Laboratory work № 5 Studying of a method of determination of content in vitamin c milk (ascorbic acid) and enzymes

Work purpose: to determine the content of vitamin C and enzymes in various samples of milk.

Task 1.To carry out high-quality reaction to vitamin C.

2. Quantitative definition of vitamin C 2,6-dikhlorfenolindofenoly (on Tilmansa)

Short theoretical data

Vitamins. Milk contains all vital vitamins, but some in insufficient quantities. Content of vitamins depends on a season of year, breed of animals, quality of forages, storage conditions and processing of milk. Fat-soluble A, D, E, K vitamins and (V-carotene are steady against heating and begin to collapse at a temperature over 120 °C (vitamin A), but aren't steady against effect of air, ultraviolet rays and acids. Vitamin A gives to butter yellow color. Vitamin E is antioxidant of fats and protects vitamin A from oxidizing destruction. Water-soluble vitamins, except for vitamins C and B12, are steady against heating. They maintain heating in the alkaline environment worse. RR vitamin almost completely remains after thermal treatment and in the course of milk storage. Most collapses at pasteurization and storage vitamin C. Vitamin C (ascorbic acid). She participates in an oxidizing voskstanovitelnykh the processes happening in an organism. The lack of a viktamin With in food can cause a scurvy. Raw milk contains 0,3 - 2,0 mg of % of vitamin C. Vitamin C sinktezirutsya by hem microflora, his content in milk depends from the inkdividualnykh of features of an animal. Usually it raises in the winter and goes down in the summer.

At milk storage the amount of ascorbic acid decreases. Light affects destructively ascorbic acid therefore at a milk hrakneniye in transparent bottles of loss of vitamin C make 50% and more. Better vitamin B bottles from dark glass and paper packages remains. It is important to consider it at production of vitaminizirovankny milk and dairy drinks.

Enzymes (Latin fermentum - ferment) - the biological katalizaktor accelerating chemical reactions in live organisms. Under a deykstviye of enzymes large molecules of proteins, carbohydrates, fats rasshchepklyatsya on smaller. Enzymes accelerate reactions in tens of thousands and millions of times. Deykstviye of enzymes it is strictly specific, i.e. each enzyme catalyzes only one chemical reaction. Enzyme corresponds to the subkstrat (substance which chemical transformation he catalyzes).

Enzymes work at a certain temperature, рН Wednesdays; their activity depends on availability of chemicals - activators and inhibitors. Opktimalny temperature, i.e. temperature at which the maximum of activity of enzymes is observed for most of them is equal to 40 - 50 °C. At further temperature increase activity of enzyme snizhaktsya. At a temperature of 60-80 °C of squirrels, the forming enzyme, denaturikrut, and enzyme is inactivated (loses the activity). The thermal denaturation of enzymes has important practical znachekny: pasteurization of raw materials promotes destruction of enzymes and predokhranyat foodstuff from enzymatic damage.

The important factor influencing activity of enzymes is рН Wednesdays. Enzymes differ on optimum for their action to znachekniya рН. At too sour or alkaline reaction of the environment the enzyme denaturation proiskhokdit, and it loses the activity.

By the chemical nature enzymes represent proteinaceous substances. They can be simple and complex proteins. Enzymes nakzyvat on that substance which they affect, adding the termination "az" to a name root: lipase, lactase, peptidaza and so forth.

In milk enzymes are in a free state, and also are connected with casein micelles and covers of fatty balls.

Devices and equipment

Conic flasks with a capacity of 250,100,50 ml; scales; pipettes on 5, 10,25 ml; burette; microburette; a funnel, the folded filter, solution with a mass fraction of metaphosphoric or hydrochloric acid of 2%; saturated solution of oxalic acid; saturated solution of chloride of sodium, 2,6-dikhlorfenolindofenol; methylene blue, a reduktaznik, a water bath, 0,1% solution of a fenolftaleinfosfat of sodium in ammoniac buffer mix.

Work performance order

Essence of a method: the chemical method of definition of vitamin C 2,6-dikhlorfenolindofenoly (Tilmans's paint) is based by the specific indicator on properties of this indicator to change the coloring depending on reaction of the environment. In acidic environment the indicator gets pink coloring, in alkaline and neutral - blue. Being restored, the indicator becomes colourless. Decolouration of the indicator in the presence of vitamin C is explained by easy oxidability of vitamin.

Carrying out experience: To part 5 ml of milk by 3 times with water. At cultivation to measure milk a pipette, to pour the distilled water from the burette. To bring 1 ml of 2% in a conic flask with a capacity of 50 ml hydrochloric acid of 5 ml of the received milk solution, to bring water to the total amount of 15 ml. Carefully shaking up, contents of a flask titrut from the microburette 0,001 N solution of a 2,6-dikhlorfenolindofenol, flowing him drops before emergence of the weak-pink coloring remaining 0,5-1 min. To make two parallel definitions of one portion of the divorced milk and to find average size.

To determine the content of vitamin C by a formula:

х = мг %,

where  - the amount of working solution 2,6 dikhlorfenolindofenol which has gone for titration, ml; k - the amendment on a solution 2,6 caption dichlorine-fenolindofenola; V- the volume to which the milk hinge plate at addition to her of water and hydrochloric acid is brought, by ml; V₁ - the volume of the analyzed liquid taken for titration, ml; q - milk hinge plate; 0,88 - amount of vitamin C, corresponding 1 ml 0,001 N of solution 2,6 dikhlorfenolindofenol, mg.

The simplified vitamin C definition method

In a flask pour 50 ml of milk and add 4 ml of saturated solution of oxalic acid, shake up, flow 10 ml of saturated solution of chloride of sodium, shake up and leave at the room temperature for 5 min. Then contents of a flask are filtered via the paper folded filter, measure a pipette 20 ml of a filtrate and titrut him from the burette with a capacity of 5 ml solution of a 2,6-dikhlorfenolindofenol before emergence of the weak-pink color remaining 30 seconds.

For definition of a mass fraction of L-ascorbic acid in milk use a formula:

,

where To – a mass fraction of L-ascorbic acid, mg/kg;

V – solution of the 2,6-dikhlorfenolindofenol spent for titration, ml;

T – correction coefficient to an indicator caption;

0,088 – mass of L-ascorbic acid, corresponding 1 ml2,6-dikhlorfenolindofenol, mg;

m - the mass of milk corresponding to the taken filtrate, (m=19,5 of)

1000-coefficient recalculation on kilogram of milk.

After substitution of the known values in a formula receive a final settlement formula:

К= 4,51_*V_*Т (мг/кг)

Test on reductase. Reductase – the enzyme produced by microorganisms. The more in milk of microbes, the it is more also enzymes. This enzyme is capable to decolour paints – a methylene blue or резазурин. The quicker will occur decolouration, the it is more in milk of microorganisms. The method of definition of a class of milk on bacterial impurity is also based on this regularity, it is necessary to follow sanitary and hygienic rules. Reduktazny test can be carried out with a methylene blue and with rezazuriny.

With a methylene blue. Test can be carried out by two methods: standard and accelerated. The method is based on property of the enzyme of reductase emitted by microorganisms. To restore a methylene blue in her colourless leykoforma. The more microorganisms in milk, the quicker there is a decolouration of a methylene blue. Optimum temperature of restoration of a methylene blue enzyme reductase makes from 38 to 40 ˚С.

1. In a test tube to pour 1 ml of solution of a methylene blue and 20 ml of milk, to close a stopper and it is good to mix.

2. To place a test tube with milk in a bath (or in a reduktaznik) with water temperature 38-40 ˚С. Water level in a bath has to be higher than the level of milk in a test tube, it is necessary to maintain constant water temperature.

3. To check time of decolouration of tests in 20 minutes, in 2 hours and in 5,5 hours. The end of test on reductase to consider the moment when milk in test tubes becomes colourless. Existence of the small painted ring-shaped layer on top or coloring of a small part below aren't taken into consideration.

4. If milk is investigated by the accelerated method, then standard solution of a methylene blue needs to be diluted by 10 times and for the analysis to take 10 ml milk.

On the basis of change of coloring of milk and duration of observation there is table 5.1, using which it is possible to establish a class of bacterial impurity of milk.

Table 5.1 – Definition of a class of bacterial impurity of milk (with a methylene blue)

Decolouration duration		Quantity of bacteria in 1 ml (one million)	Amount of milk	Class milk
by standard method	By the accelerated method
Over 5,5 h	Over 3 h	before 0,5	good	1
after 2 before 5,5 h	after 1 before 3 h	before 4	Satisfactory	2
after 20 minbefore 2 h	after 8 min before 1 h	before 20	Bad	3
Less than 20 min.	Less than 8 min.	More than 20	Very bad	4

With rezazuriny. This test allows to define quickly all complex of bacteriological and hygienic qualities of milk (existence of microorganisms – streptococci, stafilokokk, bacteria of group of colibacillus, leukocytes – especially at a disease of cows of mastitis). The method is based on property of the enzyme of reductase emitted by microorganisms to restore резазурин, easily giving oxygen atom, in oxazones. At the same time milk slowly changes the color (from blue through all shades lilac to pink, and then and to white).

1. In a test tube to pour 1 ml of 0,005% of solution of a rezazurin and 10 ml of milk.

2. To close a test tube a stopper and slowly from 3 to 4 times to turn, without allowing stirring.

3. To put a test tube in the closed water bath or a reduktaznik at a temperature from 38 to 40 ˚С.

4. In 20 minutes and 1 hour to watch change of coloring of contents of a test tube. Using table 5.2, to define a class of bacterial impurity of milk.

Table 5.2 – Definition of a class of bacterial impurity of milk (with rezazuriny)

Test coloring	Quantity of bacteria in 1 ml (one million)	Quality of milk	Class Milk
Blue and steel	Before 0,5	Good	1
Lilac or blue-violet	Before 4,0	Satisfactory	2
Pink or white	Before 20	Bad	3
White	More	Very bad	4

Test on phosphatase

Add 1 ml of 0,1% solution of a fenolftaleinfosfat of sodium in ammoniac buffer mix (80 ml 1 N of solution of ammonia and 20 ml 1 N of solution of chloride of ammonium) to 2 ml of milk, heat on a water bath (40 — 45 °C) and check coloring of contents of a test tube in 10 and 60 min. If phosphatase is destroyed, i.e. milk is pasteurized, his color doesn't change. Light-or bright pink coloring demonstrates violation of the mode of pasteurization.

The report on the done work

To present results of work in the form given in tab. 5.3 and to write conclusions about the content of vitamin C and enzymes in various samples of milk.

Table 5.3

Research technique	Mass fraction in milk
	Vitamin С	Fermentov

Control questions:

1. What vitamins contain in milk?

2. Essence of a technique of definition of enzymes in milk

3. What is enzymes?