Furukawa Keiji, Yamada Tasuku, Mizoguchi Haruhiko, Hara Shodo
General Research Laboratory of Kiku-Masamune Sake Brewing Co. Ltd., 1-8-6 Uozaki-nishimachi, Higashinada-ku, Kobe 658-0026, Japan.
J Biosci Bioeng. 2003;96(4):380-6. doi: 10.1016/S1389-1723(03)90141-1.
Sake mash was prepared using rice with polishing ratios of 70%, 80%, 90% and 98%. At a polishing ratio of 70%, the highest isoamyl acetate/isoamyl alcohol (E/A) ratio in sake was obtained, and inositol addition caused a decrease in E/A ratio. In several strains tested, inositol addition to the mash decreased isoamyl acetate content and E/A ratio in sake Inositol addition significantly decreased alcohol acetyltransferase (AATase) activity which is responsible for the synthesis of acetate esters from alcohols and acetyl coenzyme A. The results of Northern blot analysis and disruption of the OPII gene, an inositol/choline-mediated negative regulatory gene, showed that the decrease in AATase activity following inositol addition is not due to a transcriptional event. Inositol addition increased phosphatidylinositol (PI) content 3-fold in sake mash yeast cells, while it had no effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyl-serine (PS) contents. When cell-free extracts prepared from sake mash yeast cells were treated with chloroform or phospholipase C to remove PI, no difference in AATase activity in sake mash between with (Ino+) and without (Ino-) inositol addition was observed. PI prepared from sake mash yeast cells inhibited AATase activity more strongly than PC and PE. Furthermore, when PI, PC, PE and PS at a ratio (1.0:1.28:0.70:0.09) corresponding to the phospholipid composition of Ino+ sake mash yeast cells were added to a reaction mixture, the AATase activity decreased to 26-55% that of yeast cells from the Ino- mash with a phospholipid composition of 0.34:1.28:0.7:0.09. Approximately all of the PI was recovered in the ammonium sulfate precipitate of the cell-free extract, while only half of the PC and PE was recovered. The acidic phospholipid, phosphatidylglycerol, as well as PI inhibited AATase activity more strongly than PC, despite its having the same fatty acid composition as PC. These results suggest that the strong inhibition of AATase activity by PI is due to its high adsorptive capacity for the AATase protein. Therefore, rice polishing can remove inositol from rice leading to an increase in AATase activity, and resulting in a high E/A ratio in sake.
使用精米率分别为70%、80%、90%和98%的大米制备清酒醪。在精米率为70%时，清酒中乙酸异戊酯/异戊醇（E/A）的比例最高，添加肌醇会使E/A比例降低。在测试的多个菌株中，向醪中添加肌醇会降低清酒中乙酸异戊酯的含量和E/A比例。添加肌醇会显著降低醇乙酰转移酶（AATase）的活性，该酶负责由醇和乙酰辅酶A合成乙酸酯。Northern印迹分析结果以及肌醇/胆碱介导的负调控基因OPII基因的破坏表明，添加肌醇后AATase活性的降低并非由转录事件引起。添加肌醇使清酒醪酵母细胞中的磷脂酰肌醇（PI）含量增加了3倍，而对磷脂酰胆碱（PC）、磷脂酰乙醇胺（PE）和磷脂酰丝氨酸（PS）的含量没有影响。当用氯仿或磷脂酶C处理从清酒醪酵母细胞制备的无细胞提取物以去除PI时，添加（Ino+）和未添加（Ino-）肌醇的清酒醪中AATase活性没有差异。从清酒醪酵母细胞制备的PI对AATase活性的抑制作用比PC和PE更强。此外，当以与Ino+清酒醪酵母细胞的磷脂组成相对应的比例（1.0:1.28:0.70:0.09）将PI、PC、PE和PS添加到反应混合物中时，AATase活性降至具有0.34:1.28:0.7:0.09磷脂组成的Ino-醪中酵母细胞活性的26 - 55%。几乎所有的PI都在无细胞提取物的硫酸铵沉淀中回收，而PC和PE仅回收了一半。尽管酸性磷脂磷脂酰甘油与PC具有相同的脂肪酸组成，但它对AATase活性的抑制作用比PC更强。这些结果表明，PI对AATase活性的强烈抑制是由于其对AATase蛋白具有高吸附能力。因此，大米抛光可以去除大米中的肌醇，导致AATase活性增加，并使清酒中的E/A比例升高。
