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Whereas the Parliament of India has set out to provide a practical regime of right to information for citizens to secure access to information under the control of public authorities, in order to promote transparency and accountability in the working of every public authority, and whereas the attached publication of the Bureau of Indian Standards is of particular interest to the public, particularly disadvantaged communities and those engaged in the pursuit of education and knowledge, the attached public safety standard is made available to promote the timely dissemination of this information in an accurate manner to the public.

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“Step Out From the Old to the New”

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“The Right to Information, The Right to Live”

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“Invent a New India Using Knowledge”

है”ह”ह

IS 4684 (1975): Edible Groundnut Flour (Expeller Pressed)[FAD 16: Foodgrains, Starches and Ready to Eat Foods]

IS 4684 : 1975( RNftlnned 1183'

Indian Standard

SPECIFICATION FOR EDIBLE GROUNDNUTFLOUR ( EXPELLER PRESSED )

( First Revision)

Second ReprintAUGUST 1996

UDC 664.641.2 :634.58

C Copyright 1976

BUREAU OF INDIAN STANDARDSMANAK BHAVAN, 9 BAHADUR SHAH ZAFAR. MARa

NEW DELHI 110002

Gr6

IS I 4684 • 1975( Reaf'nnned 1983 )

Indian Standard

SPECIFICATION FOR EDIBLE GROUNDNUTFLOUR (EXPELLER PRESSED)

(First Revision)Nutrition Sectional Committee, AFDC 37

ChairmanD. M. S. SWAMINATHAN

MembersDR K. T. AO.AYA

SURI A. ARORASUBI P. C. JOSHI (Altern.t,)

DR A. AUSTIN

RepresentingIndian Council of Agricul rural Research, New

Delhi

Protein Foods and Nu trition Development Associa­tion of India, Bombay

Modern Bakeries ( India) Limi ted, New Delhi

Indian Agricultural Research Institute (leAR),New Delhi

DR M. S. NAIK (Alt,rnall )DB B. P. BALIGA Tata Oil Mills Company Limited, Bombay

SRRI M. C. BADAMI ( Alternate)DR S N. BANERJKE Food and Nutrition Board, Ministry of Agriculture

& Irrigation, New DelhiSnRI T. V. SUBRAMANIAN ( Alternate)

DB (SMT) BHAVANI BEr..A.VADY National Institute of Nutrition, HyderabadLT-GEN S. N. CHATTERJEE Directorate General of Armed Forces Medical

Services, New DelhiAlB VICE MARSHAL J. H. F

MANEK SHAW ( Alternate)DR J. D. CONTRACTOR Coca-Cola Export Corporation, New Delhi

SURI P. C. VIN ( Alternat« )DB (SMT) RIJAMAL P. DEVADA8 Home Science Association of India, Coimbatore

SMT JALAJA SUNDARAM (Alternate)DB K. HALDER Indian National Science Academy, New Delhi

DR J. NAGCHAUDHUBI (Alternat,)SIIRI R. S. IVER Glaxo Laboratories ( India) Limited, Bombay

DR V. S. MOHAN ( Altemat« )DB K. K. G. MENON Hindustan Lever Limited, Bombay

DB P. J. THOMAS (Altemat')

( Conli1Wld onpOI' 2 )

@ Copyright 1976

BUREAU OF INDIAN STANDARDS

This publication II protected under the 111dill1l Copyrighl Ad (XIV of 1957) andreproduction in whole or in part by any means except with written permiuion of thepubliaber ,hall be deemed to be aD IDfriDgement of copyright under the aaid Act.

181~684 • 1975

( COIl'illUltlfrom /Ja" 1 )

Central Food Technological Research Institute(CSIR), My.ore

DB D. V. S. K. RAOSaRI R. B. aAO (Alt'rllGl,)

DB S. V.NEATA R.AO

M,m6" I R."slftli",Da H..NATK Defence Food Retearch Laboratory, My.ore

Da P. K. VIJAYABAGBAVAN (AlI"na',)Da T. M. PAUL National Dairy Research Institute (leAR).

KarnalDa R. BBIKA81lNA RAO ( AlI"1Ia', )DBS. V. PINGALII Food Corporation of India, New Delhi

SHal J. NBBLKANTHA (AI'''"II,)Sual A. PaAIAD Nutrition Panel, Planning Commiuion (Ministry

of Planning), New DelhiSHBI S. N. PANIJtKAB (AII"1IIII,)

SBBI S. RAIIA.WAIIY Directorate General of Technical Development,New Delhi

The Britannia Blacuit Company Limited, Bombay

DR P. B. RAMA aAO (Allmla,,)Snal M. G. SAT.. Sathe Biscuits and Chocolate Company Limited,

PoonaSBal U. R. Kulkarni (AII,rrull')

8mu V. H. SHAH Kaira Distric,t Cooperative Milk Producers UnionLimited, AnanC!

DR I. M. PATIIL (.41t'nttll')DB M. C. SWAMINATHAN Directorate General of Health Services, New Delhi

SSBI D. S. CHADHA (AII""al')KUMARI M. S. UIRA G. B. Pant University of Agriculture and Techno-

logy, Pantn_gar (Dilt Nainital )Saal N. K. VI88ANJI Roller Flour MUler. Federation of India, New

DelhiS8.1 SAN'.rANU CBAUDBUBI (.AI''''"J1')

SSRI T. PuaNAXAKDAII, Director General, lSI (&-oJI"io M'mlHr )Deputy Director ( Alrl &. Food)

$","a,)D. R.. B. MA'lBUB

Deputy Director ( Agri & Food), lSI

Oilseed Protein ConcentrateS Subcommittee. AFDC 37 : 3

C""'IIIf'DB K. T. AOBA'I'.A. Protein Food. and Nutrition nlvllopmlDt

Aaaociation of India, BombayAI",."

SOl S. N. Aa~.wALDa B. P. BALIOA

DB K. S. ROLLA (AII",..,,)SOl D. S. CB.lDBA

Prallee &, Oil Milia, AU.arbTata Oil Mill. Company Limited, Bombay

Central Committee for Food Standarda, NewDelhi

8.1f DaDI MUKBDJU (.401'''"01')

( Co,,'illlUd '"~" 21 )

2

IS : 4684 • 197~

( Co"'Uau,J from page 2)

Memhers RepresentingDIBECTOR (VANA8PATI) Directorate of Sugar & Vanaspa ti, New Delhi

DEPUTY DIRECTOR ( VANASPATI) (Alternate)SBBI S. C. GUPTA Food Corporation of India, New DelhiDR G. LAK8HMINARAYA~A Regional Research Laboratory ( CSIR ),

HyderabadSHBI P. L. NARAYAN RAO ( Alternate)

SBRI S. RAMASWAMY Directorate General of Technical Development,New Delhi

BRIG D. S. RAO Technical Standardization Committee ( Foodstuffs):New Delhi

SHBI J. K. jAO'l'IANI ( Alternate)SURI R. B. RAO Britannia Biscuit Company Limited, BombayDB U. Y. REOE Raptakos Brett & Company Limited, BombaySBJU HARI8H SETHI Directorate of Oilseeds Development, HyderabadSaBI DINESll SHAHRA General Foods Private Limited, Indore

SUBI SANTOSH SIIABRA ( Alternate)Snnr L. J. T ANN A Solvent Extractors' Association of India, BombaySHRI M. KANTHARAJ URS Central Food Technological Research Institute

( CSIR ) J MysoreSrrar N. SUBRAMANIAN (Alternate)

21

A~NDMENT NO. 1 FEBRUARY 1986

TO15:4684-1975 SPECIFICATION FOR EDIBLE GROUNDNUT

FLOUR (EXPELLER PRESSED)

(First Revision)

(Page 4, cLause 2.2.1, line 3) - S*bstitute '11 and12 respectively of IS:7814(Part 1)-1975" for '11 orIS:1714-1960* and in Appendix A of IS:1712-1972§'.

(Page 4, foot-notejj with '*' and I§ I marks) _Substitute the following for the existing foot-notes:

'*Method of sampling and test for animl feeds andfeeding stuffs: Part 1 General methods.'

[Page 5, TabZe i, si No. (vi). col: (3)] - Substitute'6.0' for '4.0'.

(AFDe 31)

Printed It NewIndiaPrintu. Pre-. Khurja. Iactia

.('!e",.. Copy (~'J.~ ... ,1~)ttlItt In ", [ssur d (,,~ i.;y i~ ;1~;;' .

AMENDMENT NO. 2 JULY 1999TO

IS 4684: 1975 SPECIFICATION FOR EDIBLEGROUNDNUf FLOUR (EXPELLER PRESSED)

( First Revision )

[ Page5, Table 1, 51 NQ~ (viii). col3 ) - Substitute C30' for '60'.

I'~f:\'~\ ,', !~~.f \~~";">'"( FAD 15) I ..':.L:;'\,", ,,~,\ ,. . ~ \ ' .\ ',: '). "

If ,.~ .. ).. O' ·L ,\; \ '; \ '. 't I •• :). ," .' . 't.'. \ ..,

\ \ f. /' . ., I ' I -+-Re--b-U-' UIS N I'\--lh7 -. - .; :-I .l:-',;:' \'::1\ J, .i\l. " ' ., .:~ ,.1,' iJprograp y mt, ,ew I.J"lC I, .........

\... . 'Jo .'~ /,\," . .' .~ 'J v \., .If

-, .. .. ' \:\ '\ "\. ,.<; I'I .'

IS I 4684 • 1975

Indian StandardSPECIFICATION FOR EDIBLE GROUNDNUT

FLOUR (EXPELLER PRESSED)

( First Revision)

o. FOREWORD

0.1 This Indian Standard (First Revision) was adopted by the IndianStandards Institution on 30 October 1975, after the draft finalized by theNutrition Sectional Committee had been approved by the Agricultural andFood Products Division Council.

0.2 Edible groundnut flour is well recognized as a rich source of dietaryprotein. Its properties, such as taste, odour and colour have led to its use inmany food products like biscuits, BALAHAR and other snack foods.

0.3 This Indian Standard was first published in 1968. Consequent uponimprovement in the quality of the edible groundnut flourproduced commer­cially, it was felt that the standard required to be modified. Accordingly,this revision is being issued. In this revision the requirement for availablelysine has been excluded, limit for aflatoxin reduced and description of rawmaterial preparation elaborated.

0.4 One of the toxicants which is usually present in edible groundnut proteinproducts is aflatoxin, produced by a fungus (Asp"gillru jlavus). It isnow well-established that aflatoxin is harmful to human beings when ingestedeven in minute quantities. It, therefore, becomes imperative that the edibleflour is produced under strictly controlled hygienic conditions. Underoptimum conditions of growth, harvesting, drying, the toxin content can bealmost negligible. At the same time several methods have been developedfor detoxification of the aflatoxin present in edible groundnut flour, bytreatment with chemicals like ammonia, hydrogen peroxide and certainoxidizing agents. Simultaneously, these treatments bring about a loweringof the nutritive value of the edible groundnut protein products throughdestruction of the sulphur amino acids. Therefore, safe methods of makingedible groundnut protein products of good nutritive value and low aflatoxincontent are either by treating groundnut pods in the field to avoid fungalcontamination, or by manually removing fungus-affected kernels beforeprocessing the remainder, or by using groundnut naturally resistant tofungus. At present, manual removal of fungus-affected kernel is adopted for

3

IS : 4684 - 1975

commercial production of edible groundnut flour, and products with anaflatoxin content well below the prescribed limit are regularly obtained.

0.5 A separate Indian Standard (IS: 1875.1975*,) for solvent extracted( variety of groundnut flour) has also been formulated.

0.6 This standard has been formulated in close collaboration with theProtein Foods and Nutrition Development Association of India. Whileformulating this standard due consideration has been given to relevant Rulesissued by the Government of India under the Prevention of Food Adultera­tion Act, 19.14. This standard is, however, subject to the restrictions imposedunder that Act, wherever applicable.

0.7 For the purpose of deciding whether a particular requirement of thisstandard is complied with, the final value, observed or calculated, expressingthe result of test or analysis, shall be rounded off in accordance wi thIS: 2-1960t. The number of significant places retained in the rounded offvalue should be the same as that of the specified value in this standard.

1. SCOPE

1.1 This standard prescribes the re~uircnlcnts, and the methods of samplingand test for edible groundnut flour ( expeller pressed).

2. REQ,UmEMENTS

2.1 Raw Material - Expeller pressed edible groundnut flour shall be madeby expelling in a screw press fresh, clean degermcd groundnut kernels whichhave been decuticled after mild roasting. In order to reduce the proportionof immature, shrivelled and mouldy kernels which could carry high levels of .aflatoxin, the kernels shall be selected either by visual inspection, inspectionunder ultraviolet light, electronic sorting or by other means. The kernelsshall be free from insect or fungal infestation.

2.2 Description - Expeller pressed edible groundnut flour shall be whitishto light brown in colour, uniform in composition and shall be free frominsect or fungal infestation, objectionable odour and rancid taste. I t shallnot contain added flavouring and colouring agents or any other extraneousmatter.

2.2.1 Expeller pressed edible groundnut flour shall be free from castorhusk or MAHUA oilcake when tested according to the methods prescribedin 11 of IS : I 714..1960+ and in Appendix A of IS : 1712- I 972§, respectively.

·Specification for edible groundnut flour ( solvent extracted) (first revision).tRules for rounding off numerical values ( reoised],:Methods of sampling and test for oilcakes as livestock feed.§Specification for cottonseed oilcake as livestock feed (first revision).

4

IS I 4684 • 1975

2.3 Particle Size - Unless otherwise specified the materla l shall be of suchfineness that, when tested by- the method prescribed in Appendix A, notmore than 5 pcrcent by Inass shall be retained on a 710-micron IS Sieve( see IS : 460-1962* )and not more than 20 percent by mass shall be retainedon a 500-micron IS Sieve.

2.4 Premises - Place where manufacture, packing and storage of thematerial is done, and the eqipmcnt ernpolyed, shall be maintained underhygienic conditions (see IS : 2491-1972t ).

2.5 Edible groundnut flour (expeller pressed) shall also comply with therequirements given in Table 1.

TABLE 1 REQUIREMENTS FOR EDIBLE GROUNDNUT FLOUR(EXPELLER PRESSED)

F

J

GH

E

D

METHOD OF TEST( REF TO ApPENDIX)

(4)

BC

s-o

0·35

4·05·0

60

(3)s-o

45-0

REQUIREMENTCHARACTERISTIO

(2)

Moisture, percent by mass, Max

Protein (N X 6·25) (on drybasis), percent by mass, Min

"iii) Total ash (on dry basis),percent by mass, Max

iv) Acid-insoluble ash (on drybasis), percent by mass, Max

v) Fat ( on dry basis), percent bymass, Max

Acid value of extracted fat, Max

Crude fibre (on dry basis),percent by mass, Max

Aflatoxin, (.L g/kg, Max

vi)

vii)

viii)

SLNo.(1)

i)ii)

2.6 Bacteriological Requirements -1'he edible groundnut flour( expeller pressed ) shall be tested periodically to comply with the require­ments given in Table 2.

3. PACKING AND MARKING

3.1 Packing - The material shall be' packed in polyethylene or polyethy­lene-lined jute bags, or in clean tinplate containers. When packed in bagsthe mouth of each bag shall be either machine- or hand-stitched. If hand..stitched, the mouth shall be rolled over and stitched. Stitches shall be intwo cross-rows with at least 14 stitches in each row.

*Specification for test sieves ( revised).tCodc for hygienic conditions for food processing units (first rtv;s;on)•

s

18 I 4684. 1915

3.2 MarldDl - The following particulars shall be marked or labelled oneach container :

a) Name of the material,b) Name and address of the manufacturer,c) Batch or code number,d) Net mass, ande) Date of manufacture.

TABLE 2 BACTERIOLOGICAL REQUIREMENTS FOR EDIBLEGROUNDNUT FLOUR ( EXPELLER PRESSED)

(Claus, 2.6)

IS : 5401.1969t

METHOD 01' TEST,RJa.. TO

(4)

IS : 5402·1969·

10

(3)50000

REQumEMBNTCBABAOTBRISTIOSLNo.(1) (2)

i) Total bacterial count per g,Ma

Ii) Coliform bacterial count perI. Ma

iii) Salmo"llltJ bacteria Nil IS : 5887.1970~tMethod for standard plate count of bacteria in foodstuffs.tMethods for detection and estimation of coliform bacteria in foodstuffs.: Method. for detection of bacteria responsible for food poisoning and food-borne

dileases.

3.3 DIS Certlftcatlon Marking

The product may also be marked with Standard Mark.

'.3.1 The use of the Standard Mark is governed by the provisions of theBureau of Indian Standards Act, 1986 and the Rules and Regulations madethereunder. The details of conditions under which the licence for the use ofStandard Mark may be granted to manu{acturers or producers may be obtainedfrom the Bureauof IndianStandards.

4. SAMPLING4.1 Representative samples of the material shall be drawn and tested forconformity to this standard as prescribed in IS :5315·1969*.

S. TESTSS.t Tests shall be carried out as prescribed in 2.2 and 2.3, and Tables 1and 2.

-Methods of sampling for milled cereals and pulses products.

6

18 I 4684• 1'75

s.% Q,uallty of RealeDt. - Unless specified otherwise, pure chemicals anddistilled water ( see IS : I070-1960t ) shall be employed in tests.

NO'rIl- ( Pure chemicals' shall mean chemicals that do not contain impurltielwhich affect the test reaults..

APPENDIX A( Clause 2.3)

DETERMINATION OF PARTICLE SIZE

Co",sporadi"1 Si,v,BS T~lt Sieve 22, ASTM SIeve 25 or

Tyler Teft Sieve 24BS Telt Sieve SO, ASTM Sieve 35 or

Tyler Telt Sieve 52SOO-micron

A-I. TEST SIEVESA-I.I Make a nest of two test sieves, the upper being 710-micron IS Sieveand the lower SOO-micron IS Sieve provided with a cover and receiver.

NOTB - In cue the prescribed IS Sieves are not available, the following correl­ponding sieves, which have their apertures within the limitl Ipecified for the prtlcribedone, may be used:

IS Si,v,'IO-micron

A-2. PROCEDUREA-2.1 Weigh accurately about 100 g of the material and transfer to theupper sieve. Fit the upper sieve with the cover, place the nest of sieves In asuitable mechanical sieve shaker and sieve the material continuously for fiveminutes. Stop sieving, remove the nest of sieves and transfer the residue oneach sieve separately to two tared weighing dishes using a brush. Weigheach dish.

A-3. CALOULATIONA-3.1 Calculate the percentage of the material retained on the respective­sieves as follows :

a) Material retained on 710-micron100 u.

IS Sieve, percent by mass - M

whereM 1 - mass in g of the material retained on 710-rnicron IS Sieve,

andM == mass in g of the material taken for the test.

tSpecificltion for w~ter, distilled quality ( "vis't!}

7

Moisture, percent by mass

IS I 4684 .1975

b) Material retained on SOO-micron100 AI..

IS Sieve, percent by mass = -- M -

where

M 2 == mass in g of the material retained on SOO-micron IS Sieve,and

M = mass in g of the material taken for the test.

APPENDIX B

[Table 1, Item (i) ]

DETERMINATION OF MOISTURE

B-1. PROCEDURE

8-1.1 Weigll accurately about 10 g of the material in a dish made ofporcelain, silica or platinum, previously dried in an air-oven and weighed.Place the dish in an air-oven maintained at 105 ± 2°C for five hours. Coolthe dish in a desiccator and weigh with the lid on. Heat again at 105 ± 2°0in the air-oven for 30 minutes. Cool the" dish in the desiccator and weigh.Repeat this process of heating for 30 minutes, cooling and weighing till thedifference in mass between two successive weighings is less than one milli­gram. Note the lowest mass.

NOTE - Preserve the dish containinv this dried material for determination of totalash ( s" D·1.1 ).

B-2. CALCU~ATION

B-%.1 Calculate the moisture as follows:

100 (Ml - Mt)M"l- M

where

M 1 = mass in g of the dish with the material before drying,M s ::::I mass in g of the dish with the material after drying, andM = mass in g of the empty dish.

8

IS 14684 • 1975

APPENDIX C[Table 1, Item (ii)]

DETERMINATION OF PROTEIN

o-r. APPARATUS0.1.1 A recommended apparatus, as assembled, is shown in Fig. I•.

0.1.1.1 Description - The assembly consists of a round-bottom flask A of1 OOO-ml capacity fitted with a rubber stopper through which passes one end

Flo, 1 ApPARATUS FOR D~TERt.llNATION0' PROTEI~

9

IS I 4684 - 1975

of the connecting bulb tube B. The other end of the bulb tube B is connectedto the condenser C which is attached by means of a rubber tube to a diptube D which dips into a known quantity of standard sulphuric acid contain-ed in a beaker E of 250-ml capacity.

e-l.2 Kjeldahl Flask - of capacity 500 ml.

oe. REAGENTS

C-2.1 Anhyclrou. SocUam Sulphate

0.2.2 Copper Sulphate

C.%.3 Concentrated Salpharic Acid - sp gr 1·84.

•::'2.4 Sodium Hydrodde Solation - Dissolve about 225 g of sodiumhydroxide in 500 ml of water.

0-2.5 8tuaclard Sulphuric Add - 0·1 N.

0-2.6 Methyl Red laclicator Solation - Dissolve I g of methyl red in200 ml of rectified spirit ( 95 percent by volume).

0-2.7 8taadard SocIi.. Hydroslde SolutioD - O· J N.

0-3. PROCEDURE

. 0-3.1 Transfer carefully about 0-25 g of the material, accurately weighed,to the Kjeldahl flask, taking precaution to see that particles of the materialdo not stick on to the neck of the flask. Add about 10 g of anhydroussodium sulphate, about 0·2 to O~3 g of copper sulphate and 20 ml of con­centrated sulphuric acid. Place the flask in an inclined position. Heatbelow the boiling point of the acid until frothing ceases. Increase the heatuntil the acid boils vigorously and digest for 30 minutes after the mixturebecomes clear and pale green or colourless. Cool the contents of the flask.Transfer it quantitatively to the round-bottom flask with water, the totalquantity of water used being about 200 ml, Add with shaking a few piecesof pumice stone to prevent bumping.

! Add about 50 ml of sodium hydroxide solution (which is sufficient tomake the, solution alkaline) carefully through the side of the flask so that itdoes not mix at once with the acid solution but forms a layer below the acidlayer. Assemble the apparatus as shown in Fig. 1, taking care that the diptube extends below the surface of known quantity of standard sulphuric acidcontained in the beaker. Mix the contents of the flask by shaking and distiluntil all ammonia has passed over into the standard sulphuric acid. Shutoff the burner and immediately detach the flask from the condenser. Rinsethe condenser thoroughly with water into the beaker. Wash the dip tubecarefully so that aU traces of the condensate are transferred to the beaker,

10

18 I eC684 • 1975

When all the washings have drained into the beaker, add two or three dropsof methyl red indicator solution and titrate with the standard sodiumhydroxide solution,0-3.1 Carry out a blank determination using all reagents in the samequantities but without the material to be tested.0-4. CALCULAnON0.4.1 Calculate protein from the following formula:

P · ( dry basis ) b 875 ( B - A ) Nrotem on ry asis ,percent y mass == M ( 100 _ m )

whereB == volume in ml of the standard sodium hydroxide solution

used to neutralize the acid in the blank determination;A m:: volume in ml of the standard sodium hvdroxide solution

used to neutralize the excess of acid in the test with thematerial;

N = normality of the standard sodium hydroxide solution;M = mass in g of the material taken for the test; andm = moisture, percent by mass, in the material (SI' B-2.1).

APPENDIX D[Table I, Item (iii) ]

DETERMINATION OF TOTAL ASH

1).1. PROCEDURE~1.1 Ignite the dried material in the dish (8-1.1) with the flame of asuitable burner for about one hour. Complete the ignition by keeping in amuffle furnace at 550 to 600°0 until grey ash results. 0001 in a desiccatorand weigh. Repeat the process of igniting, cooling and weighillJ at half-hourintervals until the difference in mass between two successive welghings is lessthan one milligram. Note the lowest mass.

NOT. - Preserve the dish containing this alh for the determination of acidinsoluble alh ( B.2. t ).

1).1. CALCULATION0-2.1 Calculate total ash as follows :

. 100(M.- M)Total ash ( on dry baSIS ), percent by mass == Ml _ Mwhere

Ma = mass in g of the dish with ~qe ash,

11

IS I 4684 - 1915

M = mass in g of the empty dish? andM 1 = mass in g of the dish with the dried material taken for the

test ( M 2 under B-2.1 ).

APPENDIX E[Table I, Item (iv)]

DETERMINATION OF ACID.INSOLUBLE ASH

s.r, REAGENT

E-I.1 Dilate Hydrochloric Acid - approximately 5 N, prepared fromconcentrated hydrochloric acid (see IS : 265-1962· ).

E-2. PROCEDURE

E-2.1 Add 25 ml of dilute hydrochloric acid to the ash contained in thedish (D-l.l), cover with a watch-glass and heat on a water-bath for 10minutes. Allow to cool and filter the contents of the dish through a What­man filter paper No. 42 or its equivalent. Wash the filter paper with wateruntil the washings are free from the acid. Return the filter and the residueto the dish. Keep it in an electric air-oven maintained at 135 ± 2°C forabout 3 hours. Ignite in a muffle furnace at 550 to 600°0 for one hour. Coolthe dish in a desiccator and weigh. Repeat the process of igniting in themuffle furnance, cooling and weighing at half-hour intervals until thedifference in mass between two successive weighings is less than one milli­gram. Note the lowest mass.

E-3. CALCULATION

E·3.1 Calculate the ash content from the following formula:

Acid-insoluble ash (on dry basis ),percent by mass =1OO.J:1~-J'l)

where

M" = mass in g of the dish with the acid-insoluble ash,M = mass in g of the empty dish, andM 1 = mass in g of the' dish with the dried material (M. under

B-2.1 ).

•Specification for hydrochloric acid ( "vised).

IS : 4684.197~

APPENDIX F[ Table 1, Item (v) ]

DETERMINATION OF FAT

F-l. APPARATUS'.1.1 Soxhlet ExtractioD ApparatusF-2. SOLVENTF-2.1 Ethyl Ether, or Petroleum Ether --..distilling below 65°0.F-3. PROCEDUREF-3.1 Transfer about 109 of the material accurately weighed to a suitablethimble and extract with the solvent in a Soxhlet extraction apparatus forabout 16 hours. Dry the extract contained in the Soxhlet flask, whose emptymass has been previously determined at 95 to 1000 e for 30 minutes. Cool ina desiccator and weigh. Continue drying and weighing alternately atSO-minute intervals until the loss in mass between two successive weighingsis note more than one milligram. Record the lowest mass.

NOTE - Preserve the material in the thimble for estimation of crude fibre ( se«Appendix H ).

F-4. CALCULATIONF-4.1 Calculate the fat as follows:

, 100 ( M 1 - M 1 )Fat, percent by mass = M --- ._-

where

Ml == mass in g of the Soxhlet flask with the extracted fat;M2 =-mass in g of the empty Soxhlet flask, clean and dry; andM == mass in g of the material taken for the test.

APPENDIX G[Table 1,Jtem (vi)]

DETERMINATION OF ACID VALUE OF EXTRACTED FAT

G-l. APPARATUSG-l.1 Soxhlet Fat Extraction ApparatusG·2. REAGENTSG-2.1 PetroleUID Ether - distilling below 65°C,

)3

18 , 4684 • 1975

o.2.Z Benzene-Alcohol.Phenolphthaleln Solution - Add 0·4 g ofphenolphthalein to one litre of benzene and one litre of ethyl alcohol to form0'02 percent solution.

0-2.3 Standard Pota••lum Hydroxide Solution - 0'2 N, carbondioxide-free.

0-2.4 Standard Pot•••lum PermaDgallate Solution - 0·01 percent.

Q.3.PROCEDURE

0-3.1 Extract 10'00 ± 0'01 g of the sample taken in a thimble with petro­leum ether for about 16 hours in a Soxhlet extraction apparatus. Completelyevaporate the solvent from extraction flask ( weighed previously) on a steam­bath. cool and weigh the Soxhlet flask with the residue and from the diffe­rence of the mass find out the mass of fat obtained from the material.Dissolve the residue in the extraction flask with 50 ml of the benzene-alcohol­phenolphthalein solution, Titrate the dissolved extract wlth standardpotusium hydroxide solution to distinct pink colour, or in the case of yellcwsolution to orange pink colour, If emulsion is formed during titration, dispelby adding second 50-mJ portion of the benzene-alcohol-phenolphthaleinsoluqon. The end-point should match colour of the solution made by adding2·5 ml of standard potassium permanganate solution to 50 ml of potassiumdichromate solution of proper strength to match colour of orlglnalaolutionbeing titrated. (Add O'S percent potassium dichromate solution dropwise to50 ml of water until the colour matches. Then add 2'5 ml of standardpotassium permanganatc solution, )

0-3.2 Make a blank titration on 50 ml of the benzene-alcohol-phenol­phthalein solution and subtract this value from the titration value of thesample, If the additional SO-ml portion of the benzene-alcohol-phenolphtha­lein solution is added, double the blank titration.

0.4. CALCULATION

0.4.1 Calculate the acid value from the following formula:

Acid I ( I· 'd ) 56'4 VNC1 va ue as 0 ere aci ...~-

where

Y CII volume in ml of standard potassium hydroxide solutionused,

N - normality of standard potassium hydroxide solution. andM .. mass in g of the material taken for the test.

14

IS a 4684 - 197~

APPENDIX H

[Table 1, Item (vii) ]

DETERMINATION OF CRUDE FIBRE

H-l. REAGENTS

H-l.1 Dllate Salphuric Acid - 1'25 percent (m/v), accurately prepared.

H-l.~ SocIIam Hydroxide Solution - 1'25 percent (mlv), accuratelyprepared.

8-1.3" Ethyl Alcohol - 95 percent by volume,

a-l.4 Petroleam Ether - distilling below 65°0.

H-~. PROCEDURE

B-2.1 Dry to constant mass about 5 g of the material on an electric oven at105 ± 1°C. Weigh accurately about 2'5 g of the dried material on a thimble,extract it' with petroleum ether free of fat on a Soxhlet apparatus and trans­fer it to a one-litre flask. Take 200 ml of dilute sulphuric acid in a beakerand bring to the boil. Transfer the whole of the boiling acid to the flask andimmediately connect the flask with a water-cooled reflux condenser andheat, so that the contents of the flask.begin to boil within one minute, Rotatethe flask frequently, taking care to keep the material from remaining on thesides of the flask and out of contact with the acid, Continue boiling forexactly 30 minutes. Remove the flask and filter through fine linen (about18 threads to a centimetre) held in a funnel, and wash with boiling wateruntil the washings are no longer acid to litmus. Bring to the boil 200 ml of.sodium hydroxide solution under a reflux condenser, Wash the residue onthe linen into the flask with the boiling sodium hydroxide solution,Immediately connect the flask with the reflux condenser and boil for exactly30 minutes. Remove the flask and immediately filter through the filteringcloth. Thoroughly wash the residue with boiling water, and transfer to aGooch crucible prepared with a thin but compact layer of ignited asbestos.Wash the residue thoroughly first with hot water and then with about 15 mlof ethyl alcohol. Dry the Gooch crucible and contents at 105 ± 2°0 in anair-oven to constant mass, Cool and weigh. Incinerate the contents of theGooch crucible in an electric muffle furnance at 600 ± 2°0 until all thecarbonaceous matter is burnt, Cool the Gooch crucible containing the ashin a desiccator and weigh,

15

IS : 4684 • 1975

H-3. CALCULATION

H-3.1 Calculate the crude fibre as given below:

C d fib ( b . ) b 100 ( M 1 - M g )ru e re on dry asis ,percent y mass = -----M-- ---- -where

M 1 = mass in g of the Gooch crucible and contents beforeashing;

M I ::::a mass in g of the Gooch crucible and contents after ashing;and

M = mass in g of the dried material taken for the test.

APPENDIX J[Table 1, Item (viii)]

DETERMINATION OF AFLATOXIN

J-l. PRINCIPLE OF THE METHOD

J-1.1 The method involves direct extraction of a water-wetted sample bychloroform with the usc of silica gel column for defatting and clean up forobtaining clean quantitative extracts of aflatoxin from 50 g of groundnutproduct samples for thin-layer chromatography. The low level of interferencepermits the determination and detection of as little as I llg of aflatoxin perkilogram of groundnut product sample.

J-2. APPARATUSJ-2.1 Disc MillJ-2.2 Wrist-Action Shaker -having a capacity of 1 400 to 1 600 rev/min.J-2.3 Chromatographic Column- of silica gel.J-2.4 FUDnel-having a diameter of 150 rom to fit fluted filter paper.Buchner funnel of 320 mm in diameter to fit Whatman No. 1 or equivalentfilter paper may also be used.)-2.5 Rotary Evaporator with Continuous FeedJ-2.6 Thin-Layer Chromatographic Apparatus

J-2.6.1 Glass Plates - 200 X 200 mm.J-2.6.2 Applicatorj-2.6.3 Mounting BoardJ-2.6.4 Spotting Template

16

IS : 4684 • 1975

J-2.6.5 Microsyringe - 10 (11.J-%.6.6 Desiccating Storage Cabinet)-2.6.7 Storage RackJ-2.6.8 Lone WavI 15-Watt Ultraviolet Lamp or Chromato-Vieui Cabinet­

equipped with one or two 15-watt lamps.

J-3. REAGENTS

J-3.1 Snlca Gel - for chromatography 0·15 to 0'2 mm,

J-3.2 Chloroform

)-3.3 Hexane

J-3.4 Diethyl Ether

J-3.5 Methyl Alcohol

J-3.6 Acetone

J-3.7 Sodium Sulphate (Anhydrous)

J-3.8 Bolling Chips

)-3.9 A8atoxin Standard Solution - Prepare from pure crystallineaflatoxine solutions containing O'5 fJ-g of aflatoxin Bland 0"4 -s of aflatoxinG 1 per millilitre of chloroform. If pure crystalJine aflatoxin is not available,use extracts of known aflatoxin content. Concentrations of 0'5 to I·5 fl·g ofB1 and 0·3 to 1·0 llg of 0 1 per millilitre arc satisfactory. Keep exposure tolight at minimum. Store at freezing temperature in glass stopper volumetricflask. Place in a closed jar containing a small amount of chloroform whichshould be replaced periodically. Chloroform in outside jar eliminatesconcentration of standard solution by evaporation and the jar preventscontamination with condensing water as standard solution is warm to roomtemperature each time it is used.

J-4. PROCEDURE

J-4.1 Preparation of the Silica Gel Plate - Weigh 30 g of silica gel intoa 300-ml glass stoppered Erlenmeyer flask. Add 60 rnl of distilled water.Shake vigorously for not more than a minute and pour into the applicator.Immediately coat five 200 X 200 rum glass plates with O·25-mm thicknessof silica gel suspension, and let the plates rest undisturbed until gelled. Drythe coated plates for at least 2 hours at 80°C and store in the desiccatingcabinet with active desiccant until just before use.

J-4.1 Preparation of the Sample - Groundnut butters and ground nutmeals need no preparation for extraction unless they contain large particles,in which case some type of milling operation should be used to reduce the

17

'. IS 1 4684 • 1975

particle size. A hammer mill of rotary cutter is used for meals. Samplesof raw and roasted groundnut should be ground to a paste before extraction.For this operation and for chunky-type groundnut butter, a disc-type mill isused.

J-4.3 ExtractloD - Weigh a: 50-g sample (groundnut butter, groundnutmeal, or finely ground groundnuts ) into a 500-nl1 glass-stoppered Erlenmeyerflask. And 25 ml of distilled water and mix with a spatula until visuallyuniform. Add 250 ml of chloroform and tape the stopper in place. Shakefor 30 minutes on a wrist-action shaker and filter through a fluted filter paper.Collect the first 50-ml portion of clear chloroform. This is the sampleextract. Prepare the silica gel column by placing a ball of glass wool looselyin the bottom of a 22 X 300 mm chromatographic column and add 5 g ofanhydrous sodium sulphate to give an even base for the silica gel. Addchloroform until the column is about two-thirds full; then add 10 g of silicagel ( 0·05 to 0'20 mm) with stirring to prevent air entrapment. Let silicagel settle and slowly add 15 ~ of anhydrous sodium sulphate. Draw offchloroform to the top of the sihca gel. Add the sample extract to the columnand elute at a flow rate of 10 to 20 ml per minute with 150 ml of hexanefollowed by 150 mlof anhydrous diethyl ether. Finally, elute the aflatoxinwith 150 ml of methyl alcohol or chloroform (3/97, v/v). Collect thisfraction from the time the methyl alcohol or chloroform was added until theflow stops in a vial, evaporate to near dryness and cap it.

1-4.3.1 The sample extract may now be prepared in the appropriatemanner for quantitative assay.

J-4.4 Q,uaUtadve Determination - For preparing sample extract, uncapthe vial containing dry sample extract. Add to it 500 III of chloroform, andreseal with polyethylene stopper. Puncture polyethylene stopper to accommo­date the needle of 10 III syringe. Spot as rapidly as possible in subduedincandescent light 2, 5 and 10 IJ-l on imaginary line 4 em from bottom edgeof thin-layer chromatography plate. In the presence of large quantities ofextraneous material, 10 IJ-l of the extract may not be used. Reserve the vialfor quantitative analysis. On the same plate, spot 1, 3 and 5 1'1 standardaflatoxins solution. Also spot 5 iJ'1 of standard solution containing aflatoxins BI ,

B., G1 and G, to show that the four aflatoxins are properly resolved underconditions used. Draw line across the plate 2 to 3 em from top edge as stopfor solvent front. Also draw lines 0·5 em from each side edge. Immediatelyinsert plate into unlined and unequilibrated tank containing acetone/chloro­form { 1/9) v/v) as the developing system. (Position the plate to exposecoated surface to maximum tank volume and seal.) Withdraw the platefrom tank when solvent front reaches stop line, 12 to 14 em above origin.Let solvent evaporate and illuminate plate from below by placing it flat,coated side up, on longwave ultraviolet lamp in darkened room. Alternatively,view plate in chromate-view cabinet or illuminated from above. (If

18

IS I 4684- 1915

illumination requires looking directly at lamps, protect eyes with ultravioletabsorbing filter, such as Eastman 2A filter. ) Observe pattern of 4 fluores­cent spots of qualitati ve standard, In the order of decreasing Rf theseareaflatoxins Bit BI , G 1 and Gsa' Note small colour difference, bluish fluores­cence 'of C B t contrasted with slightly green CG ' aflatoxins. Examine patternsfrom sample for fluorescent spots having identical Rf and similar appearancesto those of standards, From this preliminary plate, estimate suitable dilu­tion for quantitative thin layer chromatography analysis. Take into accounta quantity of the extract used for preliminary thin layer chromatography infinal calculations.

NOTB - If four clearly identifiable spots are Dot I visible in the qualitativestandard, repeat chromatography, correcting or adjusting conditions to obtain properresolution.

J-4.5 Q,1IUltltadve DetermmatloD - Spot successively 3'5-, 5-, and two6'5-,..,1 portions of sample extract, All spots should be of the same size andshall not be more than 0'5 em in diameter, On the same plate spot 3'5, 5,6'5 fJ'1 of B1 and G 1 standards, As internal standard spot 5",.1 of B1 andG1 on top of one of the two 6'5 (Jol portions of sample. Also spot 5 ,..,1 ofBl , Bit G J and Gsa as qualitative standard, Proceed as given under qualita-tive analysis (s" J-4.4 ). .

J-S. CALCULATIONS

J-5.1 mterpretatloD of the Chromatop-am - Examine the patternfrom the sample spot containing internal standard for aflatoxin B1 and G1spots. Rf values of B1 and G1 used as internal standard should be the sameas, or may differ only very slightly from those of respective standard aflatoxinspots. (Since' spots from sample extract are compared directly with standardaftatoxin on the same plate, magnitudes of Rf are unimportant. These varyfrom plate to plate. ) Compare sample patterns with that containing internalstandard. Fluorescent spots in sample thought to be B1 or 0 1 should haveRf values identical to end colour and similar to be B1 or G1 used as internalstandard. ldentify unknown spot of sample as B1 or 0 1 only when unknownspot and internal standard spots are superimposed. Spot from sample andinternal standard combined should be more intense than either sample orstandard alone. Compare sample pattern with qualitative standard todetermine if B. and G. are present. Compare fluorescent intensities of B1spot of the sample with those of the standard spots and determine which ofthe aample portions matches one of the standard. Ultraviolet light. maybe attenuated by moving the plate away from lamp so that any particularpair of spots ~ay be compared at extinction. Interpolate, if sample spotIntensity is found to be between those of two standard spots. If spots oflIDaUeat portion of sample are too intense to match standards, dilute sampleand rechromatograph. Compare G J spots in the same manner. Assumethat B1 and B. have same fluorescent intensity to mass relationships andcompare' B. spots of sample with B1 standard spots to make quantitative

19

IS 14684 - 1915

estimate of B,.. Likewise, assume 0 1 has the same fluorescent intensity tomass relationship as G 1 and compare G 1 spat of the sample with G, spotof the standard.

J-5.1.1 Calculation - Calculate aflatoxin from the following formula:

Aflatoxin B1 in Ilg{kg = S X r X rX M

where

S = ~l of aflatoxin B1 standard equal to unknown;r = concentration of aflatoxin B1 standard !Jog/ml;V = volume in (1.1 of final dilution of sample extract;X = III of sample extract spotted giving fluorescent intensity equal

to S, the aflatoxin B1 standard; andM:::2 mass in g of sample applied to silica column.

NOTB - For 10 g of sample extract 50 ml of aqueous methyl alcohol extract isused. If final extract dilution does not represent 10 g, calculate correct sample massand substitute.

j-5.1.1 Calculate aflatoxin G1 as given above for aflatoxin Bill

J-5.1.3 Calculate aflatoxin B2 as 'lJ,g aflatoxin B2 per kilogram based onfluorescence of aflatoxin B1 '.

j-5.1.4 Calculate aflatoxin G 2 as C flg aflatoxin G g per kilogram basedon fluorescence of aflatoxin G 1 '.

20

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