Then, the final concentrated enzyme solution was taken for comparative enzyme entrapment study. Changes in conditions/parameters, such as enzyme concentrations (< 0.15 U mL 11 Bhat, M.; Zinjarde, S. S.; Bhargava, S. Y.; Kumar, A. R.; Joshi, B. N.; J. Evidence-Based Complementary Altern. Epub 2019 Aug 2. 2021 Oct 27;6(44):29804-29813. doi: 10.1021/acsomega.1c04244. When the enzymatic activity decreases, the formation of the product also decreases; therefore, the intensity of the color is reduced. Med 2011, Article ID 810207. The authors also acknowledge to Slvia Azevedo for the valuable technical assistance. Glucose yield (average values from two determinations) measured by the described glucose oxidase/peroxidase assay that resulted from the hydrolysis of four different pure starches mediated by an amyloglucosidase from Aspergillus niger (PN: A9913, Sigma Aldrich). Likewise, actinobacteria have been shown to be excellent sources of enzyme production and, therefore, can be considered as good alternatives to the use of fungi to obtain enzymes of industrial interest. Without salivary amylase, we would not be able to digest carbohydrates properly and would not get the total nutritional value from these foods. 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). <>/ExtGState<>/XObject<>/ProcSet[/PDF/Text/ImageB/ImageC/ImageI] >>/MediaBox[ 0 0 595.32 841.92] /Contents 4 0 R/Group<>/Tabs/S/StructParents 0>> 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]. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). KI/I2 solution: KI 2.0g L1, I2 200mgL1. Universidade Federal da Bahia. 351 (2006) 146148]. For the GOPOD method under microplate format, we also performed a similar method characterization. Linearity was obtained at the acarbose concentration of 1.5 g mL-1 and 5 g mL-1. Semantic Scholar is a free, AI-powered research tool for scientific literature, based at the Allen Institute for AI. Therefore, our variables for Eq. The activity of enzymes is strongly affected by changes in pH and temperature. Metabolic disorders, including hyperglycemia, characterize type-2 diabetes. Those with normal plasma glucose had group I, those with impaired glucose tolerance had group II, and those who were diabetic had group III. The slopes of these two were compared to determine linearity using the t-test. [Crossref], 11 Finak, G.; Gottardo, R.; In Data Mining Techniques for the Life Sciences; Carugo, O., Eisenhaber, F., eds. Master degree dissertation. Miller G.L. 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. The absorbance of the product was recorded at 540 nm. Liu F, Zhu S, Ni L, Huang L, Wang K, Zhou Y. Endocrine. Slight changes in enzyme concentration, time duration prior to and during the incubation, and time in the water bath resulted in significant changes in the inhibition percentage. Achillea fragrantissima Forssk. For a blood test, blood is Recommendations and criteria presented by various international guidelines were followed to validate the optimized media.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 One negative control (absence of inhibitor) was set up to obtain 100% enzyme activity. 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. [Crossref] In this study, the values of the Z factor were 0.93 and 0.96. J Genet Eng Biotechnol 15:95101, Mandels M, Sternberg D (1976) Recent advances in cellulases technology. J. Mol. Production of Actinobacteria Amylase by Fermentation in Solid State Using Residues of Licuri Palm (Syagrus coronata). Biomol. 2011, 44, 2076. CAS Hence, the dilution factor corresponded to 2000. Transfer 50L of sample supernatant (or standard), and add 150L of GOPOD reagent to each well of the microplate. The kinetic constant maximum velocity (Vmax) and the Michaelis-Menten constant (Km) were obtained from the Lineweaver-Burk and Michaelis-Menten plots, respectively. 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. 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). Soluble starch standard solutions: prepare by stepwise dilution of the starch stock solution with water; prepare fresh daily. In this method, starch by amylase is converted into maltose. Schematic illustration of the workflow proposed for the quantification of the activity of alpha-amylase and amyloglucosidase. amylase, any member of a class of enzymes that catalyze the hydrolysis (splitting of a compound by addition of a water molecule) of starch into smaller carbohydrate molecules such as maltose (a molecule composed of two glucose molecules). From these samples, salivary amylose enzyme assays were carried out. Food Sci. 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. A comparative study towards a tailor-made enzymatic hydrolysis of starch. 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). 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. [Crossref], 2 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.; Food Chem. The .gov means its official. 2015 Apr 22;164:210-22. doi: 10.1016/j.jep.2015.02.018. [Crossref],77 Yilmazer-Musa, M.; Griffith, A. M.; Michels, A. J.; Schneider, E.; Frei, B.; J. Agric. However, extracts from other plants had no effect on the enzyme activity, suggesting that plant extracts do not contain inhibitors of the enzyme -amylase. The structure of salivary amylase is a long, spiral-shaped molecule. Unit of Microbiology, Department of Biology, University of Patras, http://www.microbiology.biology.upatras.gr. 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. 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]. [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. Basic Med. 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. 2023 Springer Nature Switzerland AG. It was observed that slight variation in the experimental conditions had a significant effect on the response. 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]. The residues used correspond to the fruit peels and the pressed cake obtained after extracting the vegetable oil from its seeds. endobj [Crossref]. The IC50 for the positive control was found to be 0.6 g mL-1. (English), https://doi.org/10.21577/0100-4042.20170919. Like most things, amylase production slows as we age, which reduces the ability of the digestive system to properly absorb nutrients. Afterwards, the produced quantity of reducing sugars released from starch is determined as described previously. The absorbance at 580nm was measured for both assay and control experiments for 10min, being the average values of 0.110 and 1.138, respectively. After obtaining informed consent, unstimulated whole saliva was collected from each subject into a sterile container for 30 minutes and subsequently centrifuged immediately. 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 ]. and transmitted securely. 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. Fig. 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. 7 0 obj Xiao Z.Z., Storms R., Tsang A. [Crossref], The phytochemical analysis indicated the presence of various metabolites such as alkaloids, flavonoids, terpenes, steroids, and phenols. Experimental conditions as described in the protocol (the incubation temperature was 45C). Optimization of the -amylase inhibition assay was performed using fractionalized factorial design (2^6-1). Acarbose was used as an enzyme inhibitor. In: Singh-Nee Nigam P., Pandey A., editors. 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. [Crossref] This state of hyperglycemia can cause complications such as nephropathy, retinopathy, atherosclerosis, and memory impairment.22 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.; Food Chem. It was experimentally established that the minimum concentration of acarbose that can be determined is 1 g mL-1. 2021 May 1;343:128423. doi: 10.1016/j.foodchem.2020.128423. 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.