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Study fermentation condition effect on the quality of green beer by using the equation to calculate reducing suger concentration of 24 diluted tubes and this experiment will look at the mechanism of yeast, and how it converts the sugars into ethanol. The concentration of reducing suger and the number of yeast will show the best optimal condition for beer performing and that lead to the apllication in industry , help reduce cost and achieve the best quality.
Laboratory experiment title: PRODUCTION OF MICROBIAL BIOMASS AND BEER PRODUCTION Date due: 23/11/2015 Lab Report I Introduction Brewing yeast Saccharmyces cerevisiae : Morphology: it is a globular-shaped, yellow-green Physiology: Energy and Carbon Source Requirements Glucose is considered the preferred carbon source of yeast, because the presence of this hexose inhibits the utilization of other carbon sources Yeast exercises this preference in part by repressing the transcription of genes required for the utilization of the alternative carbon sources Cell Composition Nutritional Requirements ©2012 Phibro Animal Health Corporation Growth Factors Effect of Temperature: the temperature effect on an enzyme-catalysed reaction, the rate of reaction is increased when it’s maximum in a specific temperature This is called optimum temperature The optimum temperature of Saccharomyces cerevisiae is 30oC- 32oC [1] Effect of pH When pH values get an optimum pH value , the enzyme activity is the highest The optimum pH of Saccharomyces cerevisiae is 5.5 Acids and alkalis cause denaturation of the structure of the enzyme , its breaking mainly hydrogen and ionic bonds lead to the substrate can't fit the active site Moreover, the charges of the amino acids within the active site are influented by pH changes, so the enzyme can not to form an enzyme-substrate complex If the ph is to high or too low compare with the optimum pH, the enzymatic activity hence the rate is reduced considerably.[2] Metabolism: Saccharmuces cerevisiae ferment sugars via the glycolytic pathway to pyruvate Glucose → pyruvate → ethanol + CO2 ( no oxygen) S cerevisiae has an intact C.A.C and a functional electron transport system to produce ATP, however,when the enviroment lack of oxygen, they can readily grow and respire under aerobic conditions Glucose is the main source of carbon and energy, glucose concentrations are high enough, lead to gene expression of enzymes used in respiration are repressed and fermentation takes over respiration Beer fermentation : Mechanism: Microbiology and Technology of Fermented Foods p 309 Before yeast is added, the wort is first aerated with sterile air Even though the beer fermentation eventually becomes an anaerobic process because oxygen is necessary element for biosynthesis of cell membrane lipids which are essential for growth of yeast in wort Influence of technology factors on the fermentation performance and beer quality II III Objective Study fermentation condition effect on the quality of green beer Meterial and method A Meterial Medium composition How was they use Germinated malt 200g Distilled water Microorganism 1000g The malt -source of the starch (becomes the sugar required in fermentation) Use for extraction Saccharomuces 1*10^7 cerevisiae cell/ml B Method Malt medium preparation Combine 200g germinated malt and 500ml water in one blender and then grind the mixed well 500ml water is poured before they are filtered into a 1000 ml beaker (3 times) The beaker is put in water balt at 52oC for 20 min, then 63oC for 30 min, next its put at 73oC , and at last 100o C for 15 Testing the sucrose concentration and pH value To enough duger for yeast to grow , the suger content of aquaous solution is around Brix (with one degree Brix is one gram surcrose in 100 gram of solution ) pH is set value at least 4.5 Calculation of spore suspension Using a haemocytometer to count cell, the purpose in order to reach 1*10^7 (cell/ml) Special pieces of equipment and the general theory of the analyses or assays used Haemocytometer is a specialized microscope slide and has an etched glass chamber which use to count cell concentration.It has a thick base and uses a special coverslip which is thick enough to stay flat under the pull of surface tension from the solution in the counting chamber.Under microscope, observation through haemocytometer can see the central area of the grid is triple ruled line Count a high density cell population Number of cell = 225 cell at ten time dilution Cell concerntration per ml = 225/5 *25*10^4 *10^2=1.13*10^9(cell/ml) Spore suspension C Counted (C) * Volume of spore suspension=Require (C) * volume of media 1.13*10^9(cell /ml)*V1(ml)=1*107(cell/ml)*200(ml)=>V1=1.78 ml Inoculate yeast erlens are prepared contain 200ml solution malt then inoculate yeast cell by adding 1.78 ml solution of spore suspension Fermentation Sample 1a Sample 1b Sample 2a Sample 2b o Temperature (t C) RT RT RT RT Cover at the top of no No yes yes the flask Inocubation time week week week week RT room temperture Determination of reducing suger The standard curve Using the standard curve to find equation to fine suger concentration Preparing tubes contain : Tube number Standard glucose 0.02% (ml) Water The concentration in each tube 20 40 60 80 100 Pipette 1ml of solution in new tubes (*)from old tubes Spectrophotometry is used to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution The basic principle is that each compound absorbs or transmits light over a certain range of wavelength Sample dilution Dilute the beer culture from flasks, each flask dilute to 10, 100 and 1000 times Preparing tubes for each diferentt concentration and pipette 1ml from each concentration in tubes, Totally, we have Each flask -3 tube with different concentrations-2 tube contain ml from each concentraion So, flask will be prepared 24 tubes with each tubes contain 1ml solution (**) For 30 (* and**)tubes : Add 1ml DNS solution Put the all tube in water bath at 100 oC for 5mins Let them cool at room temperature Add 10 ml distilled water and mix well Use spectrophotometer absorbance at 540 nm IV Results and discussion Group OD Reducing suger concentration (ppm) 0 0.109 40 0.229 80 0.369 120 0.507 160 0.648 200 OD 0.7 y = 0.0033x - 0.0164 R² = 0.9976 0.6 0.5 OD 0.4 OD 0.3 Linear (OD) 0.2 0.1 0 -0.1 50 100 150 200 250 Reducing suger concentration (ppm) The x-axis stand for reducing suger concentration and y-axis stand for ∆OD After all, the equation of ∆OD line y = 0,0033x - 0,0164 R² = 0,9976 Using the equation to calculate reducing suger concentration of 24 diluted tubes The table 1.1 show the number of reducing suger concentration and OD OD OD average Reducing suger concentration A1 A1 A1 A1 A1 A1 A2 A2 A2 A2 A2 A2 10-1 10-1 10-2 10-2 10-3 10-3 10-1 10-1 10-2 10-2 10-3 10-3 0.386 0.475 0.056 0.035 0.020 0.034 0.181 0.254 0.026 0.034 0.022 0.021 0.431 0.046 0.027 0.218 0.030 0.021 135.58 18.91 13.15 71.03 14.06 11.33 (ppm=mg/l) OD OD average Reducing suger concentration (ppm=mg/l) B1 B1 B1 B1 B1 B1 B2 B2 B2 B2 B2 B2 -1 -1 -2 -2 -3 -3 -1 -1 -2 -2 -3 10 10 10 10 10 10 10 10 10 10 10 10-3 0.365 0.377 0.030 0.033 0.021 0.028 0.322 0.324 0.027 0.032 0.020 0.021 0.371 0.032 0.025 0.323 0.030 0.020 117.40 14.67 12.55 102.85 14.06 11.03 A: aerobic B: anaerobic The table 1.2 show reducing suger concentration (g/l) Reducing suger concentration (g/l) Aerobic 10-1 Aerobic 10-2 Aerobic 10-3 Anaerobic 10-1 Anaerobic 10-2 Anaerobic 10-3 103.31× 10−3 16.49× 10−3 12.24× 10−3 110.125× 10−3 14.37× 10−3 11.80× 10−3 The table 2.0 show the yeast cell concentration in beer production A1 A2 B1 B2 Yeast cells at 41 13 diluted at 10 times Yeast cell 2.05 × 107 6.5 × 106 2.5 × 106 × 106 (cell / ml) Average of 1.35 × 107 2.25 × 106 yeast cell (cell / ml) Comparing between aerobic and anaerobic The beer culture flasks without cover at the top have less ethanol than ones covered and the flask without have more cells than the flask cover at the top Before inoculate yeast celL The sucrose concentration (o Brix) =7.67 pH= 5.94 After inoculate yeast cell The table 3.0 show the sucrose concentration after yeast were inoculated The sucrose concentration (o Brix) A1 A2 B1 B1 3.09 3.32 3.18 3.69 Average of sucrose concentration (o Brix) 3.21 3.44 The sucrose concentration (o Brix) before fermentation is higher than after fermentation Data og group The table 3.1 show the sucrose concentration after yeast were inoculated Sucrose concentration (oBrix) Aerobic Anaerobic Group 2.92 2.21 Group 3.24 2.87 Group 3.13 3.04 Group 2.89 2.65 Average 3.05 2.70 The table 1.3 show the number of reducing suger concentration and OD OD ODaverage Reducing sugar concentration (ppm) Aerobic 0.228 0.224 0.226 39.8 Aerobic 0.204 0.194 0.200 35.5 Anaerobic Anaerobic 0.261 0.267 0.264 46.2 0.283 0.265 0.274 47.8 The table 2.1 show The sucrose concentration of group A1 A2 o The sucrose concentration ( Brix) B1 15 B1 18 10 The table compare aerobic and anaerobic flasks Aerobic Anaerobic Present of alcohol Small number of smell Large number of smell Present of buble Yes, CO2 is created How Oxygen Affects Fermentation Oxygen affect on the amount of unsaturated fatty acids present in the fermentation They was decreased production of esters, the flavour compounds Compare the shape and concentration of yeast cell ( cells/ml) before and after the fermentation Shape of cell did not change, concentration of cell increase from cells × 107 to 1.35 × 107 in aerobic enviroment and decease to 2.25 × 106 in anaerobic ml Because the yeast growth well in aerobic The culture does not need to supply oxygen because it could affect on the beer quality The optimization of temperature is nescessary to the control of the production of flavour-active compouns as esters and higher alcohols An increase in temperature leads to a shorter fermentation time.In lager beers it sees an increase in higher alcohols Ethyl hexanoate is produced at an increased rate during higher temperature fermentations, however acetate esters is decreased in amount For ale strains,its increased only phenyl ethyl acetate production Most aroma-active compounds are volatile, and the increase of temperature will evaporate them, removing them effectively from the beer 11 V VI Conclusion This experiment will look at the mechanism of yeast, and how it converts the sugars into ethanol The concentration of reducing suger and the number of yeast will show the best optimal condition for beer performing and that lead to the apllication in industry , help reduce cost and achieve the best quality References [1] ,[2] : http://www.ukessays.com/essays/biology/a-study-of-the-factors-affectingsaccharomyces-cerevisiae-population-growth-biology-essay.php#ixzz3sHmS2kx2 Hutkins, R W (2006) Beer Fermentation In R W Hutkins, Microbiology and Technology of Fermented Foods (pp 301- 349) Oxford: Blackwell Publishing Ltd J Richard Dickinson, Michael Schweizer , The Metabolism and Molecular Physiology of Saccharomyces cerevisiae Second edition published 2004 by Taylor & Francis Ltd 11 New Fetter Lane, London EC4P 4EE 12 ... mechanism of yeast, and how it converts the sugars into ethanol The concentration of reducing suger and the number of yeast will show the best optimal condition for beer performing and that lead... element for biosynthesis of cell membrane lipids which are essential for growth of yeast in wort Influence of technology factors on the fermentation performance and beer quality II III Objective... concentration of group A1 A2 o The sucrose concentration ( Brix) B1 15 B1 18 10 The table compare aerobic and anaerobic flasks Aerobic Anaerobic Present of alcohol Small number of smell Large number of