面法確定了植物乳桿菌LP69產(chǎn)胞壁蛋白酶的最適培養(yǎng)基,單因素及正交設(shè)計(jì)優(yōu)化了發(fā)酵條件,分別采用鈣離子法和溶菌酶法提取了胞壁蛋白酶,研究了其酶學(xué)性質(zhì)。應(yīng)用該酶水解脫脂羊乳制備功能性羊乳飲品,研究了功能性羊乳飲品的穩(wěn)定性,獲得以下結(jié)論:1.植物乳桿菌LP69產(chǎn)胞壁蛋白酶的最適培養(yǎng)基為:葡萄糖20g/L、酵母浸粉20g/L、硫酸錳0.04g/L、吐溫80 1g/L,菊糖2g/L、酪蛋白胨4.78g/L、磷酸氫二鈉5.27g/L、亮氨酸16.36mg/L,該條件下胞壁蛋白酶的酶活為(21.46±0.81)U/mL,比活力為(1.01±0.46)U/mg。較優(yōu)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%;植物乳桿菌LP69產(chǎn)胞壁蛋白酶最佳發(fā)酵條件為:培養(yǎng)基pH 6.0,接種量為5%,39℃下恒溫培養(yǎng)22h,LP69胞壁蛋白酶活力達(dá)到(22.31±0.82)U/mL,比活力(1.17±0.06)U/mg,較優(yōu)化培養(yǎng)基后的酶活及比活力又提高了3.8%和15.8%。2.鈣離子法提取植物乳桿菌LP69胞壁蛋白酶的最佳條件為:緩沖液比例1:20、EDTA-Na2濃度60mmol/L、39℃下保溫70min、緩沖液pH6.5,其酶活和比活力分別為(26.94±0.86)U/mL和(1.33±0.03)U/mg。溶菌酶法提取植物乳桿菌LP69胞壁蛋白酶的最佳條件:裂解液比例1:41.4、溶菌酶含量0.75mg/mL、裂解液p H 8.65、37℃下保溫3h,其酶活和比活力分別為(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法用時(shí)短,該方法較溶菌酶法更為理想。3.試驗(yàn)研究植物乳桿菌LP69胞壁蛋白酶的酶學(xué)性質(zhì)表明:最適反應(yīng)pH為8,最適溫度41℃,可耐受80℃高溫。Ca~(2+)-free法提取的酶活相對(duì)穩(wěn)定,鈣離子對(duì)CEP有激活作用,鈉離子、鉀離子對(duì)CEP活性影響不顯著,鋅離子、EDTA對(duì)CEP活性表現(xiàn)出明顯地抑制作用。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應(yīng)動(dòng)力學(xué)參數(shù)Km分別為0.745 mg/mL、0.332 mg/mL和1.176 mg/mL,Vmax分別為0.479 mg/mL·min、0.228 mg/mL·min和0.798 mg/mL·min。由Km值可知,CEP與乳球蛋白的結(jié)合能力最強(qiáng),不易分離,其次依次是乳清蛋白和酪蛋白;由Vmax可知,酪蛋白的最大反應(yīng)速率大,其次為乳清蛋白和乳球蛋白。4.胞壁蛋白酶酶解脫脂羊乳的最適工藝條件為:酶加量12%,酶解溫度41℃,初始pH值8.5,時(shí)間4.5h,此條件下水解度(Degree of hydrolysis,DH)為(15.68±0.74)%,ACE抑制率為(83.25±1.05)%,DPPH自由基清除率為(64.91±1.27)%,羥自由基清除率為(89.17±1.13)%,抑制率和抗氧化性明顯。5.β-環(huán)糊精能有效改善脫脂羊乳酶解液的苦味,其最適添加量為0.8%,卡拉膠、結(jié)冷膠和蔗糖酯為影響脫脂羊乳酶解液穩(wěn)定性的3個(gè)主因子,其最適添加量分別為0.05%、0.15%和0.15%;貯存結(jié)果表明,常溫放置時(shí),其穩(wěn)定系數(shù)、ACE抑制率和抗氧化性下降快,4℃下冷藏性能穩(wěn)定。本研究表明植物乳桿菌LP69胞壁蛋白酶的制備技術(shù),用于開發(fā)具有抑制ACE活性和抗氧化性的功能性羊乳是可行的,為后續(xù)功能性羊乳產(chǎn)品的開發(fā)提供了理論依據(jù)和技術(shù)參考。
[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，

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