c

G E N E R A L INTRODUCTION
 GENERAL INTRCuUCTIQN

India, once1 the exporter of vegetable oils in the

world, has become a major importer of oils in course of the

pact two decades to cater to edible requirements. The

statistics of production of oils and fats from 1960’s to the

present time would indicate the deteriorating situation of

oils and fats in India, leading to import of substantial

quantities of oils from other countries. In 1960-61, the

demand for oils for household and vanaspati was 18.99 lakh

tonnes and 2.6B lakh tonnes for soaps and paints. The total

supply was 18.43 lakh tonnes, with a gap of 2.84 lakh tonnes.

In 10 years time (1969-70) the demand increased, to 29.34

lakh tonnes as against the local supply of 22.37 lakh tonnes,

making a gap even more of 6.97 lakh tonnes. In 1972-73, the

gap further increased to 10.72 lakh tonnes which more or

less remained same in 19'75-76 but from 1 976—78 the gap fur-
(1 )
xher widened by about another 5 lakh tonnes .

The per capita consumption of fats is rather un­

usually low in comparison with the .developed countries. Even-

to-day the per capita consumption is about 6 kg. per annum

inspite of the fact_that India is importing quite largely

vegetable oils, incurring a huge amount of foreign exchange.

The production of vegetable oils for' the year 1982-83 was

about 3D lakh tonnes, resulting in a deficit of about 15 lakh

tonnes. The Sixth Plan (1984-05) envisaged a total production

 2

of 50 lakh tonnes but the actual production appears to be

lower, thereby making the import of oils mandatory to the

tune of about 1.2 to 1.5 million tonnes. The demand for oils

for the year 2000 A.D. is estimated at 07.47 lakh tonnes

while the projected internal supply is 51.54 lakh tonnes,

making a gap of 25.93 lakh tonnes

(2 1

In order to enhance the production of edible oils

and other industrial oils, a number of integrated schemes have

been planned for implementation immediately. Among the plans,

mention may be made of the increasing use of agricultural

inputs like bringing more lands under irrigation, use of more

fertiliser, developing new hybrids (varieties) of major oil

seeds like ground nut, rape and mustard, sesame, sunflower,

etc. with a v,ew to obtaining much more acreage yield of oil

seeds, tapping of a number of tree borne oil seeds by orga­

nising the collection, and by concentrating more on the

utilisation of by product oil sources.

Among the by product sources of oil bearing mater­

ials, rice bran appears to be the most promising source for

a vegetable oil in India. The production of rice for the

year 1983-84 has been estimated to be the highest in India.

On the basis that bran constitutes 5 to 7 per cent of rice

and assuming on average 15 per cent oil content in the bran,

the potential of rice bran oil production is about 6 lakh

tonnes annually. In view of the increasing trend of pro­

duction of rice, it is expected that by 2000 A.D. about 10 ,

 3

lakh tonnes of rice bran oil will be produced. The actual

production is, however, far below the potential. The current

production of rice bran oil in the country is about 1.5 lakh

tonnes out of which only about 40,000 tonnes are edible

grade.

The main reasons for low production of edible

quality rice bran oil have been ascribed to the failure of

collection of good quality bran and fresh bran. Much efforts

are made to modernise the rice mills for obtaining bran is

not only good quality but also in greater yield and also to

ensure the deactivation of the active lipase immediately by

setting up suitable stabilisers to prevent lipolysis of the

oil.

However, the maximum rice mills are not equipped

with modern rice milling plants including stabilisers and

also there is invariably a time lag between the collection of

bran and its extraction for absolute lack of coordination. As

a result, there is always the formation of high amount of

free fatty acids in the extracted rice bran oil by the lipase

action. The refining of high f.f.a. rice bran oil is not

exploited for edible purpose because of the fact that the

vegetable oil refineries of our country are not yet equipped

with the techniques of refining of high f.f.a. oils. The

plants are processing mostly oils having f.f.a. below 10

per cent.
 4

The high f.f.a. rice bran oil is found to be quite

dark colour, making it also unsuitable for converting it into

edible quality. Rice bran oil is known for its high unsaponi-

fiable ' matter including waxes which makes its use limited as
a cooking oil and in producing vanaspati whose unsap. content

has been prescribed to a maximum limit of 2 per cent. In fact,

the uncap, matter and wax content are regarded as the second

major constraints of rice bran oil in its use in vanaspati

and in cooking medium.

For the post several years, much attention is given

to evolve a proper technology for dewaxing rice bran oil and

also reducing the total unsap. matter quite significantly.

Unfortunately, however, even to-day there does not appear to

be any satisfactory dewaxing process, although it is well

understood that dewaxing should be done to enhance the pro­

duction of edible quality rice bran oil. The dewaxing can be

achieved in the miscella phase and in the oil phase, but for

lack of information under Indian condition particularly in

the Indian industries, the choice between the miscella phase

and oil phase still remains a matter of decision in view of

the economics and the infra structures available in the

Indian industries.

As stated earlier, the major portion of rice bran

oil produced in India have high f.f.a. (15-40 per cent) and

dark colour and are therefore, used for non edible purpose,
 5

particularly in the production of soap after hydrogenation.

Considering the nutritive value of rice bran oil and the

immediate need for enhancing edible oil availability in

India, it is highly imperative that a great deal of develop­

mental research be undertaken for deacidifying high f.f.a.

rice bran oil.

The deacidification of high f.f.a. rice bran oil

by physical refininq,miscella refining with a single or a

mixed solvent, and other suitable processes are considered

for investigation for converting the high f.f.a. dark coloured

rice bran oils of commerce to edible grade for exclusive use

as cooking oil and-in the production of vanaspati. In view of

the existing facilities in most of the refineries, it is also

considered to do some basic research in the refining of high

f.f.a. rice bran oils .by the conventional alkali process in

a two stage operation. Considering the solubilities of fatty

acids in alcohols like ethanol and isopropanol, interest is also

growing in evolving a deacidification process by the liquid-

liquid extraction procedure involving the alcohols by bringing

down the f.f.a. to a level quite satisfactory for subsequent

alkali neutralisation process.

In any event before the dewaxing of rice bran oil

and its deacidification, it is always thought that degumming

of rice bran oil is essential for which a great deal 'of

information is needed to gather in order to identify an

 6

efficient degumming process that will help to produce rice

bran oil of better quality in terms of colour, oil loss and

unsap. matter content.

Considering the need for production of edible

quality rice bran oil, the present study has been aimed at

investigating : (i) characteristics and degumming of crude

rice bran oil of varying f.f.a. viz. 4 to 6' per cent normal

range and 15 per cent and above, by using a number of inorga­

nic and organic agents including the use of the selective

surface active compounds, with a view to obtaining information

that can enable to produce relatively light coloured and low

unsap. rice bran oil, (ii) the dewaxing of rice bran oil of
varying f.f.a. levnl in the oil phase as well as in the mis-

cel!a phase after degumming, by crystallisation at low temp­

erature with or without any additive including surface active

compounds, in order to develope a satisfactory dewaxing pro­

cess for a commercial adoption, (iii) a critical study on

deacidification of rice bran oil of low and high f.f.a. by

conventional alkali refining in a single or two stajsge ‘

operation, ;(iv) extraction with ethanol and isopropanol

followed by alkali neutralisation, (v) high vacuum steam stripp­

ing.process directly as well ns in combination with subsequent

alkali neutralisation, (vi) reesterification of f.f.a. with

the hydroxyl groups available in the oil or with added hydro­

xyl group from glycerol with and without catalyst and

(vii) miscella refining with a single solvent like hexane
 7

and mixed solvent refining with hexane and ethanol or iso­

propanol, with the whole object that suitable processes can

be prescribed for purifying different quality rice bran oil

in the Indian refineries according to their infra structure

and economics
REVIEW ON RICE BRAN OIL
 a

Rice bran (0ryza sativa) is a very important source

of vegetable oil3 in India for which there is a considerable

scope for exploitation. India is the largest producer of

paddy in the World. Current annual production is estimated

at about 53 million tonnes. Rice grows in Bvery state in

India and the major rice growing states are Andhra Pradesh,

Punjab, Maharashtra, Uttar Pradesh, Madhya Pradesh, Haryana,

West Bengal, Tamil Nadu, Orissa and Bihar.

The production statistics indicate taking rice bran

yield at T per cent on average, that the production of rice

bran for extraction could be around 40 lakh tonnes.

(3 ) although
Rice bran contains 15 to 20 per cent oil

the oil content can be 9 to 22 per cent depending on the

nature and quality of rice bran. Normally parboiled bran

‘contains more oil.

Taking oil at an average of 15 per cent of bran,

the potential of rice bran oil in India comes to around 6

lakh tonnes on the basis of rice production during 1983-84.

The actual production of rice bran oil has been much less.

The production of edible grade rice bran oil has

been, however, very low ca. 50,000 lakh tonnes.

The unsatisfactory product-Lon of edible quality

rice bran oil can be partly attributed'to the poor and

peculiar structure of rice mills of our country. The rice

 = y =

milling units of our country are scattered-widely that makes

organised collection of rice bran particularly from interior

areas difficult and also uneconomical. Again the rice mills

operating in the country use mostly huller mills instead of

the sheller mills. The huller units invariably contaminate

bran with broken husk, broken rice, sand and silica, making

thereby the yield of oil poor, and the deoiled bran is used

only as animal feed. On the other hand, the sheller produces

bran of good quality and greater yield resulting in much more

good quality bran oil. It is for this reason that the large

mills in the organised sector are advised to be modernised

by shellers or -by sheller-cum-huller. The number of sheller

and huller mills are, respectively, 6,000 and 80,000 out of

about 1 lakh rice mills in India. The rest of thB rice mills

are huller-cum-sheller types.

The quality of rice bran oil and yield of rice bran

oil have been reportedrto depend on also how rapidly the oil

is extracted or if the bran is subjected to some heat treat­

ment and adjusted to a critical moisture level beforeits

extraction. It is known that rice bran contains a lipase

which shows considerable activity if the bran is not imme­

diately extracted or thermally treated. This enzyniB action

invariably results in the hydrolysis of bran oil triglyceride

(4)
into free fatty acid producing high f.f.a. rice bran oil

and also imparting the colour problem. In order to deactivate

the enzyme system for purpose of making good quality rice

bran oil, it has been advised that rice bran should be stabi­

lised before its extraction. Much work has been done on the

stabilisation technique by either direct steaming and drying

or by indirect heating by hot air. Thus Central Food Techno­

logical Institute of Mysore have designed stabiliser and have

developed the working procedure for deactivation of lipase
(5) (6)
in rice bran . Srimani also stabilised ricB bran by

contacting with live steam and immediately drying in a

fluidised bed heat transfer system. Rice bran stabilisers

have also been developed by the research team of Anantapur^^

Recently, an extruder stabiliser has been developed in the

U.S.A. which has enabled to produce good quality rice bran

oil.

In spite of the indigenous development of stabi­

lisers, the quantity of edible quality rice bran oil is far

below the potential, indicating thereby that bran stabili­

sation is hardly practised yet. It is often advised and, in

fact, suggestion is made to the Ministry of Civil Supplies

of the Government of India that the stabiliser be imported

for producing edible grade rice bran oil. It may be pointed

out that unless the bran is stabilised thB f.f.a. in the oil
becomes as high as 60 per cent, and in most cases it is
around 20 to 30 per 'cent.

Rice bran oil is produced by solvent extraction

using food grade hexane after rice bran is properly cooked
 11

and pelletised.

Rice bran oil of low f.f.a. 4 to 6 is yellow to

green in colour in crude state which is due to occurrence of

pigments like cholorophyll. The colour of the oil turns

increasing in brown as f,f.a. increases and a very high f.f.a

oil assumes a dark purple colour. The crude oil has distinct

rice odour.

Rice bran oil contains waxes and other non-tri'gly-

cerides as unsap. matter, besides phospholipids and gummy

proteinacious matter, starch, water etc. The proportion of

(81
waxes varies from 1 to 6 per cent' ' generally from 3 to 6

per cent. The uns aponifiable matter varies from 3 to B per

cent in crude
(9) . The composition of rice bran wax has been
studied^*^ consisting of fatty alcohols and fatty alcohol

esters. The Waxes are of two types, hard and soft, depending

on the melting points. The hard wax has a melting range

between BO to 84°C and the soft wax having 72 to 74°C. The

hard wax is mainly composed of saturated fatty alcohols of

^24' ^26 anc* *"30 anc* saturated fatty acids of C^* ^24 anc*

anc* normal alkanes of C£g and ^31 • The soft wax is

composed of saturated fatty alcohols of C^4 and C^q and

saturated fatty acids of g and ^26 anc* normal alkanes of

and C2g* In the soft wax, lauric acid is also detected.

The oil contains hydrocarbons, sterols, sterol

esters including methyl sterols and triterpene alcohols. The

 12

hydrocarbon fraction contain 2 to 3 per cent squalene in

(3)
the raw oil

f^ice bran oil contains phenolic bodies such as

tocopherols and ferulic acids and their methyl esters, which

{3 )
give tremendous stability to the oil . Rice bran oil

appears to be one of the richest source of tocopherols that

varies from .02-.0 4 per cent in the crude. Raw oil contains .02

to .03 per cent oryzanol composed of several kinds of ferulic

acid. This substance has an effect similar to that of vitamin

E in acclerating human growth facilitating blood circulation

and stimulating hormonal secretion.

i

Rice bran oil has a fatty acid composition like

O

that of many oleic-linoleic acids rich oils. The fatty acid

composition of the oil has not been quite extensively studied.

The oil consists mainly of 15 to 30 per cent saturated acid,

35 to 50 per cent oleic acid, and 29 to 42 per cent linoleic

(31112)
acid ’ * . Among the saturated acids, palmitic acid occurs

from 12 to 20 per cent, stearic acid 1 to 4 per cent and .other

saturated acids namely, myristic, arachidic and behenic, con­

stitute 1 to 2 per cent. Linolenic occurs from trace to 1

(3)
per cent and palmitoleic from 0.2 to 0.4 per cent . The

fatty acid composition of Indian rice bran oil has not been

extensively investigated.
 The specifications for Indian rice bran oil (raw
(13)
and refined) are as fallows ' :

Iodine value varies from B5 to 105; saponification

value 175 to 195; unsaponifiable matter (% w/w) 3,0 to 6.0;

refractive index at 40°C 1.4600 to 1.4700; specific gravity

at 30/30°C 0.910 to 0.920 and flash point by Pensky-Martens

(closed) °C (min) 90 to 250.

Glyceride composition of rice bran oil is also not

adequately examined. There is only one study that gives the

distribution of fatty acids and also the types of glyceride

(14)
by pancreatic lipase hydrolysis technique ,

Rice bran oil contains phospholipids between 0.5

/ A C A C \
per cent and higher ’ ' and the phospholipids are mainly

composed of lecithin.

The refining of rice bran oil includes degumming

and its pretreatment, dewaxing by winterisation and deacidi­

fication by alkali refining and other process depending on

the f.f.a. content. Generally before degumming, the crude

oil is centrifuged to separate sand or dust. Degumming is,

however, carried out in refineries by treating the raw oil

without prior centrifugal separation.

Degumming of crude rice bran oil is invariably done

As per the literature information, agents like water, phos­

phoric acid, citric acid, oxalic acid, tartaric acid,

 14

sulphuric acid and organic acid anhydride and inorganic

chemicals like sodium pyrophosphate, sodium metaphosphate,

sodium hydrosulphite, ammonium phosphate etc. have been used.

The raw oil is generally heated to a certain specific temp­

erature and then agitated with the appropriate quantity of

the degumming agent followed by contacting for some time and

separation of the gummy material by centrifuge or by removing

the gum from the bottom after settling for a certain time

period.

There is a reference
(17) on degumming of rice bran

ail by using surface active compound along with phosphoric

acid. The report is claimed to have produced after subsequent

alkali refining and bleaching a.sample of rice bran oil con­

taining significantly lower unsap. matter content than that

acheived by phosphoric acid and Water degumming before alkali

(17)
refining and bleaching .

It is suggested that degumming should preferably be

done on the oil immediately after its extraction. The degumm -

ing of rice bran oil and other vegetable oils in our refin-<

eries is mostly a batch process and the gums are hardly

separated. The oil becomes gum conditioned and the alkali

refining on the oil _is carried out.

As stated earlier, rice bran oil contains waxes

which make the oil less acceptable to the consumers for. use
as a cooking oil. The oil is being used in India for the

manufacture of vanaspati and its use in vanaspati becomes

very much limited as the wax content affect the colour of

vanaspati and also the lustre of vanaspati crystals. It is

imperative, therefore, to dewax rice bran oil completely or

to a very low level in order to produce cooking oil and also

vanaspati without using any other liquid oil.

The dewaxing can be achieved in the oil phase as

well as in the miscella phase by winterisation without or

with suitable additives. One of the crude methods of separ­

ation of waxes' in some amouht involves the removal of the

waxes settled down in storage tank.

The separation of waxes from the pretreated oil

(degummed) is winterised directly or indirectly at low temp­

erature (8 to 10°C} followed by the separation of waxes by

filter press or by centrifuge or by bag type filters. It is

very important to maintain filtering temperature which should

be preferably 5 to 7°C higher than cloud point of the filter­

ed oil. In the winterisation step, it is necessary to have a

very control cooling instead of rapid chilling so that the

wax crystals are obtained in the form for easy separation

from the chilled oil. Dewaxing of rice bran oil after

degumming, refining and bleaching was reported by Sen et al^ ^
Q "l

by gradual chilling at 10 C for 4 hours and separating the

waxes by centrifuge of Westfalia type. It has been reported

that gradient temperature crystallisation does not yield
satisfactory dewaxing due to the fact that there is the
formation of /3 -form of fine crystals and very soon the

formation of a vitreous state resulting in one inseparable

mass. More work is needed to establish the rate of supply of
energy for formation of^-fine crystal of waxes for easy
separation^ ^ .

Calcium chloride when used in certain amount in

aqueous solution results in the separation of the waxes of

the oil and when subsequently centrifuged the oil is found

to be rid of. about 70. per cent of its wax content^^^. De­

waxing of rice bran oil has been reported to be achieved by

keeping the refined oil at low temperature in presence of

(21 )
soap stock before centrifugation .

The dewaxing of rice bran oil in the miscella phase

by winterisation has been well investigated. In Rancher's
(22)
process rice bran miscella has been gradually chilled in

a compartment fitted with the stirrer of speed 1 to 10 r.p.m.

to hasten crystal formation and the solid fat (wax fraction)

is separated by centrifuge. This process removes over 90 per

cent of rice bran oil waxes.

Rice bran" oil dewaxing in miscella phase is well

known in Japan because of the fact the solvent refining plant
is quite largB, capable of affording initial and running cost.

On the other hand, this process is not suitable in small

 17 =

refinery plants such as in the existing refineries of India.

The miscella phase dewaxing also depends onthe oil content

in the miscella and the winterisation temperature. It is
reported that the best mixture is generally.1 : 1 of degummed

rice bran oil with hexane. The final crystallisation temper­

ature is 5°C and the crystallisation cycle is standardised

at 8 to 10 hours per charge (19) .

(21 )
A detail dewaxing method has been developed by

Haraldsson which involves Separation of waxes from alkali

refined oil in an aqueous suspension by centrifuge. The crude

oil after degumming with phosphoric acid is neutralised with

caustic lye and separated off the soap stock. The oil is
brought to 30°C by regenerating cooling and further cools at
about 8°C where the oil is kept for 4 to 5 hours under gentle

stirring. The oil is then mixed with 4 to 6 per cent of

o
water and heated to 18 C by luke warm water. The .soapy water

phase formed wets the wax crystal, thus forming a heavy

suspension of wax in the soapy water. The heavy phase is cen­

trifuged in water, which is once again mixed with some water

and centrifuged to ensure a complete removal of wax. This

method has the advantage of lower operating cost duetto no

cost of filter aid, lower oil losses and lower manpower

requirements. The w-axy soap water is normally mixed with the

.oap stock before splitting but is also possible to treat

this phase separately to recover the wax and oil for further
upgrading.
 (21 )
Haraldsson also reported dewaxing of oil by
o
cooling the oil at 8 C followed by addition of 5 per cent

water containing small amount of sodium lauryl sulphate and

then agitating the mixture for about 4 hours at that temper­

ature to complete crystallisation. The wax crystals that

becomes dispersed into the water phase are separated in cen­

trifuge with the dewaxed oil going to refining.

Refining (deacidification) of rice bran oil to

edible grade is rather difficult in view of the waxes, high

unsap. matter and other impurities. Normally for rice bran

oil having f.f.a. up to about 8 per cent, the deacidification

is done by conventional alkali lye process on the gum condi­

tioned oil or degummed oil or and after dewaxing depending

on the end use of the oil. However, the refining loss

(23)
incurred is much higher ' as has been the observation also

in the refineries of India and also according to one published

(24)
report . This refining loss which is totally out of pro­

portion to the f.f.a. of oil whether the neutralisation is a

batch process or a continuous centrifugal process, A study

was made on the actual Wesson loss of the oil and it was

reported' that the Wesson loss is almost twice that of

the f.f.a. indicating much higher percentage of impurities

which might have caused excessive emulsification during

refining. One of the impurities in the oil that may be

responsible for high refining loss is the monoglyceride.

 19

In actual operation by the conventional alkali

refining process, the refiners always come across with oil

/pg\
loss of an average three times the f.f.a. . The extent of

refining loss and total process loss after degumming, refin-

(1 Q\
ing and dewaxing have been reported by Sen et al also highv

The miscella refining process is very excellent for rice bran

(27)
oil as reported by Crauer .

Refining of high f.f.a. oil with 15 to 40 per cent

f.f.a. has becomp the subject of much investigation. The

deacidification by physical refining process has been tried

by one industry in India on the thoroughly degummed, dewaxed

and bleached oil. It has been claimed that the oil ultimately

obtained is not acceptable grade of liquid refined oil as the

colour tends to concentrate in the residual oil after steam

stripping. The oil, however, can be utilised in making

vanaspati^^^®^. ThB ^acidification of high f.f.a. rice bran

oil by reesterification process with or without catalyst

under vacuum at various temperature was carried out by Anand

(29)
and Vasishtha . ThB authors did not, however, mention the

colour problem and the characteristics of the deacidified oil.

Miscella refining process has been commercialised

in Japan and a number of advantages have been claimed in the

process in respect of refining loss and colour^^^^. The

process needs explosion proof system and the cost of equip­

ment and control is some what higher than ordinary refining

 = 20

plant. In this process the extracted miscella is directly

degummed, dewaxed and refined without desolventiBing.

The mixed solvent process using hexane as main

solvent and ethanol or isopropanol as a second solvent has
been also standardised for refining of high f.f.a. rice bran
oil containing caustic lye after degumming and dewaxing in

the miscella phase. The process has been reported to be

extremely satisfactory for high f.f.a. dark colour rice bran

oil and the refining factor lies between.1.1 to 1.3' .

The process is superior to the pure physical

refining process. De-Smet Neumi process based on mixed solvent

(33)
refining is well known for high f.f.a,. rice bran oil .

Combination of physical refining and mixed solvent

(34)
refining have also been tried for high f.f.a. rice bran oil
and the refining factor is reported to be 1.1.

The refining of high f.f.a. rice bran oil by ethanol

extraction has also been claimed to have been developed by

(35)
Bhatnagar ' which is quite satisfactory but details are
completely lacking.

Rice bran oil bleaching is some what more difficult

than most of the liquid vegetable oils primarily due to pig­
ments like chlorophylls and, also in the case of high f.f.a.
oils due to oxidised products of tocopherols and metallic

salts of fatty.acids. Generally, bleaching of rice bran oil

 21

is done after degumming, dewaxing and deacidification by

alkali or by single or mixed solvent miscella refining but

in the case of steam stripping bleaching is done immediately

after degumming. Bleaching earths are of the conventional

types used between 1 and 2 per cent on the weight of oil and

generally done under a vacuum of 28 inches of Hg. at 90 to

100°C^3<^. The addition of activated carbon along with acid

activated earth has also been used in many refineries. The

quantity of earth used often exceeds 2 per cent and some time

as high as 4 per cent activated earth is used in order to

improve the colour.

Industrial grade rice bran oil is often bleached by

using the conventional chlorate bleaching for use in

(37)
soap .

Deodorisation of degummed, dewaxed and deacidified

rice bran oil is done in a normal manner just like other

vegetable oils. The general conditions of deodo^risation

includes a temperature in •dBodo'riser between 180 to 200°C and

, . (36)
a pressure of o to 10 mm Hg, .

i
EXPERIMENTAL TECHNIQUES

METHODS AND MATERIALS

 22

Crude rice bran oil of varying f±ee fatty acid

(f.f.a.) was obtained from the local industries.

The general analytical characteristics of. importance

for purification studies of rice bran oil like f.f.a#> colour,

unsap. matter and wax content, phospholipid, as well as mono-
t

glyceride and diglyceride were evaluated by adopting standard

methods. Free fatty acid, and unsap. matter in crude and

refined oil were determined by the AQC5 methods' . Colour

of the crude and refined rice bran oil was determined by

Lovibond Tintometer (39)'. Wax content was estimated by crysta­

llisation from cold acetone^ ^.

Phospholipid was estimated by combination of TLC^^^

and colour reaction (41 ) . About 20 to 30 mg of rice bran oil

was accurately weighed and spotted quantitatively in a band

fotm on preparative silica gal TLC plate. To isolate the

phospholipid band,, pure diethyl ether was used. Phospholipid

band was marked by iodine exposurs for few seconds. The band
was. scraped off and the phospholipid was extracted quantita­

tively by methanol and chloroform (2:1, v/v) mixture three

times followed by centrifugation. The extracted solvent was

then evaporated to dryness in a stoppered test tube on a

water bath to get th-e phospholipid content of the oil taken.

This phospholipid was then digested with 70 per cent

perchloric acid (A. R. grade) and the phosphorous content

was measured by colour reaction in a Varian spectrophotometer

 23 =3

at 820 nm.

Monoglyceride and diglyceride were estimated by

separation from the oil by thin layer chromatography and then
(42)
by colour reaction of glyceride glycerol . Known amount
of rice bran oil was spotted on preparative silica gel TLC
plate. To isolate the monoglyceride and diglyceride bands,

a solvent system of n-hexane diethyl ether acetic acid

(80 : 20 : 1, v/v/v) was used. Visualisation of the bands

was made by exposure to iodine vapour for fiew seconds. Then

the bands were scraped off and individual glycerides were

extracted by chloroform three times followed by centrifu­

gation. Chloroform was evaporated to dryness and glycerol of

monoglycerides and diglycerides was estimated by complete

saponification with ethanolic (spectroscopic) KOH at 60 to

65°C. Known amounts of chromatography grade monoglyceride

and diglyceride were similarly analysed by TLC and colori­

metry to prepare standard calibration curve of each neutral

g^Lyceride.

Fatty acid composition of rice bran oil samples

was determined by gas-liquid chromatography (GLC) technique

of the derived methyl esters from the oil prepared by the

procedure of Metjfcalfe' * after removing the unsaponif tables.

For the preparation of methyl ester ca. 0.1 g of

rice bran oil was taken into standard joint round bottom

flask fitted with a reflux condenser and CaO guard tube. About
5 ml of BF-j" methanol mixture (E. Merck quality) was added

and agairj/ reflux cd for about 10 minutes. The reaction was

stopped by addition of water and the mixture was trans­

ferred to the separating funnel. Then about 5 ml of n-hexane

was added to the separating funnel and the hexane layer was

washed' for few times to make it neutral and then dried by

passing thorough anhydrous sodium sulphate bed'.

Anhydrous hexane layer was taken for the fatty

acid analysis by GLC technique. GLC used in the analysis was

Hewlett-Packard H P 5840 AGO and column used was 10 per

cent DEG5, 2 ml length with 1/8 inch I.D. Conditions main­

tained were : oven temperature 190°C , injection temperature

2b0°C, Flu 300°C chart speed 0.5 cm/min. Fid signal + B, slope

sens 300.00, flow of nitrogen 30 ml/min. in A and B.

Infra-red spectroscopy for trans acids :

(44 )
Allen has developed a very rapid and easy

method for the determination of the trans acids. According

to him the measurement, of the absorption of a solution of

the methyl ester (also fatty acids and triglycerides) at two

different 'wave lengths viz. at 8.55/a.( absorption due to CQQCH^

group) and at 1U. 36/M(ab sorption due to the trans unsaturation)

were observed and the ratio of the two can be used to find

out the trans-acid content. The per cent trans-unsaturation

is given by the formula,

 25

A1 0. 36
Per cent trans-unsaturation = 121.6 X ------- - 9.18
A 8.55

for carbon disulphide solvent, where A 10.36

■ and A 8.55
• are

the respective absorptions at 10.36/Uand 8.55^M. The concen­

tration of the solution is such that the per cent absorption

does not exceed 90 per cent.

In actual practice, the rice bran oil samples after

steam-stripping were converted to their respective methyl

\

esters and dissolved in carbon disulphide (spectroscopically

pure) and absorbance at 10.3(yU- and 8,55^ were measured and
the per cent trons-ocids were calculated according to the

above formula.
•>

Ultra violet spectroscopy for measurement of diene conjugation

The extent of diene conjugation in rice bran oil

during steam stripping process is measured by ultra violBt

absorption of the oil sample in isa-octane following A0C5

. me thod ^ .