Adulterants

The ease with which milk can be adulterated has always attracted unscrupulous persons to adulterate the same with numerous compounds. The most common of them include starch or cereal products, cane sugar, glucose, urea and ammonium sulphate. Tests for their detection are now available which can easily detect these compounds. Some of these compounds are highly injurious to the health of individuals.Laws are now available which can lead to the punishment for adding these prohibited substances. These compounds are primarily added to raise the density of milk.

i. Starch or cereal flour (atta)

Starch is a tasteless, odourless and cheap adulterant which is readily available. It is often added as an adulterant because it is not sweet like sugar. It is difficult to be detected organoleptically in small quantities. Starch can be easily detected as blue coloured starch iodide complex on heating and cooling with 1% iodine solution.

The demand for liquid milk is far greater than the availability of milk. This forces the adulteration with a view to make quick profits. A large volume of milk issupplied to the consumer directly by the producers through vendors at the door step without any sort of processing, packaging or quality control. The milk supplied to the consumers is most prone to adulteration, as they do not bother about such adulteration and are under the impression that they are getting pure milk. However,such milks are often maximum adulterated. Compared to vendors the milk supplied in the organized sector is tested for the adulterants by random analysis of procured samples. The easiest way is to adulterate the milk with water and keep the fat within the PFA limit. The addition of water, however, decreases the SNF content and is compensated by adulterating milk with cheaper solid ingredients like sugar,starch, urea etc.

Starch is detected by a very simple and quick test using 10% iodine solution. In the presence of starch a voilet blue colour of starch iodine complex is obtained on boiling.

•  Take 5 ml milk in a test tube. Boil the milk over a flame. Cool the milk.
•  Add 1 to 2 drops of iodine solution.
•  Formation of voilet blue colour indicates the presence of starch or cereal flour.

ii. Cane Sugar

Cane sugar is added to raise the total solids content in milk. This adulterant is readily available and is cheaper than milk solids. When added in large quantity it is detected by sweet taste imparted to milk. It can also be detected by resorcinol reagent, which gives redish brown colour in the presence of sucrose.

•  Take 5 ml sample in a clean test tube
•  Add 5 ml resorcinol reagent and mix well
•  Place the tube in a boiling water bath for five minutes or heat directly on the flame to boiling
•  Development of red colour with or without the separation of brown red precipitate indicates the presence of cane sugar in milk.

iii. Glucose

Glucose is added to milk to increase its density. It is odorless, colourless and is not as sweet as cane sugar. It is detected by modified Barfoed’s reagent either directly from milk or clean filtrate of milk. In the presence of glucose Barfoed’s reagent gives a deep blue colour. To detect glucose in milk.

•  Take 1 ml milk sample in a test tube.
•  Add 1 ml Barfoed’s reagent. Heat for 3 minutes in a boiling water and then cool.
•  Now add 1 ml phosphomolybdic acid reagent and mix.
•  A deep blue colour shows the presence of glucose. Pure milk only gives a faint blue colour.

iv. Urea

Urea is available readily as chemical fertilizer with farmers. It is often used as an adulterant to boost total solids after dilution with water or skim milk. Urea is detected in serum part of milk after removing casein from the filtrate in alkaline medium. In alkaline medium in the presence of phenol a bluish green colour indicates the presence of urea.

•  Take 5 ml milk in a 50 ml conical flask and add 1 ml acetic acid or TCA 24% solution and heat for 3 minutes in boiling water both. Filter the precipitate. Collect the filtrate
•  Take 1 ml filtrate, add 1 ml NaOH solution, followed by 0.5 ml sodium hypochlorite solution mix and finally add 0.5 ml phenol solution.
• A bluish green colour is formed with phenol in the presence of urea.

v. Ammonium Sulphate

Ammonium sulphate is also a fertilizer, it addition boosts the solids content of milk.Detection of ammonium sulphate is carried out on casein free filtrate prepared as per urea detection.

•  Take 1 ml filtrate, add 0.5 ml NaOH, 0.5 ml sodium hypochlorite solution and mix. Now add 0.5 ml phenol and heat for 20 seconds in boiling water bath.
•  Formation of a bluish colour which changes to dark blue shows the presence of ammonium sulphate. The colour is stable for over 12 hours.

Note: In pure milk only salmon pink colour is formed, which gradually changes to bluish in course of about 2 hours.

vi. Partial Removal of Fat by Skimming

An indication of the removal of excess fat from milk give the following changes to milk:

•  Lowering of fat percentage in milk
•  Higher density of milk sample at 27 0 C
•  Higher ratio of solids-not-fat to fat in milk

vii. Addition of Skim Milk

Addition of separated milk or skimmed milk results in following changes in the milk:

•  Addition of skim milk results in lowering of fat in milk
•  Higher density of toned milk sample at 27 O C
•  Higher percentage of solids-not-fat
•  Higher ratio of solids-not-fat to fat.

viii. Dilution of milk by addition of water

Milk is commonly adulterated by adding water as it is highly profitable. It causes following effects:

•  Fat percentage is lowered
•  Density of milk is lowered at 27 O C
•  Lowering of solids-not-fat content of milk
•  Lowering of freezing point depression of milk

i)Determination of Specific Gravity of Milk by Lactometer

In routine analysis of milk the density in determined with the help of a lactometer.The lactometer is graduated at a temperature of either 15.5 0 C or 27 0 C. Lactometer consists of a long, slender glass stem of uniform diameter connected to a larger glass chamber that facilitate lactometer to float. Lower end of the lactometer is filled with synthetic material which makes the lactometer to float and also keeps it in upright position.

•  Warm the milk to 40 0 C for 5 minutes
•  Cool the milk near the temperature of 27 0 C, the temperature of lactometer graduation.
•  Pour gently the milk in a 250 ml cylinder avoiding air bubbles and place the lactometer so that it floats freely.
•  Take the lactometer reading and note the temperature Take the average of two readings
•  Correct the lactometer reading from the table.

Lactometer reading for genuine cow milk is between 26-30 and 28-32 for buffalo milk. These readings are converted to specific gravity by prefixing 1.0 for lactometer readings e.g. a reading of 28 will give a specific gravity of 1.028.
 

Calculation

% TS =(C . L . R/4) + 1.2 F + 0.14

% S.N.F. = + 0.2 F + 0.14

Where TS = Total solids in milk sample

S.N.F. = solids-not-fat in milk sample

F = Fat Percentage in sample

C.L.R.= corrected lactometer reading at 15.5 0 C

Note: If the temperature is above 15.5 0 C then to each 1 0 C rise in temperature add 0.2 to each lactometer reading. On the other hand if the temperature in below 15.5 0 C then to each 1 0 C lowering of temperature subtract 0.2 from the lactometer reading. For example, if the temperature is 16.5 and reading is 30 then the C.L.R reading will be 30.2 (30+0.0.2). On the other hand if the temperature is 14.5 0 C and the lactometer reading in 29 then the C.L.R. will be 28.8 (29.0-0.2).

ii)Fat Determination

a) Gerber Method: For routine fat analysis of milk Gerber method is commonly followed for fat estimation. It is a rapid method and results are available in short time. Gerber method is volumetric method for fat analysis.

b) Roese-Gottlieb method: In this method fat is extracted from milk with the help of fat extraction reagent i.e. solvent ether. Ammonia and alcohol are added to facilitate fat extraction. Ammonia dissolves the fat globule membrane and alcohol helps in the passage of the fat globules in the aqueous phase. This is a gravimetric method of fat estimation.

iii) Freezing Point (FP)

Milk contains upto 85 percent water and varies widely in composition. Thus a constant parameter of milk is difficult to assign for milk. As such freezing point is a fairly constant property with a freezing point value between 0.530 to 0.555 0 C.Freezing point is a colligative property which depends upon the number of solute particles present in the system or solution. The solvent is water and its freezing point is always constant. On addition of water in milk the solute particles in the solvent are diluted which affect the freezing point of milk. This results in an increase in freezing point depression of milk. With cryoscopy the percent water added is calculated as percent-added water

Neutralizers

Neutralizers are added to neutrilize the acidic sour milk. The addition of alkalis is not permissible under law. Some of these alkalis are highly injurious to health. To overcome this problem by means of a few simple test they can be detected in milk added as neutralizer. Freshly drawn milk has an acidity of 0.12-0.16 per cent expressed as lactic acid. With the passage of time the acidity increases during souring of milk. Any acidity above 0.18 per cent lactic acid coagulate milk. This is due to the formation of lactic acid from lactose. Neutralization of milk is illegal under the P.F.A act. Unscrupulous milk producers and farmers tend to neutralize the milk to avoid rejection of milk with increased shelf-life at the milk collection centers and at the dairy plants. The common neutralizers which are added to milk are caustic or sodium hydroxide, baking soda or sodium bicarbonate and washing soda or sodium carbonate. They are detected by determining the alkalinity of ash and carbonate or bicarbonate by rosalic acid test.

i. Rosalic acid test for the detection of carbonate and bicarbonate in milk
Rosalic acid test is used for the detection of carbonate and bicarbonate in milk. This is a very simple, reliable and quick test for their detection. Rosalic acid reacts with carbonate and bicarbonate and give rose red colour in their presence. The intensity of colour depends upon the amount of these chemicals present.

•  Take 5 ml sample of milk in a clean and dry test tube.
•  Add 5 ml ethanol 95% and mix
•  Now add 2-3 drops of rosalic acid solution prepared in 1% ethanol to the mixture and mix well. Note the colour change.
•  A rose red colour develops.
• Formation of rose red colour in milk indicates the presence of carbonate or bicarbonate added as neutralizer.

Note: Pure milk gives only a brownish red colour.

ii. Alkalinity Test

The presence of neutralizers can generally be detected by determining the alkalinity of ash. A known quality of milk is heated and converted into ash. The alkalinity of ash is estimated by titration against a decinormal standard hydrochloric acid in the presence of Phenolphthalein indicator. Values abnormally high would indicate neutralization of milk. To carryout this test first ash is prepared from milk so as to obtain added alkali in a concentrated form.

•  Pipette 20 ml milk in a porcelain dish and evaporate to dryness on a boiling water bath.
•  Prepare ash by keeping it over a burner or muffle furnace at 550 0 C for 1 hour.
• When using burner heat till ash becomes grey white in colour.
   Cool the basin. Add water and mix the contents with a glass rod.
   Titrate the ash solution using standard O.1N HCl in the presence of 4-5 drops of phenolphthalein indicator solution. Note the volume of HCl solution used till a pink colour is obtained.
   If the volume of O.1N HCl exceeds 1.20 ml the milk is suspected to contain neutralizers.

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 .

Preservatives, Neutralizers and Adulterants in Milk and their Detection

Preservatives, neutralizers and adulterants are often added in milk with several different aims. However, it may be emphasized that addition of these components is strictly prohibited under PFA Act. This act does not allow the addition of any external agent to milk and is punishable under law. Preservatives are also prohibited in milk except during sampling and subsequent analysis.

Preservative may be defined, as a substance which when added to food is capable of inhibiting, retarding or averting the process of fermentation, acidification or spoilage or decomposition of food. The initial quality of milk is poor in India which leads to a high bacterial load. Moreover, it takes a long time with a time gap of several hours before milk reaches the consumers or individuals and dairy plants for processing. Under such circumstances there is a tendency to use preservatives to delay or prevent microbial proliferation and spoilage of milk. Let us know more about preservatives, neutralisess and adulterants.

Enzymes in Relation to Processing

Enzymes are organic catalytst, which are found in plant and animal cells. The enzymes bring about metabolic reactions but they don’t undergo any chemical change. They are colloidal and proteinous in nature and are classified as per the reaction performed e.g., lipase, the fat splitting enzyme. The activity is affected by pH, heat, light etc. Enzyme in milk gain entry via udder or externally.Milk enzymes are technologically important. They are related with flavour (e.g.,lipase). Study and knowledge of these enzymes is essential to understand their role.

Functions of enzymes

The following functions are related to enzymes.

•  Oxidising enzymes (e.g., peroxidase)
•  Lipolytic enzyme hydrolyzing fat (e.g., lipase)
•  Decomposing H 2 O 2 (e.g., catalase)
•  Decomposes phosphorous esters.(e.g., phosphatase)
•  Lactose hydrolyzing enzyme (e.g., lactase)
•  Reductase as reducing enzyme (e.g., MBR test)
•  Proteolytic enzymes hydrolyzing protein (e.g., protease)
•  Hydrolysing aldehyde (e.g., xanthine oxidase)Peroxidase, lipase, catalase, reductase, phosphatase, xanthine oxidase, lactase are all present in freshly drawn milk. Other enzymes enter via bacterial contamination.

i)Peroxidase: Peroxidase is present in milk as lactoperoxidase enzyme. The enzyme is destroyed between 70-80 0 C. Lactoperoxidase enzyme act on H 2 O 2 in the presence of thiocyanate ions, forming hypothiocyanate ions (OSCN - ) are lethal to microbes. This enzyme has been used in milk to improve shelf life during transportation of milk from distant places to milk plant. The enzyme is also used as an index of detecting proper heating of milk as it is destroyed at 70 0 C, especially for detecting high temperature heat treatment of milk.

ii) Phosphatase: Phosphatase catalyse the hydrolysis of phosphate esters. Alkaline phosphatase is the most important milk enzyme. It is destroyed by pasteurization of milk. At the temperature of pasteurization of milk tubercle bacili bacteria present in milk are also destroyed. The inactivation of this enzyme is thus taken as the process of destruction of TB organisms. Under health consideration pasteurization of milk is mandatory in various countries. Phosphatase test has been developed to ascertain if the milk has been properly pasteurized. So as to ensure the destruction of Micobacterium tuberculosis which is destroyed at a temperature wherein alkaline phosphatase is inactivated.

iii) Lipases: Lipases hydrolyse milk fat into corresponding fatty acids and glycerol.In milk they are linked with hydrolytic rancidity of milk fat releasing butyric acid. Excessive presence of butyric acid in milk causes rancid flavour defect.This defect may also be present in butter. They are destroyed at 63 0 C when heated for 20 minutes.

iv) Proteases: Proteins are hydrolysed by proteases to simple compounds such as proteose, peptone, amino acid and other compounds. They are inactivated in the presence of salt or preservative. Proteases are destroyed by heating milk between 70-80 0 C. Proteolytic enzymes have been employed externally for preparing different varieties of cheese. These enzymes primarily hydrolyze Catalase casein.

v) Reductase: Reductase are enzymes of bacterial origin. These enzymes are capable of reducing certain dyes to their colourless leuco-compounds. It has been shown that generally speaking the reduction time at 38 0 C is approximately proportional to the number of bacteria.. They are used as measure of microbial population and determine the extent of contamination of milk by bacteria. This is possible through methylene blue reduction test (MBRT). The blue dye is reduced to a colourless compound in the presence of reductase. The earlier the dye lost its blue colour greater is the contamination.

vi) Catalase: Catalase catalyses the decomposes hydrogen peroxide as per the following reaction

2H 2 O 2      2H 2 O+O 2

Catalase content varies in milk from different animals and within the same species. It is also affected by feed given to the animal. Catalase content is high in colostrum, mastitis milk and milk contaminated with mastitis or colostrum milk or bacterial contamination. It tends to parallel leucocyte count. It increases with multiplication of bacteria in milk. It is destroyed when milk is heated to about 65 0 C or over.

vii) Xanthine oxidase: A variety of substances are oxidized by this enzyme including xanthine, hypoxanthine, aldehyde, oxypurines, etc. Thus in the presence of O 2 and an aldehyde following reaction takes place:

RCHO +H 2 O+O 2 ⎯Xanthine oxidase ⎯⎯ ⎯ ⎯ ⎯ ⎯ →RCOOH+H 2 O 2

Xanthine oxidase is a prominent enzyme of milk and was discovered as early as 1902.

Xanthine oxidase content varies from cow to cow and increases with stage of lactation. It is associated with fat globules. It can be isolated from cream or buttermilk. The following table gives the data for inactivation of the enzymes in milk.

Inactivation temperature of enzymes

Freeze Processing of Milk

Freezing has been suggested as a means for tansporting frozen concentrated milk.This is to co-ordinate supply to those areas, which are not adequately covered to supply liquid milk. The objective of freezing is to prepare frozen concentrated milk to replace liquid milk supply to distant areas, which are not well connected.

Freezing of milk and its effect on milk system: To manufacture frozen milk,the milk is first concentrated and then frozen stored. During frozen storage of milk and its subsequent thawing very fine milk particles called flocculates are formed.Initially flocculates are readily dispersible but prolonged storage period makes them difficult to disperse.

Effect of freezing on lactose and caseinate system: Lactose is the first component of milk which is affected during frozen storage. Frozen storage results in crystallization of lactose especially at very low temperatures. Lactose is present in milk in a highly supersaturated state which readily crystallize on storage. Lactose binds calcium from milk but calcium is released on crystallization. In the dissolved state lactose binds calcium but releases calcium upon crystallization. No change in protein denaturation occurs on storage even though flocculation occurs. The reason for destabilization is calcium. It has been seen that frozen stored casein remains unchanged in terms of solubility. Casein isolated from frozen stored milk has the same sensitivity to calcium precipitation as casein isolated from fresh milk. Although casein flocculates on frozen storage but protein seems to be unchanged.

Effect of Heat on Milk

I) Effect on salt system: The heat-induced changes in the milk salt system can be covered under three cateogories:

•  Readily reversible shift in salt balance by changes in temperature
•  Irreversible shift in salt balance.

Variations in temperature and concentration adversely affect salt balance. Calcium phosphate is less soluble at high temperature than at low temperature. Thus, the concentration of soluble calcium and phosphate is decreased during heating. Dissolved or soluble calcium and phosphate during heating is transferred to the colloidal state.

This transfer action occurs on the colloidal micelles of caseinate phosphate. This transfer of soluble calcium and phosphate causes extensive changes in the structure of the micelles produced by heat treatment. Dissolved calcium and phosphate tend to revert to the original system but it is not completely transferred to the original structure after heat treatment. At the same time aggregation of the caseinate-phosphate micelles may occur (reversibly or irreversibly).

II) Effect on Acidity: During heat treatment CO 2 is removed from the milk system. This causes a decerase in acidity of milk. The effect is through the release of H + ions. This process is affected by the insolubilization of calcium and phosphate.

3Ca+++2HPO 4- → Ca 3 (PO 4 ) 2 +2 H +

On the basis of available data, heat treatment lead to an increase in the dissolved citrate in milk.

III) Effect on the milk proteins: The heat-induced changes in milk are of great practical importance to the dairy industry. During denaturation the original three-dimensional structure changes. Denaturation consists of non-proteolytic changes in the structure of protein. Amongst the heat induced changes caused by denaturation of whey proteins are:

•  Development of cooked flavour
•  Development of anti-oxygenic properties
•  Impairment of clotting properties
•  Imparting of soft curd characteristic to milk
•  Prevention of age-thicking in evaporated milk
•  Improvement in the baking quality for non-fat dry milk in the bakery industry

These changes are related to whey proteins. The whey proteins are present to the extent of 0.6 to 0.7% in milk. Beta-lactoglobulin is the major whey protein of milk accounting for 50 percent of the total whey proteins. The observed changes in milk are: release of H 2 S production, of cooked flavour, development of anti-oxygenic properties and lowering of curd tension. All these changes are related to whey proteins.

a) Heat denaturation of whey proteins: Heat denaturation of whey proteins occur between 68 0 C to 80 0 C. Heat denaturation starts from 68 0 C onwards when milk is heated for 30 minutes or 71 0 C for 15 minutes. The denaturation of whey proteins occurs at a higher temperature than pasteurization. The order of denaturation of whey proteins are immunoglobulin, blood serum albumin,beta-lactoglobulin while alpha-lactalbumin is the most heat resistant whey protein.

b) Changes associated with whey protein denaturation: Above 75 0 C –SH groups are released from whey protein, which are highly reducing in nature.These groups are susceptible for oxidation. The activation of-SH groups accompanies by an important phenomenon of anti -oxygenic property of heat-induced changes in whey protein. Sulphahydryal (-SH) groups are powerful reducing agent. The ability of these groups to bind oxygen results in anti-oxygenic property. As a result it lowers the oxidation-reduction potential of milk, which shows the activation of these groups. Formation and activation of -SH also results in the liberation of volatile sulphides. These volatiles also include H 2 S. The release of H 2 S is one of the most important component responsible for cooked flavour of milk. Cysteine amino acid containing maximum number of -SH group is responsible for producing H 2 S. Whey proteins are a rich source of cysteine and are a main cause of cooked flavour. Beta-lactoglobulin is very rich in -SH group.

Another important change resulting, as a function of heat denaturation of whey proteins is the soft curd forming property of milk. It is accompanied by two important changes in curd. These are the development of a soft curd characteristic in the curd and partial loss of clotting property in cheese manufacture. These are related to changes in the flocculation of serum protein particles. The impairment of milk clotting property seems to be due to interaction of casein with whey protein(beta-lactoglobulin). The denatured whey proteins bind with casein and thus affect its lotting property.Milk contains a factor, which affect the loaf volume of bread when milk is added during bread making. As a result volume of bread is depressed and slackens dough is produced. This defect can be overcome by heating milk. This is supported by the role of added skim milk powder to dough during bread making, which contain heat denatured whey proteins. Heat denaturation of whey proteins in skim milk powder is thus used as an index of baking quality.

There is loss of creaming property and increase in whitening of milk due to denauration of whey protein. Loss of creaming property has been attributed to the interactions between whey proteins notably immunoglobulins which interact with proteins of fat globules. This interaction affect the creaming ability. Cream layer formed in such milk is shallow and indistinct from normal milk. Reflectance or improvement in whitening has been attributed to a heat denatured state of milk proteins just before browning. At this stage flocculation of whey protein occur, along with aggregation of casein and conversion of soluble calcium to insoluble salt.

c) Destabilization of caseinate system: Caseinate-phosphate particles in milk exist in a precarious equilibrium with soluble Ca ++ and Mg ++ , dissolved salts and whey proteins. Slight changes occurring as a result of heating or changes in ionic environment through pH will alter this equilibrium. Casein binds Ca ++ and Mg ++ ions very strongly. Casein is stabilized in the system by charge it carries.
 

Heating causes pH changes which affect this process. The caseinate particles are very sensitive to changes in pH. Casein start precipitating below pH 6.0 and micelles precipitation starts at pH 5.2 to 5.3 where they still contain Ca ++ and Mg ++ attached to them. The manufacture of cottage cheese is based on the phenomenon of caseinate system by heat and acidity. During this process the destabilization of the caseinate particles leads to the formations of a smooth gel occupying the entire volume originally occupied by the milk. In this system a three-dimensional type network is formed that entraps the liquid along with gel structure formation or a network and a semi-solid system is formed. On applying heat to this system at cooking stage of the process, the caseinate particles become more closely knit together, water is expelled, and the clot shrinks. A desirable product is obtained by judicious use of pH and proper heat treatment.

The calcium caseinate phosphate micelles are readily precipitable by addition of various salts such as ammonium sulphate and urea. Heating hastens the process.This is the basis of producing various fractions of casein. The effects of heat and divalent cations are important from the view point of rennet action and heat. In this phenomenon ionic concentration and heat play an important role in the stability of casein micelles. Phosphate and citrate ordinarily exert an opposite effect over Ca ++ and Mg ++ because they form undissociated complexes with Ca ++ and Mg ++ .

Some milk apparently are stabilized by added calcium and destabilized by ions such as phosphate and citrate that sequester calcium. Observations of this type are the basis of the well-known salt balance theory first suggested by Sommer and Hart(1926). This theory holds that optimum stability depends on a certain ratio of calcium and magnesium ions to those of phosphate and citrate. The concept has been of great practical utility in developing practical procedures for controlling the stability of evaporated milk during heat sterilization. In practice evaporated milk to be sterilized is treated, as a series of samples on a pilot scale with graded level of phosphate or Ca the later being rarely if ever necessary. The samples are then sterilized and after cooling the minimum level of added salt that imparted satisfactory stability is noted and used to stabilize the lot of milk to be sterilized.

IV. Forewarming process and heat stability: Before sterilization in the preparation of evaporated milk forewarming of milk provides heat stability to milk. Generally,heating milk at 95 0 C for 10 minutes provide heat stability to milk. It has been shown that a high temperature short time process of heat treatment provides a better heat stability. However, it may be stated that this phenomenon of heat stability is complex and depends upon other factors such as quality of milk, storage temperature of milk, etc.

V. Browning of milk: Browning reactions in milk and milk products are the manifestation of heat induced processing of milk. Browning reaction occur due to changes related with pH, storage conditions, moisture content, relative humidity and temperature of processing and storage of milk and milk products. Browning reaction is absent in pasteurized milk but is evident in highly heated sterilized milk on storage. Browning reaction occurs in two forms on heating. The two types of browning in relation to heating are 

(a) amino sugar or Maillard browning and 

(b) non-amino browning or caramelization. 

a) Amino Sugar or Maillard browning: Two components are responsible for this browning reaction. They are milk protein particularly casein and lactose present in milk and milk products. Phosphate salts and whey proteins make minor contribution in browning reaction. Browning reaction is complex.

The reaction occurs between aldehyde groups (-CHO) of sugars and amino groups (-NH 2 ) of amino acids. They together start the browning reaction which ultimately lead to the formation of brown pigment melanoidin.

b) Caramelization or non-amino browning: Caramelization or browning may be defined as the heat decomposition of sugar as a function of pH and buffers in the absence of amino compounds. It requires a relatively high order of heat energy. On the other hand, Maillard type browning requires a relatively low order of energy for its initiation and exhibit autocatalytic qualities once it has started. Caramelization is desirable in milk based products such as caramelized flavour which is desirable and liked.

c) Changes related to browning: Along with browning many complex reactions also occur with the formation of various compounds. In addition,fluorescent and reducing substances, various sugar fragments and flavour compounds are formed. Many of these are detected before browning starts.

These changes have great practical utility. Notable amongst these is the development of flavour especially caramelized flavour. Following changes related to browning can occur:
 

Compound formation: A large number of lactose degradationcompounds are formed. These include furfuryl alcohol, furfuryl aldehyde,
maltol, acetol, acetaldehyde, acetic, formic and pyruvic acid, NH 3 , H 2 S and CO 2
 

 Reducing substances: Heated and dried milk contain’s a complex reducing system involving –SH compounds, ascorbic acid and substances
associated with browning reaction. Heating concentrated milk for a similar period has a significant effect on browning reaction.

d) Factors affecting browning of milk: The principle factors responsible for browning in milk are:

i)pH: A pH above 6.8 favours browning reaction. This defect is predominant in evaporated milk where pH of milk plays an important role. Due to variations in pH and protein concentration in differentmilks browning is affected due to these variations. This is due to release of protons during heating. As the pH is raised above pH 6.6 browning reaction occurs at a faster rate.

ii) Storage and temperature: Higher temperature and prolonged storage period favours browning. These changes are favoured in the presence of increased humidity and moisture. Colour intensity increases with storage time and is highest at a storage temperature of 40 0 C.

iii) Total solids concentration: During concentration of milk total solids concentration increases. As the total solids concentration in milk increases the browning reaction also gains momentum. Lactose plays a major part of total solids concentration along with casein. The interaction results in increased browning.

iv) Heat treatment: Heating milk as a pre-heat treatment between 85- 100 0 C for 30 minutes or more favours browning. It is one of the most important factors of browning. Reducing the heating time such as with HTST process will reduce the browning of milk products.

v) Oxygen: Oxygen favours browning as it reacts with –SH groups released during heating. Presence of oxygen destroys these reducing
groups. Problem can be reduced by replacing O 2 with N 2 while storing heated and dried milk products.

e) Prevention of Browning: Browning can be prevented to a great extent by storing milk and milk products at low temperatures and short period of storage. In dried products moisture should be below 5%. Also N 2 packing helps in reducing browning due to replacement of oxygen. Strong and long duration heating should be avoided.