Pa ge 1 Pa ge 1 American Journal of Smart Technology and Solutions (AJSTS) Investigation into Optimal Conditions for Enzymatic Hydrolysis of Cassava Starch to Glucose by Amylase from Rice Olosunde Adebisi William1*, Onumadu Kelechi Selina1, Antia Okon Orua1 Volume 2 Issue 2, Year 2023 ISSN: 2837-0295 (Online) DOI: https://doi.org/10.54536/ajsts.v2i2.1763 https://journals.e-palli.com/home/index.php/ajsts Article Information ABSTRACT Received: June 18, 2023 Accepted: July 23, 2023 Published: July 30, 2023 The challenge of finding locally available materials in abundance to meet up with the increase in demand for glucose syrup necessitated this study. Enzymatic hydrolysis of cassava starch to glucose using glucose amylase sourced from rice was conducted. A-3 factor with 6 levels factorial viz. substrate concentration (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% w/v), pH (4, 5, 6, 7, 8 and 9) and temperature (30, 40, 50, 60, 70 and 80 °C) experiment were employed. Rice malt was prepared and enzyme activated. Starch (substrate), buffer solutions, standard glucose solution and its calibration curve were also prepared. Starch was hydrolyzed by α-amylase and tested for presence of reducing sugar using Benedict solution. Time course of the reaction was studied and enzyme activity determined. It was observed that as reaction time increased (t), amount of glucose produced [P] initially increased but soon recorded infinitesimal increase and later assumed constant. The effects of substrate concentration, pH and temperature were found to be essential on glucose production. Statistical analysis on the effect of substrate concentration [S], reaction time and their interactions showed significant impact at probability level of (p) = 0.05. Keywords Amylase, Cassava, Glucose, Hydrolysis, Rice 1 Department of Agricultural and Food Engineering, Faculty of Engineering, University of Uyo, Uyo, P. M. B. 1017, Akwa Ibom State, Nigeria * Corresponding author’s e-mail: williamolosunde@uniuyo.edu.ng INTRODUCTION Unmodified starches have diverse functional properties depending on the source of the crop. Several starch products may be made from these unmodified starches which are regarded as primary resources. The native starch has restricted applications. This is because it has high predisposition to high syneresis, retrogradation, risky processing factors such as temperature, pH, etc (Omojola et al., 2011). The modification of native starch may go a long way in curbing the limitations. This could be attained through esterification, etherification, enzymatic or acid hydrolysis, cross linking and grafting of starch. Starches possess permeable surfaces. Cassava starch has smooth surfaces which are difficult to degrade than those of corn starch (Franco et al., 1988; Jane, 2006). Starch structures are composed of two linkages: α-(1-4) and α-(1-6) linkages. Hydrolysis of starch involves the process of digestion in which enzyme hydrolysis in the digestion system break down the polymer to individual basic glucose units. Various industries extensively use starch hydrolysis in the production of several bio products. Many low molecular mass products such as sugar, brewing, spirits and textile are made by some food processing and other industries from starch. Starch hydrolysis is presently carried out using acid and enzymatic hydrolyses (Adenise et al., 2002; Odebunmo and Owalude, 2005). Milder conditions such as normal pressure, lower temperature (up to 10 0C), and medium pH of 6 to 8 are used for enzymatic hydrolysis (Kolusheva and Marinova, 2007). Enzymatic hydrolysis is considered to have a high reaction rate in terms of its potency to denature detergents, solvents and proteolythic enzymes; and lower reaction medium viscosity at higher temperatures, etc. It is often done using α-amylase which may be got from diverse sources, while β-amylase is rarely employed (Eric, 2017). The source, in which the inner part of its chain composed of polysaccharide molecules, is always attacked by bacterial α-amylase enzymes. The destruction of spiral polysaccharide chain which produces 3 to 10 units of sugar is aided by the action of starch amylose which leads to the disappearance of a typical blue colour when stained with iodine (Pontoh and Low, 1995). For the purpose of hydrolysis of starch to glucose, various grains and cereals like rice, maize, sorghum and wheat could be used as enzyme sources. Recent report from the Western Press has that this simple technology currently being used for making simple sugars from cassava starch (Tello et al., 1993). A study by Hammond and Ayernor (2000) gave maximum yield of sugars when starches obtained from various types of cereal malts were hydrolyzed. Many factors such as size of granules, source of starch, crystallinity, starch components extension of association, amylase and amylopectin reaction rates, type of polymorphism (A, B and C), enzyme type, complex of amylose lipid, and conditions of hydrolysis (concentrations, pH and temperature) may contribute to variations in the enzymatic vulnerabilities of starches (Hoover and Zhou, 2003; Li, 2004; Tester et al., 2006). Simple enzymatic reactions must take into account the factors which may affect the rate of reactions. These factors include pH, temperature, concentration of reactant, enzyme concentration, inhabitation by products, etc (WCB, 2020). Enzyme deactivation cannot be over looked on either kinetic studies or reactor engineering. In Nigeria, because of high exchange rate of naira currency to dollar it is difficult to meet the importation of certain raw materials such as enzyme (gluco amylase). Therefore, there is need to carry out more researches on the use of rice seedlings as source of amylase to Pa ge 2 https://journals.e-palli.com/home/index.php/ajsts Am. J. Smart. Technol. Solutions 2(2) 1-9, 2023 hydrolyze starch for glucose production (Bailey and Ollis, 1986; Onyenekwe, 2013). The choice of cassava (Manihot esculenta Crantz) as a substrate source in this study is its abundance in Nigeria (Adejumo and Ola, 2010; Balagopalan, 2002; Osipina and Wheatley, 2005). Due to the increase in demand for glucose syrup and high cost of importing them, there is need to fully utilize and diversify cassava crop. More so, cassava and rice are viewed as rich sources of sugar and hydrolytic enzyme (gluco amylase) respectively; may be utilized as raw materials in the production of glucose as well as other industrial productions. METHODS Reagents and Equipment The chemicals used were analytical grade from Merck, England; BDH, England and Sigma, USA. The equipment employed was spectrophotometer. Sourcing of Cassava and Rice Paddy Cassava (Manihot esculenta) roots, TMS 30572 tubers were purchased at Research Institute, Umudike, Abia State while rice paddy was sourced at Afikpo, Ebonyi State. Preparation of Rice Malt, Enzyme Activation and Buffer Solutions The rice paddy was soaked in water, drained and kept inside a container for 2 days (48 hours). It was spread on shallow bed (ridge) and allowed to germinate in the dark. Wet rice malt was harvested, cleaned, sun dried; and ground into high diastatic powder (Onyenekwe, 2013). 2g of the powder was suspended in 100 ml of distilled water at 600C for ten (10) minutes to activate enzyme amylase in the powder. The supernatant was discarded leaving the cells in the solution (Onyenekwe, 2013). The following buffer solutions were prepared using mixing adjusters and salt solutions coupled with addition of distilled water to make up to 200 ml (AnalChem- Resources, 2023): pH 4: 0.1 M potassium hydrogen phthalate (100 ml) + 0.1 M HCl (0.2 ml) pH 5: 0.1 M potassium hydrogen phthalate (100 ml) + 0.1 M NaOH (45.2 ml) pH 6: 0.1 M potassium hydrogen phosphate (KH2P04) [100 ml] + 0.1 M NaOH (11.2 ml) pH 7: 0.1 M potassium hydrogen phosphate (KH2P04) [100 ml] + 0.1M NaOH (58.2 ml) pH 8: 0.1 M tris aminomethane (100 ml) + 0.1 M HCl (58.4 ml) pH 9: 0.1 M tris aminomethane (100 ml) + 0.1 M HCl (11.4 ml) Preparation of Starch, Substrate and Standard Curve of Glucose D Concentration Cassava roots (30 kg) were peeled, washed in water and grated with a commercial grater. The pulp was screened using 25 mm aperture mesh and later suspended in water. The supernatant was decanted after allowing the pulp to sediment for about 6 hours. The white starch cake was obtained and sun dried for about 72 hours (3 days) (Oyewole and Obieze, 1995). Cassava starch concentrations of 0.5 intervals were prepared up to 3.0% (w/v) using each buffer solution obtained in Section 2.3. Each starch (substrate) concentration was gelatinized in water bath at 80 °C for 10 minutes (Nam, 2023). A stock solution of 0.1% (w/v) was prepared by dissolving 1.0 g of glucose D in 1000 ml of distilled water. The stock solution was then used to prepare glucose concentration of 50 ppm interval up to 300 ppm (Rebecca et al., 2016). For each glucose concentration, about 2 ml of Dinitrosylic acid (DNS) reagent was added and then warmed in water bath at 800C for 10 minutes to develop colour for spectrophotometer reading. A blank solution of distilled water and DNS was prepared and used to calibrate spectrophotometer to be used in absorbance readings of glucose D concentration (Nam, 2023). The results (spectrophotometer readings) were recorded. A standard glucose curve was then produced. Determination of Alpha Amylase Activity: Enzyme Assay The activity of enzyme (rice amylase from malted rice) was determined according to Silva et al. (2008). The rice enzyme was activated by incubating 3 g of the enzyme (ground malted rice) suspended in 10 ml of distilled water at 50 °C for 10 minutes. The supernatant discarded leaving the cells in solution. Enzyme solution (6 ml) was mixed with phosphate buffer (4 ml) at pH of 5.0 and 10 ml of starch solution 2% (w/v). The mixture was incubated at 40 °C for 10 minutes. Then, the reaction was discontinued after addition of 2 ml of 0.1 M HCl and colour developed by adding 0.5 ml iodine reagent. After cooling to room temperature, the amount of glucose produced was found by measuring the solution absorbance at 540 nm using Spectrophotometer. However, 1.0 mg of glucose solution reacting with coloured reagent produced an absorbance of 1.0 under the same condition. One unit of enzyme is referred to as the quantity of enzyme which produced 1.0 mg equivalent of glucose per minute under the assay condition (Edu-Enzyme, 2018; Jasco International, 2019). Assessment of Glucose Production from Starch using Enzymatic Hydrolysis Phosphate buffer (0.2 M pH 6.0) was used to disperse 15% w/v starch, with bacterial α -amylase solution (0.2% w/v) (3 ml). Exactly 1 ml aliquot sodium azide solution (10% w/v) was incubated at 37 °C for 48 hours in an orbital shaker. The quantity of reducing sugar was found after solids were decanted, and the aliquots of the supernatant removed at 6, 9, 24, 30 and 48 hours. At the expiration of incubation period of 48 hours, the dispersed enzyme was deactivated by the addition of 0.1 N HCl to reach pH of 3.0. This was followed by 15 minutes stirring. The resultant solution was neutralized with 0.1 N NaOH and Pa ge 3 https://journals.e-palli.com/home/index.php/ajsts Am. J. Smart. Technol. Solutions 2(2) 1-9, 2023 centrifuged at 2100 rpm for 20 minutes. Distilled water and ethanol were used to wash the hydrolyzed residues through filtration. The residues were dried using a hot air oven at 40 °C (Franco and Ciacco, 1992). Thereafter, Benedict test for the presence of reducing sugar was then carried out according to AOAC (1998) and Geetha (2012) methods. Experimental Design The experimental design was three (3) factors (substrate concentration, pH and temperature) as variables at 6 levels as adopted by Khan (2013). The levels selected for the glucose production were as follows: substrate concentration, Sc (0.5, 1.0, 1.5, 2.0, 2.5 and 3.0% w/v), pH (4, 5, 6, 7, 8 and 9) and temperature, tp (30, 40, 50, 60, 70 and 80° C) (Torreggiani and Bertolo, 2001; Rosa and Giroux, 2001; Nieto et al., 2001; Ozen et al., 2002; Jain and Verma, 2003). The experiment was done in replicate. Experimental Procedure The optimal values of the variables for the production of glucose were obtained based on the following experiment: Effects of pH on Concentration of Glucose (Sugar) Produced 10 ml of each of the six (6) pH level (4, 5, 6, 7, 8 and 9) was added separately to six (6) different test tubes containing 0.5% w/v of substrate prepared and gelatinized at 80 °C and cooled. They were positioned in water bath at 30 °C. About 4 ml of activated enzyme was then added to each of the six (6) contents of the test tubes. This was allowed to hydrolyze for 10 minutes. About 2 ml aliquot was used to prepare enzyme assay which was measured using spectrophotometer. Exactly 2 ml DNS reagent was added to discontinue the reaction and then heated to develop colour. This was cooled in cold water and their various absorbance readings with spectrophotometer at 540 nm were taken, recorded and tabulated. However, effects of pH on concentration of glucose (sugar) produced was evaluated. Statistical analysis analyzed was carried out using Analysis of Variance (ANOVA) at 5% level of probability embedded in Statistical Package for Social Scientists [SPSS] Version 20. Effect of Temperature and Substrate Concentrations on Concentration of Glucose (Sugar) Produced The process described in Section 2.8 (a) was carried out at 40, 50, 60, 70 and 80 °C. Also, the substrate concentrations of 0.1, 1.5, 2.0, 2.5 and 3.0% (w/v) were used separately. Their absorbance’s readings were recorded and tabulated. Evaluation of Optimal Parameters Required to Produce Glucose during Hydrolysis The optimal parameters (pH, temperature and substrate concentration) were found based on the optimum concentration of sugar obtained. Time Course of Reaction to Produce Glucose The optimal parameters were then used to study the time course of the reaction to produce glucose. Spectrophotometer reading before and after dilution, and the corresponding mean amount of glucose produced after conversion were noted. These data were used to plot several curves of glucose concentrations produced against reaction times. RESULTS Standard Glucose Calibration Curve and Test for Presence of Reducing Sugar The plot of spectrophotometer reading against glucose concentration is presented in Figure 1. Figure 1: Standard glucose calibration curve. The data for standard glucose calibration curve gave Equation 1. Y = (-4.0 ×10-11. G4) + (1 ×10-8.G3) + (4 ×10-6. G2) + (0.0004 × G) + 0.0021 (1) Where, 1 ppm = 1 mg.L-1 = 0.001 g.L-1, G = glucose concentration (ppm) as independent variable and Y = diluted value of spectrophotometer reading (nm) as dependent variable. The plot of spectrophotometer reading (nm) against glucose concentration is seen to be a polynomial function. From Figure 1, as the Pa ge 4 https://journals.e-palli.com/home/index.php/ajsts Am. J. Smart. Technol. Solutions 2(2) 1-9, 2023 spectrophotometer reading increased, the glucose concentration also increased which is an indication of a strong direct relationship with coefficient of determination (R2) of 0.9955. Similar trend was reported by Sciencell (2019), Megazyme (2019) and Tunde (2020) with R2 of 0.9962, 0.9990 and 0.9889, respectively. The α-amylase was found to hydrolyze the starch by giving a brick red colouration when applying Benedict test. This was an indication of the presence of glucose obtained. Similar studies were conducted by UkEassys (2018) and Cochran et al. (2008), and they also had the same result from the hydrolysis of starch using amylase enzyme. Effect of pH, Temperature, and Substrate Concentrations on Glucose Concentration Produced for 10 Minutes of Reaction Time The effect of pH, temperature and substrate concentrations on concentration of glucose produced was studied for 10 minutes of reaction time. Based on the data generated, the plots of glucose concentration against temperature at various pH values and substrate concentrations are presented in Figures 2 to 5, while that of glucose concentrate produced against pH values at various temperatures and substrate concentration are shown from Figures 6 to 9. Figure 2: Plot of glucose conc. produced against temperature at various pH and constant substrate concentration of 0.5 %w/v. Figure 3: Plot of glucose conc. produced against temperature at various pH and constant substrate concentration of 2.0 %w/v. Figure 4: Plot of glucose conc. produced against temperature at various pH and constant substrate concentration of 2.5 %w/v. Pa ge 5 https://journals.e-palli.com/home/index.php/ajsts Am. J. Smart. Technol. Solutions 2(2) 1-9, 2023 Figure 5:Plot of glucose conc. produced against temperature at various pH and constant substrate concentration of 3.0 %w/v. Figure 6: Plot of glucose conc. produced against pH at various temperatures and constant substrate concentration of 0.5 %w/v. Figure 7: Plot of glucose conc. produced against pH at various temperatures and constant substrate concentration of 2.0 %w/v. Figure 8: Plot of glucose conc. produced against pH at various temperatures and constant substrate concentration of 2.5 %w/v. Pa ge 6 https://journals.e-palli.com/home/index.php/ajsts Am. J. Smart. Technol. Solutions 2(2) 1-9, 2023 From Figures 2 to 9, the minimum glucose concentration produced (0.029 g DE. L-1) was recorded when pH of 9, temperature of 800C and substrate concentration of 0.5% (w/v) were used, whereas the maximum glucose concentration (0.261 g DE. L-1) was obtained, when pH of 5, temperature of 400C and substrate concentration of 0.5% (w/v) were used. Generally, lower pH favoured the higher amount of glucose produced while high pH values produced lesser amount of glucose. Increases in temperature, decreased the concentration of glucose produced. This implies that too high acidic or alkaline medium does not favour the activities of enzymes. However, different kinds of enzymes have specific range of conditions necessary for their optimal performances. At higher temperature, some enzymes might be denatured as they are made up of protein. Hence, pH of 5, temperature of 400C and substrate concentration of 0.5% (w/v) were considered as optimum condtions. These conditions were used in studing the time course of reaction or simply the production of glucose with time. In a study conducted by UkEssays (2018), the effects of pH (5 to 9) and temperature (300C to 900C) were conspicuously observed on the enzymatic hydrolysis of starch to glucose. Other researchers such as Alias- Rodinah (2009) and Karolina (2015) also reported the effect of pH, temperature, and substrate and enzyme concentrations on the production of glucose. Determination of Enzyme Activity The outcome of enzyme activity is presented in Table 1. Figure 9: Plot of glucose conc. produced against pH at various temperatures and constant substrate concentration of 3.0 %w/v. Table 1: Activity of α-amylase from rice in the production of glucose from cassava starch. Spectrophotometer Reading After Dilution [Absorbance] (nm) Amount of Glucose Produced (g DE.L-1) Molar Conc. of Glucose Produced (mol DE.L-1) Enzyme Activity 0.349 0.248 0.04468 0.0203 U.L-1 or 0.0203 μ mol.min-1.L From Table 1, 0.248 g DE.L-1 of glucose was produced when the total volume of enzyme mix in assay of 22 ml was incubated for 10 minutes at 400C and pH of 5. However, the enzyme activity in this study was found to be 0.0203 U.L-1 or 0.0203 μ mol.min-1.L. This means 0.0203 units of enzyme catalyzed the transformation of 1 μ mol of substrate into glucose in 1 minute under standard conditions. The low value of the enzyme activity might be as a result of change in optimal pH of the enzyme. This slows down the enzyme activity. However, high value might cause enzyme to denature (Cornish- Bowden, 1995; Jasco International, 2019). Production of Glucose with Time Time course of reaction was conducted using 40 0C and pH of 5. Plots of several curves of glucose concentrations produced against reaction times is presented in Figure 10. In Figure 10, initially, the amount of glucose produced at reaction time, t = 0 minute was 0 g DE.L-1. After 10 minutes, the amount of glucose produced were 0.150, 0.190, 0.194, 0.207, 0.207 and 0.227 g DE.L-1 in the six test tubes containing substrate concentrations of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 % (w/v) respectively. The production within this period increased rapidly. Test tube I with 0.5 % (w/v) of substrate concentration showed a gradual increase in glucose concentration from 10 minutes to 50 minutes of reaction time. Beyond this period, there was a decrease in glucose concentration. Test tubes II and III with 1.0 and 1.5 % (w/v) of substrate concentrations, also recorded similar increase from 0 to 0.239 and 0.247 g DE.L-1 at 50 and 60 minutes, respectively. Moreover, test tubes IV and V (contain with substrate concentrations of 2.0 and 2.5 % [w/v]), respectively; and had almost the same trend. A conspicuous increase in the amount of glucose produced was recorded between 0 – 20 minutes. This is, an increase from 0.0 to 0.246 g DE.L-1 for both samples. Test tube VI (with substrate concentration of 3.0 % [w/v]) recorded an increase in glucose concentration produced from zero to 0.253 g DE.L-1 within 40 minutes. Further increase in reaction time in both test tubes V and VI did not have any Pa ge 7 https://journals.e-palli.com/home/index.php/ajsts Am. J. Smart. Technol. Solutions 2(2) 1-9, 2023 observable increase in the amount of glucose produced. Generally, increase in substrate concentration yielded an increase the amount of glucose concentration produced. Virtually, unobservable increase or constant production might be due to that the fact the enzyme had reached its optimum activity and the substrate had been consumed completely. Therefore, maximum glucose concentration (0.253 g DE.L-1) produced was found when 2.5% (w/v) substrate concentration at 50 minutes reaction time or 3.0% (w/v) substrate concentration at 40 minutes reaction time was employed. Similar trends were observed by Cecil (1995) at optimum temperature of 500C, where glucose amylase was used to hydrolyze cassava starch / paddy rice to maltose. In addition, the variations of glucose concentration with time of reaction in this study were in accordance with the works done by many researchers (Cochran et al., 2008; Tunde, 2020; Nor, 2009). Effect of Substrate Concentration, Reaction Time and Substrate Concentration-Reaction Time Interaction on Glucose Produced at Constant pH = 5 and Temperature = 40 0C The summary of ANOVA showing the effect of substrate concentration, reaction time and substrate concentration- reaction time interaction on glucose concentration produced at constant pH = 5 and temperature = 40 0C is presented in Table 2. Table 2: Summary of ANOVA showing the effect of substrate concentration, reaction time and substrate concentration-reaction time interaction on the glucose produced. From Table 2, since p-value [0.00] < 0.05 and coefficient of determination (R2) = 1.00, F value is significant, which shows that the substrate concentration and reaction time had greater influence on glucose produced. More so, the resulting interaction between substrate concentration and reaction time had a significant impact on glucose produced. In a study conducted by Nor (2009), the effects of liquefaction temperature and saccharification pH on glucose production were very significant while the saccharification temperature and liquefaction pH, on the other hand did not influence the glucose production. The observed trend in the present study implies that these factors should not be rolled out when considering enzymatic hydrolysis of starch to produce glucose because they really influence the amount of glucose production. Figure 10: Plot of glucose concentration produced against reaction time. Table 2: Summary of ANOVA showing the effect of substrate concentration, reaction time and substrate concentration-reaction time interaction on the glucose produced Source of Variation df F P-value @ 5% Significant? Substrate concentration 6 3365.130 0.000 Yes Reaction time 5 74679.197 0.000 Yes Substrate concentration * reaction time 30 199.227 0.000 Yes R2 = 1.000 CONCLUSION Based on the outcome of the findings, the optimum conditions necessary for reasonable glucose production (0.253 g DE.L-1 ) were temperature of 40 0C, pH of 5 and substrate concentration of 2.5% (w/v) for 50 minutes or substrate concentration of 3.0% (w/v) for 40 minutes. Alpha-amylase activity was found to be 0.0203 μ mol. min-1.L. Analysis of Variance (ANOVA) results on the effect of substrate concentration, reaction time and their interaction at 40 0C and pH of 5 on glucose production showed a statistically significant impact since Pcal < Ptab at probability level (p) = 0.05. Furthermore, the use of rice amylase to produce glucose from cassava starch would complement increase in demand for glucose syrup production. REFERENCES Adejumo, B. A. and Ola, F. A. (2010). 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