Preservatives

It is a common practice by unscrupulous persons to add preservatives to liquid milk.The addition of preservatives is not permitted under law. Freshly drawn milk get contaminated with microorganisms which proliferate and multiply rapidly in milk.

The growth of these microorganisms leads to an increase in acidity and souring of milk, which leads to spoilage. The problem is acute during summer months due to high temperatures. Their use is permissible under law only where sample is stored for testing. The common preservatives added to milk are:

i)Formalin

ii) Boric acid and borates

iii) Benzoic acid and sodium benzoate

iv) Salicylic acid

v) Mercuric chloride

vi) Potassium chromate

vii) Hydrogen peroxide

i. Formalin

Formalin is a solution of 40% formaldehyde in water. By Hehner test formalin/formaldehyde can be detected in milk. Formalin is a strong preservative which is very effective even in small dose. Formaldehyde (HCHO) is a gas. Its 40 percent solution in water is known as Formalin. It contains 10% methanol to prevent polymerization. Milk samples for analysis are preserved with 0.1 ml (two drops) of formalin per 25 gm/ml samples. Formalin can be detected by three tests namely,Hehner test, Hehner-Fulton test and Chromotropic test.

i)Hehner Test

Hehner test is a very simple and quick test for detecting formalin. It can be detected with concentrated sulphuric acid in the presence of an oxidizing agent Fecl 3 . It gives intense violet colouration with this test. The detection is very easy and simple. Even a very small quantity of preservative can be detected by this test. 

• Take 5 ml sample of milk in a test tube and gently add 2 ml concentrated sulphuric acid (H 2 SO 4 ) containing a trace of FeCI 3 . Care should be taken that while adding acid it forms a separate layer at the bottom of the tube. The acid should not mix with milk 
• A coloured ring is formed at the junction of the two liquids. Note the colour of the ring formed at the junction of the milk and acid. A voilet to purple coloured ring indicates the presence of formalin/formaldehyde.

ii)Chromotropic Acid Test

Chromotropic acid test is a colour reaction test with formalin. In the presence of formaldehyde chromotropic acid develops a purple colour. The coloured reaction of Chromotropic acid test is due to reaction between chromotropic acid and formalin.Chromotropic acid solution-is prepared as a saturated solution of chromotropic acid(1,8- dehydroxy napthalene-3, 6-disulphonic acid). The solution is prepared by stirring 0.5 g of chromotropic acid in 100 ml 72% H 2 SO 4 (150 ml concentrated H 2 SO 4 in 100 ml water-mixed in cold). The solution is straw yellow in colour.

•  5 ml reagent and 1 ml milk distillate is taken in test tube. Distillate is prepared from H 3 PO 4 acidifred milk.
•  The test tube is placed in a boiling water and the developed colour is noted.
•  Development of light to deep purple colour indicates the presence of formalin.

The colour intensity depends upon the amount of formalin present in sample.

ii. Boric Acid and Borates

Like formalin, boric acid and sodium borates are also common preservatives. They are available in the form of either boric acid (H 3 BO 3 ) or its salt as sodium borate(Na 3 BO 3 ) as commercial chemicals. In acidic medium they can be conveniently detected with the help of turmeric paper. In the presence of boric acid and borates the paper turns red in acidified medium. This is due to the formation of ferric benzoate.

•  Take 20-25 ml milk in a porcelain basin. Acidify milk by adding 1.5 ml concentrated hydrochloric acid.
•  Take a strip of turmeric paper and carefully dip in the milk. Remove the strip from acidified milk and dry it in the air.
•  Presence of boric acid or borates is confirmed by noticing the change in colour of the strip. The strip changes its colour from yellow to a red colour which is characteristic for the presence of boric acid or borate.
•  On exposure of the paper strip to ammonia vapours or ammonia solution the colour changes to bluish green but reappears on re-acidification with HCl.

iii. Benzoic Acid and Sodium Benzoate (E)

These are food grade preservatives. Benzoic acid and its salt sodium benzoate are stable preservative. Benzoic acid is commonly used in the form of its sodium salt because it is more soluble than the acid but later is the active form. Sodium Salt is converted to the free acid when used as preservative. The optimum PH range for anti microbial activity of benzoic acid is 2.5 to 4.0. Benzoic acid is detected by extracting with ether from milk serum, as benzoic acid is soluble in ether. In alkaline medium with FeCI 3 it gives salmon red precipitate. In modified Mohler test a red brown ring is formed.

•  Benzoic acid is extracted from serum of milk by removing casein. Collect the clear filtrate or serum.
•  As benzoic acid is soluble in ether it is extracted by adding 50 ml diethyl ether and shaking it. The water and ether layer is allowed to separate in a separating funnel. If emulsion is formed and layers do not separate 10-15 ml petroleum ether (b.p. 60 0 C) is added. Alternatively, separate the layers by a centrifuging at 1200 r.p.m.
•  The etheral layer is carefully removed in a porcelain dish.
•  The etheral layer is carefully evaporated on a boiling electric water bath.
•  The residue so obtained is dissolved in 5 ml portion of water and divided it into two parts equally. There are two tests for detection, namely, FeCI3 test and modified Mohler test.

i) FeCI 3 Test: Make one portion of the above extract alkaline by adding a few drops of NH 4 OH solution, expel the NH 3 by evaporation and dissolve the residue in a few ml hot water. Filter if necessary and add a few drops of 0.5% neutral FeCI 3 solution. Note the change in colour. A salmon red precipitate indicates the presence of benzoic acid.
ii) Modified Mohler Test:

In another portion of the extract add 1-2 drops of 10% NaOH solution and evaporate to dryness.

•  To the residue add 1 ml conc. H 2 SO 4 and a crystal of KNO 3 . Heat for 20 minutes on a boiling water bath.
•  Cool and add 1 ml water and mix. Make ammonical by adding NH 4 OH.Boil to break any ammonium nitrate that may have formed.
•  Transfer the solution to a test tube; add a drop of freshly prepared ammonium sulphide solution without mixing.
•  Formation of a red brown ring indicates the presence of benzoic acid. The colour diffuses on mixing and give greenish yellow colour on heating.

Note: Salicylic acid also gives a reddish brown colour. However, this colour remains unchanged after heating.

iv. Salicylic Acid

Salicylic acid is an organic preservative. Like benzoic acid it is extracted from milk serum with the help of ether in which salicylic acid is soluble. To the residue in the presence of salicylic acid ferric chloride gives a voilet colour.

•  To the residue obtained after extraction add 1 drop of 0.5% neutral FeCI 3 solution and observe the colour produced.
•  A voilet colour indicates the presence of salicylic acid.

v. Mercuric Chloride

Mercuric chloride is a heavy metal salt and is highly toxic. It is also used as a preservative. Mercuric chloride is detected from milk serum by adding stannous chloride solution. A white precipitate is formed in the presence of mercuric chloride.Prepare the extract of milk as is followed for benzoic acid.

•  Dissolve the residue in 1-2 ml water. Filter if necessary.
•  Transfer the solution to a test tube and add to it 15% stannous chloride in 1:1
• HC1 solution and mix it simultaneously. 
• A silky white precipitate appears which turns grey on further addition of SnCI 2 solution if mercuric chloride is present confirms its presence in milk.

vi. Potassium Chromate

Potassium chromate is used as a preservative only for storage of milk for analysis.Its solution is yellow in colour due to chromate ions. Potassium chromate is detected by a simple test using barium chloride. Yellow precipitates are formed due to barium chromate in the presence of potassium chromate.

•  Prepare ash from 50 ml of milk by first drying on boiling water bath and then heating it in a muffle furnace at 550 0 C for two hours.
•  Add to ash 3-4 ml dil HCl and dissolve by warming.
•  To 1 ml ash solution add 2 N NaOH solutions dropwise till the solution is alkaline (test with pH paper).
•  Add 1 ml acetic acid and then 0.5 ml BaCI 2 solution and mix.
•  Formation of a yellow precipitate indicates the presence of dichromate in milk.

vii. Hydrogen Peroxide

Hydrogen peroxide is a very strong oxidizing agent, and is an efficient preservative in small quantities. It breaks into water and oxygen in the presence of natural catalase present in milk. Hydrogen peroxide plus thiocyanate also activates the native lacto- peroxidase system which can be used for prolonging shelf life of milk.

The use of hydrogen peroxide is prohibited. With paraphenylene diamine test hydrogen peroxide present to the level of 1:40,000 can be easily detected. It gives dark blue colour with paraphenylene diamine. Hydrogen peroxide can also be detected by vanadium pentoxide test which forms pink to red colour with this reagent.

i) Paraphenylene diamine test

•  5 ml milk is taken in a test tube. 
• 5 drop of 2% aqueous solution of paraphenylene diamine is added and mixed in milk. Formation of deep blue colour indicates the presence of H 2 O 2

ii)Vanadium Pentoxide (V 2 O 5 ) Test

Vanadium pentoxide test gives pink to red colour in the presence of H 2 O 2. It is a simple test and test is carried out in acidic medium first.Vanadium pentoxide reagent is prepared by mixing 1 g vanadium pentoxideV 2 O 5 in 100 ml H 2 SO 4 (6 vol conc H 2 SO 4 + 94 vol H 2 O)

•  To 10 ml milk in a porcelain dish add 10-20 drops of V 2 O 5 reagent and mix carefully with a glass rod.
•  Formation of pink to red colour indicates the presence of H 2 O 2 .