Chapter -1

ORIGIN. HKTORY& IMPORTANCE OF GHEE

1.1 Origin & History of Ghee

The origin of ghee lies far beyond the recorded history. The

word Ghee originates from the Sanskrit “ghr ” means bright or to make

bright. The “ Vedas “ which are collections of Hindu hymns placed

about the 3rd century B.C. contains numerous references to ghee and

other daily products1. Hence all the important religious or festive

Hindu rituals from weeding to the funeral find the use of ghee.

Ghee is the usual Indian name for the clarified butter fat

(anhydrous milk fat) and generally refers to the product made from cow

and buffalo milk. The invention of ghee could be the necessity of ancient

Indians to preserve the milk for longer time since the modem techniques

like refrigeration and pasteurization were not known in those days. Ghee

must have satisfied the requirement of ancient Indians since it has longer

shelf life due to absence of lactose and proteins with traces of moisture

that practically ceases the microbiological activity2.

Ghee is produced by heat desiccation of makkhan, butter or cream

at 110 to 120°C. At this temperature milk proteins and lactose are

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subjected to heat induced changes, which impart a distinctive pleasant

aroma to ghee3. Desi ghee is prepared by housewives in the villages from

makkhan, which is a source of many precursors of flavour compounds,

which are formed during the fermentation of milk. These precursors are

converted into flavour compounds during heat desiccation and gives

distinctive characteristic flavour to ghee4.

Ghee prepared from cow milk has golden yellow colour due to

presence of carotene. Where as ghee prepared from buffalo milk is white

in colour with greenish tinge due to presence of bilirubin and biliverdin5.

Ghee develops distinctive white granular crystals during slow cooling and

storage.

' In Indian diet ghee has got special importance mainly because of

its unique pleasant aroma and several medicinal properties, which are

reported, in Aayurveda. Ghee is believed to be a coolant, capable of

increasing mental power, physical appearance and curative of ulcer and

eye diseases 6. Ghee also contains Conjugated Linoleic Acids (CLAs),

which are reported as an important anti-carcinogen. The desi ghee is

reported to contain higher levels of CLAs than that of industrial ghee 7.

Ghee as an important and traditional milk product in its sacred use is

presented in Fig. 1.1

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Fig. 1.1 : Ghee as an important milk product in its sacred use

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Similar products like ghee are developed in some tropical and semi-

tropical countries. In the Middle East countries like Egypt, Israel and Iraq

in addition to cow and buffalo milk, the use of goat, sheep and camel

milk is common for the preparation of ghee like products. Such products

are known by different vernacular names in different languages. In

Arabic- Samna / Semna, Parsian - Roghan, Spanish — Mantequilla

fundida, English - Butter oil/ Butter fat/ Clarified butter/ Dehydrated

butter etc., French - Beurre fondu, Graisse beurre, German - Butteroel,

Butterschmatz, Floess butter, Gesotten butter, Italian - Burroluso,

o
Burrocotto, Burro colato etc. .

1.2 Role of Ghee As An Important & Nutritious Food

In India about 80% of the ghee produced is consumed for the

culinary purposes and rest of the quantities are used in confectionary and

religious ceremonies9. Over the centuries Indians have cultivated a liking

for the pleasant aroma of ghee and is preferred to the raw vegetable oils

and other traditional cooking medium. Ghee always had a supreme status

in the hierarchy of Indian diet due to its pleasant caramelized flavour and

granular texture unlike butter oil, which has almost bland taste, and non-

granular texture. In addition to this an association of ghee with cow might

have also played a role in giving the esteem place to ghee in the Indian
diet. As a table use it is usually served in the melted form and mixed with

rice and dal or lightly smeared on the chapatis or roties. It also serves as a

vehicle for shallow frying of vegetables, curries, paratas and dosas and

deep-ftying of materials like purees. Certain dishes that are finished by

baking and roasting such as Pulav (palau), biryani and tandoori chicken

are first roasted in or brushed with a small quantity of ghee to impart the

flavour to the food. In addition to this lots of Indian sweet dishes based

on cereals, milk solids, fruits and vegetables are preferred to be cooked in

ghee over other cooking medium10.

Ghee is an important source of fat-soluble vitamins like vitamin A,

D, E & K that are essential for the growth of the children 11. In ghee

vitamin A is known to be present in two forms, one is an ester and

another form is an orange pigment called carotene which is converted in

to Vitamin A in the body12. Since it plays a part in the series of chemical

changes by which an eye can adopt it self to dim light and hence its

deficiency can leads to night-blindness . The substantial quantity of

vitamin A comes from the green leafy vegetables, which contains

carotene, however the absorption of carotene is less if the diet is low in

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Vitamin D is known to play an important role both in lying down

of calcium and phosphorous in the bones and also in the greater

absorption of these elements from the diet. Naturally it is of special

importance to children and for pregnant women for the development of

skeleton of young ones. The important fat-soluble vitamin present in ghee

is Vitamin E. The studies carried out on laboratory animals reveals that

vitamin E is essential for normal pregnancy, birth and breast milk

production. It is also known to play an important role in several body

functions by preventing oxidation of vitamin A and unsaturated fatty

acids. In absence of vitamin E these precious material can go waste. The

main shortcoming caused by the diet which is low in fat can leads to

problems like poor resistance to stress, heat, cold, less capacity to work,

constant feeling of hunger and general undernourishment caused both by

fat shortage and by consequent poor utilization of proteins and sugar,

would all tend to occur. The nutrient from ghee and other food fats are

known to be destroyed due to rancidity and the cooking practices. In

order to get the maximum nutrients from ghee we need to control the

development of rancidity and excessive heating while cooking.

The amount of ghee or any fat absorbed by the body or system is

mainly governed by its melting point. Since the melting point of ghee is
 7

ranges from 25-45°C it get digested and absorbed faster in the system.

Except for fat in the form of milk which is partly precipitated in and

absorbed from the stomach itself, all other fats pass unchanged in to the

small intestine where they gets split by pancreatic lipase into fatty acids

and mono or diglycerides. The partial glycerides are altered and esterified

again with fatty acids present to give triglycerides, which are far different

than the fat originally ingested. Assisted by the bile salt, free fatty acids,

glycerol and mono glycerides present, such triglycerides are emulsified

into fine droplets across the wall of the intestine and are collected in the

lymphotic vessels, which empty them in to the blood. When the fat

consumed is ghee, the unique presence of glycerides of lower fatty acids

has three advantages. The initial hydrolysis of these glycerides is very

rapid. The liberated short chain fatty acids, instead of being re-esterified,

are absorbed from the intestine into the portal veins, which carries them

to the liver where they are quickly utilized as the sources of energy for

various biochemical procedures 15. Finally the residual partial glycerides,

which still contain lower acids are emulsified and absorbed comparatively

faster after re-esterification. Fats like ghee, which are absorbed more

rapidly, are quick to furnish the body energy and appeared to be liberated

in more amount than that of other fats 16. Nhavi and Patwardhan showed

that peak absorption of ghee occurred in 2-3 hours against 3-5 hours for
 8

sesame oil and 4 hours for Groundnut oil. Basu and Nath also reported

more rapid initial absorption of all, which tend to equalize at about 60%.

Other experiments to determine the digestibility of ghee also show that

eventually all the common fats tend to be equally well digested. The

presence of fat in the diet is related to the utilization of calcium and

phosphorus in the body. In the absence of fat calcium will be lost through

excretion as calcium carbonate 17. At the other extreme the presence of

excessive amount of fat in the intestine prevents the absorption of

Calcium present in food. Ghee is exceptional among fats in that large

amount can be consumed without impairment of calcium absorption 18.

Calcium is mainly required for the growth of the bone in young children

and pregnant women but is also necessaiy for ordinary metabolism of

blood clouting and normal functioning of muscles. Keher, et. al have

reported that cow ghee in the diet fed to rat resulted in higher absorption

percentage of calcium (45%) and phosphorous (57%) than did several

vegetable oils, the exception being coconut oil, which led to drainage of

calcium from the body19.

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1.3 Constituents and Fatty Acids Composition of Ghee

1.3.1 Constituents of Ghee:

Fat is a general term for a class of compounds, which are

chemically termed as triglycerides in which the hydroxyl groups of

glycerol are esterified with three fatty acids. The major constituent of

ghee or milk fat is glyceride component, which accounts for nearly 98%

of its mass. Hence the types and nature of fatty acids presents in the

triglycerides influence most of the properties of ghee. The minor

constituents of fat basically comprised of un-saponifiable matters which

are soluble in fat and includes the compounds like vitamin A, D, E & K,

carotenoids, P-carotene, xanthophylls, lycopene, tocopherols, sterols,

cholesterol and its esters, dehydrocholesterol, ergosterol, lanosterol, and

hydrocarbons etc. These trace constituents are present in widely variable

amount and some of them are very advantageous to the health of the

consumers. An average composition of ghee prepared from cow and

buffalo milk is presented in Table l.l20.

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Table 1.1: Major & Minor Constituents of Ghee:

Sl.No. Particulars of Constituents Cow Ghee Buffalo Ghee

1. Fat% 99-99.5 99-99.5

2. Moisture % <0.5 <0.5

3. Carotene (mg/g) 3.2-7.4 -

4. Vitamin A (IU/g) 19-34 17-38

5. Cholesterol (mg/g) 3-3.6 2.1-3.1

6. Tocopherol (mg/g) 26-48 18-31

7. Free Fatty Acids % 0.28 0.28

1.3.2 Fatty Acids Composition:

Ghee prepared from cow & buffalo milk have got different

physico-chemical properties due to significant difference in their fatty

acid composition. Ghee made from buffalo milk contains higher level of

long chain saturated fatty acids like palmitic & stearic acids than that of

cow ghee. The fatty acid composition of cow & buffalo ghee is presented

in the Table 1.2 21.

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Table 1.2 : Average fatty acid composition of Ghee prepared

from Cow & Buffalo Milk:

Name of Fatty Carbon Number Cow ghee Buffalo ghee

Acids (%) (%)

Butyric Acid C4:0 4.4 3.2

Caproic Acid C6:0 1.5 2.1

Caprylic Acid Q:0 0.8 1.2

Capric Acid Cl 0:0 1.3 2.6

Laurie Acid C 12:0 1.8 2.8

Myristic Acid Cl4:0

10.8 11.9

Palmitic Acid Cl6:0 ' 33.1 30.6

Stearic Acid Cl 8:0 12.0 10.1

Oleic Acid Ci8;i 27.2 27.4

Linoleic Acid Ci8;2 1.5 1.5

Linolenic Acid C 18:3 0.5 0.6

 %2

1.4 Important Quality Parameters of Ghee:

Chemically oils and fats are the triglycerides of fatty acids. Since

the glycerol molecules constitutes only 10%, it is the nature of the fatty

acids and the mode of distribution of these acids as glycerol triesters

which determines the various characteristics of fat or ghee. Ghee is

unique among natural fat that it contains a large number of fatty acids of

chain length lower than 12 carbon atoms and in consequence many of its

characteristics are quite distinctive. Among the different quality

parameters seven may be considered of importance with reference to

chemical structure and trading of the ghee22. Out of these the parameters

like Free Fatty Acids (FFA), Peroxide value, Richert - Meissl value,

Polenske value and Iodine value measures certain specific constituents of

milk fat 23‘26. Two other characteristics like Saponification value and the

Butyrorefractometer readings give indications of the over all nature of the

constituents fatty acids presents. The most important quality

parameter, which is a subjective term and cannot be

quantified, is its aroma or flavour. It is a consumer sensitive

quality parameter and has the direct impact on its acceptability

and marketing11. Hence these studies were carried out to understand

the chemical composition of ghee volatiles and impact of ghee

preparation methods on the flavour compounds..

1.4.1 The Add Value:

It is measured as the number of milligrams of potassium hydroxide

required to neutralize the free fatty acids present in 1 g of the ghee.

Alternatively the acidity of ghee is generally expressed as the percentage

of free fatty acids present in ghee calculated as oleic acid. The free fatty

acids are measured by dissolving the known amount of sample in

neutralized ethanol followed by gentle boiling to ensure that the available

free fatty acids gets dissolved in alcohol. The hot alcoholic mixture is

titrated with 0.1 N sodium hydroxide solution using phenolphthalein

indicator. The acid value of ghee is roughly twice the percentage of free

fatty acids present in ghee or any vegetable oils or fat.

1.4.2. Peroxide Value:

The most common cause of ghee deterioration is rancidity due to

oxidation and peroxide value is a measure of rancidity. It is known that

during fat oxidation first hydro-peroxides are formed which further

decomposes in to aldehydes & ketones, many of which have pronounced

 H
off flavour. Peroxide value is measured by the iodometric titration, which

measures the iodine produced from potassium iodide by the peroxide

present in fat and is expressed as mili-equivalent of oxygen per kilogram

of fat or ghee.

1.4.3 The Reichert -Meissl value:

The Reichert-Meissl value is the number of milligrams of sodium

hydroxide (0.1 N) required to neutralize steam volatile water-soluble fatty

acids distilled from 5 g of ghee/ fat under the prescribed conditions. It is a

measure of water-soluble steam volatile fatty acids chiefly butyric and

caproic acids present in ghee. While measuring this parameter ghee is

saponified by heating with glycerol sodium hydroxide solution and then

splited by treatment with dilute sulfuric acid. The volatile acids are

immediately steam distilled. The soluble volatile acids in the distillate are

filtered out and estimated by titration with standard sodium hydroxide

solution. Ghee or butter like products contains mainly butyric & caproic

acid glycerides. Since the butyric acid is volatile and soluble in water

therefore the RM value of ghee is higher (26-32) than that of other fat as

they does not contains butyric acid triglycerides.

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1.4.4. Polenske Value:

The Polenske value is the number of milligrams of sodium

hydroxide (0.1 N) required to neutralize steam volatile water insoluble

fatty acids distilled from 5 g of ghee or fat under the prescribed

conditions. It is a measure of the steam volatile and water insoluble fatty

acids like caprylic, capric and lauric acid. The principle behind the

measurement of this quality parameter remains same as that of RM value.

1.4.5. Iodine Value:

The iodine value of ghee or any fat is the number of grams of

iodine absorbed by 100 g of ghee or fat, when determined by using Wij’s

solution. The iodine value of the oil or fat is a measure of a un-saturation

or number of double bonds present in the fatty acids of the triglyceride

molecule. While measuring this parameter ghee or fat sample is dissolved

in suitable solvent like carbon -tetrachloride and treated with a known

excess of iodine mono chloride solution in glacial acetic acid (Wij’s

solution). The excess of iodine mono chloride is treated with potassium

iodide and the liberated iodine is estimated by titration with sodium

thiosulphate solution.
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1.4.6. Saponification Value: |

j

Saponification value of ghee or any fat is a measure of the number

of mg of KOH required to saponify one gm ,of fat. It is determined by

!

boiling a known weight of fat with an excess of alcoholic potassium

hydroxide dissolved. Some of the alcoholic KOH is used up in

saponifying the fat to soap of its fatty acids and glycerol and the reminder

is estimated by titrating it against the standard acids. The saponification

value is inversely proportional to the molecular weight of glycerides /

fatty acids. Since ghee contains maximum number of lower fatty acids
i

(lower than Ci2) the saponification value of ighee is found high (225 -

230).

1.4.7. The Refractive Index Or Butyaro-Refractometer Reading:

i

1
i
This is expressed as the ratio between the sign of the angle of

incidence to the sign of the angle of refraction of a ray of light of known

|
wave length usually (589.3 mji) the mean of the D lines of sodium passes
i

from air into ghee or fat. Since ghee is often semi solid or solid at

ambient temperature, the refractive index or BR readings are always

i
i

taken at 40°C. Both these reading are inter convertible with the help of
 17
standard table. The refractive index increases with decrease in the chain

length and a double bond elevates the refractive index

1.4.8 Physico - chemical Characteristics of Ghee :

An average values of important physico chemical characteristics of

ghee prepared from cow and buffalo milk are presented in Table 1.328.

Table 1.3:

SI. No. j Cow Ghee Buffalo Ghee

Particulars of Quality Parameters
1. Reichert Meissl Value ! 26.7 32.3

2. Polenske Value 1.76 1.41

3. Iodine Value 33-70 29-40

l
4. Butyaro-Refractometer Reading 42.3 42.0
1
i

5. Saponification Value I 227 230

||
6. Kirscher Value 22.1 28.4
1
7. Melting Point (°C) 28.41 32.45

8. Refractive Index 1.4465 -

9. Specific gravity 0.887 0.887

10. Unsaponifiable matter 0.40 0.40

11. Smoking Point (°C) 20.8 -

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followed by ring closure due to dehydration (lactonization) or by lipolysis

of glycerides before ring closure37.

In the last three decades several reports have brought out on the presence,

mechanism of production and role of flavour compounds mainly

carbonyls, lactones and free fatty acids. The reported carbonyls can be

grouped into two categories like monocarbonyls and dicarbonyls. Total

50 carbonyls have been detected in ghee so far out of which 40 are

monocarbonyls while the rest are polar carbonyls. Most of the

monocarbonyls have been identified using Thin Layer Chromatography

& Gas Liquid Chromatography38'39. The identified carbonyls includes

alkan-2-ones from C3 to Cjo, Ci2, alkanals from C2 to C9 , alk-2-enals

from C4 to C12 and alk-2,4-dienals from C5 to C7, C9 to C12 and C14.

Ninety percent of the reported monocarbonyls are Alkan-2-ones where as

the contribution of alkanals is nearly 6% followed by rest of the two

groups (alk-2-enals & alk-2,4-dienals) with a contribution of 2 % each.

The presence of various groups of carbonyls has been reported in cow

and buffalo ghee separately by researchers40'42. Other than carbonyls

lactones have been reported as an important contributors to the ghee

flavour. Delta & gamma lactones (8 & y) from Cg to C10 and Cjg have

been reported by Wadhwa & coworkers43'44. Delta-lactones (90-95%) are

 19
reported to be the major contributors to the ghee flavour than gamma

lactones (5-10%). The homologous series of n-saturated delta & gamma

lactones from C6-Ci6 have been identified using gas chromatography

(GLC). The free fatty acids have also been reported to contribute to the

ghee flavour and the impact of levels of free fatty acids on the flavour

quality of ghee have been studied 45.

The review of the research done on ghee flavour since two decades

reveals that most of the flavour compounds were isolated by solvent

extraction, vacuum distillation, and steam distillation. Where in there is a

risk of artifacts formation and contamination of flavour volatiles with

impurities from solvents & chemicals used. The detection of isolated

volatile compounds like carbonyls, lactones and fatty acids have been

done using analytical techniques like Thin Layer Chromatography, Gas

Liquid Chromatography and UV-visible Spectrophotometry. The

detection limits of these analytical techniques particularly TLC &

Spectrophotometry are comparatively low. The ghee volatile compounds

like carbonyls, lactones & free fatty acids were studied as a group of

compounds and there are no reports on identification & quantification of

the individual flavour compounds to the micro levels (ppb). The

comparative studies on qualitative & quantitative composition of ghee

 20
volatiles prepared by different methods eg. desi / traditional method &

industrial methods like direct cream & creamery butter have not been

reported so far.

Considering the diversity in polarity and chemical nature of ghee flavour

compounds their isolation and identification using any single technique or

method is difficult. The accuracy of identification is greatly depends upon

the isolation of flavour compounds from ghee (or any food) in their

original form. In the present studies volatile flavour compounds were

isolated using Direct Injection Chromatography & Headspace Sampling

(HSS) which are most sophisticated and highly sensitive techniques. In

these techniques the use of solvents & chemicals is avoided hence there is

minimum risk of artifacts formation and contamination. The detection

limits of Direct Injection & HSS techniques used for isolation of

volatiles from ghee prepared by different methods was further enhanced

by cryofocusing & multiple injections. The quantification of individual

volatile flavour compounds was done to the ppb levels using an internal

standard.

Headspace sampling (HSS) and Direct Injection (DI)

Chromatography have not been used earlier for the isolation of

 21
flavour compounds from ghee. Hence these methods were used for

the isolation of ghee volatiles prior to their, separation and

identification using Gas Chromatography coupled with Mass

Spectrometry (GCMS) which is one of the latest & most sophisticated

tools.

In India substantial amount of ghee is produced in the villages by

desi method whereas the creamery butter and direct cream methods are

used for ghee making by the modem dairies. Desi ghee, which is rich in

aroma than that of industrial ghee is preferred by the consumers and

fetches more price. In order to improve the acceptability of industrial

ghee, it was felt necessary to understand the flavour differences of ghee

prepared by desi and industrial methods. The present work deals with

isolation, separation and identification of volatile compounds of ghee

prepared by different methods. The objective of these studies is to

understand the qualitative and quantitative differences in volatile

compounds of ghee prepared by desi and industrial methods. As

mentioned earlier the methods of ghee preparation greatly influence its

flavour. The details of four different methods broadly used in India for

ghee preparation and their importance on ghee flavour is discussed in

detailed in Chapter II.

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1.5 Ghee Flavour (Composition and Chemistry) :

Ghee is a complex lipid of mixed triglycerides, which constitute

about 98% of the total mass. The non-glycerides constituents contribute

remaining 2% of the total material which includes sterols / sterol esters,

fat-soluble vitamins, carotenoides, volatile flavour compounds, traces of

phosphorus, calcium, iron, charred casein and moisture. The chemistry of

ghee flavour has been studied extensively. Milk fat is the source of most

of the flavour compounds in ghee and other diary products as well. Milk

proteins and lactones play an important role in producing many flavour

compounds during heat desiccation of Makkhan, Butter or Cream 29. The

ghee flavour profile is known to be affected by various technical

parameters like method of preparation, temperature of clarification,

holding time and storage period30'31. The impact of ghee flavour on its

acceptability / marketing has been the main force behind the research

being carried out to understand the chemical composition of ghee flavour.

1.5.1: Status of research on ghee flavour & value addition by

the present work:

The present knowledge of the ghee flavour reveals that it is

chemically a complex attribute and is mainly controlled by qualitative

and quantitative presence of the various flavour compounds. The key

flavour compounds are carbonyls, free fatty acids and lactones. Among

the carbonyls alkan-2-one or methyl ketones are important components of

ghee flavour and are formed either by hydrolysis of ketonogenic

glycerides followed by decarboxylation or by lipolysis of triglycerides

through penicillium moulds during fermentation of milk / cream 32"33.

Polar carbonyls like dicarbonyls; fermentation of lactose and citrate,

amino acids and browning products of lactose caramelization34 form a-

keto-acids, glyoxals and furfurals. Aldehydes like n-alkanals, alka-2-

enals, alka-2-4-dienals are known to be formed by the autoxidation of

unsaturated fatty acids of milk fat35. Free fatty acids are produced either

by hydrolysis during processing or by lipolysis during ripening /

fermentation of fatty acid glycerides36. Lactones are produced during

processing either by hydrolysis of lactogenic glycerides to hydroxy acids