Different parameters, such as enzyme concentration, substrate concentration (starch), preincubation time, incubation time, incubation temperature, and time in the water bath, were evaluated. Their age ranged between 25 65 years. All subjects were subjected to fasting blood glucose estimation followed by measurement of plasma glucose level after 75g of glucose ingestion. [1] Amylase was first described in the early 1800s and is considered one of the first enzymes in history to be scientifically investigated. J. The optimum conditions used were an enzyme concentration of 0.15 U mL-1, preincubation and incubation times of 7.2 min and 5.5 min, respectively, and a water bath duration of 15.6 min; all abovementioned conditions produced the best inhibition percentage. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. The use of soluble starch also allowed the standardization of the working conditions, avoiding any influence of the solubility of the starch in the final result. Changes in conditions/parameters, such as enzyme concentrations (< 0.15 U mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Google Scholar, Vasconcelos NM, Pinto GAS, Arago FAS (2013) Boletim de Pesquisa n. 88, Determinao de Acares Redutores pelo cido 3,5-Dinitrosaliclico: Histrico do Desenvolvimento do Mtodo e Estabelecimento de um Protocolo para o Laboratrio de Bioprocessos. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. Mid signal: Mean absorbance was recorded by the assay procedure. Starch Europe Key Figures 2016. Chem. Salivary amylase analysis is a simple procedure and the results obtained can be used as an additional criterion in the diagnostic process of diabetes. 5 ml of deionized water are added in each sample, followed by agitation. [Crossref], 30 Rahman, M. M.; Islam, M. R., Shohag, S.; Hossain, M. E.; Rahaman, M. S.; Islam, F.; Ahmed, M.; Mitra, S.; Khandaker, M. U.; Idris, A. M.; Chidambaram, K.; Emran, T. B.; Cavalu, S.; Molecules 2022, 27, 1713. [Crossref]. The absorbance of the well was monitored at 505nm, and the value recorded was 0.681. Experimental conditions as described in the protocol (the incubation temperature was 45C). State University of Santa Cruz, Ilhus, Bahia, Brazil, Milena Santos Aguiar&Elizama Aguiar-Oliveira, TechnicalCollege of Campinas, Universityof Campinas, Campinas, So Paulo, Brazil, State University of Feira de Santana, Feira de Santana, Bahia, Brazil, You can also search for this author in Lilly (2007) recommended the use of the substrate at or below the Km value. [Crossref], 31 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. Glycosidase enzymes breakdown carbohydrates into simple sugars they enter the bloodstream, producing postprandial hyperglycemia. 8600 Rockville Pike Analysis of results using the Tukey test ( = 0.05) indicated that the method is robust for two conditions, i.e., incubation temperature and preincubation time. Amylase is an enzyme, or special protein, that helps you digest carbohydrates. Screening 1999, 4, 67. Measurement of alpha-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the Ceralpha assay: collaborative study. Achillea fragrantissima Forssk. The linear range of amylase inhibition by acarbose was 1.5-5 g mL-1, with a regression equation of y = 7.5784x + 37.80, a determination coefficient of 0.9906, and an IC50 of 1.61 0.06 g mL-1 (Figure 3). 8) [8]. Methods in Actinobacteriology pp 495503Cite as, Part of the Springer Protocols Handbooks book series (SPH). This work addresses optimization, validation and application of an in vitro method for assessment of -amylase inhibition on a microscale level. Med 2011, Article ID 810207. 2021 Nov 16;10(11):2818. doi: 10.3390/foods10112818. Starch is a widespread raw material with several application fields that cover food (including confectionery and drinks), feed, pharma, chemical, and paper industries [1]. R. P.; Mol. To search for new compounds, it is necessary to carry out pharmacological screening for a large number of potential -amylase inhibitors in a short period, for which high-throughput screening can be successfully used.88 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. Universidade Federal do Recncavo da Bahia. This was followed for a second dilution of 500L of the previous solution with 4500L of universal buffer at the desired pH (enzyme solution 2, dilution factor 10). Universal buffer: o-phosphoric acid 47mmolL1, acetic acid 50mmolL1, boric acid 50mmolL1. Int J System Evol Microbiol 62:24052409, Gong Y, Bai JL, Yang HT, Zhang WD, Xiong YW, Ding P, Qin S (2018) Phylogenetic diversity and investigation of plant growth-promoting traits of actinobacteria in coastal salt marsh plant rhizospheres from Jiangsu, China. (1) are: mcontrol=2.94mg, massay=0.133mg, time=10min, volume of enzyme used in the assay=0.500mL, dilution factor=2000. 2011, 44, 2076. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. J Ferment Technol 54:267286, CAS Xiao Z., Storms R., Tsang A. Corrigendum to A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities [Anal. PubMedGoogle Scholar. For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. Read the color developed at 520 nm. It was possible to linearly (R2 0.999) measure masses of glucose up to 10.0g (this corresponds to the actual mass present in the microplate well, corresponding to 50L of a 200.0mgL1 of a glucose standard solution). However, extracts from other plants had no effect on the enzyme activity, suggesting that plant extracts do not contain inhibitors of the enzyme -amylase. Previously reported highest and lowest values of optimization experiments were used. Concerning to the measurement of soluble starch, the adjustments covered two complementary aspects: bypassing the precipitation found in microplate wells when the original volumes were used, and minimizing potential deviations of Beers law when high values of absorbance (above 1) described in the original method were found. The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. The masses of glucose represented are 0.00, 2.00, 4.00, 6.00, 8.00 and 10.0g. Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. The new PMC design is here! x=7}_ReqJ,5)tG9R/9Jj/s?Tf/8t1/.~/~rq_^8nO_",2+?EMBeg}WfOw}aq6zf'SdBfW71T? Designed by http://www.microbiology.biology.upatras.gr. Total PDF Watermark Remover 1.0.214; modified using iTextSharp 4.1.6 by 1T3XTijsr, International Journal of Science and Research, ijsr.net, International Journal of Science and Research (IJSR), www.ijsr.net The activity of enzymes is strongly affected by changes in pH and temperature. In order to monitor enzyme activity in the hydrolysis of starch, two methodological principles can be used: the formation of reducing sugars (products of the reaction) or the consumption of starch (substrate). Epub 2015 Feb 16. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. Minitab 17 (Minitab, Inc., USA) was used for data analysis. National Library of Medicine Absorbance was recorded using a microplate reader (Multiskan FC, Thermo Scientific). Because it is impossible to perform boiling in a microplate because a significant volume of liquid evaporates and negatively affects the response, the experiment was conducted in the tubes, and the microquantities were transferred to microplates and recorded. The authors also acknowledge to Slvia Azevedo for the valuable technical assistance. 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. [Crossref]-66 Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; International Journal of Advanced Engineering Research and Science 2019, 6. After preincubation, 100 L of starch was added (final concentration 0.38 mg mL-1) and incubated at 37 C for 5.5 min for enzymatic action. It was initially termed Therefore, this method should be considered as a valuable tool in clinical practice. (C6H10O5)n + H2O n For calculation of enzyme activity base on (U/ml) try according to the following stages: 1. In excel file that you have prepared standard curve, ob Process Biochem 50:21282136, Nithya K, Muthukumar C, Biswas B, Alharbi NS, Kadaikunnan S, Khaled JM, Dhanasekaran D (2018) Desert actinobacteria as a source of bioactive compounds production with a special emphases on Pyridine-2,5-diacetamide a new pyridine alkaloid produced by Streptomyces sp. MeSH Sci. Note: this is a non-interchangeable rotor centrifuge (350g) where the vials used for the activity assay can be directly centrifuged (stacked in pairs). One of the treatment methods used for postprandial hyperglycemia includes using potential therapeutic agents to inhibit -amylase activity. The degree of inhibition was evaluated in terms of the concentration of the inhibitor that inhibited half of the enzyme activity (IC50). The vial was then placed for 10min into a heating block at 45C. 2021 Oct 27;6(44):29804-29813. doi: 10.1021/acsomega.1c04244. Five plant extracts were used to determine -amylase inhibitory activity using the proposed method. FOIA However, it showed a relatively higher -amylase activity in Fig 3 d. In additio n, Fig 3 d needs control to show equal amounts of proteins were loaded. It is then important to control the activity of these enzymes in two major complementary roles: as a quality control tool, for monitoring enzyme activity along time and batch to batch reproducibility in different industrial and laboratory applications, and also as a research tool, for studying novel strategies to improve the efficiency of the overall process. The final aspect of the microplate, absorbance spectra, and illustrative calibration curve of different glucose standards (from left to right, respectively) of the glucose oxidase/peroxidase assay used for the determination of the glucose yield after hydrolysis of pure starches catalyzed by amyloglucosidase. We also analyzed the reproducibility of the method by compiling the evolution of its sensitivity (defined by the slope of the calibration curve) at both intra- and inter-day in 10 experimental days during a period of approximately two months (Fig. [Crossref]. Standard deviation was calculated, and the relative standard deviation in terms of percentage was determined (Equation 2) to check the precision values on the experimentation day and other different days. Biotechnol. The ethanol extract of A. Maxicana L. leaves possessed Europe S. 2018. Insights into the Inhibition Mechanism of Human Pancreatic -Amylase, a Type 2 Diabetes Target, by Dehydrodieugenol B Isolated from Ocimum tenuiflorum. Some examples are reported in our previous publications [7,8]. Xiao et al. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 14 Food and Drug Administration (USA); Bioanalytical method validation guidance for industry, US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research and Center for Veterinary Medicine, 2018, available at https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf, accessed June 2022. See, The calibration curve for glucose was linear for masses of glucose up to 10g (equivalent to concentrations up to 200mgL, Determination of the alpha-amylase and amyloglucosidase activities, Enzyme activity, Starch-iodine, Glucose oxidase/peroxidase, Glucose yield. Food Sci. [Crossref], Postprandial hyperglycemia can be controlled by inhibitors, such as those directed at inhibiting -amylase, which reduces the flow of glucose by digesting carbohydrates. The enzymatic reaction was stopped by adding 200 L of DNS (final concentration 19.2 mmol L-1) and subsequently placed in a boiling water bath for 15.6 min. The abovementioned protocol was followed with different concentrations of substrates. The enzyme concentration, incubation time, interaction between enzyme concentration and preincubation time, and the interaction between preincubation and boiling time showed a significant effect on the inhibition percentage. PMC 5. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). Learn more, Banerjee S, Maiti TK, Roy RN (2016) Identification and product optimization of amylolytic Rhodococcus opacus GAA 31.1 isolated from gut of Gryllotalpa africana. Maltose Basic Med. In addition, the effect of substrate concentration on enzyme activity was analyzed. [5]. Starch, acarbose, 3,5-dinitrosalicylic acid (DNS), sodium chloride, dibasic sodium phosphate, monobasic sodium phosphate, sodium, and potassium tartrate were procured from Sigma-Aldrich, USA. The values in parentheses are the limits of the 95% confidence levels intervals, and starch mass is expressed in mg. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro. [Crossref], 21 Subramanian, R.; Asmawi, M. Z; Sadikun, A.; Acta Biochim. ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. Factors significantly contributing to the response were selected by the Pareto graph. The linear range of the measurements included masses of soluble starch up to 3.00mg (see, The amount of hydrolyzed starch was the difference between the masses calculated from the calibration curve obtained by plotting absorbance. The .gov means its official. A test can be done to measure the level of this enzyme in your blood. This concentrated buffer should be diluted 200 times (e.g. The protocol applies to two new strains of actinobacteria, isolated in Northeastern Brazil, using residues from the processing of a local fruit of a palm tree (licuri) as a cultivation substrate for solid state fermentation (SSF). Mina Karimi-Avargani kindly mail me saidurga48@gmail.com. I need your help for calculation of data. Acarbose, an inhibitor of the amylase enzyme, was evaluated at six different concentration levels by regression analysis. Preparation of soluble starch standards for quantification of amylolytic enzyme activity. These protocols and guidelines are followed in this work. Leaf starch degradation comes out of the shadows. The variability test is conducted at three response levels with varied signal intensities.1515 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Magnetic stirrer/heater plate (MAG-H, Gerhardt, Knigswinter, Germany), for preparing the soluble starch solution. Amylase activity from partially purified samples was assayed by DNS method. In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. Mntele W., Deniz E. UVvis absorption spectroscopy: Lambert-Beer reloaded. Please note that if you reject them, you may not be able to use all the functionalities of the site. [Crossref], 6 Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; International Journal of Advanced Engineering Research and Science 2019, 6. Prepare EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. For measuring the amylase activity with DNS solution (Miller Method): 1. The evaluation of the matrix effect showed linearity at 1 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Set up the standards of different test tubes and repeat the experiment as per the test and measure the color developed at 520nm absorbance. [Crossref], a determination coefficient of 0.9947, a slope of 11.493, and an IC50 value of 2.9 0.03 g mL-1 (data not shown). Int. Additionally, different solvents, such as ethanol (25%) and dimethyl sulfoxide (DMSO) (final concentrations of 0.15% and 0.25%), were included. When the substrate concentration is increased, the inhibition by the competitive inhibitor is reversed. [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. and transmitted securely. 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