比色法快速测定发酵液中γ-聚谷氨酸含量
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摘要
建立了发酵液中γ-聚谷氨酸(γ-PGA)含量快速测定的比色检测方法.其原理为γ-聚谷氨酸可以与亚甲基蓝溶液发生反应,并且使亚甲基蓝溶液颜色发生变化.配制不同浓度的γ-PGA标准品溶液,使其与亚甲基蓝溶液形成反应体系,并对亚甲基蓝溶液浓度、反应温度以及反应时间对反应体系的影响分别进行对比优化.结果显示,亚甲基蓝溶液质量浓度为10 mg/L,反应温度为30℃,反应时间为5 min,线性回归方程y=0.002 4x+0.328 9,R~2=0.996 5,空白加标平均回收率为106.8%,平均RSD值为1.51%,发酵液中加标平均回收率为118.3%,平均RSD值为1.89%.利用比色法测定发酵液中γ-PGA的含量简便快捷、重现性好、灵敏度较高、准确度好,可用于发酵液中γ-PGA浓度的检测.
A colorimetric method for the rapid determination of γ-PGA in fermentation broth was established. The principle is that poly-γ-glutamic acid can react with the methylene blue solution and change the color of the methylene blue solution. Different concentrations of γ-PGA standard solutions were prepared to form a reaction system with methylene blue solution,and the effects of methylene blue solution concentration,reaction temperature,and reaction time on the reaction system were compared and optimized. The results showed that the mass concentration of methylene blue solution was 10 mg/L,the reaction temperature was 30 ℃,the reaction time was 5 min,the linear regression equation was y=0.002 4x+0.328 9,R~2=0.996 5,and the blank average recovery rate was 106.8%. The average RSD value was 1.51%,the average recoveries in the fermentation broth were 118.3%,and the average RSD was 1.89%. The determination of γ-PGA in fermentation broth by colorimetry is simple and rapid,with good reproducibility,high sensitivity and good accuracy. It can be used to detect γ-PGA concentration in fermentation broth.
A colorimetric method for the rapid determination of γ-PGA in fermentation broth was established. The principle is that poly-γ-glutamic acid can react with the methylene blue solution and change the color of the methylene blue solution. Different concentrations of γ-PGA standard solutions were prepared to form a reaction system with methylene blue solution,and the effects of methylene blue solution concentration,reaction temperature,and reaction time on the reaction system were compared and optimized. The results showed that the mass concentration of methylene blue solution was 10 mg/L,the reaction temperature was 30 ℃,the reaction time was 5 min,the linear regression equation was y=0.002 4x+0.328 9,R~2=0.996 5,and the blank average recovery rate was 106.8%. The average RSD value was 1.51%,the average recoveries in the fermentation broth were 118.3%,and the average RSD was 1.89%. The determination of γ-PGA in fermentation broth by colorimetry is simple and rapid,with good reproducibility,high sensitivity and good accuracy. It can be used to detect γ-PGA concentration in fermentation broth.
引文
[1] 刘婷.发酵液中γ-聚谷氨酸的分离纯化及初步鉴定研究[D].西安:陕西科技大学,2016.
[2] 桑秀梅.利用聚谷氨酸高产菌NS-18制备γ-聚谷氨酸[D].南京:南京师范大学,2017.
[3] 董健,吕颖,方丽,等.γ-聚谷氨酸的发酵制备及快速鉴定分析方法研究[J].中国酿造,2012,32(5):148-150.
[4] MAKOTO A,KAZUYA S,HISAAKI N,et al.Enzymatic Synthesis of High-Molecular-Mass Poly-γ-glutamate and regulation of its stereochemistry[J].Applied and environmental microbiology,2004,7:4249-4255.
[5] 张庆庆,金鑫强,陈剑翔,等.发酵液中γ-聚谷氨酸含量快速测定方法研究[J].食品工业科技,2012,19:294-296.
[6] ING-LUNG S,YI-TSONG V.The production of poly-(γ-glutamic acid)from microorganisms and its various applications[J].Broresource technology,2001,79(3):207-225.
[7] 朱凡,吕忠良,杨叶东,等.高黏发酵液中γ-聚谷氨酸的分离纯化工艺[J].化学工程,2013,41(12):9-11.
[8] EZZELL J W,WELKOS S L.The capsule of bacillus anthracis,a review[J].J Appl Microbiol,1999,87(2):250.
[9] 李德衡,赵兰坤,李树标.γ-聚谷氨酸的生物合成及应用研究进展[J].发酵科技通讯,2012,41(3):12-16.
[10] 李江涛,郑涛.介孔碳材料对亚甲基兰的吸附特性研究[J].西安文理学院学报(自然科学版),2010,13(1):48-53.
[11] 肖敏,李丽,钟龙飞,等.活性炭吸附法处理印染废水的研究[J].辽宁化工,2009,38(8):537-539.
[12] 王静心,李政,张秋亚,等.亚甲基蓝染液的γ-PGA水凝胶脱色处理[J].印染,2013,24:1-5.
[13] MEHMET D,MAHIR A,AYDM T,et al.Kinetics and mechanism of removal of methylene blue by adsorption onto perlite[J].Journal of hazardous materials,2004,109:141-148.
[2] 桑秀梅.利用聚谷氨酸高产菌NS-18制备γ-聚谷氨酸[D].南京:南京师范大学,2017.
[3] 董健,吕颖,方丽,等.γ-聚谷氨酸的发酵制备及快速鉴定分析方法研究[J].中国酿造,2012,32(5):148-150.
[4] MAKOTO A,KAZUYA S,HISAAKI N,et al.Enzymatic Synthesis of High-Molecular-Mass Poly-γ-glutamate and regulation of its stereochemistry[J].Applied and environmental microbiology,2004,7:4249-4255.
[5] 张庆庆,金鑫强,陈剑翔,等.发酵液中γ-聚谷氨酸含量快速测定方法研究[J].食品工业科技,2012,19:294-296.
[6] ING-LUNG S,YI-TSONG V.The production of poly-(γ-glutamic acid)from microorganisms and its various applications[J].Broresource technology,2001,79(3):207-225.
[7] 朱凡,吕忠良,杨叶东,等.高黏发酵液中γ-聚谷氨酸的分离纯化工艺[J].化学工程,2013,41(12):9-11.
[8] EZZELL J W,WELKOS S L.The capsule of bacillus anthracis,a review[J].J Appl Microbiol,1999,87(2):250.
[9] 李德衡,赵兰坤,李树标.γ-聚谷氨酸的生物合成及应用研究进展[J].发酵科技通讯,2012,41(3):12-16.
[10] 李江涛,郑涛.介孔碳材料对亚甲基兰的吸附特性研究[J].西安文理学院学报(自然科学版),2010,13(1):48-53.
[11] 肖敏,李丽,钟龙飞,等.活性炭吸附法处理印染废水的研究[J].辽宁化工,2009,38(8):537-539.
[12] 王静心,李政,张秋亚,等.亚甲基蓝染液的γ-PGA水凝胶脱色处理[J].印染,2013,24:1-5.
[13] MEHMET D,MAHIR A,AYDM T,et al.Kinetics and mechanism of removal of methylene blue by adsorption onto perlite[J].Journal of hazardous materials,2004,109:141-148.