Precautions for laboratory works

The development of biotechnology allows significant intensification of manufacturing, improve the efficiency of natural resources, solve environmental problems and create new sources of energy. The possibilities of biotechnology in case of international cooperation experts can address the world's critical problems caused by deficiency of protein and energy, prevention of dangerous diseases, environmental protection.

Due to analytical methods of biotechnological industries society has the opportunity to consciously control its economic and technology policy, seeking release of products of high quality. With increasing the competition should increase product quality, but it requires strict control at all stages of obtaining raw materials, production, storage and sales.

The main purpose of laboratory guidance is mastering the basic methods of analysis in biotechnology industries, sequence of raw materials and finished products control, the study of factors and conditions affecting the quality of products and methods of assessment. Themes of laboratory works are chosen to prepare the expert to work on modern biotechnology industries to future specialist able to use the methods of determining quality indicators and assess its compliance with specified requirements; identify indicators of quality of raw materials and finished products according to the requirements of existing standards; identify factors that influence the measurement results.

Laboratory classes of training course “Analytical Methods for Biotechnological Industry” are carried out according to the curriculum for bachelors full-time and distance learning direction 6.051401 "Biotechnology" specialties 7 / 8.05140103 "Pharmaceutical Biotechnology" and 7 / 8.05140105 "Environmental Biotechnology and Bioenergy".

In biochemical laboratory it is necessary to follow the rules of behavior and work to ensure the safety of people. Therefore on the first lesson, student must read the rules of safety and sign the register.

At the classes in the laboratory the duty is assigned, who is responsible for the maintenance of order and cleanliness. On the desktops there should be no unnecessary objects, bags, briefcases. Reagents occasionally scattered on the table and solvents spills should be immediately removed. It is strictly forbidden to eat in the laboratory. Do not use substances present in glasses without labels.

Laboratory work can begin only after careful studying of techniques and learn how to work on the appropriate manner. You can work only with proper equipment and appliances.

Careless handling of acids and alkalis can lead to accidents, damage to the eyes, skin face and respiratory tract. For eye protection when working with concentrated acids and alkalis should wear safety glasses. When diluting concentrated acids must follow the order of mixing, slowly pour acid into water.

Experiments with caustic, toxic or highly aromatic substances permitted to hold only in a laminar box.

Special care requires working with arsenic, mercury and other poisons. Residues containing toxic substances cannot be thrown in a bowl or basket. They are collected in special glass. After the experiments it is necessary to wash your hands thoroughly.

Glassware and instruments (including thermometers) should be handled with care, do not put on the edge of the table, do not touch with elbows. Pieces of broken glassware should be removed immediately. After the classes it is necessary to clean workplace close all gas and faucets, turn off electrical appliances.

Duty student report to laboratory technician (engineer), the laboratory is in the proper order.

Modern methods for raw materials analysis at the biotechnological industries

Detemination of plant originated raw material acidity. Plant extracts and juices acidity definition

1 To familiarize with methods of determining the acidity of raw materials and finished products in biotechnological industries

In the normal state grain is slightly acidic, this is caused by the presence of acid salts of phosphoric acid and a small amount of organic acids (lactic, formic, citric). When the grain comes out of dormancy it increases the action of enzymes and this in its turn leads to increased titrated acidity.

Titrated acidity is expressed in degrees, 1 degree of acidity equal to 1 ml of 1N sodium hydroxide solution, taken to neutralize the acid in 100 g of grain.

During the titration alcali is associating not only with dissolved acidic substances, but diffusing the flour solid particles that is resulting in measured acidity increase.

The magnitude of acidity is usually considered as defining a quality of raw material. Increasing of grain acidity during the storage time is an indicator of the raw material damaging. Acidity of healthy grain defined in the limits 1,8-2,5°.

Active acidity of plant raw material affects the fermentation process. Concentration of hydrogen ions in solution is defined as active acidity. The pH magnitude is a hydrogen ions factor. It is changing from 1 to 14. Determination of active acidity is usually conducted with the help of pH-meter.

Determination of plant extracts and juices acidity with the help of pH-meter

Materials and reagents: pH-meter, chemical glasses vol. 50 ml, glass funnel, measure flasks vol. of 250 cm³, fixanals for preparation of the buffer solutions, distilled water, filtering paper, absorbent cotton.

For determination of pH universal ionometers or pH-meters with a measuring glass electrode and chlor-silver reference electrode are used.

For carrying out of measurements of active acidity from the prepared test in a glass vol. 50 ml take such quantity of a product which would provide immersing of electrodes. Readout of

pH level count by the scale when the device scale will stop. Readout of results takes to accuracy of 0,1.

Measurement of pH repeats by three-four times, each time taking out electrodes from a solution and immersing them again in a solution for measurement.

2 Give the definition to the total or titrated and active acidity and alkalinity.

3 What are the different indicators? How they are used in determining the alkalinity and acidity of foods?

From average test take 10 g yeast and weigh in a porcelain cup to within 0,01 g, add 50 ml of the distilled water, carefully mix for homogeneity and titrate with 0,1 N sodium hydroxide solution at presence of phenolphthalein till the pink coloring appearance.

5 g of the flour, weighed to within 0,01 g, transfer to a dry conic flask vol. of 100-150 ml and add 50 ml of the distilled water. Flask contents mix to disappearance of lumps of a flour and add three drops of phenolphthalein (for rice flour - 5 drops). Titrate with 0,1 n solution of sodium hydroxide to occurrence of pink coloring which does not disappear throughout 20-30 s. At the moment of coloring disappearance in specified time add to 3-4 more drop of phenolphthalein. Occurrence of pink coloring is signaling to the titration termination.

Flour charge 25 g, weighed to within 0,01 g, transfer in a conic flask vol. of 300-500 ml, add 250 mlof the distilled water from a measured flask, carefully mix and leave for 2 hours for extraction of water-soluble substances. Then filter in a dry flask and transfer with pipette 25 cm³ of obtained filtrate into a conic flask of 100 ml, add 3-4 drops of phenolphthalein and titrate with 0,1 N solution of sodium hydroxide.

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.

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 %.

2. How significant are variety classes of enzymes for technological processes of manufactures?

Take a glass cylinder of diameter allowing alcoholmeter to fall freely into the liquid without touching the walls of the cylinder and thermometer. Wash and rinse the cylinder thoroughly with analyzed ethanol. Alcoholmeter wipe with a soft cloth slightly moistened with ethanol.

Pour ethanol 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 lowered carefully into the cylinder alcoholmeter, holding it by the top rod thumb and forefinger and does not touching hands the part that is immersed.

Hold alcoholmeter and thermometer in the liquid 3... 5 min while counting alcoholmeter 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 must be determined with different thermometers: two 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-ethanol solution is determined by the special tables.

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

Ethanol is mixed with water in any ratio. This process is exothermic and is accompanied by change in volume of aqueous-alcohol fluid (adiabatic compression), which depends on the concentration of alcohol in the solution. The content of ethanol is determined at 20 °C.

1. What is the principal difference between scale of areometric alcoholmeters and scale of areometric saccharimeters?

2. What factors can affect the result of measuring the concentration of water-alcohol solution?

3. Check the accuracy of measurements with a solution of known concentration of maltose.

1.2 g of malt extract weigh in a porcelain cup on industrial scales to the nearest 0.01 g sample of the product taken from a calculation of sugar concentration in the filtrate 0.8 - 0.4%.

Sample of the test product taken transfer to the volumetric flask of 250 ml, washing a cup several times with distilled water. The amount of water is equal to 100 ml. The flask containing hot water bath and then make 3-4 ml of sodium hydroxide solution. The content of the flask and stirred add 5 cm of lead nitrate. If the solution is not transparent for 5-7 minutes, add another 1-2 ml of both solutions. We're sure that there was no excess of alkali that promotes the oxidation of sugar. To remove excess lead to the deproteinised solution, heated to 60 ° C, add 3.4 ml of a saturated solution of sodium sulfate and left at the same temperature for 10 min in a water bath, then cool. It forms large crystals of lead sulfate, which are could be easily filtered. The resulting solution test for complete precipitation of excess lead, adding to bulb by the wall a few drops of sodium sulfate. In the absence Turbidity touch and place liquid flask topped up with distilled water to the mark, mix and contents of the flask after 1-2 min filtered through a filter. In the case of turbidity to the resulting solution add another 5-7 ml of a solution of sodium sulfate. The content of the flask is stirring to allow settling and re- check for deposition completeness. Filtered colorless solution can be used to determine the mass fraction of sugars by chemical method.

For determination of reducing sugar by Max-Müller pour water extract prepared to analyze into conical flask 150-200 ml consistently take by 25 ml of solutions Fehling I and Fehling II with pipette. The resulting mixture heat to boiling and pour with a pipette 50 ml of solution prepared for analysis. Boil exactly 2 minutes, counting the time from the first appearance of the surface bubbles. After boiling, allow the copper precipitate to settle down.

A solution of the residue should be blue from excess Fehling solution. If the solution discolored, indicating partial oxidation of sugar, the analysis should be repeated, slightly decreasing, but exactly measure the volume of the filtrate analyzed. Add distilled water in an amount of 50 ml together with a new volume to the flask. Carefully decant test solution to Bunsen flask without transferring the precipitate to the filter. The precipitate always must be covered with liquid to prevent oxidation Cu₂O with oxygen. This residual solution after draining the filter to precipitate copper oxide is added 5-10 ml of pre- boiled (to remove oxygen) hot water, and after re- deposition of copper oxide the water decant through a filter. Operations of washing, sedimentation, decantation with hot water repeat several times until the disappearance of the blue color of water.

After washing liquid is poured from the flask, and wash the flask thoroughly with water. Then the conical flask with sediment copper, located under a small layer of washing water, pour in small portions 30 ml of iron- ammonium alum solution to dissolve the precipitate. Oxidized copper oxide and iron oxide is reduced to iron. The resulting light- green liquid is poured carefully on the glass stick to the same filter in earlier prepared flask. It dissolves copper oxide particles that got into the filter before.

For better dissolution of copper particles surface filter and fluid carefully stirred with a glass rod. After dissolution Su2O conical flask and the filter washed well with distilled water, and the liquid from the bulb passed through a filter. The filtrate was titrated by 0.1 M solution of potassium permanganate to stabile for 30 s light pink color.

10FеSО₄+ 2KMnO₄ + 8Н₂SO₄ = 5Fе(SО₄)₃ + К₂SО₄+ 2МnSО₄ + 8Н₂О

Take 25 ml of 0.3 N solution HCl in dry volumetric flask of 100 ml and add through a funnel with constant stirring a sample of tested product (shredded potatoes) weighing 5 g, weighed with an accuracy of ± 0,01 g When the material is well mixed washed funnel and upper portion of the new bulb (25 ml) of the same acid. Stirring constantly, immerse the flask in boiling water and shake for 3 min. (with a stopwatch). Hold it in the bath 12 minutes more. After 15 min. after immersion in a bath remove the bulb, pour by cylinder 40 ml of cold distilled water and rapidly cool under the water flow to 20 ° C.

For the deposition of protein and lighting solution in the flask pour reagents cylinder - coagulants: the 2ml of reagents Karrez I and II, or 5ml of 4% solution of phosphotungstic acid, or 6 ml solution of ammonium molybdate. After 5 min. prove the contents of the flask to the mark with distilled water, shake and filter through a folded filter. The first portion of the filtrate (10 ml) must not be used. Fill the polarimetric tube of 200 mm length with transparent filtrate at 20 ° C and measure the polarization on polarimeter to receive polarimeter indication P₁.

In parallel control experiment is carried out for correction on the optically active water-soluble substances sedimented by coagulant reagents and that are present in solution (mainly carbohydrates).

Weigh 10 g of crushed potato, transfer it to a volumetric flask of 100 ml, add 70 ml cylinder with water and with frequent shaking stand for 40 minutes. Then wash the top of the bulb 10 ml of distilled water, deproteining reagent, which was used in experiment. Add 5 ml of 10% solution of tannin, 5 ml of Pb acetate, shake for 5 min. and prove the contents of the flask to the mark with distilled water, mix and filter. 50 ml of filtrate take with pipette and transfer to volumetric flask of 100 ml, add 25 ml of 2% solution of HCl, stand 15 minutes in a boiling water bath, cooled to 20 ° C, measured in quvette of 200 mm length. Obtain polarimeter readout P₂.

Sample of crushed corn 15 g (14,48 -normal sample) weighed on technical scales and transfer with distilled water into the flask 200 ml. Add 45 ml of hydrochloric acid in a volume of 200 ml, add about 180 ml of water and place in a boiling water bath for 25 min.

After hydrolysis cool the flask rapidly under the water flow to 20 °С, add water to the mark, mix and filter. Deproteinise the filtrate with 0.3 g of dry phosphotungstic acid and 0.25 g of dry activated charcoal for 5 minutes, filter again. The filtrate polarize in polarimetric cuvette at 400 mm. Obtain the readout of polarimeter P₁.

In parallel control experiment conducted for adjustment by the soluble carbohydrates.

Weigh 15 g of crushed grain transfer into the flask and leave for 20 minutes, adjust the contents of the flask to the mark with distilled water, mix and filter. 100 ml of filtrate transfer into a flask of 200 ml, 45 ml of HCl is added to the relative density of 1.19 to bring the volume to 180 ml. Flask placed in a boiling water bath for 25 min. After hydrolysis flask was cooled to 20 ° C, bring water to the mark, deproteinise with 0.3 g of dry phosphotungstic acid and 0.25 g of dry activated charcoal, stirred, filter, polarize in polarimetric cuvette of 400 mm. Obtain the readout of polarimeter P₂.

1. What requirements must meet the drug prepared for polarimetric analysis carbohydrates?

3. Give the comparative analysis of the method for carbohydrates determination by Evers and Arkhypovych?

Prepare the working solution of vitamin B₂. Prepare 10 - 20 ml of 0.01% solution from a dry preparation.

Determine the pH of solutions using indicator paper or pH-meter. Simultaneously with pH, determine refractive index of the solutions with refractometer. The results of measurements and color of the solution record to the table.

Using transparent to UV light glassware to assess the ability of samples for fluorescence, the data record to the table.

Add to fluorescence solution a few drops of 0.1 M KOH solution, to observe the disappearance of fluorescence. Measure the pH again and the index of refraction.

Extraction. To prepare the sample extract samples weighing from 5 to 50 g weighed with deviation ± 0,01 g

For extraction of vitamin C with dry food samples 5-10 g sample is ground in a mortar with a small amount of the mixture or solution ekstrahuvalnoho acids (at least 1 ml of solution per 1 g of sample) and sand transferred to a volumetric flask or graduated cylinder capacity of 100 ml, washing the mortar and small portions pestyk ekstrahuvalnoho solution until the volume reaches the mark. Stand for 10 minutes, stirred and filtered.

For extraction of vitamin C from foods thick consistency sample of 5-10 g homogenized sample of no more than 2 minutes with a little ekstrahuvalnoho solution (at least 1 ml of solution per 1 g of sample) and transferred to a volumetric flask or cylinder capacity of 100 ml, washing the homogenizer small portions ekstrahvalnoho solution until the volume reaches the mark. Contents to stand for 10 minutes, stirred and filtered.

Visual titration. In the flask 50 or 100 ml pipette made from 1 to 10 ml extract, dilute with water to 10 ml and titrated with a solution of sodium 2,6-dyhlorfenolindofenolate to light pink color that persists for 15-20 sec.

At the same time control experiment is carried out on the contents of the product substances reduces. To this flask is placed in a same volume of extract, as well as to determine (1 to 10 ml) was added an equal volume of acetate it buffer formaldehyde solution in a volume equal to half the volume of buffer solution, stirred and allowed to stand for 10 minutes, closing.

1. Advances in Biochemical Engineering/Biotechnology, Vol. 66 Editor: Th. Scheper/ Springer-Verlag Berlin Heidelberg 1999 – 230 p.

2. Джигирей В.С. Екологія та охорона навколишнього природного середовища – К.: Знання, 2004 – 230 с.

3. Медико-биологические требования и санитарные нормы качества продовольственного сырья и пищевых продуктов – М.: Минздрав СССР, 1990.

4. В.Л. Яровенко, В.А. Мариниченко, В.А.Смирнов и др. Технология спирта. – М.: Колос «Колос-Пресс», 2002. – 464 с.

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6. Грачева И.М., Мосичев М.С. Лабораторный практикум по технологии ферментных препаратов. – М.: Легкая и пищевая промышленность, 1982. – 239 с.

7. Лисицын А.Б., Иванкин А.Н. Методы практической биотехнологии – М.: Издательство ВНИИМП, 2002. – 408 с.

8. Родопуло А.К. Основы биохимии виноделия – М.: легкая и пищевая промышленность, 1983. – 240 с.

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11. Кислухина О.В. Ферменты в производстве пищи и кормов – М.: Делипринт, 2002. – 336 с.

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INTRODUCTION........................................................................ 3

Chapter 1. Modern methods for raw materials analysis at the biotechnological industries.......................................... 5

Laboratory work 1. Detemination of plant originated raw material acidity. Plant extracts and juices acidity definition............... 5

Laboratory work 2. Starch, yeast and flour acidity determination. Titrated acidity. 10

Laboratory work 3 Enzymes activity determination. Determining of malt of α-amylaze enzymatic activity. Malt extractives definition.....................................................................................................16

Laboratory work 4. Quantification of water-ethanol solutions, areometric method. Definition of ethanol aqueous solution strength........................................................................................................21

Chapter 2. Modern methods for analysis of integrated and waste products at the biotechnological plants.......................................... 29

Laboratory work 5. Chemical methods for carbohydrates determination. Determination of reducing sugars by Max-Müller............26

Laboratory work 6. Water extracts preparing for carbohydrates quantification. Determination of starch content in potato by Evers............................................................................... 29

Laboratory work 7. Polarimetric method for carbohydrates analysis. Starch content determination in cereals by Archipovich..........................32

Laboratory work 8. Analysis of vitamins. Physical and chemical properties of vitamins 34

Literature................................................................................ 43

Laboratory work can begin only after careful studying of techniques and learn how to work on the appropriate manner. You can work only with proper equipment and appliances.

Experiments with caustic, toxic or highly aromatic substances permitted to hold only in a laminar box.

Duty student report to laboratory technician (engineer), the laboratory is in the proper order.

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