MODERN TRENDS IN FERMENTATION TECHNOLOGY
LABORATORY
Immobilization of yeast cells by adhesion – application for ethanol fermentation in brewing
Bogumiła Kroczyńska
Anna Iwaśków
Agnieszka Kubacka
Dominika Kochanek
Joanna Kołacińska
Theoretical Part
Beers are commonly categorized into two main types—
bottom (lager) fermented with Saccharomyces pastorianus yeast
top (ale), fermented with Saccharomyces cerevisiae yeast
Brewing yeasts may be classed as "top cropping" or "top fermenting” and "bottom cropping“ or "bottom-fermenting” .
Top cropping yeasts are so called because they form a foam at the top of the wort during fermentation. They can produce higher alcohol concentrations and in higher temperatures, typically 16 to 24 °C (61 to 75 °F), produce fruitier, sweeter beers.
Bottom cropping yeasts are typically used to produce lager-type beers. These yeasts ferment more sugars, creating a dryer beer, and grow well at low temperatures.
Realization
First part
Prepared two different wort samples to compare ethanol efficiency:
Standard brewing fermentation – brewing wort inoculated with yeast cells suspension,
Modern brewing fermentation – brewing wort inoculated with yeast immobilized on the carriers
Standard fermentation of brewery wort
We made the microscopic observations of the yeast cells using x40 magnification lens. The yeasts were pullulating.
We counted an average number of cells in 40 squares (116 cells in 40 squares).
We calculated the number of cells in 1 ml of medium:
L = 4 • 106 • n • a = 4 • 106 • 4 • 2, 95=4,72*106
n- dilution of cell suspension, n=4
a –average cell number for each square, a=$\frac{118}{40}$=2,95
(Bottom fermentation)
We calculated the amount of inoculum for brewery pitching preparation:
y = $\frac{25*10^{8}}{47,2*10^{6}}$=53ml
We took the calculated volume of yeast suspension and centrifuged for 5 minutes in 20°C. We transferred the biomass quantitatively to 250 ml of wort in sterile conditions. We closed the flasks with fermentation tube filled with parafine.
Modern brewery fermentation
After adhesion process, we transferred 50 carriers (previously prepared) to 50ml of brewing wort in sterile conditions. We closed the flasks with fermentation tube.
To achieve staining of the adhered yeast cells, we took out of flask two carriers using sterile tongs. We placed the carriers on the porcelain plate, flooded with ether-ethanol (1:1) mixture and left for evaporation of reagent. Then we performed the 15 minutes incubation of the carriers in methylene blue at 20°C. After staining we washed carriers with water and left them to dry. Then we took photos of each carrier by Olympus BX41 microscope equipped with digital camera.
To perform alcoholic fermentation conditions we incubated the wort pitching for 1 week in 20°C (high-) and in 10°C (bottom fermentation).
Control of fermentation process
We made macroscopic observations of fermenting wort.
Standard fermentation wort - formation sediment, production of foam
Modern fermentation wort- production of foam, turbidity
Second Part
Young beer analysis
Before the analysis we degassed 200ml of beer sample by filtering through cotton cloth and repeated this operation three times.
To determine total acidity we took 50ml of degassed beer in a Erlenmayer flask and titrated with 0,1m NaOH until 4 drops of titrated sample mixed with 2 drops of phenolphthalein on a porcelain plate turned pink.
After setting the approximated amount of NaOH, we composed the extract sample by taking 50ml of degassed beer and adding 1ml less than the approximated amount of 0,1m NaOH. Then we checked the end of titration on porcelain plate.
TA=$\frac{\mathbf{a*2*m}}{\mathbf{10}}$
a-volume of NaOH [ml]
m-coefficient for exact 0,1n NaOH, m=1
I Modern fermentation (Immobilization)
TA=$\frac{\mathbf{9,8*2*1}}{\mathbf{10}}$ =1,96
II Standard fermentation (Traditional)
TA=$\frac{\mathbf{19,5*2*1}}{\mathbf{10}}$= 3,9
To determine content of alcohol and total extract we took 100ml of beer in a volumetric flask and transferred it quantitatively to a distilling flask (bulb flask). We used the volumetric flask as a receiver and collected about 75ml of distillate.
For alcohol determination: We filled the volume of receiver up to the line with water, mixed and measured alcohol concentration at 20°C using Tralles alcoholometer. We quoted the alcohol content as % v/v.
For total extract determination: We transferred the residue from bulb flask to volumetric flask. We filled the volume up to the line with water, mixed and measured extract using refractometer. We quoted the total extract as °Bx.
Practical part
Parameters of top and bottom fermentation
| BOTTOM FERMENTATION |
|---|
Fourth group |
| TOP FERMENTATION |
|---|
| 2 |
Summary
Alcohol content is higher in top fermentation than the bottom fermentation
Amount of sugar is higher in bottom fermentation than the top fermentation
There are differences between amount of alcohol depending on methods of fermentation. In bottom fermentation method the alcohol content was higher in the modern method than traditional method but amount of sugar and acidity was higher in traditional method.
In the top fermentation metod the alcohol content was similar in both methods.