Investigating Enzymatic Reactions To Determine The Concentration Of Glucose Of Coke And Gatorade

Abstract

The purpose of this experiment was to investigate the glucose concentration in popular drinks coke and Gatorade and to identify how a changing environment can impact the hydrolysis of sucrose. This was examined by a series of mutli- enzymatic reactions of glucose resulting in NADPH, measured by a spectrophotometer at 340nm. The results showed the glucose concentration of coke 2.30 g/100ml and Gatorade 1.06g/100ml.These results in contrast to advertised nutritional values of glucose didn't correspond as they were lower for Coke and for Gatorade which was advertised to not have glucose content.

Introduction

Within modern-day society, sugar is an essential component within humanities diet. High amounts of sugar such as sucrose(sugar cane) present within confectionery energy and soft drinks consumed daily by some, can lead to increasing insulin resistance thus leading to low amounts of high- density lipoproteins and an increasing calorie intake leading to Non- alcoholic fatty liver disease(Nasir, Nassar & Assay, 2010).This condition is caused by an excess build-up of fat within the liver, predominantly seen in those who are overweight or obese and correlated to diabetes, high blood pressure and kidney disease. (Nasir, Nassar & Assay,2008)

Sugars are the primary components of many sport or soft drinks. Within these drinks the main molecule that is carried within the bloodstream is glucose. Glucose is one of the simplest sugars and important monosaccharides which is dextrose. It is an essential molecule in the formations of disaccharides and polysaccharides that can be broken down into monosaccharides (Hein, Pattison & Arena,2015).

The glucose concentration can be examined via the spectrophotometer which measures solutions absorbances at certain wavelengths by utilises a multi-step enzymatic reaction to produce an NADPH concentration. This is illustrated in the beer-lambert law that exhibits a positive linear correlation between the absorbance of light and the concentration of the molecule (Rodger, 2013). Glucose 6 – phosphate doesn’t contain chromophore; therefore, it requires a range of enzymatic reactions to produce NAPDH that contains chromophore, allowing the absorption of the 340nm wavelengths, in turn allowing the spectrophotometric to measure the glucose concentration. (Pearson, 2019)

Enzymes are molecules typically proteins which are catalysts that speeds up the catalyse biochemical reactions. This is accomplished by lowering the activation energy necessary for a reaction to occur. Enzymes contain active sites to which certain types of substrate bind to, thus accelerating the reaction. Specific enzymes need assistance to trigger the reaction and require the presence of co-factors or co-enzymes(Hein et al., 2015).

D glucose is phosphorylated by the enzyme hexokinases and adenosine 5’ triphosphate (ATP) to glucose 6 phosphate and ADP. Glucose-6 phosphate dehydrogenase is then oxidised by nicotinamide-adenine dinucleotide phosphate ( NADP+) to gluconate 6 phosphate and reduced nicotinamide-adenine dinucleotide phosphate ( NADPH)(Pearson, 2019) .Each enzyme contains an optimal operating temperature and pH, by altering these components it can impact the rate of reaction in which the enzymes reacts under as well as its hydrolysis. This affects the frequency of enzyme-substrate collision or impacts the capacity the enzyme and substrate bind to another (Hein et al., 2015)

The purpose of this experiment to investigate enzymes activity co- enzymes (NADPH) by utilising the spectrophotometer to measure the glucose concentration of the hydrolysis of sucrose of drinks. By determining the concertation, it can be analysed whether the advertised labeling is accurate and not misleading consumers, thus impacting their daily intake.

Methods

The spectrophotometer was set at 340mm and zeroed before measuring glucose standards. Utilising a pipette glucose standard of 5 difference concentrations, coke, Gatorade and sucrose solution were made using a 1/100 dilution factor. The glucose assay regent containing ATP, hexokinase, NADP+, and buffer were added to the standard 2 minutes apart, over 18 minutes.

Measurements were conducted twice from the spectrophotometer. Refer to lab manual section for further detail.

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Discussion

Through the experimental results, the determination of glucose concentration within the two drinks (Gatorade and Coke) were able to be deduced. These calculations were conducted to analyse whether advertised glucose concentration labels on these drinks given to the public are accurate and are valid. It was demonstrated that the glucose concentration in coke was higher (2.39g/100ml) than within Gatorade 1.06g/100ml. It can be suggested that the hydrolysis of glucose was more predominately seen within the coke rather than the Gatorade.

The experimental value for glucose concentration found was lower than the literal value, which may have been influenced by a variety of factors, such as the pH, temperature, and concentration of the solution that may have not been at its optimal level to promote hydrolyses of sucrose without the enzyme of sucrase. The impact temperature and pH contain on sucrose can be seen in figure 1.

The advertised levels of sugar cane(sucrose) for Coke within Australia as of 2019 was 10.6g per 100ml. ( Coco-cola,2019). The hydrolysis of sucrose through invertase( sucrase) results in an equal molar ratio of the two monomers glucose and fructose (Clemens et al, 2016), hence, by halving the nutrition value the glucose concertation can be found. Therefore, coke contains 5.3g per 100ml of glucose. The experimental glucose concentration discovered was 2.3 per 100ml, this value lower to the actual literacy value. However, there a variety of variables that should be considered, attributing to the difference in the two values. Coke is considered as a strong acid as it is composed of carbonic, phosphoric and citric acid, giving it an acidic nature with a pH of approximately 2.35. (Reddy et al 2016).

The hydrolyses of sucrose can be affected by changing pH and temperature conditions(Costa,Mendes& Santana,2005). Acidic environments are optimal for the complete hydrolyses of sucrose, allowing enzymes an easier opportunity to bind to the substrate and lead to the breakdown into glucose and fructose. Therefore, the hydrolyses of sucrose can occur without the enzyme sucrase and utilising other reagents to produce glucose concentration by altering the temperature and pH. This is supported by figure 1 by observing the sucrose concentration, the more basic solutions contained an absorbance rate of zero, in which the solution had a pH of 5 at 50Thus, not the optimal and environment for the enzyme reactivity for the hydrolyses of sucrose. Therefore, the rate of sucrose hydrolyses is increased in low pH and higher temperatures. The coke was also diluted with water hence, perhaps hindering the full catalysation to glucose.

Gatorade as of 2019 was advertised to contain 5.5 g of sucrose and maltodextrin 0.5 g (Gatorade,2019). Both sucrose and maltodextrin were hydrolysed through the glucose assay regnant catalysing them to produce a mixture concentration of glucose. The results produced in the experiment established a glucose concentration of 1.06g/100ml. Gatorade is composed of citric acid which is a weak acid giving approximately pH of 3.5 ( Gatorade, 2019). Although an acidic environment should theoretically increase hydrolysis, the conditions for sucrose and maltodextrin hydrolysis weren't as optimal as seen within coke thus, inhibiting the complete hydrolyses into glucose.

This highlights that depending on how long since the production of the drink, and the temperatures it’s stored in the more sucrose units can undergo hydrolysis thus, increasing the amount of glucose present within the solution. Thus, further explaining the high glucose concentration observed in the experiment results, therefore suggesting incompletely hydrolysis.

The experiment results collected were also compared to previous literature that identified the glucose concentration by using a high-pressure liquid chromatography in Coke( 3.5-9 g/100ml) and Gatorade(2.4g/100ml) ( Ventura, Davis and Goran,2011). This increase the experimental results as the values were close and indicates that hydrolyses of sucrose did occur within the drinks however, it is slightly lower, which can be attributed to discrepancies made. The reliability of the data can as be examined by viewing the r2 value (0.9652), the relationship between glucose standards and sample concentration has a strong association. This correlates with the Beer- lambert law as the close it was to 1 the more accurate the data is, as the equation was superimposable.

The discrepancies observed throughout the conduction of the experiment can be classified as random or systematic. During the preparation of the glucose standards of the dilution, incorrect pipetting technique/mixing with the glucose assay reagent may have resulted in an inaccurate concentration, impacting the gradient of the absorbance curve calculated by the spectrophotometer, possibly attributing to the slightly lower readings. Furthermore, the glucose was left on the side of the cuvettes which may have altered the results. Also, the wait time of 2 minutes before placing the glucose assay reagent in the glucose standards affected the validity and reliability (random error) of the results as some samples could’ve started hydrolysing before even placed in the spectrophotometer. A systematic error that was identified was that the spectrophotometer was improperly cleared to zero before placing glucose standards, as well as when starting the experiment, it didn’t clear at zero as well. Although, this may have not heavily impacted the results it would've made a small difference in the final data collection.

There was a lack of repetitive measures conducted within the experiment, therefore, reducing the reliability and validity of the results. By repeating the experiment several times, it would generate a wider range of results, hence a larger average would've resulted, reducing the impacts that potential outlier would’ve had on the data. As previously mentioned, there was a short waiting time, so permitting a longer waiting time after placing the glucose assay reagent and before placing it within the spectrophotometer, may allow complete hydrolysis, hence a higher concentration of glucose. The last improvement identified as seen within the literature reviewed is liquid chromatography, an analytical technique that measures the separation of ions that hydrolysis within a solvent ( Pitt, 2009) may result in a more accurate way to determine sugar concentration. In further investigation fructose, sucrose or maltodextrin concentrations should also be accounted not only glucose as they greatly impact the composed amount of total sugar which results in illness such as non- alcoholic liver disease.

Through research conduct glucose concentration within coke and Gatorade were able to be determined. This was verified with literature valves as advertised on drinks nutritional information, and there were evident differences viewed. Despite this, the differences weren't as large as thought to be and can be attributed to experimental error. Therefore, the advertisement of these drinks is accurate but still contains high sugar concentration. High levels of intake of sugar within a diet are associated with diseases such as non-alcoholic liver disease. Companies should find a way to create an alternative with a reduce amount of sugar concentration, to prevent modern society illnesses associated with high intakes of sugars.

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