Ethanol fermentation experiment

Summary

Under anaerobic conditions, the role of yeast in the fermentation of hexose to produce ethanol and CO2 is called ethanol fermentation. The yeast protozoa used in production are generally solid slant test tube strains, due to the number of starting yeast is too small, not enough to produce the needs of the slant strains must be a number of times the expansion of the culture, in order to obtain a yeast culture containing a sufficient number of yeast.

Operation method

fermentation

Principle

The fermentation of hexose by yeast to produce ethanol and CO2 under anaerobic conditions is called ethanol fermentation. Currently the microorganisms used in ethanol fermentation are mainly yeasts. The yeast used in production is generally a solid slant test tube strain, due to the number of yeast is too small, not enough to produce the needs of the slant strain must be a number of times to expand the culture, in order to obtain a sufficient number of yeast containing yeast cultures (usually liquid cultures, but also solid cultures) for ethanol fermentation, so it is known as the mother of wine.

Materials and Instruments

Bacteriophage K. Yeast Yeast Wheat teeth Sweet potato flour Tapioca flour
Bacterial amylase Agar
Inoculation cups Alcohol lamps Test tubes Triangular flasks Beakers Water baths Constant temperature incubators Distillation flasks Condensate tubes Bullhorn tubes Volumetric flasks Measuring cylinders Electric stoves Asbestos wire mesh Ethanol Thermometers

Move

I. Expanded culture of yeast
1. Preparation of culture media
The base for test tube and triangular flask culture in yeast expanded culture is usually made of maitake juice, which is prepared as follows: grind dry maitake, add 3.5 times the amount of hot water at 65 ℃, and saccharify it for 3-4 h at 58-60 ℃, and after checking the saccharification completely with iodine solution, boil it, filter it, and adjust the concentration of the filtrate to ^13oBx. Divide the maitake juice prepared above into portions and sterilize it.
(1) solid slant embassy medium: take 100 ML of wheat tooth juice, add 2% agar, melt and split the test tube, generally the amount of medium is 1/5 of the capacity of the test tube. 121 ℃ steam sterilization for 20min, take out and set up into a slant while hot, cool, the length of the slant for the length of the test tube 1/2 ~ 3/5.
(2) Liquid test-tube medium: adjust the pH of the wort with dilute sulfuric acid to 4~4.4, and dispense 5 ML into test tubes; steam sterilize at 121 ^oC for 20 min.
(3) Small triangular bottle medium: the pH adjusted to 4 ~ 4.4 of the wheat tooth juice was divided into triangular bottles with a capacity of 150ML, each bottle containing 50ML, and steam sterilized at ^131oC for 20 min.
2. Inoculation and expanded culture
(1) Transfer the slant test tube yeast protozoa to a solid slant test tube and incubate for 48 h at 28～30 ℃.
(2) Connect a loop of the above cultured slant-activated yeast strains in a liquid test tube and incubate at 28～30 ℃ for 24 h. The yeast strain was then incubated at 28～30 ℃ for 24 h. The yeast strain was then incubated in a liquid test tube for 24 h.
(3) Inoculate the above liquid in vitro yeast in small triangular vials (one triangular vial connected to each test tube) and incubate at 28-30°C until the end of the logarithmic period (about 16 h).
3. Quality requirements for seeds (brewers)
The cultured triangular vials of seeds should be inspected for quality. Microscopic examination requires that the yeast morphology is robust and neat, the intracellular protoplasm is thick and free of stray bacteria, the number of cells (0.8-1.0) × ^108/ML , the teething rate of 20%-30 %, and the mortality rate should be less than 1 %.
II. Cooking and saccharification of starch raw materials
1. According to the sweet potato flour or cassava flour 3.5 times the amount of water ratio, with 50 ~ 55 ℃ of warm water and starch raw materials stirring mix, add raw materials amount of 0.1% of the bacterial amylase, heating and stirring on the electric stove, heating to 90 ~ 93 ℃, 5 ~ 10 min, liquefaction; continue to heat and boil for 1h, so that the starch is fully pasteurized and liquefied, and the amount of water supplementation.
2. Cool the above cooking mash to 60～62 ℃, add 180 u/g of saccharification enzyme of raw material, adjust the pH of the mash to 4～4.5 with sulfuric acid, hold the saccharification on the water bath for 30 min, and then dispense the mash into 1,000 mL triangular flasks, each flask containing 500 mL of saccharification mash.
3. Quality requirements for the mashed sugar mash: Brix 15 to 17 °Bx, reducing sugar 6 % to 9 %, Ph 4 to 4.5.
III. Fermentation of the saccharification mash
The cultured matured yeast seeds (brewer's yeast) from small triangular vials were connected to large triangular vials containing 500 mL of saccharification mash by castorless operation (one large triangular vial was connected to each small triangular vial, and fermented for 68-72h at 30 ℃.
IV. Measurement of _CO2 generation
The apparatus for determining _CO2 production in ethanol fermentation is shown in Figure 5-1. The method of determination is as follows:
1. Prior to fermentation, dry the outside of the triangular flask and weigh it on a 1/100 g scale, noting the mass as _m1.
2. When fermentation is complete, the triangular flask is gently shaken to allow as much _CO2 as possible to escape. Weigh again on the same balance and note the mass as _m2.
3. _CO2 production = _m1 - _m2.
V. Determination of Ethanol Degree
1. Install the distillation unit.
2. Accurately measure 100 mL of fermented mature mash into a 500 Ml distillation flask and add an equal amount of distilled water. Distillation bottle with a thermometer inserted into the rubber plug tightly, connected to the condensing tube, do not make leakage. Heating with an electric stove, while connected to the cooling gas, distillate collected in 100mL volumetric flask (such as no volumetric flask can also be collected directly with a 100 mL cylinder). When the distillate reaches the scale, immediately stop collecting (note: not more than the scale), pour into the cylinder, slightly stirred, so that it is uniform, the alcohol meter and thermometer at the same time into the cylinder, determine the alcohol and temperature.
3. According to the measured degree of ethanol and temperature, check the conversion table to get the concentration of ethanol at a temperature of 20 ℃.

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