De Angelis Maria, Mariotti Liberato, Rossi Jone, Servili Maurizio, Fox Patrick F, Rollán Graciela, Gobbetti Marco
Sezione di Microbiologia Agro-Alimentare, Dipartimento di Scienze degli Alimenti, Università degli Studi di Perugia, Italy.
Appl Environ Microbiol. 2002 Dec;68(12):6193-201. doi: 10.1128/AEM.68.12.6193-6201.2002.
The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.
最初对70株最常分离出的酸面团乳酸菌的细胞质提取物进行了筛选，以检测精氨酸脱亚氨酶（ADI）、鸟氨酸转氨甲酰酶（OTC）和氨基甲酸激酶（CK）的活性，这三种酶构成了ADI（或精氨酸双水解酶）途径。只有专性异型发酵菌株，如旧金山乳杆菌CB1；短乳杆菌AM1、AM8和10A；希氏乳杆菌51B；以及果糖乳杆菌DD3和DA106，表现出所有三种酶的活性。植物乳杆菌B14未表现出CK活性。旧金山乳杆菌CB1表现出最高的活性，通过几个色谱步骤从这种微生物中纯化出这三种酶，使其达到同质。ADI、OTC和CK的表观分子量分别约为46、39和37 kDa，其等电点在5.07至5.2范围内。OTC、CK，尤其是ADI对酸面团发酵过程中通常出现的pH（酸性，pH 3.5至4.5）和温度（30至37摄氏度）条件具有良好的适应性。这三种酶的内部肽序列与清酒乳杆菌的ADI、OTC和CK具有最高水平的同源性。旧金山乳杆菌CB1在含有17 mM精氨酸的MAM肉汤中或在添加1至43 mM精氨酸的酸面团发酵过程中表达ADI途径。二维电泳显示，与未添加精氨酸培养的细胞相比，在含有17 mM精氨酸的MAM肉汤中生长的细胞的全细胞提取物中，ADI、OTC和CK分别被约10、4和2倍的因子诱导。旧金山乳杆菌CB1中的精氨酸分解代谢取决于碳源和精氨酸的存在；高达约54 mM的葡萄糖没有抑制作用，pH对诱导也没有影响。由旧金山乳杆菌CB1发酵的酸面团的pH取决于添加到面团中的精氨酸量。在旧金山乳杆菌CB1进行酸面团发酵期间，低量的精氨酸供应（6 mM）促进了细胞生长、在7摄氏度储存期间的细胞存活以及对酸性环境胁迫的耐受性，并有利于鸟氨酸的产生，鸟氨酸是面包皮香气化合物的重要前体。
