PS: 3189 - 1992

PAKISTAN STANDARD SPECIFICATION

FOR FLAVOURED MILK

0. FOREWORD 0.1 This Pakistan Standard was adopted by the Pakistan Standards Institution on

25th , 26th August, 1992, after the draft finalized by the Milk & Dairy Products Sectional Committee had been approved by the Agricultural and Food Product Divisional Council.

0.2 Conversion of milk into flavoured milk is a recognized form of its marketing. The

product is similar to bottled pasteurized milk except that it is flavoured and sweetened to taste. It has consumer acceptability and sale appeal. Overall nutritive value of the milk used is retained and enhanced by the addition of sugar. Manufacture of the product fits in with the routine milk handling and processing procedures in the dairy. The organized dairies in the country have, therefore, started the production of flavoured milk. This standard is being issued to help these dairies in controlling the quality and also for guiding other dairies in taking up the production of flavoured milk.

0.3 For preparation flavoured milk, raw milk (see 2.1.1) is tested for quality and

clarified or filtered to eliminate extraneous matter, dust, etc and standardized. To the milk required ingredients (see 3) and flavour (see 2.2) are added and the mix and then homogenized at a suitable pressure and temperature. The mix is then pasteurized at a suitable temperature and time, cooled and filled in glass milk bottles and capped. Sterilized milk is filled in glass milk bottles with crown-cork or in sterilized cans and sealed air-tight. The containers are placed in a sterilizer where they are heated to a suitable temperature and then cooled.

0.4 For the purpose of deciding whether a particular requirement of this standard is

complied with the final value observed or calculated, expressing the result of a test or analysis shall be rounded off in accordance with PS:103-1991(1st Rev.) Methods for Rounding Off Numerical Values, the number of significant placed retained in the rounded off value shall be the same as that of the specified value in this standard.

1. SCOPE 1.1 This standard prescribes the types, the requirements and the method of sampling

and test for flavoured milk. 1.2 DEFINITION – Flavoured milk means a beverage or confection consisting of

milk to which has been added syrup of flavour made from whole some ingredients.

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2. TYPES 2.1 Flavoured milk, pasteurized or sterilized, may be prepared from any of the types

of milk given below.

a) Cow b) Buffalo c) Standardized d) Skimmed e) Toned, and f) Milk Powder g) Condensed Milk.

2.1.1 The types of milk mentioned in 2.1 shall be as defined in the Prevention of

Pakistan Pure Food Laws 1965. 2.1.1.1 Milk Product should not be less than SNF 8.5 % and Fat not less than 3 %. 2.2 The product shall also be classified according to the flavour added as chocolate

milk, coffee milk, fruit flavour milk, etc. 3. INGREDIENTS: Any of the types of milk given in 2.1.1 may be used in the product. 3.1 Milk and fluid milk products shall be handled in clean and sterilized containers,

stored at temperature not exceeding 10oC, till required for use. 3.2 The chocolate/cocoa/coffee/fruit juice shall be of good quality and stored in a

clean and cool place. 3.2.1 Chocolate/cocoa/coffee should be mixed with sugar and stirred into milk

thoroughly before homogenization and pasteurization/sterilization. Sediment, if any, should be strained before processing by using suitable strainers.

3.3 Fruit juices of any suitable variety of food shall be used. They shall be prepared

form properly mature fruits free from piths, seeds, skin and core. They shall be pasteurized at 63oC for 30 minutes for destroying pathogens or 72oC for 15 second or sterilized at 140oC to 3 seconds. Fruit juices shall be used immediately after preparation; if required to be stored, they should be stored in clean containers at a temperature not exceeding 10oC.

3.4 Sugar may be added according to the requirements of the consumer. 3.5 Colouring matter and flavouring assences shall be selected from those permitted

by the Pakistan Pure Food Laws.

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3.5.1 Flavoured extracts and various artificial or imitation flavour shall be pasteurized at 63oC for 30 minutes before use.

3.5.2 Colour solution shall be added to the milk after pasteurization. Aqueous solution

of colour shall be pasteurized in to by heating at 63oC prepare their solution in the plant as and when required. The dry colours may be dissolved in hot water (80oC) or 45 percent cane-sugar solution and boiled for 2 minutes. They shall be stored at a temperature below 5oC in sterile glass jars provided with screw-caps. Old solutions shall be pasteurized by heating at 63oC for 30 minutes.

3.5.3 Stabilizers permitted under the Pakistan Pure Food Laws exceeding 0.2 percent by

weight of milk may be used. They shall be clean and free from any bad taste or smell and shall be protected from dust and contamination during storage. The stabilizer is added to the milk before pasteurization. Gelatin, whenever used, shall be soaked in cold water for 15 to 20 minutes and then heated to about 70oC for 10 minutes before adding to the milk.

NOTE:- Chocolate / coffee milk and milk flavoured with fruit juice need the addition of stabilizer.

4. REQUIREMENTS: 4.1 Hygienic Requirements - The product shall be processed, packed stored and

distributed under strictly hygienic conditions (PS:1825) contamination should be avoided. Good Manufacturing Practice in processing, packing or holding human food.

4.2 Pasteurized at a suitable temperature Milk – The mix ilk, sugar, stabilizer, or shall

be pasteurized at a suitable temperature and for a period which ensures the destruction of all pathogenic organism. After pasteurization the milk, no ingredient other than fruit juice, essence and / permitted colour should be added. The mix shall be cooled to 5oC immediately after heat treatment .

4.3 Sterilized Flavoured Milk – The mix (milk, stabilizer, sugar, flavour, colour, etc.)

shall be sterilized at a suitable temperature and for period which ensures the destruction of all pathogenic organisms and cool to room temperature.

4.4 Flavour - The product shall conform to the designated flavour (2.2). It shall have

no off-flavours. No. visible sediment of the added natural flavouring materials is desirable.

4.5 Keeping Quality – Variation of the pH on 7 days incubation of the sterilized milk

shall not exceed more than 0.3 when tested by the method given in Appendix. A. 4.6 The flavoured milk shall also conform to the requirements given in Table – 1.

PS: 3189 - 1992 4

TABLE -1 REQUIREMENTS FOR FLAVOURED MILK

SL.# CHARACTERISTICS REQUIREMENTS METHOD OF TEST.

REF. TO APPENDICES Sterilize

Pasteurize

i Creaming index, Max 20 20 B ii Bacterial spores per ml,

Max. 5 - C

iii Total colony count , per ml, Max

-

50 000 D

iv Coliform count per ml, Max.

- 10 E

v Phosphatase Test - Negative F 5. PACKING AND MARKING 5.1 Pasteurized Flavoured Milk - The pasteurized flavoured milk

shall be filled in glass milk bottles or any other suitable container and capped or any other cartons packing.

512 Sterilized Flavoured Milk - The sterilized flavoured milk shall be

filled in glass bottles or sanitary cans properly sterilized. The containers shall be capped or sealed air-tight and placed in a sterilizer where they shall be gradually heated to a suitable temperature and cooled to room temperature

5.2.1 Marking - The following information shall be marked legible on each

container : a. Name of the material b. Type of the product

1. Pasteurized sterilized milk; 2. Cow/buffalo/standardized/skimmed/toned/double

toned milk and milk Powder, Condensed milk 3. Flavoured-chocoloate milk/coffee milk/fruit milk,

etc. c. Name and address of the manufacturer; d. Batch or code number; and e. Net content f. Date of Expiry

PS: 3189 - 1992 5

g. This Pakistan Standard no. PS Mark & Licence no.

6.1 Representative sample of flavoured milk f o r testing conformity to this standard shall be drawn as described in PS: 968 Method of Sampling and Analysis -for Milk Produces.

7. TESTS; 7.1 Testing shall be carried out as prescribed in appropriate appendices of Pakistan Standards given in col. 5 of Table - 1. 7.2 Quality of Reagents – Unless specified otherwise, pure chemicals shall be

employed in tests on water for analytical Laboratory use . PS: 593* shall be used where the use of water as a reagent is intended. NOTE:- ‘Pure chemicals’ shall mean chemicals that do not contain Impurities which affect the test result. * Water for Analytical Laboratory use (1st Rev.)

APPENDIX – A (Table 1, Item (iii)

DETERMINATION OF pH A.1 APPARATUS A.1.1 Incubator – adjusted at 55o C ± 1oC A.1.2 PROCEDURE A.2.1 Determine pH of 50 ml of the sample in the flask, with a glass ml sample at 55oC ± 1oC

for 7 days. Examine the flask each day, then shake and replace it in the incubator. If any physical alteration of the content is observed (coagulation with or without exudation, grittiness, flocculation, formation of bubbles or scum, peptonization or proteolysis) the result of the test shall be considered positive and the sample as non-sterilize.

A.2.1.1 If no alternation takes place during the 7 days incubation at 55o C remove the sample from the incubator and cool to room temperature. Take a small porting of it and measure the pH in the pH meter with glass electrode at 20o C From this pH value subtract the initial pH value (A.2.1)

A.3 INTERPRETATION OF RESULTS:

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A.3.1 Sample which does not show any physical alteration during incubation at 55oC ± 1oC for 7 days and where the pH does not show a difference of more than 0.3 unit from the initial pH , is considered sterile.

APPENDIX – B (Table 1, Item (i)

DETERMINATION OF CREAMING INDEX B.0 GENERAL: B.0.1 Low creaming index is an indication of good homogenization. Sterilized milk

may be graded as under for the quality of homogenization: Quality of Homogenization Creaming Index Excellent up to 10 Good 11 to 20 Fair 21 to 30 Bad Over 30 B.1 APPARATUS B.1.1 Glass Tubes – three, with ground glass stoppers, outside diameter 24 l length

with stoppers 245 mm (suitable for use with a 24-tube Gerber centrifuge), and graduated from 0 to 50 ml.

B.1.2 Pipette – three, fine pointed, connected to a suitable vessel and to a vacuum pump.

B.1.3 Apparatus for the Determination of Fat as specified in Appendix E of PS : 363 – 1082 (1st rev.)

B.2 PROCEDURE B.2. Place 50 ml of the material at 20 o C ± 1oC in each of the three tubes. Centrifuge

for 15 minutes at 1000 rev/min. B.2.2 Using the separate pipette, take 5 ml from the upper part of each of the three

tubes, carefully taking the cream that adheres to walls of the tube and transfer into a container (sample I) then empty the three tubes into a separate container ( sample II). Measure the fat content in the samples I and II by the fat content in the samples I and II by Gerber method taking all the precautions required to be taken for homogenized milk that is, heating the sample at 65oC ± 20oC for 5

PS: 3189 - 1992 7

minutes in a water-bath after each centrifuging before taking the reading till identical reading are obtained. Usually three centrifuging are required each lasting for at least 5 minutes * Pakistan Standard Specification for Milk Powder (1st rev.)

B-3 CALCULATION Creating index = A – B X 100 B Where A = fat content of the Sample I, and B = fat content of the sample II.

APPENDIX - C (Table 1, Item (iv)

DETERMINATION OF TOTAL BACTERIAL SPORES C.1 APPARATUS C.1.1 Dairy Bacteriological Pipettes – to deliver one milliliter of milk.

NOTE 1 – Use of improperly cleaned pipettes may cause incomplete deliveries of test portions. To facilitate removal of milk solids, preferable rinse the pipettes immediately after use with water. After rinsing thoroughly, wash the pipettes with suitable detergents (such as alkaline sulphate type or an alkaline phosphate) and then rinse until all detergent residues are removed. Exercise caution when using wetting agents for glass-were washing as some of these have been found to adhere tenaciously and result in inhibiting growth.

NOTE 2 – To ensure complete cleaning, soak the pipettes for 24 hours at

weekly intervals, in strong cleaning solution and rinse several time with clean water. A cleaning solution may be prepared by dissolving 50 g of sodium or or potassium bichromate in 200 ml of water in a glass or glazed earthen container, and then by adding cautiously with stirring 300 ml commercial sulphuric acid.

NOTE 3 - Before use, test a few pieces of glass water in each batch for the presence of residual acid of alkali with appropriate indicator, such as bromothymol blue.

C.1.2 Pipette Container - preferably of metal, may be round, square or rectangular,

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length.about 400 ram,

C.1.3 Petri Dishes - outside diameter 98 mm, inside diameter about 94 mm; depth 15 mm, with flat bottom and free from bubbles, scratches or other defects.

C.1.4 Petri Dish Containers -metal boxes with covers, cylindrical or square, for protection and convenient handling of dishes both before and after sterilization.

C.1.5 Hot Air Oven -capable of giving uniform and adequate temperatures, equipped with a thermometer calibrated to read up to 220°C, and with vents suitably located to ensure prompt and uniform heating.

C.1.6 Autoclave - capable of providing uniform temperatures within the chamber -up to the sterilizing temperature of 121°C, equipped with accurate mercury-filled thermometer with bulb properly located so as to register the minimum temperature within the sterilizing chamber (with or without temperature recording instrument), pressure gauge and properly adjusted safety valve.

C.1.7 Incubators - two, either water jacketed or anhydric type, electri- cally heated, thermostatically controlled and provided with shelves so spaced as to ensure uniformity of temperature; one at maintained at 30°± 1°C and other 55° ± 1°C.

C.1.8 Hand Tally - a mechanical counting device.

C.1.9 Potentiometer or Comparator with Standard Colour Disce - for accurate determination of pH of media. .

C.1.10 Media Making Utensils ~ Natural heat resistant glass ware or other suitable non-corrosive equipment, clean and free from foreign

residues or foreign material which may contaminate media, such as, chlorine, copper, zinc, aluminum, antimony and chromium.

C.2 REAGENTS . . . . . . .

C.2.1Prepare the slid media, by dissolving the following ingredients in 1 000 ml of distilled water.

g

Trypton 10

Yeast extract 13

Glucose 1

Soluble starch 1

PS: 3189 - 1992 9

Agar, bacteriological grade 8

After dissolving the above ingredients, adjust the pH to 7. Place in each 200 X 20 mm tubes, about 20 ml of medic. Sterilize in the autoclave for 15 minutes at 120o C (or 1 kg cm2steam pressure). Media may be stored at 40o C for not more than one month.

;

C.3 PROCEDURE:

C.3.1 Pouring Plates - Add aseptically one millilitre of the sample to the petri dishes. Melt the agar medium in the boiling water-bath

and cool to 45°C. Introduce 20 ml of the medium aseptically in to the petri dishes within 5 minutes and mix by rotating and tilting the dish without splashing over edge. Spread the mixture evenly over the bottom of the plat. Allow solidify.

C.3.2 Incubation – Invert the dishes and incubate at 30 ± o C and 55 o C ± 1 o C separately for 4 days in the incubator. C.3.3 Counting – Count the colonies with the aid for hand tally.

APPENDIX – D (Clause 5.1)

SAMPLINGN OF STERILIZED MILK D.1 GENERAL REQUIREMENTS OF SAMPLING D.1.1 Samples are required for chemical and bacteriological examination . All

precautions shall be taken to prevent contamination and adulteration D.1.2 The sampling instrument shall be clean and dry. D.1.3 For bacteriological purposes, all equipment including plungers, sample bottles

and rubber stoppers, shall be sterile and the samples shall be drawn under aseptic conditions. All equipment shall be sterilized by either of the following methods.

a. Heating in a hot air oven for not less than 2 hours at 160o C or b .Autoclaving for not less than 15 minutes at 12o C. D..2 SCALE OF SAMPLING

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D.2.1 Lot – In a single consignment all the bottles or cans containing one type of milk drawn from a single bathc of manufacture shall constitute a lot.

D.2.2 Sample shall be tested from each lot separately for ascertaining the

conformity of the material to the required specification. D.2.3 If milk of uniform quality I s applied in containers, the number of units to be

selected at random for sampling shall be in accordance with col 1, 2 and 3 Table 2

TABLE – 2

NUMBER OF CONTAINERS TO BE SELECTED FOR SAMPLING

NUMBER OF CONTAINER IN THE LOT

NUMBER OF CONTAINERS TO BE SELECTED FOR

Up to 25 1 1 26 to 100 2 5 101 to 500 3 7 501 to 1000 3 9

1 001 to 5000 4 11 5 001 and above 4 13

D.2.4 the containers shall be selected at random and for this purpose a random

number table shall be used. In case such a table is not available is available the following procedure shall be adopted. Starting from any container count all the containers in one order as 1,2,3…….

Etc, up to r, and so on. Every rhe container so counted shall be withdrawn where r is the integral part of N/n (N being the number of containers in the lot and n being the number of container to selected).

D.3 TEST SAMPLES AND REFREE SAMPLES: D.3.1 Preparation of Sample fro Chemical Analysis – Mix thoroughly the content

of the container selected according to col. 3 of Table – 2. Taking equal amount of sterilized milk from each selected container, collect about 200 g of the material which shall be mixed and divided into three equal parts. Each part is transferred to a clean land dry container land labeled. One of these sample shall be for the purchaser, the other for the vendor and the third for the referee.

D.3.2 Preparation of Sample for Bacteriological Analysis – From the selected containers according to col. 2 of Table 2, draw with suitable sampling instrument, which is sterilize,

PS: 3189 - 1992 11

at least 100 g of the material and mix thoroughly under aseptic conditions. Divide the sample (taking care not to bring in microbiological contamination) into three equal parts. Each part shall be transferred to sterile glass containers, sealed air-tight and labeled of r bacteriological examination. One of these samples shall be for the purchaser, the other for the vendor and the third for the referee.

D.3.3 Referee Sample – Referee samples shall consist of sample for chemical analysis and

sample for bacteriological analysis. These shall be kept at a place agreed to between the purchaser and the vendor.

D.4 CRITERIA FOR CONFORMITY D.4.1 the lot shall be considered as conforming to the standard if the test samples taken in

D.3.1 and D.3.2 satisfy all the requirements of this specification.

APPENDIX – E E.0 COLIFORM BACTERIA E.1 General – this group of bacteria is important in quality control of milk as it is indicative

of possible facial contamination and due to it ability to produce acid and taints in milk. The presence of this group in mil considered to be an indicator of the degree of unhygienic practices during production, processing or storage and is intended to measure general care taken in handling this product. The members of this group are generally destroyed during pasteurization treatments and, therefore, a positive coliform test in the case of pasteurized milk is considered to indicate post-pasteurization contamination.

E.2 The coliform bacteria in milk shall be determined in accordance with th4e procedure

laid down in Appendix G of PS: 636. * Pakistan Standard Specification for Milk Powder (1st Rev.). E.2.1 Since organisms giving positive presumptive tests are practically absent from properly

processed pasteurized milk collected directly from pasteurizers, a confirmatory test generally is not needed. Therefore, in routine and official control of pasteurized samples, testing for organisms of the coliform group is limited to the presumptive test.

E.2.2 In presumptive test with liquid media, for routine plant control of pasteurized products

where results are likely to be negative, use not less than 3 tubes , each inoculated with 1 ml or, preferably, 10 ml of samples . Where frequent tests for quality control on samples taken at successive are almost always negative, use of one tube inoculated with 1 ml , or

PS: 3189 - 1992 12

preferably, 10 ml., may sufficient. In this work, as in obtaining estimates indicating total bacterial contamination, simple examination of routine samples, taken frequently, furnish more useful information than a complete examination of samples taken less frequently.

E.3 Interpretation E.3.1 The following coliform contents are suggested as a guide for grading of raw milk

supplies Raw Milk : ‘Coliforms’ absent in 1 : 100 a dilution-Satisfactory Pasteurized Milk : ‘Coliforms’ absent in 1:10 dilution-Satisfactory E.3.2 In case of milk containing added sugar, the results of the presumptive coliform te4st

shall be interpreted with caution because of the possible risk of a false positive.

APPENDIX – F PHOSPHATE TEST

F.0 General – The test is used to judge the efficiency of pasteurization of milk. According to

the accepted practice in this country, milk for liquid trade is pasteurized by the holding method ( 63o C to 66 o C for 30 minutes), or by the High Temperature – Short time (HTST) method (holding at 72.2 o to 72 8o C for 15 seconds ). To test whether the heat-treatment by eight of these methods was properly carried out, the treated milk is subjected to the phosphates test which helps to indicate the presence or absence of phosphate enzyme. Phosphates present in milk is destroyed by jut about the same heat-treatments necessary for the destruction of Mycobacterium tuberculosis, the most heat resist5ance pat6hogen likely to be –treatment is less than that specified above, some of the phosphates remains active and will liberate phenol from disodium phenylphosphate in a buffered solution at a pH of approximately 9.5 . The presence of free phenol is determined by a colorimetric test.

The sample of pasteurized milk shall be tested within 48 hours after

pasteurization. The sample shall be stored at a temperature not exceeding 5o C . Several modifications of phosphates test are in use. The method described here

is based on the Aschaffenburg and Mullen technique. The test yields valuable information about the pasteurization of milk after incubation for only 30 minutes, and results comparable with those of the Kay and Graham phosphatase test are obtained after the incubation period of 2 hour instead of 24 hours. Faulty heat-treatment may thus be quickly detected and remedied. Moreover, the new test is simple to carry out, requires

PS: 3189 - 1992 13

the minimum of equipment and, being highly specific, is unlikely to be adversely affected by interfering lsubstances.

In the test, milk is added to a suitably buffered solution of disodium p-

nitroghenyl phosphate which is readily hydrolyzed by the enzyme phos-phatase to form p-nitrophenyl phosphate which is readily hydrolyzed by the enzyme phosphatase to form p-nitrophenol which is yellow in alkaline solution.

F.1 Apparatus F.1.1 Lovibond All-Purphose Comparator with Standard F.1.2 Standard Discs – giving 0,6,10,18,42, or 0,6,10, 14, 18, 25, 42 readiang F.1.3 Fused Glass cell – 25 mm. F.1.4 Test Tubes – 15 X 1.9 cm with ring at 10 ml fitted with rubber stoppers. F.2 Reagents F.2.1 Buffer Solution – 3.54 g of sodium carbonate analytical reagent grade and 1.5 g of

sodium biucarbonbate analytical reagent grade dissolved in one litre of water. F.2.2 Substrate – disodium p-nitrophenylphosphate not less than 95 percent pure. F.2.3 Buffer Substrate – Transfer 0.15 g of the substrate into a 100-ml measuring cylinder or

stoppered graduated flask and make up to the mark with the buffer solution. The solution should not be stored for long periods but may normally be kept in a refrigerator for up to one week. The solution is practically colourless; when viewed through a 25-mm cell in the all-purpose comparator, it should give a reading of less than 10 on the disc.

F.3 PROCEDURE Fill 210m ml (5 ml may be used) quantities of the buffer substrate solution into test-

tubes marked at 10 ml and bring to 37 oC to 38o C in a water-bath. Add ml (one milliliter if 5 ml of buffer substrate are used) of the milk to be tested, close the tubes with rubber stoppers and invert to mix. Prepare in the same way a blank from a boiled milk of the same type as that under test. Incubate all the tubes at 37 oC to 38o C. Read the yellow colur after 30 minutes, return to the bath, and take a second reading after incubation for a further 90 minutes. The yellow colour is read in a Lovibond all purpose comparator on a reasurin stand, fitted with the disc calibrated in microgram p-nigrophenol. The blank is placed on the left of the stand and the sample on the right. Readings are taken by looking down on to the two operators with the comparator facing a good source of north daylight; the disc is revolved until the sample is matcfhed; reading falling between two standards are recorded to the nearest reading.

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F.4 Interpretation of Result Disc reading After 30 Minutes incubation Interpretation 0 or trace Properly pasteurized 6 Doubtful 10 over Under pasteurized Disc reading After 2 Hour incubation 0 to 10 Properly pasteurized Over 10 Under pasteurized The 30-minutes test will reveal any serious fault in pasteurization but to enable minor

errors to be detected, readings shall be taken after further incubation for 90 minutes _____________________