Production of baker's yeast by fermentation

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Turn on the data sampling to computer: turn on the computer and start the program Biostat B Data Collection. The computer starts to sample the data only after the Biostat relays the signal to it. The sampling rate could be altered only on Biostat in subprogram MAINTENANCE. Enter the data sampling rate and then alter the mode by “ALTER” and confirm by “ENTER”. MAINTANANCE 2 nd page Printer CYCLE : 1 min MODE: Off PARAM : Data sampling interval Start: Data collecting is turned on Off: Data collection is turned off Setting of parameters Software: PROCESS VALUES – it displays the actual values of monitored process quantities TRENDY – it displays the development of monitored quantities during fermentation process PRINTED VALUES – it displays the printing layout The program continuously stores the data into file printer.txt. For use in program EXCEL, it is better to export given data to a file in form of a database. Data exporting: Within the window TRENDY, press the icon of a diskette located in lower right corner “Copy to file” and store the data in a file “name.dbf”. 3.5 Sampling Sample extraction: 1. During cultivation, take an approximately 6 ml sample every hour; during exponential phase of growth, take sample every 45 minutes. Sample processing: Use 5 ml of sample for measurement of optical density. Centrifuge 0,8 ml of sample in an Eppendorf vial (300 rpm, 10 minutes). After centrifugation, use micropipette to carefully draw clear supernatant into a clean Eppendorf vial and freeze it. At the end of cultivation, all sampled supernatants would be used to determine the concentration of the substrate – the glucose. 8
3.6 Measurement of optical density 1. 5 ml of sample centrifuge (3000 RPM min -1 , 10 minutes). Wet biomass rinse gradually 3 times with isotonic solution and centrifuge at 3000 RPM min -1 , 10 minutes. Then add accurately 5 ml of isotonic solution, mix well a measure optical density of cells against isotonic solution at wavelength of 600 nm. If the sample absorbance is greater than 0.7, the sample would be properly diluted by isotonic solution. The concentration of wet biomass would be determined during growth using a calibration line. Construction of calibration line 1. Prepare 100 ml of standard biomass solution of concentration of 3 g/l in a isotonic solution. Dilute the standard biomass solution with physiological solution to reach concentrations of: 0.3, 0.15, 0.12, 0.075, 0.06, 0.05 g/l (each solution with volume of 10 ml). Measure absorbance of all solutions in comparison with absorbance of isotonic solution at 600 nm. Using the measurements of absorbance of standard biomass solutions, construct a calibration dependence cx = f (A600), calculate the parameters of calibration equation and calculate the regression coefficient. 3.6 Determination of glucose concentration by a glucose test Principle The product of the enzymatic reaction – the glucose – is quantified using a glucose test GLU GOD 6 x250. The basis of glucose quantification is formation of colored complex, whose absorbance is measured spectrophotometrically. This complex is formed by a selective oxidation of glucose to hydrogen peroxide and gluconate using enzyme glucose-oxidase. Formed hydrogen peroxide is quantified by an oxidation reaction with substituted phenol and 4-aminoantipyrine catalyzed by enzyme peroxidase; a red colored product is formed. Principle of quantification of released glucose: β − D − glucose + H O + O ⎯⎯⎯⎯⎯→ D − gluconate + H O (2) glucose−oxidase 2 2 2 2 2 H O + dye ⎯⎯⎯⎯→ dye + H O (3) peroxidase 2 2 reduced oxidized 2 9
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Production of baker's yeast by fermentation

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