红茶菌发酵罗城毛葡萄13种有机酸的同时检测

郝俊光; 陈婉玲; 杨丽; 陈霜梅; 梁振荣; 银书; 祁岑

doi:10.13386/j.issn1002-0306.2020020252

红茶菌发酵罗城毛葡萄13种有机酸的同时检测

Simultaneous Determination of 13 Organic Acids during the Fermentation of Luocheng Vitis quinnquangularis Rehd by Kombucha Consortium

摘要

摘要: 为解决目前尚无对红茶菌发酵液中多种有机酸同时检测方法的问题,本文利用液相色谱建立红茶菌发酵液13种有机酸的准确定量方法,并对发酵过程中的典型有机酸变化进行跟踪。优化后的色谱条件:Atlanis T3色谱柱,流动相为0.02 mol/L KH₂PO₄(pH=2.6),流速0.4 mL/min,等度洗脱,柱温30 ℃、检测波长210 nm。此色谱条件下,葡萄糖醛酸和草酸完全重合(只能用葡萄糖醛酸标准曲线相对定量),但其它11种有机酸均能有效分离。定量方法线性范围较宽,R²>0.990,定量限0.080~4.789 mg/L,加标回收率92.68%~107.35%,相对标准偏差<5%,葡萄糖酸、乙酸、苹果酸在发酵过程中一直增加,而其它酸类呈先升后降趋势。葡萄糖酸、乙酸是主要的有机酸,在17 d的发酵液中所测有机酸占75.03%和10.46%。本研究建立的有机酸检测方法适合红茶菌发酵样品的检测,为红茶菌发酵毛葡萄生产酸性功能软饮品提供了基础依据。

Abstract: In order to get a feasible means to simultaneous detect organic acids in the fermentation process of kombucha bacteria, the method of accurate quantitation 11 organic acids and 2 relative quantitation in kombucha bacteria was established via liquid chromatography, and the changes of the organic acids in the fermentative kombucha broth of Luocheng Vitis quinnquangularis Rehd were also tracked. The analytical conditions of organic acids were optimized using the Atlanis T3 chromatographic column as follows:The mobile phase was 0.02 mol/L KH₂PO₄ (pH=2.6) with a constant flow rate of 0.4 mL/min.The column temperature and UV detection wavelength were set as 30℃ and 210 nm, respectively. Glucuronic acid and oxalic acid were completely merged into a co-elution peak, and the coelution peak was relatively quantified by standard curve of glucuronic acid. Meanwhile, the other 11 organic acids including gluconic acid and quinic acid were effectively separated in the fermentative kombucha broth of Luocheng Vitis quinnquangularis Rehd. The method has wide linear with correlation coefficients were all above 0.990. The quantitative limits were in the range of 0.080~4.789 mg/L, while he spiked recoveries ranged from 92.68%~107.35% with RSDs less than 5%. The fermentation process of Luocheng Vitis quinnquangularis Rehd was tracked and the synergistic evolution of organic acids was investigated. The contents of gluconic acid, acetic acid and malic acid keep on increasing during the fermentation process, while the changes of other acids turn to rise first and then fall later. Gluconic acid and acetic acid were the main organic acids, accounting for 75.03% and 10.46% of the measured organic acids in the fermentation broth of 17 d, respectively. This method is suitable to be applied to simultaneously determine the main organic acids in the fermentation broth of kombucha. The traced data during fermentation provide basic data for further development of acidic functional soft beverage from Luocheng Vitis quinnquangularis Rehd fermented by Kombuch consortium.