Glycolysis and Fermentation of Grape Juice - Assignment Example

Glycolysis and Fermentation of Grape Juice [Name] [Institution] Glycolysis and Fermentation of Grape Juice Aim the main objective of the experiment is to establish parameters of wine alcohol content and glucose level during the progress of fermentation. Glycolysis happens prior to fermentation of grape juice. Further, to establish the amount of glucose present in grapes in order to harvest it for glycolysis and fermentation so as to process the wine. This is achieved through estimating the levels as well as the content of sugar in the grape juice. The agenda behind all this is to establish the generation of energy by foods in form of ATP that drives all the processes. Introduction Glycolysis is a Greek term with glucus meaning sweet while lysis stands for rupture. The process itself comprises of intracellular changes of glucose as well as fructose into pyruvate. This is the first biochemical pathway in which carbohydrates are catabolised within the cytoplasm. Fully description of the pathway was performed in 1940 partly due to the contribution of Gustav and Meyenhorf. As a result, the process is also known as Embdem-Meyerhoff pathway although others who participated are excluded in this name (Kresge et al. 2005 p. 126). The main pathway for yeasts is glycolysis which they use to catabolise their sugars. The process of glycolysis utilizes a series of 11 reactions which chemically break down hexoses and in return produce energy in form of ATP (Barnett, 2003 p. 513). The first thing prior to start of the process involves transportation of hexoses inside the cell enhanced by diffusion (Lagunas, 1993 p.227). That process does not need energy because of the concentration of the inner sugar which exceeds the external concentration. The initial step is phosphorylation through the use of hexokinanes that add a phosphate group to glucose. In the process, ATP is consumed although the inner concentration remains low. The low concentration enhances the progressive exportation of sugars inside the cell via the transporters inside the plasma membrane. The product yielded in the first process is isomerised by phosphor-glucose. There are numerous processes that follows until the last step whereby a substrate level phosphorylation occurs yielding another molecule of ATP. Pyruvate is formed as the last product in gylcolysis and two molecules of energy are formed. In the process NAD+ is reduced to NADH whereas in some other steps the reverse happens. Fermentation of grape juice starts with glycolysis as explained above. The process above yields pyruvate that is capable of being converted to ethanol as well as carbon dioxide. The enzyme involved in this conversion is pyruvate decarboxylase. Afterwards, the ethanol can be converted into ethanol in a reduction process known as alcoholic fermentation, through which NAD+ is regenerated from the consumption of glycolysis. At the same time, yeast obtains two ATP molecules via metabolized hexose (Barnett & Entian, 2005 p. 845). Pyruvate can be converted by pyruvate dehydrogenase to acetyl-CoA as well as carbon dioxide. In the process NAD+ is reduced to coenzyme A and NADH. The major aim of alcoholic fermentation here is to transform grape juice into wine through numerous treatments. M & M: the first step in the procedure is to inoculate grape juice for day zero. Afterwards, sampling is done of the fermenting grape juice from day 1 to day 7. Then, grape juice is analysed by reading the baume hydrometer. Calibration temperatures are also recorded for the baume. Glucose levels are then determined with different assays which is done in duplicates. Graphs of Glucose (M) and ethanol (M) vs. day of culture Baume Hydrometer result for ethanol (M) Beer Lambert Law states that A = .c. l Rearranged c = A/ (.l) A= Absorbance at 340nm [unit: mol.L-1]  is M-1.cm-1 and l is cm, the units are: 1/ (M-1.cm-1 x cm) i.e. M, i.e. mol.L-1] ε.l A c  A= 0.854 at absorbance of 340nm for duplicate 1 Ethanol readings from the hydrometer= 0.854 mol.L-1 Hence the reading = 0.854 mol.L-1/ 1/ (M-1.cm-1 x cm) = 0.854 M/ mol.L-1 Converting this value in terms of percentage of the alcohol yields 13.2% v/v, this is close to the required level- 12.6% v/v. The determination has been achieved via the use of enzymes that checks the NADH yielded in the step catalysed by ADH. The hydrometer at 340nm then detects the NADH. Ethanol / Glucose ratio Ethanol/Glucose ratio=Glucose disappearing (M) / Ethanol appearing (M) Calculations are for day 0-7 ratio = [glucose concentration (M) day 1 – glucose concentration (M) day 7] divided by [ethanol concentration (M) day 7 - ethanol concentration (M) day 0] Ratio= {[0.665-0.928]/ [0.0011-0.0056]} = [-0.263/ -0.0045] = 58.44 Discussion It is expected that 2 moles of ethanol would be yielded for every mole of the catabolised glucose that is when all glucose is transformed into ethanol via fermentation. However, the ratio between ethanol and glucose shows that 58 moles of glucose are catabolised to yield a mole of ethanol. The variance can be explained by the difference in concentrations of the substances used during their treatments. Alcohol is in a high concentration whereas glucose is in a low concentration. This shows that fermentation is taking place to convert grape juice into wine. The graphs plotted for ethanol versus days of culture reveals a similar pattern for the two duplicates used. This reveals the accuracy of the experiment. The curves reveal that on day zero, alcohol had a concentration of 0.005 M and 0.008 M whereas it remains below 0.002 M for the remaining 7 days. This is different for the curves yielded by plotting glucose against days of culture. From day zero, the concentration increases consecutively until day 7. This means that all the differences observed even in their ratio is affected by this difference in their concentrations. The absorbance of alcohol for both duplicates is non-liner although between the first and fourth day the curve appears partly linear and these are the valid measurements. Therefore, the Beer-Lambert law holds for only that measurement which is linear. These are the only valid estimates of the standard curve. This result means that alcohol is an absorbing substance in high concentrations. On the other hand, the absorbance of glucose for duplicate 1 is linear hence the Beer-Lambert law holds. This means that glucose is a low concentration absorbing substance. Measurement for duplicate 1 here becomes valid estimates. For duplicate 2, the curve appears linear although at day 1, it is non-linear. Going by the majority, the law also holds here and the estimates became valid. The Baume readings show that the grape juice has been fermented. Conclusion Evidently, glycolysis and fermentation of grape juice has parameters that determine the content of wine alcohol as well as the level of glucose. It is very evident that absorbance is affected by the concentration of the substance. The experiment was successful because we are able to infer the valid estimates produced from the absorbance. The ratio difference and other results show that the differences in concentrations of glucose and alcohol affect they way they behave. Hence, it is recommended that a lower concentration of alcohol should be used in order to attain the expected 2:1 ratio. This experiment has enhanced the understanding how secondary metabolites are synthesised de novo. References Barnett, J.A 2003, A history of research on yeasts 5: the fermentation pathway. Yeast, 20, 509–543. Barnett, J.A., & Entian, K.D 2005, A history of research on yeasts 9: regulation of sugarmetabolism, Yeast, 22 , 835–894 Kresge, N., Simoni, R.D., & Hill, R.L 2005, Otto Fritz Meyerhof and the Elucidation of the Glycolytic Pathway, Journal Biological Chemistry, 280, 124–126. Lagunas, R 1993, Sugar transport in Saccharomyces cerevisiae. FEMS Microbiological Review, 16, 229–242 Read More