Chemical Quality Assurance

Module 3. Chemical analysis of milk and milk products

Lesson 8
DETECTION OF NEUTRALIZERS, PRESERVATIVES AND ADULTERANTS IN MILK AND MILK PRODUCTS

8.1 Introduction

8.1.1 Adulteration of food products

As per the definition of the Prevention of Food Adulteration Act, 1954 (PFA) Rule 2, "adulterant" means any material, which is or could be employed for the purposes of adulteration.

Any food article is deemed to be "adulterated"

• if the article sold by a vendor is not of the nature, substance or quality demanded by the purchaser and is to his prejudice, or is not of the nature, substance or quality which it purports or is represented to be;
• if the article contains any other substance which affects, or if the article is so processed as to affect, injuriously the nature, substance or quality thereof;
• if any inferior or cheaper substance has been substituted wholly or in part for the article so as to affect injuriously the nature, substance or quality thereof;
• if any constituent of the article has been wholly or in part abstracted so as to affect injuriously the nature, substance or quality thereof;
• if the article has been prepared, packed or kept under insanitary conditions whereby it has become contaminated or injurious to health;
• if the article consists wholly or in part of any filthy, putrid, rotten, decomposed or diseased animal or vegetable substance or is insect-infested or is otherwise unfit for human consumption;
• if the article is obtained from a diseased animal;
• if the article contains any poisonous or other ingredient which renders it injurious to health;
• if the container of the article is composed, whether wholly or in part, of any poisonous or deleterious substance which renders its contents injurious to health;
• if any coloring matter other than that prescribed in respect thereof is present in the article, or if the amounts of the prescribed coloring matter which is present in the article are not within the prescribed limits of variability;
• if the article contains any prohibited preservative or permitted preservative in excess of the prescribed limits;
• if the quality or purity of the article falls below the prescribed standard or its constituents are present in quantities not within the prescribed limits of variability, but which renders it injurious to health;
• if the quality or purity of the article falls below the prescribed standard or its constituents are present in quantities not within the prescribed limits of variability but which does not render it injurious to health provided that, where the quality or purity of the article, being primary food, has fallen below the prescribed standards or its constituents are present in quantities not within the prescribed limits of variability, in either case, solely due to natural causes and beyond the control of human agency, then, such article shall not be deemed to be adulterated within the meaning of this sub- clause.
  

Further under rule 44, PFA states that no person shall either by himself or by any agent sell milk which contains any added water and also milk containing a substance not found in milk.

Amongst the food items, milk is a complex mixture of nutrients and a liquid food which, can be easily adulterated by the unscrupulous persons. According to PFA definition, “Milk is the normal mammary secretion derived from complete milking of healthy milch animal without either addition thereto or extraction therefore. It shall be free from colostrum. Milk of different classes and different designations shall confirm to the standards laid down for them.

8.2 Adulteration of Milk

8.2.1 Common adulterants

The most of the adulterants added to milk can be grouped in the following categories:

a) Carbohydrates

Sugar, glucose, starch, malto-dextrin, etc.

b) Salts and fertilizers

Urea, ammonium sulphate, ammoniacal fertilizers, potassium sulphate, sodium chloride etc.

Fig. 8.1 Sugar

c) Neutralizers

Sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium hydroxide, etc.

d) Preservatives

Hydrogen peroxide, formalin etc.

e) Detergents

Liquid detergents, washing powders etc.

f) Oils and Paints

Vegetable fats and oils, mineral oil / cutting oil, white paint etc.

Most of these adulterants and preservatives can be detected by the following tests

8.2.2 Carbohydrates

8.2.2.1 Sugar

Take 1ml of milk sample and add 1 ml of 0.5% resorcinol solution (0.5% resorcinol solution in 3N Hydrochloric acid). Mix the contents and heat in boiling water bath for 5 min. If sugar is present in the sample, the red color will be produced. Blank shows pale yellow or brownish color.

8.2.2.2 Starch

Starch being cheaper, is some times added in the milk by adulterators to raise the S.N.F of milk. However, it can be easily detected by using iodine solution. Take 3.0 ml of suspected milk sample in a test tube. Add one drop of 1% iodine solution (1gm of iodine crystals and 5gm Potassium iodide in 100 ml distilled water, mixed by warming to dissolve the iodine crystals). Development of blue color indicates the presence of starch.

Fig. 8.2 Starch

8.2.2.3 Glucose

Reagents

a) Barfoed’s reagent ( Prepared by dissolving 24 gm of cupric acetate in 450 ml boiling distilled water and immediately adding 25 ml of 8.5% lactic acid to the hot solution and cooling and diluting the contents to 500 ml).

Fig. 8.3 Glucose

b) Phosphomolybdic acid reagent. (Dissolve 35gm of Ammonium molybdate and 5 gm of sodium tungstate in 400ml of 5% (W/V) Sodium hydroxide solution. Boil the contents vigorously for 20 – 40 min so that the reactants are dissolved properly. While boiling, the ammonia is released. To check the ammonia escaping in the vapours, put red litmus paper in the path of vapours, if it turns blue means reagent is still not free from ammonia. No change in the color of litmus paper indicates that reagent is free from ammonia. As a result of boiling, water gets evaporated, now cool the contents and dilute to about 350 ml and add 125ml of concentrated (85%) phosphoric acid. Finally make the volume to 500ml with distilled water).

Procedure

Glucose is also easily available in commercial form as concentrated syrup, so adulteration of milk with this concentrated syrup is very much prevalent in the industry. To detect glucose in milk, take 1ml of adulterated milk sample in a test tube and add 1 ml of Barfoed’s reagent. Heat the mixture for 3 min in a boiling water bath and cool for 3min under tap water. Add 1ml of phosphomolybdic acid reagent and mix the contents. Formation of blue color indicates the presence of glucose.

8.2.2.4 Dextrin/Maltodextrin

Take 1ml of suspected milk sample in a test tube. Add 1 ml of iodine solution (0.005N Iodine solution prepared using 3% Potassium iodide solution in water). Mix the contents. Development of chocolate red brown color indicates the presence of dextrin/ maltodextrin.

8.2.3 Neutralizers & preservatives

I. Neutralizer

In milk, sodium hydroxide, sodium carbonate and sodium bicarbonate are added by adulterators to neutralize the developed acidity in milk. Their detection can be carried out by employing the following tests:

A) Rosalic acid test

Take 5 ml milk in a test tube of capacity 20 ml. Add 5 ml ethanol (95%,v/v) and 4 drops of Rosalic acid solution (0.05%; w/v in ethanol). Mix the contents. If carbonate or bicarbonate is present a pink color will appear. If NaOH is present a deep rose red color will appear.

Fig. 8.4 Neutralizers

B) Alkalinity of ash test

Take 20 ml milk in a silica crucible. First evaporate the water to dryness, and then burn the content to ash in a muffle furnace at 550°C. Disperse the ash in 10 ml distilled water and titrate the ash content against N/10 HCl using phenolphthalein as indicator. If the volume of N/10 HCl exceeds 1.20 ml, then the milk contains the added neutralizers.

II. Formaldehyde

Formalin (40% water solution of formaldehyde) is generally used by Public Health Departments to preserve the milk samples for chemical analysis purpose. Formaldehyde is very poisonous chemical. Though, it can preserve the milk for very long time, it should never be added to milk meant for human consumption due to its poisonous property. Moreover, it affects the quality of the milk products. If milk kept at room temperature (25 to 35°C) for longer time, did not sour, then that milk must be suspected for the presence of formaldehyde. Formaldehyde added to milk can be tested by the following simple test.

Hehner Test:Take 5 ml of the suspected milk sample in a test tube. Add gently 2 ml of sulfuric acid containing trace of Ferric chloride along the side of the tube ( the added acid should form a bottom layer of the mixture without mixing the milk). Formation of a violet to purple colored ring at the junction of the two liquids indicates the presence of formalin in the milk sample.

Chromotropic acid test: Take one ml of milk in a test tube and add one ml of chromotropic acid reagent (reagent is prepared by dissolving 500 mg of chromotropic acid (1, 8-dihydroxynapthalene-3, 6 disulphonic acid), in 72 per cent sulphuric acid). Mix the contents well. Presence of formalin in milk is cofirmed by the appearance of yellow color; whereas, control sample remains colorless.

Leech test: Take about 5.0 ml of milk in a test tube. Add to it equal volumes of concentrated HCl containing 1.0 ml of 10% ferric chloride solution to each 500 ml of the acid. Heat over a flame for about five minutes. Rotate the tube to breakup the curd, and observe the color. Development of violet color indicates the formaldehyde added as a preservative.

III. Hydrogen peroxide

Take 5 ml milk in a test tube and then add 5 drops of 2% paraphenylene diamine solution and shake it well. Change of the color of milk to blue confirms that the milk is added with hydrogen peroxide.

8.2.4 Fertilizers and salts

A) Urea

i) Take 5 ml of milk in a test tube and add 5 ml of 1.6% DMAB reagent (prepared by taking 1.6 g of p-dimethyl aminobenzaldehyde in 100 ml of ethyl alcohol containing 10 ml of concentrated hydrochloric acid). Appearance of distinct yellow color indicates the presence of added urea whereas development of slightly yellow color is due to natural urea in milk.

Fig. 8.5 Urea test

ii) Take 5 ml of milk in a test tube and add 0.2 ml of urease (20 mg/ml). Shake well at room temperature and then add 0.1 ml of bromothymol blue solution (0.5%). Appearance of blue color after 10-15 min indicates the adulteration milk with urea.

B) Pond water

This method actually detects nitrates present in the pond water. In the pond water nitrates may come from fertilizers used in the fields.
Rinse a test tube with the suspected milk sample. Along the side of the test tube add about 1 or 2 drops of 2% solution of diphenylamine. The sides of the test tube will turn blue if the milk sample contains pond water.

Fig. 8.6 Pond water

C) Ammonium compounds

Ammonium compounds are detected by using Nessler’s reagent.

Nessler’s reagent

Dissolve 50gm of potassium iodide in the smallest possible amount of distilled water, add a saturated solution of mercuric chloride until an excess is indicated by the formation of reddish precipitate. Add 900 ml of 50% solution of potassium hydroxide. Make up the volume to one litre. Allow the contents to settle down and take the clear supernatant.

Take 1ml milk adulterated with ammonium compounds. Add 1 ml of Nessler’s reagent. Mix the contents and observe the color after 1.5 min and before 2 min. Development of characteristic red brown color is an indication of the presence of ammonium compounds in milk.

D) Common salt

Detection of common salt is based on the principle of argentometric titrations. In this case excess of silver nitrate is made to react with potassium chromate and resulted in the formation of reddish brown precipitates of silver chromate.

Take 5ml of milk in a test tube. Add 1 ml of silver nitrate solution (4.3 gm/100ml double distilled water). Mix it and add few drops of 5% potassium chromate indicator in water and mix the contents. Observe the color change. Development of yellow color indicates the presence of excess salt and red color indicates the absence of excess sodium chloride.

8.3 Detergents in Milk

i) Take 2.0 ml of milk in a test tube and add 1.0 ml of 0.025% methylene blue solution in water. Mix the contents and add 5.0 ml of chloroform and shake the contents vigorously. Keep the tubes undisturbed for 15 min or centrifuge the contents for 10 min. and observe the color in lower layer. Intense blue color in lower layer indicates the presence of detergent.

Fig. 8.7 Detergent

8.4 Test for Detection of Ammonium Sulphate

Take 1.0 ml of milk; add 0.5 ml of 2% sodium hydroxide, 0.5 ml of 2% sodium hypochlorite and 0.5 ml of 5% phenol solution. Heat for 20 seconds in boiling water bath, bluish color turns deep blue in presence of ammonium sulphate. The development of pink color shows that the sample is free from Ammonium sulphate.

8.5 Adulteration of Milk Products

The most frequently adulterated milk product is Ghee along with other milk products. The reason is the price of milk fat. It is commonly adulterated with animal body fats, vegetable oils and vanaspati.

8.6 Detection of Vanaspati in Ghee

Isolate the fat from milk by heat clarification method. Take about 5 g of the melted fat in a test tube. Add 5 ml of concentrated HCl (AR grade). Add 0.4 ml furfural solution (2% in alcohol) and shake the tube thoroughly for 2 min. Allow the mixture to separate. The development of pink or red color in the acid layer indicates presence of vanaspati. Confirm by adding 5 ml distilled water and shaking again. If the color in acid layer persists, vanaspati is present. If the color disappears, it is absent SP:18 (1987).

8.7 Detection of Animal Body Fats and Vegetable Oils/Fats by the Opacity Test in Ghee

Melt the sample of fat (5 gm) isolated by heat clarification method at 50 +1°C in a test tube and maintain for 3 min to equilibrate. Then transfer the test tube at 23 + 0.2°C water bath and record the opacity time (Time taken by fat sample to acquire either O.D. at 570 nm between 0.14-0.16 or Klett reading using red filter between 58-62 after adjusting the instrument to 100% transmittance). The opacity time of pure buffalo ghee is 14-15 min, cow ghee is 18-19 min and that of ghee from cotton tract area is 11-12 min. The opacity time of buffalo ghee adulterated at 10% level with vanaspati is 10-11 min, with pig body fat is 8-9 min, with buffalo body fat is 2-3 min, with cow body fat is 3-4 min and with refined oils is 20-25 min (Singhal, 1980).

8.8 Test for Skimmed Milk Powder in Natural Milk (Cow, Buffalo, Goat, Sheep)

Take 50 ml of milk in a 60 ml centrifuge tube. Place the tube in the centrifuge and balance it properly. Centrifuge at 3000 rpm for 15 minutes. Decant the supernatant creamy layer carefully. Add 0.5 ml of 4 % acetic acid for coagulation and then add 2 ml of 1 % phosphomolybdic acid in water. Mix the contents thoroughly and heat in a water bath at boiling temperature for 15 minutes and then cool. The curd obtained from pure milk shall be greenish in color whereas the curd of sample containing skimmed milk powder shall be bluish in color. The intensity of bluish color depends on the amount of the skim milk powder present in the sample.

8.9 Detection of Vegetable Oils in Milk Using Modified Gerber Butyrometer

Fig. 8.8 Modified gerber butyrometer

Isolate the fat from milk by Gerber method using specially designed milk butyrometer, which is open at both ends. Close the stem side opening with a good quality acid resistant silicon cork. Add 10 ml of 90% H₂SO₄, 10.75 ml milk and 1 ml amyl alcohol. Close the neck side with lock stopper; mix the contents and centrifuge for 5 min to get clear fat in the column. Remove the silicon cork and take out fat from the stem of butyrometer with the help of a capillary tube or a syringe. Place the fat on the prism of the butyrorefractometer maintained at 40°C and note down the reading. Since, B.R. reading is depressed due to hydrolytic effect of H₂SO₄ on the fat. Therefore, observed B.R. reading is corrected as follows

Corrected B.R. = 1.08 × Observed B.R.

The Corrected B.R. reading of milk fat thus obtained should be consistent with the values given for ghee as per PFA standards. Any deviation from the standard value indicates adulteration of milk with vegetable oils. However, this method has limitation of detection of adulteration with two oils i.e. coconut oil and palm oil whose values are close to that of milk fat (Arora et al, 1996).