In this experiment, acarbose at a concentration of 2.5 g mL-1 was used to record the desired mean or mid signal. The error percentage was calculated to be 5.46%, which indicates that the method is accurate. J Genet Eng Biotechnol 14:133141, CrossRef Some examples are reported in our previous publications [7,8]. The basic function of an enzyme is to increase the rate of a reaction. Estimation of Tyrosine by Folin-Ciocalteau Method, Estimation of Proteins by Lowry method (Quantitative Analysis). 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. where AVGmax is the average of the maximum absorbance values, SDmax is the standard deviation of the maximum absorbance values, AVGmin is the average of the minimum absorbance values, SDmin is the standard deviation of the minimum absorbance values, n is the number of tests performed. Chem. 2021 Nov 16;10(11):2818. doi: 10.3390/foods10112818. This assay was also very precise, with RSD values below 3% in all cases. Unable to load your collection due to an error, Unable to load your delegates due to an error. The reaction was stopped by adding 200 L of DNS (final concentration 19.2 mmol L-1) and boiling in a water bath for 15.6 min. Different experiments were performed to evaluate the robustness of the method. In the present method of Millers reaction, the product to be analyzed was obtained by heating it in a boiling water bath. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. Xiao Z., Storms R., Tsang A. Corrigendum to A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities [Anal. 1 ml of D.N.S. Learn more 2023 Springer Nature Switzerland AG. 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. Just download this Color Reactions of Carbohydrates file, make a print, and distribute it to the students. EMBRAPA Agroindstria Tropical, Fortaleza, Brazil. This section illustrates an example of the application of the method that includes the corresponding calculations for both methods herein described. The use of a microscale method offers several advantages such as 1) fewer chemicals needed, 2) reduction in material cost, 3) ease of disposal of the reaction mixture after experimentation, 4) reduction of the safety risk, and 5) multiple experiments can be performed at the same time. [Crossref]. Chem. The first approach relies on the measurement of reducing sugars, being the dinitrosalicylic acid (DNS) method [3] the classic approach. The -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. The .gov means its official. Required fields are marked *. Enzyme activity is defined as the amount of product formed in 1 min under defined conditions, and its unit is moles min-1. [. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Also, depending on the temperature-activity relationship, the alpha amylase may not be active at 25C but is somewhere <100C. Do you think that woul Three different concentrations (1, 2.5, and 5 g mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. CAS Microbiol Res 207:116133, Srinivas A, Rahul K, Sasikala C, Subhash Y, Ramaprasad EVV, Ramana CV (2012) Georgenia satyanarayanai sp. Oliveira H.M., Correia V.S., Segundo M.A., Fonseca A.J.M., Cabrita A.R.J. This article will discuss the different applications of amylase. In contrast to the DNS method, the staining is instantaneous with the simple addition of a staining solution containing I3 that results from the stepwise dissolution of KI and I2 in water. Relative standard deviation (expressed in percentage, RSD%) measured the precision of the assay. 2017, 109, 962. Salivary amylase is an enzyme that is found in saliva. 2011, 136, 305. 1979 Nov;17(11):705-8. Amylase Test. Accessibility An example of the application of this method was the determination of the glucose yield after the hydrolysis of four pure starches assisted by an amyloglucosidase from A. niger (Fig. The obtained results are shown in Table 2. DA3-7. The proposed method relies on principles that allow a fast, simple, and versatile measurement of the activity of alpha-amylase or amyloglucosidase. Additionally, the optimized assay was applied to extracts from five different plants. nov., an alkaliphilic and thermotolerant amylase-producing actinobacterium isolated from a soda lake. (1) the calculated activity for this enzyme was 1123UmL1. Part of Springer Nature. In: Singh-Nee Nigam P., Pandey A., editors. Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Thus, it is important to optimize these two parameters to obtain a fixed value. -Amylase is an endoglycosidase that catalyzes the cleavage of internal -D-(1-4) glycosidic bonds in starch to produce oligosaccharides, dextrins, and maltose.44 Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Iran. This method is based on the binding of iodine to terminals of the starchs polymeric chain [4] that results in a blue colored complex that can be also quantitatively monitored by UVvis spectrophotometry [5]. Semantic Scholar is a free, AI-powered research tool for scientific literature, based at the Allen Institute for AI. It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus. Currently, there are different protocols and guidelines to perform the process of bioassay validation, such as Food and Drug Administration, USA (2018), Eli Lilly Guide (2007), and the 1033 Chapter of United States Pharmacopeia (1980).1414 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.https://www.fda.gov/files/drugs/publishe Place the vial in the block heater (or other alternative heating source) previously set at the target temperature of the study during the desired time of the assay (, Immediately stop the hydrolysis process by adding 750L of 1.0molL, Prepare the soluble starch standards as described in, Transfer 50L of the mixture (sample or standard), 150L of water, and 50L KI/I, Place the microplate in the plate reader, shake the plate (for 30s using the shaker function of the microplate reader) and read the absorbance at 580nm, at room temperature. [Crossref], 11 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. [Crossref]. 5 ml of deionized water are added in each sample, followed by Dissolve 0.2M (2.7218 grams) of KH2PO4 in 100ml of distilled water to this solution add 0.5M (2.8053 grams) KOH drop by drop till the pH is set to 6.8. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Federal government websites often end in .gov or .mil. In vivo studies of species of Zanthoxylum revealed that the phenolic content of the plant had a potential hypoglycemic effect.3131 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. It is sensible to include a blank sample for analysis, especially when matrix inference is obtained. results of inhibition of alpha amylase enzyme by the selected plant extracts have been represented in Figure 1. Absorbance was recorded at 540 nm using a microplate reader. <>>>
Measurement of alpha-amylase activity in white wheat flour, milled malt, and microbial enzyme preparations, using the Ceralpha assay: collaborative study. Federal government websites often end in .gov or .mil. endstream Hugo M. Oliveira and Margarida R. G. Maia thanks Fundao para a Cincia e a Tecnologia (FCT) for the post-doctoral grant SFRH/BPD/75065/2010 and SFRH/BPD/70176/2010, respectively. ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. The Z factor that measures both the variability in the assay and the dynamic range between maximum and minimum controls should be in the acceptable value of Z > 0.4.2929 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. The Z factor was computed for each plate (Equation 4). ; Humana Press: New York, 2016 [Crossref] Bioassays are challenging in terms of determining accuracy. Place the vial in the block heater (or other alternative heating sources) previously set at the target temperature of the study during the desired time (, Immediately stop the hydrolysis by adding 200L of 1.0molL. Centrifuge the vials for 5min at 350g, at room temperature. Chem. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. If necessary, the solution should be prepared fresh for daily use. The method is convenient, reliable and well-suited to the analysis of large numbers of malt samples, and the results are closely correlated to the addition of 2 ml. Boric acid (PN: A79-212, 99.5%) and NaOH (PN: S/4920/60) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). The site is secure. One hundred twenty five patients were included in the present study. When the substrate concentration is increased, the inhibition by the competitive inhibitor is reversed. For the calculation of the glucose yield assay promoted by amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich), 1500L of water were added to a vial containing of 3.00mg of a pure rice starch (mstarch) weighed in an analytical scale. 2019 Oct;18(4):2531-2539. doi: 10.3892/etm.2019.7840. 3 0 obj
An illustrative linear (R2=0.999) calibration curve is abs580nm=0.061 (0.025)+0.366 (0.014) mstarch. Therefore, the glucose yield corresponded to the ratio between mglucose (483.4g) and the total mass of starch mstarch (2400g), which resulted in a value of 20.14%. Validation of the method was performed according to international guidelines by analyzing linearity, precision, accuracy, robustness, plate uniformity, and signal variability.1414 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.https://www.fda.gov/files/drugs/publishe Oliveira H.M., Pinheiro A.Q., Fonseca A.J.M., Cabrita A.R.J., Maia M.R.G. The authors would like to thank the Professors Ana Paula TrovattiUetananbaro and Andra Miura da Costa, from the Laboratory of Applied Microbiology (LABMA, UESC), and the State University of Santa Cruz (UESC, Ilhus, Bahia, Brazil) for their crucial academic and experimental support. Xiao et al. Determination of kinetic constants was performed by determining the effect of substrate concentration on enzymatic activity. For measuring the amylase activity with DNS solution (Miller Method): 1. Cruz das Almas, Bahia, Brazil, Rodrigues RCB (2017) Produo de lipase e pectinase por fermentao em estado sido utilizando resduo de licuri como substrato. Humana, New York, NY. Additionally, different solvents, such as ethanol (25%) and dimethyl sulfoxide (DMSO) (final concentrations of 0.15% and 0.25%), were included. [Crossref]. We used a US bath to facilitate the complete solubilization of I2 and subsequent formation of triiodide (please note that the concentration of KI described in the original report of Xiao et al. These findings demonstrated that our optimized and validated method is highly selective, enabling the identification of samples with or without potent activity. Food Chem. For the control experiment, the vial contained 1500L of soluble starch solution 2.00gL1 and 1500L of universal buffer. Mina Karimi-Avargani kindly mail me [email protected]. I need your help for calculation of data. By intercepting this absorbance values in the calibration curve (e.g. The long-term use of the method also showed a high-precision for both intra-day and inter-day measurements. Plant extracts contain various phytochemicals, such as phenols, saponins, and alkaloids, that contribute to many observed pharmacologically relevant effects. [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. % [Crossref] Additionally, it is necessary to validate the assay method to demonstrate that it is acceptable for determining the pharmacological activity of a new chemical entity by a detectable signal that allows the biological process to be quantified. This method intends to facilitate the implementation of a new routine determination of alpha-amylase and amyloglucosidase activities in any laboratory that could deal with amylolytic enzymes by providing all the necessary experimental details and conditions that could maximize the performance of the method. 3,5-Dinitrosalicylic acid ( DNS or DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which strongly absorbs light at 540 nm. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. Copyright 2016 Elsevier Ltd. All rights reserved. Place the microplate in the plate reader, shake the plate (for 30 to 60s using the shaker function of the microplate reader), incubate for 30min at 37C, and read the absorbance at 505nm. The inhibition percentage was calculated using Equation 1: where A is the absorbance of the negative control, and B is the absorbance of the solution containing an inhibitory agent. PN: 27217, Sigma-Aldrich). Simultaneously setup the blank as per the test by adding DNS prior to the addition of enzyme simultaneously. Constructing a standard curve / graph for maltose helps us to estimate concentration of reducing sugars present in an unknown sample and for determining the activity of amylase enzyme in Then, the final concentrated enzyme solution was taken for comparative enzyme entrapment study. It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. The xylanase was immobilized for 4 h on 10 BCL aldehydeagarose gel by multicovalent attachment in 100 mM bicarbonate buffer at 25 C and pH 10 (Guisan, 1988).Periodically, the The authors are also grateful for the financial support of the Coordenao de Aperfeioamento de Pessoal de Nvel Superior (CAPES, Brazil) under grant number 451408/2019-01 and the Fundao de Amparo Pesquisa do Estado da Bahia (FAPESB, Brazil) under grant number RED0023/2014. It comprises two chains of amino acids, which are held together by weak bonds. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). To evaluate linearity, amylase inhibition studies were performed using different concentrations of the inhibitor acarbose (0.5 to 12 g mL-1). The RSD value for inter-day precision was 2.5%. From these samples, salivary amylose enzyme assays were carried out. Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; de Sales, P. M.; de Souza, P. M.; Dartora, M.; Resck, I. S.; Simeoni, L. A.; Fonseca-Bazzo, Y. M.; de Oliveira-Magalhes P.; Silveira, D.; Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; Rahimzadeh, M.; Jahanshahi, S.; Moein, S.; Moein, M. R.; Valentina, P.; Ilango, K.; Chander, S.; Murugesan, S.; Paiva, R. V. N.; Chaves, A. C. T. A.; de Paula, V. F.; de Macedo, G. E. L.; Queiroz, R. F.; Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; Granados-Guzmn, G.; Castro-Ros, R.; de Torres, N. W.; Salazar-Aranda, R.; Satpathy, G.; Tyagi, Y. K.; Gupta, R. K.; Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y.; Subramanian, R.; Asmawi, M. Z; Sadikun, A.; Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Iversen, P.; Beck, B.; Chen, Y.; Dere, W.; Devanarayan, V.; Eastwood, B. J.; Farmen, M. W.; Iturria, S. J.; Montrose, C.; Moore, R. A.; Weidner, J. R.; Sittampalam, G. S. In. It is also crucial to accurately use well-defined conditions for laboratory transfer. Warm the assay buffer to room temperature before use. Acta Part A-Mol. Get this protocol in PDF format. These extractions are commonly performed with buffer or salt solutions at mild temperatures (from room temperature to 40C). [Crossref], 10 White, R. E.; Annu. The positive control used was acarbose (0.6 g mL-1). Bookshelf More details and information about the extraction of other amylolytic enzymes from different natural matrices can be found in the method AOAC 2002.01, commonly named in the literature as the Ceralpha method [9]. grown on orange waste powder. Determination of amylose/amylopectin ratio of starches. [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). In addition, the assay method could be applied to a wide variety of molecules, and the obtained results should be reproducible and achieve the same results every time.1010 White, R. E.; Annu. Importance of Assay of Salivary amylase enzyme, Estimation of Carbohydrates by the Anthrone Method, Assay of Acid Phosphatase enzyme activity from Potatoes, Assay of Urease Enzyme Activity (Enzymology Practical Protocol), Effect of Temperature on Amylase activity (Enzymology Protocol). The use of a suitable volumetric scale for the assay was also an important concern for our development work. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The development work herein reported sought to answer to the major methodological difficulties that we experienced for implementing the protocols for measuring enzyme activity (for both amylase and amyloglucosidase) and glucose yield (for amyloglucosidase), starting from the report of Xiao et al. This methodological approach can be used for measuring the activity of both alpha-amylase and amyloglucosidase [5]. strains showed the amylolytic activity. The results of the experiment include RSD values of <3.13 (maximum signal), <3.19 (mid signal), and <5.02 (minimum signal), which are in the acceptable range (RSD < 20%). Rev. After weighing 3.0g of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 (diluted from the concentrated solution). Department of Microbiology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India, 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature, Aguiar, M.S., Maldonado, R.R., Carvalho, A.L., Aguiar-Oliveira, E. (2022). <>stream Accessibility It helps you protect your students from spelling mistakes and volumetric errors. The diagnosis of saliva amylase enzyme activity deficiency requires an oral test with a standard protocol. [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. xZmoEna?T [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. 2020-04-29T17:28:43+05:302017-05-24T07:28:08ZMicrosoft Office Word 2007 As an example, we summarize in this section the extraction protocol for the measurement of alpha-amylase activity in wheat flour [9]. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> The method was ideal with respect to linearity, accuracy, precision, selectivity, and reliability. 12 Stevenson, R. A.; American Laboratory, 2011, available at https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, accessed June 2022. A higher substrate concentration (more than Km) will interfere with the competitive inhibitor present in the extract and hinder the identification of the inhibitor in the extract. Without salivary amylase, we would not be able to digest carbohydrates properly and would not get the total nutritional value from these foods. Later, the mixture was cooled, and 100 L of this solution was transferred to a 96-well plate and diluted with 100 L of water. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. Amylolytic enzyme working solutions: prepare by stepwise dilution of the commercial solutions with universal buffer at the desired pH to fit the linear range of soluble starch determination. Like most things, amylase production slows as we age, which reduces the ability of the digestive system to properly absorb nutrients. Based upon the plasma glucose value, all subjects were divided into three groups. Starch is a widespread raw material with several application fields that cover food (including confectionery and drinks), feed, pharma, chemical, and paper industries [1]. Microbiol Res 192:192202, CrossRef It is defined as the combinatorial effect of all components of the analysis other than the analyte that is to be measured.2828 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. [Crossref]. Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. When the enzymatic activity decreases, the formation of the product also decreases; therefore, the intensity of the color is reduced. For enzyme inhibition studies, the absorbance was recorded at a concentration with 50% inhibition (IC60). Hence, the dilution factor corresponded to 2000. Furthermore, this improved method is very flexible and allows a broadband application, being able to be easily adjusted according to the requirements of each study or the equipment and instrumentation available at each laboratory. Standards for quantification of glucose yield. A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. Forty-One Plant Extracts Screened for Dual Antidiabetic and Antioxidant Functions: Evaluating the Types of Correlation between -Amylase Inhibition and Free Radical Scavenging. The test is done in microtitre plates with a total volume of 260L and an assay time of 40min including the pre-incubation steps. Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. Positive controls are compounds with proven activity in humans, whereas negative controls are those without activity. For enzymes, denaturation can be defined as the loss of enough structure, rendering the enzyme inactive.
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