Enzymes activity determination. Enzymatyc activity of α-amylaze malt. Malt extractivity. Malt sugarising duration.

Biochemical reactions are taking a place in many processes of biotechnological manufactures, which catalises by various enzymes. So, the big attention is payed to the quantitatyve control of enzymes and determination of optimal conditions of their action.

Problem on work performance:

1. To define amylolytic enzyme activity of fresh malt by visual method.

2. To define extract content of malt (extractivity).

3. To define sugarising duration of malt.

The determination of amylolytic enzyme activity of fresh malt by visual colorymetric method

Devices, laboratory ware and reactants: thermostate, water bath, thermometer, technical scales, a porcelain plate, pipettes volume 10 cm³, chemical bottles volume 200 cm3, a glass stick, test tubes volume 25 ml , a funnel for filtering, a filtering paper, the phosphatic buffer with pH 4,7... 4,9, 2% solution of potato starch, an iodine solution.

Determination course

At first prepare a malt extract, transferring amylolytic enzymes to solution. For this purpose take a charge weight 10,00 g the crushed malt, transfer it to the measured flask volume 100 ml, add 10 ml of a phosphatic buffer solution and add the distilled water up to a mark. Carefully shake the mix and place for 30 minutes to the thermostat at temperature 30 °С. Received extract filter through the paper filter with following use of a filtrate for analysis carrying out.

For carrying out of enzymatic reactions mix a 25 ml of a solution of starch in a wide test tube about 23 ml of the distilled water and 2 ml of a malt extract. A mix put in the thermostat at temperature 30 °С. Through every minute transfer a drop from the test tube on a white porcelain plate and mix it with a drop of a solution of iodine. Mark time when iodine ceases to change the colouring. The analysis takes 10-20 minutes, if iodine colouring disappears less than in 10 minutes, determinations carry out with about 1 ml of a malt extract and 24 ml of water. If reaction lasts longer 20 minutes, increase the quantity of the malt extract. But under each conditions total amount of a reactionary mix there should be 50 ml. Amount АА , units/g, calculate by such formula:

АА = 0,25 × 60 / (a t)

Where 0,25 - weight of starch in 25 ml of a solution, g; 60 - recalculation on a time unit; a - weight of malt in the reactionary environment, g; t; - time of sugarising, min.

Example

Analyzed fresh barley malt. To 25 ml of a substratum added 23 ml of the distilled water and 2 ml of a malt extract (0,2 malts). sugarising Time - 12 minutes

АА=0,25*60 / In terms of a photocolorimetric method it makes (0,2*12 =6,2 units: 6,2*5=31 unit

Determination of extract content of malt

The method essence consists in transferring to solution extractive substances of malt under the influence of its own enzymes under the conditions close to optimum, with following isolation of a solution and determination of its concentration by one of known methods.

Devices, laboratory ware and reactants: a water bath, refractometer or densitometer, the thermometer, technical scales, chemical bottles volume 200 and 1000 ml , a glass stick, the cylinder volume 200 cm3, a funnel for filtering, a filtering paper.

Determination course

In preliminary weighed glass take charge in weight 50,00 g malt and add 200 ml of the distilled water in temperature 47 °С. Bottle contents stir for receiving of homogeneous suspension and put it on a water bath with temperature 45 °С. At such temperature and periodic hashing a mix 30 minutes then maintain temperature gradually raise to 70 °С.

When the temperature reach 70 °С add the 100 ml of the distilled water heated to 70 °С. At this temperature suspension sugarise throughout 1 h then cool to a room temperature.

In the ml all portions of the distilled water wash off the thermometer and a glass stick. On the technical scales bottle contents pour the distilled water till 450 ml. Mix carefully mix and filter through the folded filter to the dry glass.

The first muddy portions of a filtrate return on the filter, the transparent solution (mash) will not go yet. In a filtrate with the help refractometer or densitometer define contents of solids е, %. Extract content malt Е₁, and also its recalculation on a solid of malt Е₂ count under such formulas:

E₁ = e (800 + W) / (100 - e); E₂ = E₁ × 100 / (100-W)

Where W - humidity of the mash, %.

Example

Analyzed malt humidity of 5,2 %. The maintenance of solids in a mash made 8,2 %. Then extract content malt

Е₁=8,2 (800+5,2) / (100-8,2 =71,92 %, and in recalculation on a malt solid extract content:

Е₂=71,92 (100 / (100-5,2) = 75,86 %.

Determination of malt sugarising duration

The method is based on ability of starch and products of its hydrolysis to give a colour reaction with iodine. As a result of enzymatic hydrolysis of the starch, low-molecular dextrines, maltose and glucose are accumulates in a solution and they are not painted by iodine.

As sugarising duration understand time in the minutes, necessary for transformation of starch of suspension to connections which are not painted by iodine. The analysis carry out at temperature 70ºC, that answers optimum temperature conditions of action α-amilase and at the same time is experience by determination of extract content of malt.

Devices, laboratory ware and reactants: a water bath, the thermometer, technical scales, a porcelain plate, a chemical bottle volume 1000 cm3, a glass stick, an iodine solution.

Determination course

After achievement of temperature of suspension 70ºC, through each 5 minutes a glass stick take a drop of suspension and place it with a drop of a solution of iodine on a white plate. A plate incline, both drops mix and watch colouring change.

It is considered, that sugaring has come to the end, if iodine colouring does not change.

Questions for self-examination

1. What are the enzymes? How to classify enzymes by reaction type?

2. What value of separate classes of enzymes for technological processes of food manufactures?

3. What the fresh sprouted malt amylolytic enzyme activity amount characterises?

4. What products of hydrolysis of starch are not painted by iodine?

5. What understand as sugarising duration malt? How it define?

6. In what the determination technique of extract content malt consists?

Laboratory work 4

In raw and rectified alcohols containing 4-5% by volume of water at 20 ° C directly determine the actual strength of vodka determine the apparent strength, and if they are pre distilled and dilute with distilled water to the original volume, are true competitors. The strength of alcoholic drinks, alcoholic juices berry juices, beer, mash, infusions is determined after distillation.

The method is based on measuring the relative density of water-alcohol solution through a glass alcoholometer.

Task to perform work

1. Determine the strength of water-alcohol solution.

2. Calculate the amount of waterless alcohol.

3. Determine the actual alcohol content and extract by distillation of beer.

4. Perform test for the purity of alcohol using sulfuric acid.

Determination of strength aqueous alcohol solutions by using

a glass alcoholometer

Instruments, laboratory glassware and reagents: glass alcoholometer, thermometer, glass cylinder, ethanol, aqueous alcohol solution.

Progress definitions

Take a glass cylinder of diameter to alcoholometer freely fell into the liquid without touching the walls of the cylinder and thermometer. Cylinder wash and rinse thoroughly with analyzed alcohol. Alcoholometer wipe with a soft cloth slightly moistened with alcohol.

Pour alcohol in the cylinder To speed up the removal of air bubbles that appear in the cylinder while the liquid is stirred with thermometer. After complete removal of all bubbles, without removing the thermometer gently lowered into the cylinder alcoholometer, holding it by the top rod thumb and forefinger and do not touching hands part that is immersed.

Hold alcoholometer and thermometer in the liquid 3 ... 5 min while counting alcoholometer immersion and the thermometry. In this case the eye must be located just below the liquid level in a position clearly visible the line crossing the liquid level of the scale.

Temperature determine thermometers of scale interval 0,1 ° C in the range -30 ... +20 C and 0 ... +50 ° C and thermometer with scale interval of 0,5 ° C in the range -25 .. . +30 ° C.

If the solution temperature is different from 20 ° C, actual strength of aqueous-alcoholic solution is determined by the special tables.

Knowing the temperature of the solution and indications of alcoholometer, alcohol content in water-alcohol solution are finding in a table

Examples of the use of tables to determine the strength of water-alcohol solutions

If the temperature and indications of alcoholometer are expressed by integers, the actual strength of of water-alcohol solution is found in the relevant columns of the table. In case of small parameters, except for indications of alcoholometer multiple of 0.5, using the method of linear interpolation.

Example 1. Indications a glass of alcoholometer - 89.5; solution temperature - 12 ° C. In the table we find the strength of alcohol at 20 ° C - 0.9152 volume fraction.

Температура, °С	Вміст спирту, показання спиртоміра
	42,0	42,4	42,5
22,0	41,2		41,7
22,6	X	Y	Z
23,0	40,8	-	41,31

Calculation of absolute alcohol

Instruments, laboratory glassware and reagents: alcoholometer glass, thermometer, glass cylinder, ethanol.

Progress definitions

Ethanol is mixed with water in any ratio. This process is accompanied by a change in the amount of heat and aqueous-alcoholic fluid (adiabatic compression - contracting), which depends on the concentration of alcohol in the solution. Alcohol content in water-alcohol solution measured in dry year. The unit take 1 dl of waterless alcohol.

Content waterless alcohol Sb.s. in the water-alcohol solution at 20 ° C are finding by multiplying the volume of water-alcohol solution on the alcohol content in the C volume fractions, dl:

С_б._с=VC

When counting the number of water-alcohol solutions have their volumes measured at arbitrary temperatures. But it is well known that temperature influences the alcohol content per unit volume.

To transfer different volumes of water-alcohol solutions having different temperatures, in anhydrous alcohol at 20 ° C using a table of factors. Multiplier are finding the content of alcohol in volume fractions and the temperature at which the measurement was performed.

Volume of water-alcohol solution, which is found at a given temperature, multiplied by factor K and found getting the volume of waterless alcohol in the liquid at 20 ° C dl:

С_б._с=VК

Examples of the use of tables to determine the strength of water-alcohol solutions

Example 1. Volume of water-alcohol solution at a temperature of 14 ° C is equal to 4000 liters with alcohol content of 0.963 volume of shares (96,3% vol.).

Find the amount of of waterless alcohol.

As Table factors for conversion on the anhydrous alcohol intended for integer values ​​of alcohol and temperature, when the calculations are fractional numbers, the multiplier are finding by linear interpolation.

In table factors for the determination of alcohol with a volume fraction of alcohol are: 0.9662 and 0.9762. For finding the multiplier to determine the content of alcohol with a volume fraction of 0.963, using the following equation:

(0.9662-x) / (96,0-96, ZYO,9662-0, 9762) / (96,0-97,0), x = 0.9692% vol.

Thus, the amount of waterless alcohol 4000 * 0.9692 = 3876.8 liters.

Example 2. Volume of water-alcohol solution of 20,000 liters at a temperature of 17,5 ° C when the content of alcohol by volume 0.9558 shares (95.58% vol.).

Find the amount of of waterless alcohol.

In the table find the value multipliers for neighbors integers alcohol content and temperature in the table.

Alcohol content at different temperatures

Temperature, ° C multiplier to determine the amount of alcohol in its content, the volume fraction

0.96 0.9558 0.95

18.0 0.9620 - 0.9520

17,45 X Y Z

17.0 0.9631 - 0.9530

Find the value of X:

(0.9620-x) / (18,0-17,45) = (0,9620-0,9631) / (18,0-17,0);

     X = 0,96126

Calculate the value of Z:

(0,9620 - Z) / (18,0-17,45) = (0,9520-0,9530) / (18,0-17,0);

     Z = 0,95255

Find the value of Y:

(0,96261 - Y) / (96,0-95,58) = (0,96261-0,95255) / (96,0-95,0);

     Y = 0,96263

Number of waterless ethanol is:

20000 * 0.96263 = 19252.6 l

Запитання для самоперевірки

1. Чим має відрізнятись шкала ареометричного приладу-спиртометра від шкали ареометра-цукрометра?

2. Які фактори можуть впливати на результат вимірів концентрації водно-спиртового розчину?

3. Для чого в спиртометрі є вбудований термометр?

Laboratory work 5