Katrin Reichenberger, Sabine Lutz-Wahl, Lucas Kettner, Lutz Fischer
{"title":"一种极灵敏的淀粉酶活性测定方法及其在面包中残留淀粉酶活性测定中的应用","authors":"Katrin Reichenberger, Sabine Lutz-Wahl, Lucas Kettner, Lutz Fischer","doi":"10.1007/s12161-025-02781-3","DOIUrl":null,"url":null,"abstract":"<div><p>An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the <i>α</i>-amylase from <i>Bacillus subtilis</i> in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL<sup>−1</sup>), a GOD from <i>Aspergillus niger</i> (1550 nkat mL<sup>−1</sup>) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- (<i>α</i>-amylase from <i>B. subtilis</i>) and exo-acting amylase (maltogenic amylase from <i>Geobacillus stearothermophilus</i>), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL<sup>−1</sup> for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of <i>α</i>-amylase from <i>B. subtilis</i> in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined. </p></div>","PeriodicalId":561,"journal":{"name":"Food Analytical Methods","volume":"18 6","pages":"1149 - 1161"},"PeriodicalIF":3.0000,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1007/s12161-025-02781-3.pdf","citationCount":"0","resultStr":"{\"title\":\"An Extremely Sensitive Amylase Activity Assay and its Application for the Determination of the Residual Amylase Activity in Bread\",\"authors\":\"Katrin Reichenberger, Sabine Lutz-Wahl, Lucas Kettner, Lutz Fischer\",\"doi\":\"10.1007/s12161-025-02781-3\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the <i>α</i>-amylase from <i>Bacillus subtilis</i> in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL<sup>−1</sup>), a GOD from <i>Aspergillus niger</i> (1550 nkat mL<sup>−1</sup>) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- (<i>α</i>-amylase from <i>B. subtilis</i>) and exo-acting amylase (maltogenic amylase from <i>Geobacillus stearothermophilus</i>), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL<sup>−1</sup> for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of <i>α</i>-amylase from <i>B. subtilis</i> in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined. </p></div>\",\"PeriodicalId\":561,\"journal\":{\"name\":\"Food Analytical Methods\",\"volume\":\"18 6\",\"pages\":\"1149 - 1161\"},\"PeriodicalIF\":3.0000,\"publicationDate\":\"2025-03-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://link.springer.com/content/pdf/10.1007/s12161-025-02781-3.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Food Analytical Methods\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s12161-025-02781-3\",\"RegionNum\":3,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"FOOD SCIENCE & TECHNOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Food Analytical Methods","FirstCategoryId":"97","ListUrlMain":"https://link.springer.com/article/10.1007/s12161-025-02781-3","RegionNum":3,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"FOOD SCIENCE & TECHNOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
采用天然底物淀粉和葡萄糖氧化酶和过氧化物酶两种辅助酶，建立了一种极灵敏的淀粉酶活性测定方法，测定了白面包中枯草芽孢杆菌α-淀粉酶的残留活性。首先，对电子受体DA-67 （50 μM）、辣根过氧化物酶（681 nkat mL - 1）、黑曲霉GOD （1550 nkat mL - 1）和天然底物淀粉（0.01% (w/v)）的浓度进行优化，以获得较高的灵敏度。用枯草芽孢杆菌的内α-淀粉酶和嗜脂嗜热地杆菌的外显淀粉酶检测该方法的线性关系，并考察和优化培养时间对检测灵敏度的影响。优化后的检测方法能够测定两种淀粉酶的最低淀粉酶活性为0.33 pkat mL−1，检测运行时间为7.5 h。与优化后的商用Ceralpha（测定内淀粉酶活性）和Betamyl3（测定外淀粉酶活性）相比，这种新的DA-67淀粉酶检测方法的灵敏度分别提高了4.7倍和4.2倍。采用新的DA-67淀粉酶测定法测定白面包中枯草芽孢杆菌α-淀粉酶的残留活性。残留活性为2.26±0.15%。
An Extremely Sensitive Amylase Activity Assay and its Application for the Determination of the Residual Amylase Activity in Bread
An extremely sensitive amylase activity assay was developed using the natural substrate starch and two ancillary enzymes: a glucose oxidase (GOD) and a peroxidase, to measure the residual activity of the α-amylase from Bacillus subtilis in white bread. Firstly, the concentrations of the assay components: electron acceptor DA-67 (50 μM), horseradish peroxidase (681 nkat mL−1), a GOD from Aspergillus niger (1550 nkat mL−1) and the natural substrate starch (0.01% (w/v)), were optimized to achieve high sensitivity. The linearity of the assay was then tested with both an endo- (α-amylase from B. subtilis) and exo-acting amylase (maltogenic amylase from Geobacillus stearothermophilus), and the effect of the incubation time on the assay sensitivity was investigated and optimized. The optimized assay was capable of determining a minimal amylase activity of 0.33 pkat mL−1 for both amylases tested with an assay run time of 7.5 h. This new DA-67 amylase assay demonstrated 4.7- and 4.2-times higher sensitivity, respectively, compared to optimized versions of the commercial Ceralpha (determination of endo-amylase activities) and Betamyl3 (determination of exo-amylase activities) assays. The new DA-67 amylase assay was used to determine the residual activity of α-amylase from B. subtilis in white bread. A consistent residual activity of 2.26 ± 0.15% was reliably determined.
期刊介绍：
Food Analytical Methods publishes original articles, review articles, and notes on novel and/or state-of-the-art analytical methods or issues to be solved, as well as significant improvements or interesting applications to existing methods. These include analytical technology and methodology for food microbial contaminants, food chemistry and toxicology, food quality, food authenticity and food traceability. The journal covers fundamental and specific aspects of the development, optimization, and practical implementation in routine laboratories, and validation of food analytical methods for the monitoring of food safety and quality.
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号
