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Basic theoretical informationThe basis of many biotechnological production processes laid in biochemical reactions catalyzed by different enzymes.
Enzymes - are biological organic catalysts increasing speed of the reaction between various substances, while remaining without significant changes. Enzymes are consist of long chains of amino-acids connected by peptide bonds. They are present in all living cells, where taking an important place in control of metabolic processes in which nutrients are converted to energy and materials to build new cells. All enzymes are divided into six classes: oxydoreductases, transferases, hydrolases, lyases, isomerases, ligases (or synthetases). Favorable conditions for enzyme action - temperature (30-70° С) and the pH close to neutral medium (pH 7,0) In cereals nutrients for microorganisms are present, but not in the form that they could consume, macromolecular compounds present in the grain necessary to cleave under enzymes action to compounds of low molecular weight that are suitable for consumption by microorganisms. Therefore, the biological manufactures using malt as a base raw material and source of enzymes. The purpose of mashing malt is to transfer maximum number of substances to solution using enzymes. In the process of mashing is necessary to create optimal temperature conditions for the action of enzymes. Therefore, provided for maintaining the mash at the most favorable temperature for the action of peptytases, cytolytic and amylolytic enzymes. Under the action of malt amylolytic enzymes starch is hydrolyzed to dextrines of various molecular weight. The speed and completeness of the enzymatic hydrolysis of starch affects the activity of amylolytic enzymes malt mashing temperature, pH and concentration of mash. The unit of amylase activity is accepted as an amount of enzyme that catalyzes the hydrolysis of 1 g of soluble starch in 60 min. At 30 ° C and pH 4.7
The determination of amylolytic enzyme activity of fresh malt by visual colorimetric method Materials and reagents: thermostat, water bath, thermometer, technical scales, a porcelain plate, pipettes volume 10 ml, chemical bottles volume 200 ml, 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, iodine solution.
Work procedure At first prepare a malt extract, transferring amylolytic enzymes to solution. For this purpose take a charge weight 10,00 g of 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 carrying out the analysis. For carrying out the enzymatic reaction mix a 25 ml of starch solution in a wide test tube with about 23 ml of the distilled water and 2 ml of malt extract. The mixture is placed in thermostat at 30 °C.. 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 coloring. The analysis takes 10-20 minutes, if iodine coloring 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 than 20 minutes, increase the quantity of the malt extract. But under each conditions total amount of a reactionary mix should consist 50 ml.
Experimental data processing Amount АА, units/g, calculate by such formula:
АА = 0,25 × 60/(а 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: Fresh barley malt were analyzed. To 25 ml of a substratum added 23 ml of the distilled water and 2 ml of a malt extract (0,2 malts). Time of sugarising - 12 minutes АА=0,25*60/(0,2*12)=6,2 units/g
Determination of extract content of malt
Materials and reagents: water bath, refractometer or densitometer, thermometer, technical scales, chemical bottles volume 200 and 1000 ml, glass stick, cylinder volume 200 ml, a funnel for filtering, filtering paper.
Work procedure Take 50,00 g weight charge of malt to preliminary weighed glass and add 200 ml of the distilled water of 47 °С. Glass contents mixing for receiving the homogeneous suspension and set it on a water bath with temperature 45 °С. At such temperature and periodic mixing maintain 30 minutes then temperature gradually raise up 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. Wash off the thermometer and a glass stick with small portions of the distilled water. On the technical scales glass contents pour the distilled water till 450 ml. Mix carefully mix and filter through the folded filter to the dry glass. The first portions of a filtrate return on the filter, till the transparent solution appearance. Define contents of solids in a filtrate with the help of refractometer (shown on fig 3.1) in %.
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