The relative standard deviation and Z value were found to be <4% and >0.93, respectively. When we eat foods that contain carbohydrates, the salivary amylase begins to break down the complex carbohydrates into simpler sugars. Leaf starch degradation comes out of the shadows. LOD was calculated as the starch mass equivalent to the absorbance that resulted from the addition of the blank signal (standard solution without soluble starch) to 3 times the corresponding standard deviation [14]. 2, Fig. 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 2020 Jan;67(1):87-94. doi: 10.1007/s12020-019-02118-1. However, these methods share a common problem of being labor intensive, time consuming and require substantial amount of reagents and samples. government site. For a urine test, its likely a two-hour or 24-hour sampling. Alpha-amylase is found in saliva and is responsible for the initial breakdown of starch into maltose. endobj Salivary amylase analysis is a simple procedure and the results obtained can be used as an additional criterion in the diagnostic process of diabetes. Humana, New York, NY. Huang Y, Deng Y, Shang L, Yang L, Huang J, Ma J, Liao X, Zhou H, Xian J, Liang G, Huang Q. Exp Ther Med. A total of 64 experiments were performed. The new PMC design is here! The masses of glucose represented are 0.00, 2.00, 4.00, 6.00, 8.00 and 10.0g. The slopes of these two were compared to determine linearity using the t-test. Dissolve 134.1g of malic acid, 70g of NaOH, and 58.4g of NaCl in 900mL of water (deionized or ultra-pure). MeSH [Crossref], 23 Ha, K. S.; Jo, S. H.; Kang, B. H.; Apostolidis, E.; Lee, M. S.; Jang, H. D.; Kwon, Y. I.; J. Tukeys test ( = 0.05) was used to evaluate the robustness of the individual methods. Add slowly 30.0 gms sodium potassium tartrate tetrahydrate. Afterwards, the produced quantity of reducing sugars released from starch is determined as described previously. where SD is the standard deviation of each measurement, and AVG is the average of such measurements. 15 Markossian, S.; Coussens, N. P.: Dahlin, J. L.; Sittampalam, G. S.; SLAS Discovery 2021, 26, 1241. sharing sensitive information, make sure youre on a federal Relative standard deviation (expressed in percentage, RSD%) measured the precision of the assay. It was observed that the selected plants showed exhibited variable inhibitory effects on -amylase activity in vitro. 2010, 75, H97. The use of a suitable volumetric scale for the assay was also an important concern for our development work. 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. In: Singh-Nee Nigam P., Pandey A., editors. Schematic illustration of the workflow proposed for the determination of the glucose yield when pure starches were submitted to the action of amyloglucosidase. Acarbose, an inhibitor of the amylase enzyme, was evaluated at six different concentration levels by regression analysis. 37% (w/w) HCl (PN: 131020) was purchased from Panreac (Barcelona, Spain). D-Glucose stock solution 1.000gL1: dissolve 100.0mg of D-glucose in water to a final volume of 100mL; keep the solution at 4C up to three days. abs505nm=0.042 (0.005)+0.0846 (0.0008) mglucose), we obtained a mass of glucose of 7.56g that is contained in the 50L of sample transferred to the microplate. This corresponds to a total mass of 483.4g of glucose (mglucose) in the 3200L total volume. The results were subjected to regression analysis to determine R2 and the regression equation for the plot. Anal. Sci. reagent is added in each tube and the mixture is agitated for a few seconds on vortex mixer. According to the obtained results, the sensitivity of salivary amylase assay was found to be 95% and specificity was 100%. 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. [Crossref], 18 Apostolidis, E.; Lee, C. M.; J. In all cases, a sample blank was analyzed to demonstrate that there were no matrix interferences caused by the extract. Salivary amylase is the enzyme produced by the salivary glands. 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. [Crossref], 25 Vadivel, V.; Nandety, A.; y Biesalski, H. K.; Plant Foods Hum. An amylase (/ m l e s /) is an enzyme that catalyses the hydrolysis of starch (Latin amylum) into sugars.Amylase is present in the saliva of humans and some other mammals, where it begins the chemical process of digestion.Foods that contain large amounts of starch but little sugar, such as rice and potatoes, may acquire a slightly sweet taste as they are chewed The monitoring of the activity of alpha-amylase and amyloglucosidase is an important tool for studying their role in the hydrolysis of starch. Universidade Federal do Recncavo da Bahia. It is a reversible, competitive inhibitor. 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. is incorrect [10]). In each plate, the location of each solution was changed with respect to its column or row of wells. E)CQb_w)33;3:'jOjd_nx~xln%x=,syv:'{xq70r,ugN'@5TzFPD:1$K$d This article will discuss the different applications of amylase. Otherwise, the extract from Chrysactinia mexicana had an IC50 value of 9.09 g mL-1, indicating that the extract from this plant also contained bioactive metabolites, which have an inhibitory effect on the enzyme -amylase. Because acarbose is structurally similar to the substrate (starch), it competes for the active site on the enzyme. Biotechnol. Pharmacol. Biomol. This methodological approach can be used for measuring the activity of both alpha-amylase and amyloglucosidase [5]. The pancreas is an organ located behind your stomach. [Crossref] Although other protocols are based either on the glucose oxidase reaction or on the determination by fluorescence, they are commonly used in microplates. The reduced 3-amino-5-nitrosalicylic acid is red-brown in color and can be quantified spectrophotometrically with an absorption maximum at 540 nm. The analytical protocol can also be conducted in mild conditions (room temperature), bypassing the heating steps and the protocol complexity that is present in the DNS method, as well in other alternative methods for the same analytes [6]. Springer Nature is developing a new tool to find and evaluate Protocols. The .gov means its official. Then, we have partially purified these amylases employing three conventional methods: (a) cold acetone precipitation, (b) ion-exchange chromatography, and (c) gel filtration chromatography. One negative control (absence of inhibitor) was set up to obtain 100% enzyme activity. D-Glucose (PN: 8337) and KI (PN: 105043) were purchased from Merck (Darmstadt, Germany). Epub 2020 Oct 26. PEst-C/EQB/LA0006/2011) and co-financed by FEDER (UID/QUI/50006/2013 NORTE-01-0145-FEDER-00011) under the Partnership Agreement PT2020. In these experimental conditions, an illustrative calibration curve is abs505nm=0.042 (0.005)+0.0846 (0.0008) mglucose. 5). Med 2011, Article ID 810207. 2008, 55, 391. Chem. 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. 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. The RSD value for inter-day precision was 2.5%. Salivary amylase is an enzyme that is found in saliva. The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4mol L1) NaOH or HCl. In general, they have a poor design of experiments and an inadequate statistical significance.1111 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. Warm the assay buffer to room temperature before use. Linearity was determined using the graph of concentration and percent inhibition. Determination of kinetic constants was performed by determining the effect of substrate concentration on enzymatic activity. The Km value is the substrate concentration needed to reach half the maximum velocity. Therefore, the objectives of this work were: (i) the consistent preparation of some reagents, especially the soluble starch and KI / I2 solution that generates I3, to improve batch to batch reproducibility and maximize the precision and accuracy of the of the starch-iodine method; (ii) the rescale of the activity assay, in order to minimize evaporation issues typical from microplate assays and to adapt it to different experimental requirements (in our particular case this was related with the US-assisted hydrolysis of starch); (iii) the adjustment of sensitivity by modifying the volumes of the starch-iodine microplate assay, to avoid precipitation and to minimize potential deviations to the Lamberts-Beer law of the absorbance measurements; and (iv) the measurement of glucose yield for amyloglucosidase through the glucose oxidase / peroxidase (GOPOD) method replicating the sample handling protocol used for measuring its activity, but using pure starches from different botanical sources instead of soluble starch. 4 0 obj ; Eli Lilly & Company and the National Center for Advancing Translational Sciences: Bethesda, 2004. Evaluation of precision and accuracy was performed using the extracted sample of J. dioica in the presence or absence of acarbose (positive control). Soluble starch standard solutions: prepare by stepwise dilution of the starch stock solution with water; prepare fresh daily. After 10min, the reaction was stopped with 200L of HCl 1.0molL1 and the vial was centrifuged for 5min at 350g. Next, 50L of supernatant were transferred to a 96-well microplate, followed by the addition of 150L of GOPOD reagent. 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). A catalyst is a substance that hastens a chemical reaction but does not become part of the end product. Food Chem. [Crossref]. Europe S. 2018. endobj The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. Analytical validation was performed based on different international guidelines. Additionally, the optimized assay was applied to extracts from five different plants. Amylase, like other enzymes, works as a catalyst. The -amylase activity was assessed by Millers method by estimating the colored product formation by the action of enzyme and substrate starch (Scheme 1).99 Miller, G. L.; Anal. [Crossref], 22 Wu, T.; Zhou, X.; Deng, Y.; Jing, Q.; Li, M.; Yuan, L.; J. Ethnopharmacol. Int. for potential alternative wavelengths). 1979 Nov;17(11):705-8. 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. Proceed as described in steps 48 of the amylolytoc enzyme activity assay protocol. Transfer 1500L of water and 1500L of universal buffer (control) or of amyloglucosidase solution (assay) into a 4mL amber glass vial. 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. 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. 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.). [Crossref]. 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). The residues used correspond to the fruit peels and the pressed cake obtained after extracting the vegetable oil from its seeds. 4 % and > 0.93, respectively was determined using the graph of concentration percent... And AVG is the standard deviation of each amylase activity by dns method protocol was changed with respect to its or. Value is the standard deviation and Z value were found to be %. Blank was analyzed to demonstrate that there were no matrix interferences caused by the amylase! This methodological approach can be quantified spectrophotometrically with an absorption maximum at 540 nm 5 ] Eli &! 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