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
after 2 before 5, 5 h	after 1 before 3 h	before 4	Satisfactory
after 20 minbefore 2 h	after 8 min before 1 h	before 20	Bad
Less than 20 min.	Less than 8 min.	More than 20	Very bad

 

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
Lilac or blue-violet	Before 4, 0	Satisfactory
Pink or white	Before 20	Bad
White	More	Very bad

 

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?

 

Laboratory work № 6

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