Toxicol. The use of a suitable volumetric scale for the assay was also an important concern for our development work. The reagent has a dark orange colour and is stable for several days in room temperature. The https:// ensures that you are connecting to the }$q6/vo~\eT9 Qu|Sn\tY?n}O65nnu:jqbWNVY6/`By[%"*"/^ry ~0*zp1= >Iu*#OR2ou8#jV|6i)UJ*G2ZE Wm^nVd~TaswO],+:h;, Screening on the Amylase Producing Activity of some Bacterial Strains Isolated from Hot Spring, International Journal of Science and Research (IJSR). Med 2011, Article ID 810207. 29 Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; J. Biomol. Google Scholar, Mounaimen O, Mahmoud K (2015) Statistical optimization of cultural conditions of an halophilic alpha-amylase production by halophilic Streptomyces sp. Salivary amylase is present in high concentrations in the saliva of young children and decreases as we age. endobj Enzyme immobilization Preparation of alginate beads Alginate gel beads were prepared according to Awad et al. For measuring the amylase activity with DNS solution (Miller Method): 1. where SD is the standard deviation of each measurement, and AVG is the average of such measurements. The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. 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. Hence, the comparison of the two assays becomes difficult. (+)-Catechin hydrate (PubChem CID: 9064); 3,5-Dinitrosalicylic acid (PubChem CID: 11873); Amylase inhibitors; Anhydrous dextrose (PubChem CID: 5793); DNSA; Gallic acid (PubChem CID: 370); Glucose oxidase; Maltose; Maltose monohydrate (PubChem CID: 16211336); Potassium sodium tartrate tetrahydrate (PubChem CID: 2724148); Rutin hydrate (PubChem CID: 45479757); Sodium phosphate dibasic (PubChem CID: 24203); Sodium phosphate monobasic (PubChem CID: 23672064); Starch soluble (PubChem CID: 439341); -Amylase. https://doi.org/10.1007/978-1-0716-1728-1_74, Shipping restrictions may apply, check to see if you are impacted, Tax calculation will be finalised during checkout. High intra-day and inter-day reproducibility. 50mL of concentrated solution for a final volume of 1000mL) in order to be used in the extraction protocol. These inhibitors delay the rate of absorption of glucose, thereby maintaining glucose levels in hyperglycemic individuals. [Kinetic determination of alpha-amylase in serum and urine with an oligosaccharide as substrate--modification for a fully mechanized enzyme measuring device (author's transl)]. 27 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. Extract from Zanthoxylum fagara showed maximum inhibitory activity with an IC50 value of 4.75 g mL-1. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. After 10min, the hydrolysis was stopped with the addition of 750L of 1molL1 HCl. This extract was the closest to the acarbose positive control. Chem. Salivary amylase is essential for the digestion of carbohydrates. The flask should be vigorously swirled and placed in a temperature-controlled water bath for 1520min at 40C with occasional swirling. [Crossref], 3 Moein, S.; Pimoradloo, E.; Moein, M.; Vessal, M.; BioMed Res. Regarding the starch-iodine method, it was possible to measure masses of starch up to 3.00mg (this mass respects to the total amount of starch contained in the vial, corresponding to a maximum mass of 42.9g in an individual microplate well). Food Chem. Epub 2020 Oct 26. Simultaneous, rapid, spectrophotometric determination of total starch, amylose and amylopectin. 12 Stevenson, R. A.; American Laboratory, 2011, available at https://www.americanlaboratory.com/913-Technical-Articles/19109-Bioassays-2011/, accessed June 2022. % Other equipment can be used with adjustments of the centrifugation time and force. 5. Due to the presence of free carbonyl groups in sugars, they can reduce DNS and are oxidized to carboxyl groups. [Crossref], 25 Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Plant Foods Hum. (EN), 1 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. This also means that the volumes could be easily adjusted in order to match the requirements of each particular enzyme analysis or study. For a urine test, its likely a two-hour or 24-hour sampling. An inhibition percentage near 50% was chosen as the best response, while the absorbance of the control at 0% inhibition (100% activity) resulted in the smallest photometric error. Clipboard, Search History, and several other advanced features are temporarily unavailable. It was observed that slight variation in the experimental conditions had a significant effect on the response. for potential alternative wavelengths). <>/Font<>/ExtGState<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI]>>/Contents[19 0 R 20 0 R]/Group<>/Type/Page/Tabs/S>> Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. ; Humana Press: New York, 2016 [Crossref] Bioassays are challenging in terms of determining accuracy. endobj Zhang, J. H.; Chung, T. D.; Oldenburg, K. R.; 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.; Quan, N. V.; Anh, H.; Lam, V. Q.; Takami, A.; Teschke, R.; Khanh, T. D.; Xuan, T. D.; Analytical Chemistry Department, School of Medicine, Autonomous University of Nuevo Len, Madero y Aguirre Pequeo, Mexico, Text Method. 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. 4 0 obj The higher is the intensity of color, the higher is the concentration of reducing sugars in the solution. [Crossref], 20 Hou, W.; Li, Y.; Zhang, Q.; Wei, X.; Peng, A.; Chen, L.; Wei, Y.; Phytother. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. Acarbose was used as an enzyme inhibitor. Absorbance was recorded at 540 nm using a microplate reader. The in vitro antidiabetic activity was studied by using -amylase inhibitory assay modified 3,5-dinitrosalicylic acid (DNS) method. The robustness of the method was analyzed by estimating the response obtained by deliberately changing the conditions. The -amylase assay was performed using Millers method, i.e., the DNS method. Additionally, the optimized assay was applied to extracts from five different plants. Our developments initially aimed the monitoring of the effect of ultrasound (US) in the activity of these two enzymes [7,8], and sought the improvement / addition of methodological features that lead to complementary information (glucose yield) about amyloglucosidase using a similar experimental protocol. Mid signal: Mean absorbance was recorded by the assay procedure. [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. -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. Amylase activity from partially purified samples was assayed by DNS method. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The new PMC design is here! Please note that if you reject them, you may not be able to use all the functionalities of the site. This is a preview of subscription content, access via your institution. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. 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. J Genet Eng Biotechnol 15:95101, Mandels M, Sternberg D (1976) Recent advances in cellulases technology. J. 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 enzyme activity should be determined within one hour. Bashkin A, Ghanim M, Abu-Farich B, Rayan M, Miari R, Srouji S, Rayan A, Falah M. Molecules. The slopes of these two were compared to determine linearity using the t-test. Biochem. Universidade Federal da Bahia. After obtaining informed consent, unstimulated whole saliva was collected from each subject into a sterile container for 30 minutes and subsequently centrifuged immediately. Unable to load your collection due to an error, Unable to load your delegates due to an error. Regarding reagents, we describe in detail the preparation of the solutions of soluble starch and triiodide (from the KI / I2 solution), which are key elements for the starch-iodine method. 2021 May 1;343:128423. doi: 10.1016/j.foodchem.2020.128423. 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. When the substrate concentration is increased, the inhibition by the competitive inhibitor is reversed. Federal government websites often end in .gov or .mil. This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007265) and National funds (FCT through grant no. Achillea fragrantissima Forssk. The first approach relies on the measurement of reducing sugars, being the dinitrosalicylic acid (DNS) method [3] the classic approach. 10 g of 3,5-dinitrosalicylic acid are gradually dissolved under heating conditions, in 700 ml of NaOH solution 0.5 N. Then 300 g of sodium potassium tartrate (NaKC4O6.4H2O) are added and distillated water was added in the mixture until a final volume of 1000 ml. In addition, the effect of substrate concentration on enzyme activity was analyzed. 2021 Oct 27;6(44):29804-29813. doi: 10.1021/acsomega.1c04244. On the other hand, the measurement of the substrates consumption is possible using the starch-iodine staining. [Crossref], Postprandial hyperglycemia can be controlled by inhibitors, such as those directed at inhibiting -amylase, which reduces the flow of glucose by digesting carbohydrates. [Crossref]. Hi Mahmoud, I am not exactly sure about this specific method. So, I have some questions for clarity. You are using soluble starch and adding differ From a quantitative point of view, the determination of amylase activity is generally carried out by a spectrophotometric method using DNS (3,5-dinitosalicylic acid) in a colorimetric reaction with reducing sugars released as a result of the hydrolysis of starch, although other colorimetric methodologies are possible [ 1, 3, 4, 5, 6 ]. Rev. Acarbose and miglitol are conventional inhibitors used to inhibit the activity of enzymes. Two plant extracts (Zanthoxylum fagara and Chrysactinia mexicana) inhibited -amylase activity. 96-well flat-bottom microplates (well volume 340L, Thermo Fisher Scientific). and transmitted securely. 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. [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. Saliva can be collected using a calibrated collection device, and the resulting sample should ideally be analyzed within 15 minutes after collection. 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. Save my name, email, and website in this browser for the next time I comment. ; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, 2004. 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. Basic Med. Dilute to a final volume of 100 ml with reagent grade water. Chem. 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. Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycemia (high blood sugar levels) due to insulin resistance or absolute lack of insulin production. [Crossref], 22 Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; J. Ethnopharmacol. This study utilized fractional design and the simplex method to optimize in vitro microscale assay inhibition conditions using Millers reaction. 1959, 31, 426. This assay was also very precise, with RSD values below 3% in all cases. water baths) and containers (e.g. ),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=; zuQp>$C(7X>/tV s5hcD!8-)~uJB$0NmzIuBR3X"@I53]M}BF:HFuK$dEQesCaxw9h5$SLA[_m}4#S?0&g~g^>*'kHI$J(YUr03hv;y5tt &TRsB#C[E,mI=6XFv64on6NCD7og. @fl9w8 XgUT>{LBGD"?og oY]LCxn(n?(Kp{D\:bB#kV!45 d!n;$w d\|:f. It is an important enzyme and has wide range of functions like decomposing starch molecules in the human body. Part of Springer Nature. Note: a clear solution shall be obtained. 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). The samples are placed in a water bath (T=100C) for 5 min and then they are left to cool at room temperature. Learn more Acta Part A-Mol. The plant extract solutions were in the concentration range of 6 g mL-1 to 200 g mL-1. Another potential application of the proposed method is the evaluation of the activity of amylolytic enzymes extracted from different natural matrices such as flours and microbial enzyme preparations. Proceed as described in steps 48 of the amylolytoc enzyme activity assay protocol. Additionally, the significant factors were optimized using the rules of the basic sequential simplex method and the combination of conditions that maximized the inhibition percentage with the lowest photometric error. Food Sci. Glycosidase enzymes breakdown carbohydrates into simple sugars they enter the bloodstream, producing postprandial hyperglycemia. will also be available for a limited time. This study optimizes and validates microscale in vitro methods for the assessment of -amylase inhibition.
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