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है”ह”ह

IS 7235 (1974): Method for Estimation of Tocopherols(vitamin E) in Foodstuffs [FAD 16: Foodgrains, Starches andReady to Eat Foods]

Indian Standard

IS : 7235 - 1974

METHOD FOR ESTIMATION OF TOCOPHEROLS (VITAMIN E) IN FOODSTUFFS

Food Hygiene, Sampling and Analysis Sectional Committee, AFDC 36

Chairman Representing

MAJ-GEN M. S. BOPARAI Directorate General Armed Forces Medical Services ( Ministry of Defence ), New Delhi

Members

AGRICULTURAL M AR IZ E T I N G Directorate of Marketing & Inspection ( Ministry ADVISER TO THE GOVERNMENT of Agriculture ), Faridabad OB INDIA

SARI T. V. MATHEW (Alternate) SHR~ V. N. ADZBLE Institute of Agricultural Research Statistics

( ICAR ), New Delhi SERI K. S. KRISHNAN ( Alternate )

SHRI K. BALASUBRAMANIAN Public Analyst, Government of Tamil Nadu, Madras

LT S. L. CHADHA Health Department, Municipal Corporation of Delhi

DR A. J. AJWANI ( Alternate ) DR G. C. DAS Corporation of Calcutta DR P. K. DATTA All India Institute of Hygiene and Public Health,

Calcutta SHRI SUKUMAR DE National Dairy Research Institute ( ICAR ), Karnal

DR C. A. MULAY ( Alternate) DIRECTOR Central Food Laboratory, Calcutta EXECUTIVE HEALTH OFFICER Bombay Municipal Corporation

MUNICIPAL ANALYST ( Alternate ) HEALTH OFFICER Corporation of Madras DR ( SMT) S. KHOSLA Department of Health & Family Planning, Govern-

ment of Punjab, Chandigarh DR P. K. KY~WAL Food & Nutrition Board ( Ministry of Agriculture ),

New Delhi DR R. C. SINRA ( Alternate)

SHRI Y. N. LAKSHMAN Defence Food Research Laboratory (Ministry of Defence ), Mysore

DR G. N. VERMA ( Alternate) SHI~I F. G. T. MENEZES Directorate of Sugar & Vanaspati (Ministry of

SHRI D. VENKATAPPAIA ( Alternate ) Agriculture ), New Delhi

( Continued on page 2 )

@ Copyright 1974

INDIAN STANDARDS INSTITUTION

This -publication is protected under the Indian Copyright Act ( XlV of 1957) and reproduction in whole or in part by any means except with written permission of the publisher shall be deemed to be an infringement of copyright under the said Act.

IS:7235-1974

( Continued from page 1 )

Members Representing

PUBLIC ANALYST (FOOD AND Government of West Bengal, Calcutta WATER )

DR T. N. RAMCH~NDR~ RAO Central Food Technological Research Institute (CSIR ), Mysore

SRRI C. T. DWARKaNATR (Alternate) DR RANJIT SEN Directorate General of Health Services ( Ministry

of Health & Family Planning), New Delhi SENIOR COM~XERCIAL OFFICER, Chief Catering Officer, Northern Railway,

CATERING New Delhi COL K. N. SRARMA Quarter Master General’s Branch, Army Head-

LT-COL 0. P. KAPUR (Alternate) quarters

DR S. B. SINQH Public Analyst, Government of Uttar Pradesh, Lucknow

DR M. C. SWAMINATHAN Central Committee for Food Standards, New Delhi SHRI D. S. CHADHA (Alternate)

SHRI P.G.VIN The Coca-Cola Export Corporation, New Delhi SHRI J. D. CONTRACTOR (Alternate )

DR HARI BHAGWAN, Director General, IS1 (Ex-ogicio Member) Director ( Agri & Food )

Secretary

SSRI SOHRAB Assistant Director ( Agri & Food ), IS1

Vitamin and Amino-Acids Assay Subcommittee, AFDC 36 : 3

Convener

DR T. A. V. SUBRAMANIAN

Members

DR(SMT)BHAVANIBELAVADY DR P.K.DATTA

SHRIH.S.GURUDAS DR V. S. KRISHNAnlACIIAl? SRRID.MITRA

DR N. S.NAIK

DRN. A. NITYANANDA RAO SRRI S. S. ARYA (Alternate)

DRP.B.RAMARAO

Vallabhbhai Pate1 Chest Institute, Delhi

National Institute of Nutrition, Hyderabad All India Institute of Hygiene and Public Health,

Calcutta Voltas Limited, Bombay National Chemical Laboratory, Poona Public Analyst, Government of Uttar Pradesh,

Lucknow Indian Agricultural Research Institute (ICAR),

New Delhi Defence Food Research Laboratory, Mysore

Central Food Technological Research Institute, Mysore

DR S. VENKATA RAO (Alterrlate) Da R. V. RAO National Dairy Research Institute, Karnal DR V. K. MOHAN RAO Central Drug Research Institute ( CSIR ), Lucknow SHRI RUP KTSHOIXG Directorate of Sugar & Vanaspati, New Delhi

SHIU D. VENIL4TAPlfA~AH (Alternate)

1S : 7235 - 1974

Indian Standard METHOD FOR ESTIMATION OF

TOCOPHEROLS ( VITAMIN E) IN FOODSTUFFS I

0. FOREWORD

0.1 This Indian Standard was adopted by the Indian Standards Institution on 2 1 January 1974, after the draft finalized by the Food Hygiene, Sampling and Analysis Sectional Committee had been approved by the Agricultural and Food Products Division Council.

0.2 Vitamins are required to be assessed in a large number of foodstuffs, such as processed cereals, dairy products, animaI feeds and other natural or manufactured foodstuffs. Moreover, diierent methods of vitamin assay are used in different laboratories. Therefore, with a view to establishing uniform procedures and also for facilitating a comparative study of results, IS1 is bringing out a series of standards on vitamin assays. These would include chemical as well as microbiological methods, wherever applicable.

0.3 Keeping in view the facilities available in the country, onIy the chemical method has been included in this standard and the biological method has not been covered.

0.4 In reporting the result of an analysis made in accordance with this standard, if the final value, observed or calculated, is to be rounded off, it shall be done in accordance with IS : Z-1960*.

1. SCOPE

1.1 This standard prescribes a chemical method for estimation of tocopherols ( vitamin E ) in foodstuffs.

2. QUALITY OF REAGENTS

2.1 Unless specified otherwise, pure chemicals and distilled water ( see IS : 1070- 1960t ) shall be employed in tests,

NOTE - ‘ Pure chemicals ’ shall mean chemicals that do not contain impurities which affect the test results.

*Rules for rounding off numerical values ( revised). jSpecification for water, distilled quality ( reuised ).

3

IS: 7235-1974

3. FORMS OF THE TOCOPHEROLS

Structure ~omenclature

5, 7, 8-trimethyl a -tocopherol

5, 8-dimethyl ~-tocopherol

7, 8-dimethyl y-tocopherol

8-methyl $-tocopherol

4. PREPARATION OF ASSAY SAMPLE

4.0 The technique used for preparing the material for the analysis is commonto every vitamin determination. It should be ensured that the sample takenfor the assay is representative of the whole, and that any deterioration of thevitamin to be examined is prevented.

4.1 Powders and liquids are mixed thoroughly until homogeneity is achieved.Dry materials, such as bread, biscuits, and rice, are ground and mixedthoroughly.

4.2 Butter may be melted under constant stirring. Samples drawn frommargarine or cheese or other such foods should contain portions of thesurface as well as of the interior.

4.3 Wet or fresh material may be minced with a knife or scissors, or homo-genized in a blender, if necessary, in the presence of the extracting solvent.

5. METHOD

5.0 General — The determination of vitamin E is carried out by calori-metry. Vitamin E is oxidized by ferric chloride and the ferrous chloride soformed gives, on reaction with K, a-dlpyridyl, a red colour which is measuredat 515 nm.

5.1 The procedure allows separation and estimation of a- and ~-tocopherolfrom the other tocopherols, and can also determine the total tocopherolcontent.

5.2 Principle

5.2.1 u- Toco@erol — The extract containing total tocopherol is purfledfrom interfering substances, such as vitamin A and carotenoids by reductionwith zinc and treatment with antimony trichloride, and fats are eliminatedby saponification. a. and ~-tocopherols are separated together fi-om the othertocopherols by chromatography on alumina and determined calorimetricallywith iron-dipyridyl. Calculation is carried out with the aid of a calibrationcurve and a recovery test.

4

..——---------- -,1

.,f ““:

‘&“<L

‘

IS : 7235 - 1974

5.2.2 Total Tocopherol- The p- , y- , 6- , E- , and q-tocopherok stiIl adsorbed on the alumina are consequently eluted together and determined calorimetrically ( see 5.2.1 ). The sum of the results obtained by both assays gives the total tocopherol content.

5.3 Apparatus

5.3.1. Chromatographic Column - See Fig. 1.

T- 197

I 14$ -

SINTERED GLASS

GROUND GLASS J Tn

w All dimensions in millimetres.

FIG. 1 CHROMATOGRAPHIC COLUMN

5.3.2 Photoelectric Colorimeter - for taking reading at 515 nm.

5.3.3 Blender

5.3.4 Stop-Watch

IS : 7235 - 1974

5.4 Reagents

54.1 Methanol - Distil 10 litres of methanol over 5 g of potassium permanganate and 10 g of potassium hydroxide.

5.4.2 Petroleum Ether - Boiling point 30 to 40°C or 40 to 60°C or 90 to 115°C distilled over concentrated sulphuric acid.

5.4.3 Ethanol -Absolute, purified like methanol ( 5.4.1), and distilled repeatedly until no fluorescence can be detected in ultra-violet light.

5.4.4 Diethyl Ether - free from peroxide, treated with ferrous sulphate.

5.4.5 El&ion Mixtures

5.4.5.1 Mixture of 100 ml of diethyl ether ( 5.4.4 ) and 100 ml of petroleum ether ( 5.4.2 ).

5.4.5.2 Mixture of 99 ml of cyclohexane ( 5.4.16 ) and 1 ml of diethyl ether ( 5.4.4 ),

5.4.5.3 Mixture of 15 ml of diethyl ether ( 5.4.4 ) and 85 ml of petroleum ether ( 5.4.2 ) .

5.4.6 Sodium Ascorbate - pure, powder,

5.4.7 Methanolic Potassium Hydroxide - approximately 1 N, prepared shortly before use.

5.4.8 Aluminium Oxide for Chromatography - Activate the aluminium oxide powder by heating at 400°C for 2 to 4 hours and allow to cool while being protected from moisture. Shake 100 g of the powder with 10 to 15 ml water till all clots disappear and the powder flows freely. The de-activated alumina is stable for one day and should be prepared at Ieast one hour before use.

NOTE Y It is essential to standardize the activity of each lot of alumina. The test is carried out with vitamin A which is added to column and the column is developed with benzene or hexane. Vitamin A is located either by antimony chloride reagent or under ultra-violet light. Suitability of alumina should be checked with pure a-tocopherol and with b- or y-tocopherols.

5.4.9 Ferric Chloride - 0.2 percent alcoholic solution. ferric chloride in 100 ml of ethanol ( 5.4.3 ).

Dissolve 0’2 g of The solution is sensitive to

light. It should be prepared shortly before use and stored in brown bottle and kept away from light.

5.4.10 a+-Dipyridyl- 0.5 percent, alcoholic solution, 0’5 g a,a-dipyridyl is dissolved in 100 ml of ethanol ( 5.4.3 ) .

5.4.11 Antimony Trichloride - Add 22 g of antimony trichloride to 100 ml of chloroform and reflux until complete dissolution is achieved. When cold, keep the reagent in a brown bottle over highly activated aluminium oxide. Agitate and filter before use.

5.4.12 Hydrochloric Acid

5.4.12.1 Hydrochloric acid, concentrated - 38 percent.

6

IS : 7235 - 1974

5.4.12.2 Hydrochloric acid- ApproximateIy 1 N. Add 94 ml of concentrated hydrochloric acid to 1 Iitre of distilled water.

5.4.13 zinc - powder, pure.

5.4.14 Ammonium Hydroxide Solution - 10 percent.

5.4.15 Enzymes

5.4.15.1 Lipase

5.4.15.2 Clarase 900

5.4.15.3 Pig pancreatic powder

5.4.16 Cyclohexane - pure, distilled.

5.4.17 Water - The distilled water used for washing the diethyl ether- petroleum ether extracts should be saturated with a mixture of equal parts of the two solvents.

5.4.18 Vitamin E Test Solution - Rapidly weigh a known amount (90 to 100 mg ) of pure a,a-tocopherol in a 100 ml volumetric flask, immediately dissolve in ethanol ( 5.4.3 ), and make up the solution to volume with the same solvent. The solution can be kept for 5 to 10 days at 4°C in the dark, Dilute ten times with ethanol before use.

5.5 Procedure for the Extraction of Vitamin E from Preparations Containing the Vitamin in Water Dispersed Form

5.5.1 Vitamin E is extracted from these products according to the Roese-Gottlieb procedure as follows:

40 ml of the liquid is mixed in a 250-ml glass-stoppered cylinder with the following reagents and shaken thoroughly after each addition:

10 ml of 10 percent ammonia solution 40 ml of ethanol, absolute 80 ml of diethyl ether 80 ml of petroleum ether

The mixture is let stand for at least 15 minutes in the dark. When complete separation of the layers is achieved, the total volume of the ether-petroleum ether solution is determined, and an aliquot thereof is evaporated carefuhy, and the residue further treated as described in 5.6.2.4.

5.5.2 An aliquot of the ether-petroleum ether extract containing at least 100 micrograms of vitamin E is evaporated in vacuum and the residue, depending on its content of fat, is further treated as described in 5.6.2.1 or 5.6.2.3.

5.6 Procedure

5.6.1. Calibration Curve - Measure increasing amounts ( 0 to 200 micro- grams of vitamin E ) ‘of the test solution ( 5.4.18) each into a lo-ml volumetric

7

f

IS:7235 -1974

flask and dilute with ethano1 up to a volume of 8 ml. Add 1 ml of a,a-drpyridyl solution ( 5.4.10) and 1 ml of ferric chloride solution ( 5.4.9) consecutively to the flask. Immediately on the addition of the ferric chloride solution, set the stop-watch in motion. Set the calorimeter to the zero point with ethanol ( 5.4.3 ) and after two minutes measure the absorption of the vitamin E solution at 5 15 nm.

NOTE- It is essential that the calibration curve runs concurrently with the sample. The procedure should be carried out in non-actinic light.

5.6.2 Extraction of Tocopherols from Foods

5.6.2.1 Dry products with low total lipid content - Weigh accurately 10 g ( if a larger sample is needed, the quantity of the solvents should be increased accordingly ) of sampIe, containing at least 1 mg of vitamin E, in a non-actinic glass vessel. After addition of about 100 mg each of sodium ascorbate ( 5.4.6) and of the three enzymes ( 5.4.15 ), stir the sample into a paste with 35 ml of warm ( 70°C ) distilled water and let the flask stand for 20 minutes on a water-bath at 45°C in the dark. Then agitate the mixture with 6’5 ml of ammonia ( 5.4.14 ) and with 35 ml of ethanol ( 5.4.3 ) and cool to room temperature. Shake thoroughly with 70 ml of diethyl ether (5.4.4 ) followed by gentle agitation with 70 ml of petroleum ether ( 5.4.2 ), and allow to stand until separation of the layers occurs. Separate the layers. Remove the alcohol contained in petroleum ether layer by washing approximately 100 ml of the later twice each time with 10 ml of water ( 5.4.17). Measure an aliquot of the washed extract containing at least 200 micrograms of total tocopherol into each of two round-bottomed flasks. To one of the flasks add an amount of standard vitamin E solution ( 5.4.18 ) corresponding to its expected cc-tocopherol content,

5.6.2.2 Dry products containing lipids- Extract the foods as given in 5.6.2.1 and evaporate the extract in the recovery flask in vacuum or under nitrogen and saponify the samples ( see 5.6.2.4).

5.6.2.3 Fats and oils -Weigh exactly ( butter, etc ) 5 to 10 g of sample in a lOO-ml volumetric flask, dissoIve in petroleum ether ( 5.4.2 ) and bring up to volume with the same soIvent. Measure an aliquot of the solution, corresponding to a total tocophero1 content of at least 100 micrograms and to a fat amount not exceeding 3 g ( if a larger amount of fat is needed, the quantity of reagents and solvents necessary for further treatment should be increased accordingly), twice into two separate round-bottomed flasks and treat with about 100 mg of sodium ascorbate ( 5.4.6 ) . In one of the flasks ( Recovery Test ), add an amount of vitamin E test sohrtion ( 5.4.18 ) corresponding to the a-tocopherol content of the assay solution. Subsequently, treat both normal and recovery tests in the same way. Evaporate the solvent in avcuum and saponify the residue ( see 5.6.2.4 ) .

f$ : 7234 - 1974

NOTE - Peroxides eventually present in fats may destroy the vitamin E during the assay and should be removed as follows : Take up the residue in 10 ml of elution mixture ( 5.4.5.3 ), filter the solution through a 5-cm high column of alumina ( 5.4.8), and wash out total tocopherol out of the column with 80 ml of elution mixture ( 5.4.5.3 ). Saponify the residue obtained after evaporation of the filtrate in vacuum and treat further as described in 5.6.2.3.

5.6.2.4 SaponiJcation - Add 15 ml of methanolic potassium hydroxide solution ( 5.4.7 ) to the residue obtained from 5.6.2.2 and 5.6.2.3 and add 100 to 200 mg sodium ascorbate powder ( 5.4.6 ) . Reflux for 15 minutes in a water-bath at 60 to 70°C. The mixture should be stirred from time to time ( a magnetic stirring is suitable ) .

5.6.2.5 Extraction of the unsaponifiable matter - Cool the soap solution to approximately 40°C and transfer quantitativeIy into a 250-ml glass-stoppered cylinder with 50 ml of methanol ( 5.4.1) . Then cool it to room temperature and extract by thoroughly shaking it with 100 ml of petroleum ether ( 5.4.2 ) . As soon as the layers have separated add 10 ml of distilled water and repeat the shaking. ( Only after the addition of water, the quantitative separation of the methanol-petroleum ether mixture is achieved. ) Remove the aqueous layer by suction and wash the extract first with 20 ml of distilled water, then

, with 20 ml of hydrochloric acid ( 5.4.12.2 ).

5.6.2.6 Purijication of the extract - The extraction obtained directly from dry products containing low amount of lipids ( 5.6.2 ) of extract of unsopanifiable matter is evaporated in vacuum or under nitrogen. Mix the residue thoroughly with 3 ml of antimony trichloride ( 5.4.11). After 5 minutes, add 10 ml of ethanol ( 5.4.3 ), 10 ml of concentrated hydrochloric acid ( 5.4.12.1)) and about 50 mg of zinc powder ( 5.4.13 ) . Repeat the addition of zinc powder two or three times until the solution becomes colourless. ( During this operation the flask should be cooled in order to prevent overheating of the mixture above 40°C. ) After this treatment quantitatively transfer the mixture to a 250-ml glass-stoppered cylinder with 30 ml of ethanol ( 5.4.3 ) and thoroughly shake with 50 ml of diethyl ether ( 5.4.4 ). Then agitate it with 50 ml of petroleum ether (5.4.2 ) and, after the layers have separated, with 50 ml of distilled water. The impurities ( products formed after reaction with antimony trichloride and some zinc powder) contained in the extract should be removed before the assay is continued. Therefore, after separation of the layers has occurred filter an aliquot of the ether-petroleum ether extract corresponding to 50 to 150 micrograms of total tocopherol, through a 5-cm high column of alumina ( 5.4.9 ) previously soaked with eluting mixture ( 5.4.5.1). Wash out total tocopherol of the column with 80 ml of eluting mixture ( 5.4.5.1) .

5.6.3 Determination of cr-Tocopherol

5.6.3.1 Chromatography -Evaporate the filtrate obtained under 5.6.2.3 or an aliquot containing 50 to 100 micrograms of total tocopherol in vacuum

9

IS : 7235 - 1974

and take up the residue in 20 ml of cyclohexane ( 5.4.16 ). Fill up the chromatographic tube to a height of about 12 cm by pouring slurry of alumina in cyclohexane ( 5.4.16 ). Add cyclohexane ( 5.4.16 ) through until it covers the alumina for about 1 cm.

The cyclohexane solution of the sample is adsorbed on the alumina and washed with 60 ml of cyclohexane ( 5.4.16 ). Replace this cohecting flask by a clean one and elute the fraction containing u- and P-tocopherol with 130 ml of elution mixture ( 5.4.5.2 ) . The other tocopherols remain adsorbed on the alumina ( see 5.6.3 ) .

5.6.3.2 Colour reaction and measurement - Evaporate the eluate under reduced pressure or in a stream of nitrogen and take up the residue in 8 ml of ethanol ( 5.4.3 ). Carry out the addition of 1 ml of a,a-dipyridyl ( 5.4.10 ) and 1 ml of ferric chloride solution ( 5.4.9 ) and measure the absorption exactly as described in 5.6.1.

5.7 Calculation

5.7.1 The calibration curve obtained by measuring the prescribed amounts of a-tocopherol is linear but does not pass through the origin, Therefore the vitamin E content cannot be calculated directly from extinction readings found for the normal and the recovery tests.

5.7.2 Micrograms of vitamin E/ 100 g of sample = 100xcxb

(a--b) where

c = amount in micrograms of vitamin E added in the recovery test per gram of sample,

b = micrograms of vitamin E per gram of sample found in the normal test by means of the calibration curve, and

a = micrograms of vitamin E per gram of sample found in the recovery test by means of the calibration curve.

5.8 Accuracy - The accuracy of the method is f 5 percent.

5.9 Sensitivity - 10 micrograms vitamin E/g. NOTE - The recovery test may be omitted when products of the same type have

to be assayed frequently, provided that the loss of vitamin E during the whole assay is previously determined~and does not exceed 7 percent.

5.10 Determination of Total Tocopherol

5.10-O The total tocopherol content can be determined by continuing the chromatography after elution of the a- and p-fraction. The collecting flask is changed and the other tocopherols are eluted with 80 ml of elution mixture ( 5.4.5.2 ). This eluate, which contains p- , y- , S- , E- , and q-tocopherol, is

10

I IS:723501974

evaporated in vacuum and the residue is taken up in 8 ml of ethanol ( 5.4.3 ) and treated with ferric dipyridyl as described in 5.5.1. However, measure-

) ment of the absorption is carried out 10 minutes after addition of the ferric chloride solution because the tocopherols mentioned above react more slowly than a-tocopherol with ferric dipyridyl. calculated by reference to the calibration

The content of these tocopherols is curve obtained with a-tocopherol.

5.10.1 Calculate the total tocopherol content by adding this result to the one obtained for the a- and p-fraction,

x

INDIAN STANDARDS

ON

FOOD HYGIENE, SAMPLING AND ANALYSIS

IS:

2491-1972

5059-1969

53981969

5399-1969

5400-1969

5401-1969

5402-1969

5403-1969 5404-1969

5835-1970

5837-1970

5838-1970

5839-1970

5886-1970

5887-1970

Code for hygienic conditions for food processing units (Jirst revision)

Code for hygienic conditions for large scale biscuit manufacturing units and bakery units

Methods for estimation of thiamine ( vitamin Bt ) in foodstuffs

Methods for estimation of riboflavin ( vitamin Bs ) in foodstuffs

Methods for estimation of nicotinic acid ( niacin ) in foodstuffs

Methods for detection and estimation of coliform bacteria in foodstuffs

Method for plate count of bacteria in foodstuffs

Method for yeast and mould count in foodstuffs

Code of practice for handling of food samples for microbiological analysis

Method for estimation of vitamin D in foodstuffs

Code for hygienic conditions for soft drinks manufacturing units

Method for estimation of vitamin C in foodstuffs

Code for hygienic conditions for manufacture, storage and sale of ice-creams

Methods for estimation of carotenes and vitamin A ( retinol ) in foodstuffs

Method for detection of bacteria responsible for food poisoning and food- borne diseases

6540-1972

6541-1972

65421972

6850-1973

6851-1973

6852-1973

6853-1973

6854-1973

6968-1973

6969- 1973

Code for hygienic conditions for manufacture and handling of ice for human consumption

Code for hygienic conditions for establishment and maintenance of midday school meal programmes

Code for hygienic conditions for fruit and vegetable canning units

Agar, microbiological grade

Meat extract, microbiological grade

Bile salts, microbiological grade

Petone, microbiological grade

Methods of sampling and test for ingredients used in media for micro- biological work

Code for hygienic conditions for PAN ( betel ) stalls and vendors

Code for hygienic conditions for handling and sale of refrigerated drinking water

7003-l 973

7004-1973

7005-1973

Code for hygienic conditions for sago (SABOODANA ) manufacturing units

Yeast extract, microbiological grade

Code for hygienic conditions for production, processing, transportation and distribution of milk

7127-1973 Tryptone, microbiological grade

7128-1973 Proteose peptone, microbiological grade

7203-1973 Casein hydrolysate (acid digested ), microbiological grade

7219-1973 Method for determination of protein in foods and feeds

7234-1974 Method for estimation of folic acid in foodstuffs

I