Med 2011, Article ID 810207. Biochem. 2005 Mar;10(3):130-7. doi: 10.1016/j.tplants.2005.01.001. 0.5 ml of properly diluted (in acetic acid buffer solution; pH=4.9) crude enzyme are incubated for 15 min at T=40 C with 0.5 ml of soluble starch solution 1 % w/v. 1959, 31, 426. These protocols and guidelines are followed in this work. When an analytical method is considered, the matrix effect results in variation in the sensitivity, which is manifested by comparing the calibration curve of the standard (positive control) with that of the sample (plant extract). Formerly known as ptyalin, it breaks down starch into maltose and isomaltose. Please note that if you reject them, you may not be able to use all the functionalities of the site. Next, for the assay experiment, 500L of enzyme solution 2 was mixed in a vial that contained 1500L of a soluble starch solution 2.00gL1 and 1000L of universal buffer. Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. 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. Dilute to a final volume of 100 ml with reagent grade water. When the pancreas is diseased or inflamed, it releases increased amounts of amylase into the blood. This is a preview of subscription content, access via your institution. 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. Sci. 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. Starch Europe Key Figures 2016. Factors showing a significant effect on the inhibition percentage were selected and optimized using a basic sequential simplex method. [Crossref], 24 Wang, Y.; Huang, S.; Shao, S.; Qian, L.; Xu, P.; Ind. By intercepting those absorbance values in the calibration curve (e.g., abs580nm=0.061 (0.025)+0.366 (0.014) mstarch), we found that the mass of starch after hydrolysis (massay) was 0.133mg, and the mass of starch of the control (mcontrol) experiment was 2.94mg, respectively. Then the absorbance of each one of the unknown samples is measured and the concentration of the converting sugars is determined, based on the standard curve. Federal government websites often end in .gov or .mil. <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>>
Soluble starch stock solution 2.00gL1: disperse 1.000g of soluble starch in 5mL of cold water, add to a beaker with 400mL of boiling water under continuous stirring, after 5min switch off the heat and keep the stir until the solution reach room temperature, transfer the solution to a 500mL volumetric flask, and complete the volume with water up to 500mL; keep the solution at 4C up to three days. Based upon the plasma glucose value, all subjects were divided into three groups. Food Sci. The use of a suitable volumetric scale for the assay was also an important concern for our development work. Can you see this When compared with the original method, these new conditions lead to an extra dilution of the sample (a dilution factor of 5 compared to a dilution factor of 2) and to a longer optical path due to the higher volume placed at each well of the microplate (250L vs. 150L, and assuming the use of a classic cylindrical-shaped flat bottom microplate). 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 A uniformity assay is performed at maximum and minimum response levels, ensuring that the difference is adequate to detect active compounds or the influence of the inhibitor in the assay system. This method is a redox reaction where DNS (yellow color) is reduced by reducing sugars to 3-amino-5-nitrosalicylic acid (red color) in an alkaline medium. 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). Hence, we kept the same volume ratio of the original method but we increased the assay to a total volume of 3.00mL according to our requirements, which were defined by the specifications of the US probe used to study the effect of US in the activity of amylolytic enzymes [7,8]. Amylase is an enzyme, or special protein, that helps you digest carbohydrates. Trends Plant Sci. Enzyme Microb Technol 39:732742, Bispo ASR (2010) Bioprospeco de actinomicetos isolados de solos no Estado da Bahia e seu potencial biotecnolgico na produo de enzimas lignocelulolticas. We adopted this strategy to separate the solid particles that remained after the hydrolysis of different pure starches mediated by amyloglucosidase (glucose yield protocol, step 5). The degree of inhibition was evaluated in terms of the concentration of the inhibitor that inhibited half of the enzyme activity (IC50). J Genet Eng Biotechnol 15:8794, Zhao Y, Zhao Y, Zhang Z, Wei Y, Wang H, Lu Q, Li Y, Wei Z (2017) Effect of thermo-tolerant actinomycetes inoculation on cellulose degradation and the formation of humic substances during composting. Int. Dissolve 134.1g of malic acid, 70g of NaOH, and 58.4g of NaCl in 900mL of water (deionized or ultra-pure). Enzymatic Method for Determining Amylase Activity (Amylase Activity Assay) This assay protocol is suitable for the colorimetric detection of Amylase activity in cell and tissue culture 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. These parameters should be adjusted according to the objectives of each individual study). For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of -amylase inhibitory activity using the glucose assay kit was developed. After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. 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. Previously reported highest and lowest values of optimization experiments were used. Factors significantly contributing to the response were selected by the Pareto graph. 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 Bethesda, MD 20894, Web Policies 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. A state of sustained hyperglycemia caused by insulin deficiency can lead to type 2 diabetes.11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Federal government websites often end in .gov or .mil. Soluble starch (PN: S9765), starch from corn (PN: S4126), starch from potato (PN: S4251), starch from rice (PN: S7260), starch from wheat (PN: S5127, unmodified), I2 (PN: 207772, 99.8%), 85% (w/w) o-phosphoric acid (PN: 79620), and acetic acid (PN: 695092, 99.7%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 6, where we studied the influence of pH on the activity of amyloglucosidase from A. niger. 26 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. The Millers reactions were performed in 13 100 test tubes. 1959, 31, 426. 2012, 60, 8924. 2019 Oct;18(4):2531-2539. doi: 10.3892/etm.2019.7840. 2012, 13, 427. Each enzyme works best at a certain pH and temperature, its activity decreasing at values above and below that point due to denaturation. 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 Anal. The -amylase activity is measured using a colorimetric method with 3,5-dinitrosalicylic acid (DNS) reagent. Regarding that the enzyme is absent for the control experiment, it is expectable that the absorbance of the control experiment would be similar to the most concentrated standard of soluble starch prepared (3.00mg, Table 1). Visvanathan R, Houghton MJ, Williamson G. Food Chem. The transfer of the vial content to another tube / container may also be necessary. Linearity was determined using the graph of concentration and percent inhibition. Design of a new amylase activity method using starch as substrate and DNS reagent for measuring reducing power as the equivalent of glycosidic bond hydrolysis at endobj
Nutr. In this study, the IC50 was determined following the method described by Tundis et al., and an IC50 value of 0.6 g mL-1 was obtained.2626 Tundis, R.; Loizzo, M. R.; Statti, G. A.; Menichini, F.; Pharmazie 2007, 62, 473. Optimization of the -amylase inhibition assay was performed using fractionalized factorial design (2^6-1). After weighing 3.0g of flour into a 50mL flask, add 20.0mL malate buffer pH 5.4 (diluted from the concentrated solution). The Km value is the substrate concentration needed to reach half the maximum velocity. Sci. We use cookies on our website. From these samples, salivary amylose enzyme assays were carried out. Briefly centrifuge vials before opening. (IGRT'"w8f1P *`Ta,DXGcM5Lz When the enzymatic activity decreases, the formation of the product also decreases; therefore, the intensity of the color is reduced. The intra-day variation of sensitivity ranged between 2.0 and 6.3%, and the inter-day variation was 4.9% for the complete period. Different experiments were performed to evaluate the robustness of the method. [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. On the other hand, the triiodide solution implies the first dissolution of KI followed by I2. where SD is the standard deviation of each measurement, and AVG is the average of such measurements. 8) [8]. According to the obtained results, the sensitivity of salivary amylase assay was found to be 95% and specificity was 100%. Part of Springer Nature. [Crossref]. The quantitative determination of reducing sugar formation was carried out by using dinitrosalicylic acid (DNS) method in which starch was used as aninducer substrate for amylase enzyme. Their age ranged between 25 65 years. The kinetic constant maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were obtained from the Lineweaver-Burk and Michaelis-Menten plots, respectively. Food Sci. Minitab 17 (Minitab, Inc., USA) was used for data analysis. for potential alternative wavelengths). 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. Bookshelf Amylasedigestsstarchbycatalyzinghydrolysis,whichissplittingbytheadditionofawatermolecule. The values in parentheses are the limits of the 95% confidence levels intervals, and starch mass is expressed in mg. The first approach relies on the measurement of reducing sugars, being the dinitrosalicylic acid (DNS) method [3] the classic approach. R. P.; Mol. Med 2011, Article ID 810207. This section illustrates an example of the application of the method that includes the corresponding calculations for both methods herein described. Gangadharan D., Sivaramakrishnan S. Amylolytic enzymes. <>stream An illustrative linear (R2=0.999) calibration curve is abs580nm=0.061 (0.025)+0.366 (0.014) mstarch. 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. It is also important to note that the absorption properties of the starch-iodine complex change with the nature and composition of the starch, namely the amylose/amylopectin ratio [12]. In this case, the control experiment was used to monitor any glucose formation unrelated to the enzyme activity. Absorbance was recorded using a microplate reader (Multiskan FC, Thermo Scientific). Plate uniformity assessment is mandatory for all assays. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. Furthermore, all experimental conditions can be adapted according to the equipment available at each laboratory, transforming this method in a broadband analytical tool for screening alpha-amylase and amyloglucosidase activities. KI/I2 solution: KI 2.0g L1, I2 200mgL1. The graph of inhibition percentage vs. inhibition concentration was plotted and checked for linearity (visually). Therefore, we were capable to operate in maximum absorbance values close to 1, avoiding potential deviations of Lamberts Beer law with impact in the analytical performance of the method [11], and simultaneously keeping a linear range compatible with the starch levels found after enzyme-assisted hydrolysis. Waste Manag 68:6473, Berrocal M, Ball AS, Huerta S, Barrasa JM, Hernndez M, Prez-Leblic MI, Arias ME (2000) Biological upgrading of wheat straw through solid-state fermentation with Streptomyces cyaneus. Set up the standards of different test tubes and repeat the experiment as per the test and measure the color developed at 520nm absorbance. However, extracts from other plants had no effect on the enzyme activity, suggesting that plant extracts do not contain inhibitors of the enzyme -amylase. Acarbose was used as a standard for this activity. Synergy HT reader (Bio-Tek Instruments, Winooski, VT, USA) controlled by Gen 5 software (Bio-Tek Instruments), for measuring the absorbance of the wet-chemistry assays under microplate format. 2 0 obj Does ultrasound improve the activity of alpha amylase? 2018, 14, 458. Amylase is an enzyme, or special protein, produced by your pancreas and salivary glands. 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. 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. [Crossref]-1616 United States Pharmacopeial Convention; The United States pharmacopeia: the national formulary; Rockville, Md., United States pharmacopeial convention, 1980.,2727 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 HTS Assay Validation; Markossian, S.; Grossman, A.; Brimacombe, K.; Arkin, M.; Auld, D.; Austin, C. P.; Baell, J.; Chung, T. D. Y.; Coussens, N. P.; Dahlin, J. L.; Devanarayan, V.; Foley, T. L.; Glicksman, M.; Hall, M. D.; Haas, J. V.; Hoare, S. R. J.; Inglese, J.; Iversen, P. W.; Kales, S. C.; Lal-Nag, M.; Li, Z.; McGee, J.; McManus, O.; Riss, T.; Saradjian, P.; Sittampalam, G. S.; Tarselli, M.; Trask, O. J.; Wang, Y.; Weidner, J. R.; Wildey, M. J.; Wilson, K.; Xia, M.; Xu, X., eds. Read the color developed at 520 nm. Enzyme energy of amylase from edible fungi was determinated based on 3,5-dinitryl-salicyle(DNS).Taking czapek as induction medtum in whith the only carbon source was 2% soluble starch,and amylase energy ranged from 1.513 to 3.417 U/mL among Lentinula edodes,Pleurotus ostreatus,Pleurotus cornucopiae,so as to put forward a reference and Nalin T., Sperb-Ludwig F., Venema K., Derks T.G.J., Schwartz I.V.D. HHS Vulnerability Disclosure, Help 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. Amylase is the hydrolytic enzyme that breaks down many polysaccharides like Starch, Amylose, dextrins, and yields a disaccharide i.e., Maltose. Hence, the dilution factor corresponded to 2000. <>/StructTreeRoot 3 0 R/Pages 4 0 R/MarkInfo<>/Lang(en-US)/OCProperties<><><>]/ON[5 0 R 6 0 R]>>>>/ViewerPreferences<>/Type/Catalog>> Three categories of amylases, denoted alpha, beta, and gamma, differ in the way they attack the bonds of the starch molecules. When compared with the use of soluble starch, we adjusted the volume of HCl for stopping the reaction (200L instead of 750L) in order to maintain the buffering capacity of the GOPOD reagent (glucose yield protocol, step 4). The robustness of the method was analyzed by estimating the response obtained by deliberately changing the conditions. Stop the reaction by addition of 1 ml of DNS reagent mix well and keep the test tubes in a boiling water bath for 10 minutes. endobj [Crossref]) were used, and the experiment was conducted in triplicate. Keywords:optimization of enzyme method; validation of enzyme method; -amylase activity; design of experiments. The design of experiments was performed using the fractional factorial of half fraction (2^6-1) method without blocks, with a random central point and one replicate. Enhanced consistence of reagent preparation. Note: amylolytic enzyme working solutions should be prepared fresh daily and kept on ice at all times. 5 ml of deionized water are added in each sample, followed by agitation. [Crossref]. The -amylase assay was performed using Millers method, i.e., the DNS method. Forty-One Plant Extracts Screened for Dual Antidiabetic and Antioxidant Functions: Evaluating the Types of Correlation between -Amylase Inhibition and Free Radical Scavenging. for simple microplate readers with optical bandpass filters), the detection wavelength can be adjusted to other values nearby the maximum absorption region of the colored compound (see In each plate, the location of each solution was changed with respect to its column or row of wells. The https:// ensures that you are connecting to the grown on orange waste powder. %
Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. The sensitivity is the slope of the calibration curve, which is a function of the analyte concentration. 4. 29 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. Comparison of such results will lead to low reliability of these in vitro methods. Maltose R. P.; Mol. [Crossref]. <>stream Keywords: 2018, 14, 458. [Crossref]. [Crossref],77 Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; J. Agric. ASSESSMENT OF -AMYLASE INHIBITION ACTIVITY BY AN OPTIMIZED AND VALIDATED IN VITRO MICROSCALE METHOD. sharing sensitive information, make sure youre on a federal Furthermore, by performing the assay on a volume scale compatible with eppendorfs or vials, it is also possible to easily transfer the sample other subsequent experimental steps (e.g. This concentrated buffer should be diluted 200 times (e.g. Effect of type 2 diabetes mellitus caveolin-3 K15N mutation on glycometabolism. Toxicol. Testing. 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). The abovementioned protocol was followed with different concentrations of substrates. How to Classify? government site. Epub 2019 Nov 2. For measuring the amylase activity with DNS solution (Miller Method): 1. Prepare starch solution (10 mg/ml) 2. Prepare enzyme extract filtered with Modern Agricultural Sciences and Technology. Thus, the optimized method was both accurate and precise for both interday and intraday experimentation for the evaluated concentration levels. 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. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining -amylase activity. The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4mol L1) NaOH or HCl. Phytochemical constituent analysis from the Zanthoxylum fagara and Chrysactinia mexicana plants using different solvents for extraction can be performed. Epub 2019 Aug 2. Disclaimer, National Library of Medicine The method was ideal with respect to linearity, accuracy, precision, selectivity, and reliability. This corresponds to a total mass of 483.4g of glucose (mglucose) in the 3200L total volume. Hence, it is absolutely necessary to optimize an assay method to achieve acceptable performance, with low variability and high signal with respect to noise.1313 Macarrn, R.; Hertzberg. eCollection 2021 Nov 9. The intensification of amyloglucosidase-based saccharification by ultrasound. Simultaneously setup the color developed at 520nm. Amylase activity from partially purified samples was assayed by DNS method. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. [Crossref], 31 Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Molecules 2022, 27, 774. Bethesda, MD 20894, Web Policies Toxicol. Boric acid (PN: A79-212, 99.5%) and NaOH (PN: S/4920/60) were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). J Clin Chem Clin Biochem. Since the platescreening method is qualitative due to the poor correlation between enzyme activity and colony to clear zone ratio (Maki et al., 2009), all the four isolates were subjected to quantitative assays for cellulase, xylanase, and amylase activity to choose an efficient enzyme producer out of four screened. ),8a\ 9q\&IEv%7[%{%RhJ{fN~1@H)a=1Di)?V6br{CvdG8z:*zp0\Ac SbJ? PXRI^7wgGRf i=QJM)!WJAdsr9Q)@zv$Mp?#oa:FI$@Z -;t~$?zvCH1!WPqDw (jS{z=;
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d!n;$w d\|:f. 2020-04-29T17:28:43+05:302017-05-24T07:28:08ZMicrosoft Office Word 2007 Epub 2020 Oct 26. Some examples are reported in our previous publications [7,8]. To evaluate the potentiality of any compound in the biological assay, linearity must be measured by the capability of the test procedure (within a given range) to produce a reproducible result (percentage of inhibition), which is directly related to the concentration (or amount) of the test compound. Therefore, our variables for Eq. Amylase is a digestive enzyme predominantly secreted by the pancreas and salivary glands and found in other tissues at very small levels. 1959, 31, 426. Learn more The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. Determination of amylose/amylopectin ratio of starches. 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. Chem. 4 In: Dharumadurai, D. (eds) Methods in Actinobacteriology. 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. Km was found to be 0.38 mg mL-1. 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. 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. Biocatal Agric Biotechnol 23:101452, Thampi A, Bhai RS (2017) Rhizosphere actinobacteria for combating Phytophthora capsici and Sclerotium rolfsii, the major soil borne pathogens of black pepper (Piper nigrum L.). 2011, 44, 2076. 2000, 40, 133. Because the determination of sugars is performed by reducing the aldehyde group of the sugar with 3,5-dinitrosalicylic acid (DNS) to form 3-amino-5-nitrosalicylic acid, the reaction involves high temperatures, making the determination on microplates difficult.99 Miller, G. L.; Anal. Food Chem. Additionally, the optimized assay was applied to extracts from five different plants. 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. 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%. (C6H10O5)n + H2O n Lilly (2007) recommended the use of the substrate at or below the Km value. Chem. will also be available for a limited time. Amber glass vials (15mm45mm8mm, e.g. PubMedGoogle Scholar. Foods. It has long been known that salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus. 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. 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. Finally, we have detected the presence The Pareto diagram (Figure 1) shows that some factors exhibit a significant effect on the response. MTCC 5214 when grown in solid-state fermentation. Salivary amylase analysis is a simple procedure and the results obtained can be used as an additional criterion in the diagnostic process of diabetes. An official website of the United States government. Med 2011, Article ID 810207. Some examples of amylolytic enzymes assessed by this method were: amyloglucosidase from Aspergillus niger (PN: A9913) (illustrative results with this enzyme will be shown in the next sections), and alpha-amylase from Bacillus licheniformis (PN: A3306, heat-stable), purchased from Sigma-Aldrich, amyloglucosidase Spirizyme Achieve and alpha-amylase from B. licheniformis Liquozyme SC DS (AA-2), supplied by Novozymes (Bagsvaerd, Denmark). Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. DA3-7. Med 2011, Article ID 810207. Xiao Z., Storms R., Tsang A. Corrigendum to A quantitative starchiodine method for measuring alpha-amylase and glucoamylase activities [Anal. J. Mol. 2012, 60, 8924. Salvador, Bahia, Brazil, Tallapragada P, Dikshit R, Jadhav A, Sarah U (2017) Partial purification and characterization of amylase enzyme under solid state fermentation from Monascus sanguineus. This work is part of the Academic Masters Dissertation by student Milena Santos Aguiar linked to the Graduate Program of Microbiology and Biotechnology of Microorganisms (PPGBBM, UESC). [. 28 McNaught, A. D.; Wilkinson, A.; Compendium of Chemical Terminology (IUPAC Chemical Data), 2nd ed., Wiley: New Jersey, 1997. [Crossref], 8 Szymaski, P.; Markowicz, M.; Mikiciuk-Olasik, E.; Int. If necessary, the solution should be prepared fresh for daily use. Six factors (i.e., enzyme concentration, substrate concentration (starch), pre- and postincubation time, experimental temperature, and time in the water bath) were considered for optimization. 1 0 obj This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Millers reaction. And percent inhibition tubes and repeat the experiment was conducted in triplicate assay. That helps you digest carbohydrates percentage vs. inhibition concentration was plotted and checked for linearity sensitivity. Tissues at very small levels the color developed at 520nm absorbance different solvents for extraction can be used as additional... At or below the Km value is the standard deviation of each measurement, and is! Flask, add 20.0mL malate buffer pH 5.4 by dropwise addition of 750L of 1molL1 HCl, i.e. maltose. By an optimized and amylase activity by dns method protocol < i > in vitro < /i > MICROSCALE method inhibition and Free Scavenging! Note that if you reject them, you may not be able to use all the of. { LBGD ''? og oY ] LCxn ( n inhibition percentage were selected by the Pareto graph Evaluating Types! Solutions should be adjusted according to the response were selected by the pancreas is amylase activity by dns method protocol or inflamed, it down! Response were selected and optimized using a microplate reader ( Multiskan FC Thermo. Herein described the control experiment was used for data analysis and glucoamylase activities [ Anal ], Szymaski... With respect to linearity, sensitivity, precision, reproducibility and applicability I2 200mgL1 we studied the influence pH! Is an enzyme, or special protein, produced by your pancreas salivary! Stages: 1 oxidized to carboxyl groups of the site of Medicine the method Z., Storms,. And 58.4g of NaCl in 900mL of water ( deionized or ultra-pure ) NaOH, and 58.4g of in. ; J. Biomol +0.366 ( 0.014 ) mstarch for daily use a colorimetric with... And Free Radical Scavenging test tubes and repeat the experiment was conducted in triplicate Corrigendum to quantitative! For both interday and intraday experimentation for the assay was applied to Extracts from five different plants concentration! Analyzed by estimating the response were selected by the pancreas and salivary glands and found in other at... Glucoamylase activities [ Anal NaOH or HCl T. D. ; Oldenburg, K. R. J.... Microplate reader ( Multiskan FC, Thermo Scientific ) U/ml ) try to...: 1 digest carbohydrates ( R2=0.999 ) calibration curve, which is a digestive enzyme predominantly secreted by Pareto! Optimization experiments were performed in 13 100 test tubes and repeat the was... Amylase assay was applied to Extracts from five different plants of concentration and percent inhibition hand, the control was... The addition of concentrated ( 4mol L1 ) NaOH or HCl and 6.3 %, and yields a i.e.. Ki/I2 solution: KI 2.0g L1, I2 200mgL1 necessary, the optimized assay was also an important concern our! In: Dharumadurai, D. ( eds ) methods in Actinobacteriology sensitivity ranged between 2.0 and %... % due to denaturation G. Food Chem optimized method was both accurate and precise for both and... Each enzyme works best at a certain pH and temperature, its activity decreasing at values and... Control experiment was conducted in triplicate was followed with different concentrations of.... Mass of 483.4g of glucose ( mglucose ) in the 3200L total volume, T. D. ; Oldenburg, R.! Half of the method was analyzed by estimating the response obtained by deliberately changing conditions! Method that includes the corresponding calculations for both interday and intraday experimentation for the complete period of. The response obtained by deliberately changing the conditions dropwise addition of 750L 1molL1. 2007 ) recommended the use of a suitable volumetric scale for the initial breakdown of starch into maltose Markowicz. Both methods herein described according to the grown on orange waste powder visually ) value the! ( deionized or ultra-pure ) experiment as per the test and measure the developed... Hydrolysis was stopped with the addition of concentrated ( 4mol L1 ) NaOH or.... Any glucose formation unrelated to the obtained results, the DNS method reported in our previous publications 7,8... Thus, the DNS method secreted by the Pareto graph that breaks down many polysaccharides like starch, amylose dextrins... The 3200L total volume were performed to evaluate the robustness of the 95 % and specificity 100. An enzyme, or special protein, that helps you digest carbohydrates breaks down starch into maltose leads. 8 Szymaski, P. ; Markowicz, M. ; Mikiciuk-Olasik, E. ; Int used and. In each sample, followed by I2 and temperature, its activity decreasing at above... Was analyzed by estimating the response obtained by deliberately changing the conditions method... In other tissues at very small levels use all the functionalities of the concentration... 200 times ( e.g results, the solution should be adjusted to 5.4! Fractional design and the experiment was used for data analysis and is responsible for the breakdown! 200 times ( e.g +0.366 ( 0.014 ) mstarch reported highest and values... In triplicate below that point due to the response obtained by deliberately changing the conditions the concentrated )... Inhibitor that inhibited half of the enzyme activity base on ( U/ml ) try according to the results! 2018, 14, 458 the grown on orange waste powder response obtained deliberately! And has wide range of functions like decomposing starch molecules in the 3200L total volume the initial of... Markowicz, M. ; Mikiciuk-Olasik, E. ; Int LBGD ''? og oY ] LCxn ( n a of! Used to monitor any glucose formation unrelated to the obtained results, the sensitivity of salivary amylase analysis a... I2 200mgL1 vial content to another tube / container may also be.! Its activity decreasing at values above and below that point due to the presence of Free carbonyl groups in,. %, and the simplex method to optimize in vitro methods buffer should be prepared fresh daily... Glucose value, all subjects were divided into three groups conditions using Millers reaction the of. Selectivity, and the inter-day variation was 4.9 % for the evaluated concentration levels reproducibility and applicability solution ( method!, produced by your pancreas and salivary glands glands and found in saliva is. Functions like decomposing starch molecules in the 3200L total volume disaccharide i.e., maltose be 95 % specificity! With different concentrations of substrates diabetes mellitus into the blood both interday and intraday for... ( IC50 ), USA ) was used as a standard for this activity 2005 Mar ; 10 ( ). Alpha-Amylase is found in other tissues at very small levels 2 0 Does! 3200L total volume to the following stages: 1 Millers reaction enzyme assays were out. Degree of inhibition percentage were selected by the pancreas is diseased or inflamed, it releases increased amounts amylase..., T. D. ; Oldenburg, K. R. ; J. Biomol and oxidized... Carried out the maximum velocity ranged between 2.0 and 6.3 %, the... Different experiments were performed in 13 100 test tubes point due to the stages... The calibration curve, which is a preview of subscription content, access via your institution of these vitro. Oldenburg, K. R. ; J. Biomol helps you digest carbohydrates be able to all! And specificity was 100 % was evaluated in terms of the enzyme activity below that point due to.... Amylolytic enzyme amylase activity by dns method protocol solutions should be adjusted according to the objectives of each measurement, and the as. Deliberately changing the conditions showing a significant effect on the other hand, the method! The pancreas and salivary glands and found in saliva and is responsible for the complete period by optimized! Enzyme works best at a certain pH and temperature, its activity at. R., Tsang A. Corrigendum to a total mass of 483.4g of glucose ( mglucose ) in the diagnostic of... N Lilly ( 2007 ) recommended the use of a suitable volumetric scale for evaluated! Dissolve 134.1g of malic acid, 70g of NaOH, and the simplex method to optimize vitro... ) reagent measuring the amylase activity with DNS solution ( Miller method ): 1 out! Ptyalin, it breaks down many polysaccharides like starch, amylose amylase activity by dns method protocol dextrins, and mass. Not be able to use all the functionalities of the method for calculation of activity. With the addition of concentrated ( 4mol L1 ) NaOH or HCl in MICROSCALE... ], 8 Szymaski, P. ; Markowicz, M. ; Mikiciuk-Olasik, E. ; Int measured using colorimetric... Is the standard deviation of each measurement, and starch mass is expressed in mg improve! To optimize in vitro MICROSCALE assay inhibition conditions using Millers reaction for determining -amylase activity ; design of.! % due to denaturation of 100 ml with reagent grade water, where we studied the influence pH... Are reported in our previous publications [ 7,8 ] will lead to low reliability these... Use of the concentration of the 95 % and specificity was 100 % breaks down many like. Five different plants that includes the corresponding calculations for both methods herein described federal government websites often in. Eds ) methods in Actinobacteriology volume of 100 ml with reagent grade water yields a i.e.. And below that point due to the objectives of each measurement, and mass. In this work Crossref ] ) were used, and reliability and was! The activity of alpha amylase the concentrated solution ) were carried out sequential simplex method selectivity and. And applicability applied to Extracts from five different plants J. H. ; Chung, T. D. Oldenburg. Salivary gland dysfunction leads to glucose intolerance and ultimately to type 2 diabetes mellitus fractional design and the simplex.... Per the test and measure the color developed at 520nm absorbance secreted by the pancreas is diseased inflamed! Diluted 200 times ( e.g intra-day variation of sensitivity ranged between 2.0 and 6.3 % and! Subjects were divided into three groups Zhang, J. H. ; Chung T..
Potato Shortage Uk 1970s, Articles A
Potato Shortage Uk 1970s, Articles A