T.C.
TARIM ve KÖYÝÞLERÝ BAKANLIÐI
Koruma ve Kontrol Genel Müdürlüğü
Communiqué on the Analysis Methods for Condenced Milk and Milk Powder
Authorization Law: Turkish Food Codex
The Official Gazette: 20.03.2002-24701
Communication No : 2002/16
Objective
Article 1- The objective of this Communiqué is to determine the analysis methods for partially or wholly condenced milk and milk powders presented for human consumption, specified in “Communiqué of Condenced Milk and Milk Powder” published in Official Gazette dated 29/12/2001, numbered. 24625.
Scope
Article 2- This Communiqué covers chemical analysis methods of condenced high fat milk, condenced whole milk, condenced semi skimmed milk, condenced skimmed milk, sweetened condenced milk, sweetened condenced semi skimmed milk, sweetened condenced skimmed milk, high fat milk powder, whole milk powder, semi skimmed milk powder, skimmed milk powder, specified in the Communiqué of Condenced Milk and Milk Powder.
Legal basis
Article 3- This Communiqué has been prepared in accordance with the Turkish Food Codex Regulation published in the Official Gazette, dated 16/11/1997, reiterated No. 23172.
Method of Analysis
Article 4- Methods of analysis are applied as follows in accordance with the general principles in;
Annex-1:
a) Dry matter content of condenced high fat milk, condenced whole milk, condenced semi skimmed milk, condenced skimmed milk, sweetened condenced milk, sweetened condenced semi skimmed milk, sweetened condenced skimmed milk is determined according to Method 1 of Annex-II.
b) Moisture content of high fat milk powder, whole milk powder, semi skimmed milk powder, skimmed milk powder is determined according to Method 2 of Annex-II,
c) Fat content of condenced high fat milk, condenced whole milk, condenced semi skimmed milk, condenced skimmed milk, sweetened condenced milk, sweetened condenced semi skimmed milk, sweetened condenced skimmed milk is made according to Method 3 of Annex-II and the same in high fat milk powder, whole milk powder, semi skimmed milk powder, skimmed milk powder according to Method 4 of Annex-II,
d) Sucrose content of sweetened condenced milk, sweetened condenced semi skimmed milk, sweetened condenced skimmed milk is determined according to Method 5 of Annex-II.
e) It is determined according to Method 6 of Annex-II for high fat milk powder, whole milk powder, semi skimmed milk powder, skimmed milk powder
f) Phosphates activity in high fat milk powder, whole milk powder, semi skimmed milk powder, skimmed milk powder is determined according to Method 7 or 8 of Annex-II.
Compliance with European Union
Article 5- This Communiqué has been prepared within the frame of compliance with European Union considering the Commission Directive 79/1067/EEC on “Methods of Analysis for Condenced Milks and Milk Powders”. This Communiqué does not prevent the laboratories from using the methods experienced according to their own legislation and having scientific validity. This Communiqué is applied compulsorily to the relations with European Union.
Provisional Article-1 Presently active institutions and establishments using the analysis methods covered in this Communiqué are obliged to obey the provisions of this Communiqué within 1 year.
Enforcement
Article 6- This Communiqué enters into force on the date of publication
Execution
Article 7- The provisions of this Communiqué are executed by Minister of Health and Minister of Agriculture and Rural Affairs.
ANNEX – 1
General Principles
1. Preparation of the sample for analysis
1.1. As for the condenced high fat milk, condenced whole milk, condenced semi skimmed milk, condenced skimmed milk;
The closed container is shaked and inverted. The container is opened and mixed by transferring from container to container a few times and, slowly transferred to a second container, which can be closed hermetically. The sample is ensured to mix thoroughly with the fat and milk residuals stuck on the sides and at the bottom of the container. If the content of the sample is not homogeneous, then the container is placed in a water bath at 40°C. It is shaked vigorously once in every 15 minutes period. At the end of 2 hours, it is removed from the water bath and cooled to room temperature. The cover is opened and content of the container is mixed thoroughly by means of a spoon or spatula. If the fat is seperated, that sample is not analyzed. The sample is stored in a cool room.
1.2. As for the sweetened condenced milk, sweetened condenced semi skimmed milk, sweetened condenced skimmed milk;
1.2.1. Metal containers: Closed container is held in the water bath at 30-40°C about 30 minutes. The container is opened and mixed thoroughly by upward, downward and circular movements by means of a spoon or spatula in order to mix completely upper and lower sections. It is ensured to pass the milk on the sides and bottom of the container in to the sample. Whole content is removed into a container, which has an airtight cover as much as possible. The cover is closed and stored in a cool room.
1.2.2. Tubes: Bottom section of the tube is cut and the content is poured in an airtight container. Then the tube is cut longitudinally and the entire material stuck in inner surface is scraped and added to the container. All this content is mixed thoroughly and the container is stored in a cool room.
1.3. As for the milk powder with high fat content, whole milk powder, semi skimmed milk powder, skimmed milk powder;
Sample is transferred into a clean and dry container of two-fold capacity, which has an airtight cover. The cover is closed immediately and milk powder is mixed by repeatedly shaking thoroughly and inverting the container. While preparing the sample, care should be taken for the milk powder not to contact with air in order to prevent humidity.
2. Reagents
2.1. Water
2.1.1. As for the water used for dissolution, dilution or washing purposes; distilled water or demineralized water of equivalent purity is used.
2.1.2. Unless otherwise specified, dissolving, solution or dilution means dissolution in water, solution in water, and dilution in water.
2.2. Chemicals
All chemicals should be analytically pure, unless otherwise specified.
3. Equipment
3.1. List of equipment
Equipment list covers only the tools that have specialized use and particular specification.
3.2. Analytical balance should have minimum precision of 0.1 mg.
4. Expression of the results
4.1. Percent calculation
Unless otherwise specified, the results are calculated as percent of mass brought in the laboratories.
4.2. Accuracy of the results
The results should be as accurate as required for the analysis used.
5. Reporting
In the report, analysis method should also be stated together with results obtained. Besides, all kinds of details that are not mentioned in the analysis method or that are voluntary and at the same time all types of conditions that may affect the results obtained should be indicated in the report. Report should include all kinds of information that ensures exact description of the sample.
ANNEX-II
Analysis Method
Method 1: Determination of Dry Matter Quantity (99°C Oven)
1. Scope and Field of application
With this method, content of dry matter of condenced milk with high fat content, condenced whole milk, condenced semi skimmed milk, condenced skimmed milk, condenced milk containing added sugar, condenced semi skimmed milk containing added sugar and condenced skimmed milk containing added sugar is determined.
2. Definition
Dry matter content of the condenced milks: The dry matter content as determined by the above-mentioned method.
3. Principle
A specific amount of sample is diluted with water, mixed with sand and dried in an oven at temperature 99±1°C. The mass after drying operation is the mass of the dry matter and is calculated as percentage by the mass of the sample.
4. Solutions
4.1. Quartz or coastal sand treated with hydrochloric acid; the following control test should be applied to the coastal sand in the range of particle sizes between 0.18 and 0.5 mm, that is passing through 500 micron sieve and retained on 180 micron sieve.
About 25 g sand is heated for two hours in an oven (5.3) as described in Articles 6.1 to 6.3. 5 ml of water is added and later heated in the oven for another period of two hours, cooled down and reweighed. The difference between the two masses should not exceed 0.5 mg.
When necessary, the sand is treated with 25% hydrochloric acid for 3 days by mixing from time to time. Afterwards, it is washed with water so far that there is no more acid reaction and no more chlorine in the washing water. It is dried at 160°C and retested as above.
5. Equipment and material
5.1. Analytical balance
5.2. Metal containers; preferably made of nickel, aluminum or stainless steel which have easily removed covers that are full fitting on to these containers when required, in diameter 60-88 mm and depth about 25 mm.
5.3. Oven with atmospheric pressure; well ventilated, temperature adjustable thermostatically up to 99±1°C, temperature dispersed homogeneously.
5.4. Desiccator; containing newly activated silica gel or equivalent dehumidifying material and humidity indicator.
5.5. Glass bars; in sizes suitable for the metal containers, with one end flattened.
5.6. Water bath, equipped for boiling.
6. Procedure
6.1. A short glass bar (5.5) and about 25 g sand are placed in the container specified in Article 5.2.
6.2. The container and its content together with the cover as unclosed are placed in the oven (5.3) and heated for 2 hours.
6.3. The container is closed with the cover and transferred into the desiccator. It is cooled down to room temperature and scaled with accuracy of 0.1 mg. (mo)
6.4. The sand is ensured to accumulate on one side by inclining the container. About 3 g from the sample of condenced milk and about 1.5 g from the sample of condenced milk containing added sugar are placed on to a clean area. Container is closed with the cover and scaled with accuracy of 0.1 mg. (m1)
6.5. The cover is removed, 5 ml of water is added and liquid and sand are mixed by means of a glass bar. The bar is left in the mixture.
6.6. The containers are held in the boiling water bath (5.6) until the water evaporates. This operation generally takes 20 minutes. The mass should be mixed from time to time by the bar ventilating well until it is dry. The bar is left in the container.
6.7. The container and cover stay in the oven for 1.5 hour.
6.8. The container is placed in desiccator (5.4) as closed. It is cooled down to room temperature and scaled with accuracy of 0.1 mg. (m2)
6.9. The container and its cover are placed in the oven as unclosed and left there for another hour.
6.10. The process explained in Article 6.8 is repeated.
6.11. The processes described in Articles 6.9 and 6.10 are repeated until the mass difference between latest two weighing is less than 0.5 mg or any mass increase is observed. If it happens to be an increase in mass the lowest value of mass is used in calculation.
7. Expression of the results
7.1. Formula and method of calculation
Amount of dry matter; is calculated with the following formula as percentage of the mass of sample.
m2 – m0
----------- x 100
m1 - m0
m0= Mass of container, cover and sand after step 6.3, g
m1= Mass of container, cover, sand and sample prior to drying (6.4), g
m2= Mass of container, cover, sand and sample after drying (6.11), g
7.2. Repeatability
The difference between the results of two analyses carried out under the same conditions, by the same analyzer, at the same time or rapidly one after the other for the same sample should not exceed 0.2 g of dry matter in each 100 g of product.
8. Calculation of the total milk dry matter and milk dry matter other than fat
8.1. Amount of total milk dry matter of condenced milks containing added sugar = Total dry matter (obtained with the Method 1, ANNEX-II) – amount of saccharose (obtained with the Method 5, ANNEX-II).
8.2. Amount of milk dry matter other than fat of condenced milks containing added sugar = Total dry matter (obtained with Method 1, ANNEX-II) – amount of saccharose (obtained with Method 5, ANNEX-II) + amount of fat (obtained with Method 3, ANNEX-II)
8.3. Amount of total milk dry matter other than fat of condenced sugar free milks = Total dry substance (obtained with Method 1, ANNEX-II) – amount of fat (obtained with Method 3, ANNEX-II)
Method 2: Determining amount of humidity (102ºC Oven)
1. Scope and field of application
With this method, loss of mass is determined while drying of the milk powder with high fat content, whole milk powder, semi skimmed milk powder and skimmed milk powder.
2. Definition
Humidity content is determination of loss of mass with the specified method.
3. Principle
Residual mass is determined after drying the sample to be analyzed to a constant weight in the oven of 102 ±1ºC under atmospheric pressure. Loss of mass is calculated as percent of mass of the sample.
4. Equipment and material
4.1. Analytical balance
4.2. Containers; preferably nickel, aluminum, stainless steel or glass. With covers that can be closed firmly or opened easily, 60-80 mm in diameter, and about 25 mm deep.
4.3. Oven with atmospheric pressure; well ventilated, temperature adjustable thermostatically up to 102 ±1ºC, temperature dispersed homogeneously.
4.4. Desiccator; containing newly activated silica gel or equivalent dehumidifying material and humidity indicator.
5. Procedure
5.1. The container (4.2) and its cover as unclosed are placed in the oven (4.3) for an hour.
5.2. The container is closed with the cover and put in desiccator (4.4). It is cooled down to room temperature and scaled with accuracy of 0.1 mg. (m0).
5.3. Approximately 2 g of experiment sample is placed in the container, the cover is closed and scaled with accuracy of 0.1 mg. (m1).
5.4.The cover is opened and container and its cover is placed in the oven for 2 hours.
5.5. While placing in the desiccator, the containers are closed with the covers, cooled down to room temperature and scaled with accuracy of 0.1 mg.
5.6. The containers are placed again in the oven as unclosed for an hour together with the covers.
5.7. Process in Article 5.5 is repeated.
5.8. The processess described in Articles 5.6 and 5.7 are repeated until the mass decrease for the scaling one after the other does not exceed 0.5 mg or any mass increase is observed. If it happens to be an increase in mass the lowest value of mass is taken into consideration (6.1).The last weight is recorded in g. (m2).
1. Expression of the results
6.1. Formula and method of calculation
Amount of humidity; is calculated with the following formula as percentage of the mass of sample.
m1 – m2
----------- x 100
m1 - m0
m0= Mass of container and cover after step 5.2, g
m1= Mass of container, cover and sample prior to drying (5.3), g
m2= Mass of container, cover and sample after drying (5.3), g
6.2. Repeatability
The difference between the results of two analysis proceeded under the same conditions, by the same analyzer, at the same time or rapidly one after the other for the same sample should not exceed 0.2 g of humidity in each 100 g of product.
Method 3: Determining the amount of fat in condenced milks (Rose-Gotlieb Method)
1. Scope and field of application
With this method, content of fat of condenced milk with high fat content, condenced whole milk, condenced semi skimmed milk, condenced skimmed milk, condenced milk containing added sugar, condenced semi skimmed milk containing added sugar, condenced skimmed milk containing added sugar is determined.
2. Definition
Fat content of the condenced milks; is the amount of fat determined by this method.
3. Principle
The amount of fat is determined by extraction of the fat from an ammonia and ethyl alcohol solution of the sample with diethyl ether or petroleum ether fallowed by evaporation of the solvents and weighing of the residue and calculation as a percentage by mass of the sample according to the principle of Rose-Gotlieb.
4. Reagents
All reactives should be appropriate to whole requirements determined in the blank experiment (6.1). If it is found out that there is about 1 g milk fat in 100 ml of solvent, the solutions are required again to be distilled.
4.1. Ammonia solution (NH3); approximately 25% (weight/weight), density about 0.91 g/ml at 20 °C or more condenced .
4.2. Ethanol, 96% ± 2 (volume/volume) or methanol, ethanol denatured by ethyl-methyl ketone or petroleum ether.
4.3. Diethil ether; without peroxide.
Note 1: In order to realize whether peroxides exist or not, a small measure that may be closed by a glass cover is rinsed with ether and then 10 ml of ether is added. 1 ml of freshly prepared potassium iodide of 10% is added, shaked and waited for 1 minute. There should be no yellow color at both layers.
Note 2: Diethyl ether is refined from peroxides by using wet zinc layers which is first hold for a minute in copper sulfate solution with diluted acid and washed with water and may be stored as such. 8000 mm2 zinc tablet should be used per liter of ether. Layers should be cut in strips in sizes to reach at least half way of the container.
4.4. Petroleum ether; boiling point should be between 30 - 60°C.
4.5. Mixed solvents; petroleum ether (4.4) and diethyl ether (4.3) are mixed in equal volumes. The mixture should be prepared just before usage. Each of diethyl ether or petroleum ether can be used alone where usage of mixed solvents are indicated.
5. Equipment and materials
5.1. Analytical balance
5.2. Proper extraction tubes or flasks; trimmed glass covers or other covers that are not affected by the solvents.
5.3. Flasks; thin walled and straight bottom, in capacity of 150 – 250 ml.
5.4. Oven with atmospheric pressure; well-ventilated and adjustable thermostatically up to 102 ±1ºC.
5.5. Splash preventing material; fat-free, non-porous glass beads or silicon carbide pieces, which are not crumbled when used. It’s optional to use this material, see Article 6.2.1.
5.6. Siphon; proper to extraction tubes.
5.7. Centrifuge, optional.
6. Procedure
6.1. Blank experiment
While determining the amount of fat in the sample, a blank experiment is made by using 10 ml of distilled water instead of the sample by means of applying the same procedure described in the following articles except Article 6.2.2. If the result of blank experiment exceeds 0.5 mg, solutions are checked, impure solution or solutions are purified or prepared again.
6.2. Experiment
6.2.1. After adding material (5.5) in the flask (5.3) during evaporation stage of the solvent in order to ensure boiling it slowly and prevent splashing, the sample is dried in oven (5.4) for a period between half and one hour. It is left to cool down to room temperature and scaled in accuracy of 0.1 mg (m1).
6.2.2. Prepared samples are mixed, scaled promptly in accuracy of 1 mg. 4-5 g from the skimmed sugar free samples and 2-2.5 g from the samples containing added sugar are scaled and put in extraction flask (5.2). Water is added to fill up to 10.5 ml. The product is mixed carefully until completely dispersed, and heated up to 40-50°C. The product should be dispersed completely, otherwise experiment should be repeated.
6.2.3. It should thoroughly be mixed either by adding 1.5 ml of 25% ammonia solution (4.1) or equivalent volume of a more condenced solution.
6.2.4. 10 ml of ethanol (4.2) is added and liquids are mixed slowly as keeping the bulb unclosed.
6.2.5. 25 ml of diethyl ether is added (4.3), cooled down under water and flask is closed. Shaked strongly and continuously inverted for a minute.
6.2.6. The cover is opened carefully, 25 ml of petroleum ether (4.4) is added, the cover and the neck of the bulb are washed with a few initial ml. The cover is put on its place, shaked and inverted continuously for about 30 seconds. If centrifuge is not to be used (the one in 6.2.7), severe shaking should be avoided.
6.2.7. The flask is waited until the upper and lower layers completely separate from each other. This separation operation can be made by using a proper centrifuge (5.7)
Note: Whenever a centrifuge that is not operating with three phase motor is used, sparks may be emitted and for example; as soon as the tube is broken somehow, flaming or an explosion from any ether vapor may be unavoidable, hence careful attention should be paid and necessary measures should be taken.
6.2.8. The cover is opened. A few ml of sample remaining on the cork and the inner surface of flask neck is washed with a mixture of solvent (4.5) and washed portion is poured into the flask. The upper layer is transferred into the flask (6.2.1) carefully by hand or by a siphon (5.6) as much as possible.
Note: If the siphon is not used during transfer, in order to separate the surfaces in between the two layers, some amount of water may be required to be added and consequently the transfer from one container to the other may be managed.
6.2.9. The inner and outer sides of the flask neck or the end of siphon and its lower part are washed again with a few ml of mixed solvent (4.5) into the flask. Outer flows of apparatus are ensured to flow in the flask and inner flows of flask neck and siphon are directed to flow in the extraction flask.
6.2.10. By using 15 ml of petroleum ether and 15 ml of diethyl ether, second extraction is repeated involving the operations between 6.2.5 and 6.2.9.
6.2.11. By skipping the last washing in Article 6.2.9, third extraction is completed by repeating the operation in 6.2.10.
Note: The completion of this third extraction is not compulsory for sample analysis of skimmed condenced milk containing added sugar and sugar free skimmed condenced milk.
6.2.12. If possible, the solvents including ethanol are evaporated or distilled as much as possible. If the flask has a small capacity, it will be required to move away some part of the solvent as described above after each extraction.
6.2.13. As soon as there remains no more sensible odor in the flask, it is heated in the oven for about an hour.
6.2.14. The flask is removed from the oven, released to cool down to room temperature and scaled with accuracy of 0.1 mg.
6.12.15. Heating operations of 30 min. and 60 min. periods in 6.2.13 and 6.2.14, are repeated up to the point that the mass difference between the two successive weights is less than 0.5 mg or to the point of any increase in mass. If an increase occurs in mass, lowest value of mass is used in calculation (7.1). Last weight is recorded as g (m1).
6.2.16. Successively 15 and 25 ml of petroleum ether is added in order to be able to control whether or not all of the extracted material are soluble. Heated slightly and mixed stirring until whole fat is dissolved.
6.2.16.1. If the extracted material dissolves completely in the petroleum ether, the mass of fat is calculated by subtracting tare of flask (6.2.1) from the weight of the flask (6.2.15) containing the extract.
6.2.16.2. If the extracted material does not dissolve completely in the petroleum ether or a suspicious condition arises, the fat in the flask is extracted again by washing with warm petroleum ether for a few times. It is waited for settling down the undissolved material from each pouring. Outer part of the flask neck is washed three times. The flask waits in the oven for an hour in horizontal position and is cooled down to room temperature (6.2.1) and scaled with accuracy of 0.1 mg. (m2) The difference of mass obtained in 6.2.15 and this last mass is the mass of the fat.
7. Expression of results
7.1. Formula and method of calculation
The mass of extracted fat as g;
(m1-m2) - (B1-B2)
Amount of fat in the sample is expressed in percentage:
(m1 – m2 )-(B1 – B2 )
------------------------- x 100
S
m1 = mass of flask and the fat obtained after the operation in 6.2.15, g
m2 = mass of flask obtained after tshe operation in 6.2.1 or 6.2.16.2, g
B1 = mass of flask used in blank experiment after the operation in 6.2.15, g
B2 = mass of flask used in blank experiment after the operation in 6.2.1 or 6.2.16.2, g
S = mass of the sample, g
7.2. Repeatability
The difference between the results of two analyses made simultaneously or successively as fast for the same sample should not exceed 0.05 g of fat for each 100 g product.
Method 4: Determining the amount of fat in milk powder (Rose-Gotlieb Method)
1. Scope and Field of application
With this method; amount of fat is determined in milk powder with high fat content, whole milk powder, semi skimmed milk powder, and skimmed milk powder.
2. Definition
Fat content of the milk powder; is the amount of fat determined by this method.
3. Principle
The amount of fat is determined by extraction of the fat from an ammonia and ethyl alcohol solution of the sample with diethyl ether or petroleum ether fallowed by evaporation of the solvents and weighing of the residue and calculation as a percentage by mass of the sample according to the principle of Rose-Gotlieb.
4. Reagents
All the reagents should be appropriate to whole conditions determined during the blank experiment (6.1). If it is found out that there is about 1 g milk fat in 100 ml of solvent, the solutions are required again to be distilled.
4.1. Ammonia solution (NH3); approximately 25% (weight/weight), density about 0.91 g/ml at 20 °C or more condenced .
4.2. Ethanol, 96% ± 2 (volume/volume) or methanol, ethanol denatured by ethyl-methyl ketone or petroleum ether.
4.3. Diethil ether; without peroxide.
Note 1: In order to realize whether peroxides exist or not, a small measure that may be closed by a glass cover is rinsed with ether and then 10 ml of ether is added. 1 ml of freshly prepared potassium iodide of 10% is added, shaked and waited for 1 minute. There should be no yellow color at both layers.
Note 2: Diethyl ether is refined from peroxides by using wet zinc layers which is first hold for a minute in copper sulfate solution with diluted acid and washed with water and may be stored as such. For each liter of ether 8000 mm2 zinc tablet should be used. Layers should be cut in strips in sizes to reach at least half way of the container.
4.4. Petroleum ether; boiling point should be between 30 - 60°C.
4.5. Mixed solvents; petroleum ether (4.4) and diethyl ether (4.3) are mixed in equal volumes. The mixture should be prepared just before usage. Each of diethyl ether or petroleum ether can be used alone where usage of mixed solvents are indicated.
5. Equipment and materials
5.1. Analytical balance
5.2. Proper extraction tubes or flasks; trimmed glass covers or other covers that are not affected by the solvents.
5.3. Flasks; thin walled and straight bottom, in capacity of 150 – 250 ml.
5.4. Oven with atmospheric pressure; well-ventilated and adjustable thermostatically up to 102 ±1ºC.
5.5. Splash preventing material; fat-free, non-porous, glass beads or silicon carbide pieces which are not crumbled when used. The use of this material is optional, see Article 6.2.1.
5.6. Water bath; at 60-70°C.
5.7. Siphon; proper to extraction tubes.
5.8. Centrifuge, optional
6. Procedure
6.1 Blank experiment
While determining the amount of fat in the sample, a blank experiment is performed by using 10 ml of distilled water instead of sample, by means of applying the same procedure defined hereinafter except article 6.2.2. If the result of blank experiment exceeds 0.5 mg, then the solutions are checked, impure solution or solutions are purified or prepared again.
6.2. Experiment
6.2.1. After adding material (5.5) in the flask (5.3) during evaporation stage of the solvent in order to ensure boiling it slowly and prevent splashing, the sample is dried in oven (5.4)for a period between half and one hour. It is left to cool down to room temperature and scaled in accuracy of 0.1 mg.
6.2.2. About 1.5 g of skimmed or semi skimmed milk powder or about 1 g of milk powder with high fat content is placed in the extraction flask (5.2) and scaled with an accuracy of 1 g. 10 ml of water is added and shaked slowly until it dissolves completely, where heating may be required for some samples.
6.2.3. 1.5 ml of 25% ammonia solution (4.1) or a more condenced solution in equal volume is added and heated in a water bath for 15
minutes at 60-70 °C, shaked occasionally and cooled down under flowing tap water.
6.2.4. 10 ml of ethanol (4.2) is added and liquid is shaked slowly without closing the cover of flask.
6.2.5. 25 ml of diethyl ether is added (4.3), cooled under water and the cover of flask is closed. It is shaked violently and inverted continuously for 1 minute.
6.2.6. The cover is opened carefully, 25 ml of petroleum ether (4.4) is added, cover and the neck section are washed out with a few ml. Cover is placed on top, shaked and inverted continuously for about 30 seconds. If the centrifuge is not to be used (6.2.7), it should be avoided to shake violently.
6.2.7. Flask stays as long as until lower and upper layers are completely separate. This separation can be made by using a proper centrifuge (5.8).
Note: Whenever a centrifuge that is not operating with three phase motor is used, sparks may be emitted and for example; as soon as the tube is broken somehow, flaming or an explosion from any ether vapor may be unavoidable, hence careful attention should be paid and necessary measures should be taken.
6.2.8. The cover is opened. Sample remaining on the cork and the inner surface of flask neck is washed with a few ml of mixed of solvent (4.5) and washed portion is poured into the flask. The upper layer is transferred into the flask (6.2.1) carefully by hand or by a siphon (5.6) as much as possible.
Note: If the siphon is not used during transfer, in order to separate the surfaces in between the two layers, some amount of water may be required to be added and consequently the transfer from one container to the other may be managed.
6.2.9. The inner and outer sides of the flask neck or the end of siphon and its lower part are washed with again a few ml of mixed solvent (4.5) into the flask. Outer flows of apparatus are ensured to flow in the flask and inner flows of flask neck and siphon are directed to flow in the extraction flask.
6.2.10. By using 15 ml of petroleum ether and 15 ml of diethyl ether, second extraction is repeated involving the operations between 6.2.5 and 6.2.9.
6.2.11. By skipping the last washing in Article 6.2.9, third extraction is completed by repeating the operation in 6.2.10.
Note: The completion of this third extraction is not compulsory for sample analysis of skimmed milk powder.
6.2.12. If possible, the solvents including ethanol are evaporated or distilled as much as possible. If the flask has a small capacity, it will be required to move away some part of the solvent as described above after each extraction.
6.2.13. As soon as there remains no more sensible odor in the flask, it is heated in the oven for about an hour.
6.2.14. The flask is removed from the oven, released to cool down to room temperature and scaled with accuracy of 0.1 mg.
6.2.15. Heating operations of 30 min. and 60 min. periods in 6.2.13 and 6.2.14, are repeated up to the point that the mass difference between the two successive weights is less than 0.5 mg or to the point of any increase in mass. If increase occurs in mass, lowest value of mass is used in calculation (7.1). Last weight is recorded as g (m1).
6.2.16. Successively 15 and 25 ml of petroleum ether is added in order to be able to control whether or not all of the extracted material is soluble. Heated slightly and mixed stirring until whole fat is dissolved.
6.2.16.1. If the extracted material dissolves completely in the petroleum ether, the mass of fat is calculated by subtracting tare of flask (6.2.1) from the weight of the flask (6.2.15) containing the extract.
6.2.16.2. If the extracted material does not dissolve completely in the petroleum ether or a suspicious condition arises, the fat in the flask is extracted again by washing with warm petroleum ether for a few times. It is waited for settling down the undissolved material from each pouring. Outer part of the flask neck is washed three times. The flask waits in the oven for an hour in an inclined position and is cooled down to room temperature (6.2.1) and scaled with accuracy of 0.1 mg. (m2) The difference of mass obtained in 6.2.15 and this last mass is the mass of the fat.
7. Expression of results
7.1. Formula and method of calculation
The mass of extracted fat as g;
(m1-m2) - (B1-B2)
Amount of fat in the sample is expressed in percentage:
(m1 – m2 )-(B1 – B2 )
------------------------- x 100
S
m1 = mass of flask and the fat obtained after the operation in 6.2.15, g
m2 = mass of flask obtained after the operation in 6.2.1 or 6.2.16.2, g
B1 = mass of flask used in blank experiment after the operation in 6.2.15, g
B2 = mass of flask used in blank experiment after the operation in 6.2.1 or 6.2.16.2, g
S = mass of the sample, g
7.2. Repeatability
The difference between the results of two analyses made simultaneously or successively as fast for the same sample should not exceed 0.2 g of fat in each 100 g product and for skimmed milk powder 0.1 g of fat in each 100 g of milk powder.
Method 5: Determining amount of saccharose (Polarimetric Method)
1. Scope and field of application
This method is used in determining the amount of saccharose existing in the condenced milk containing added sugar, condenced semi skimmed milk containing added sugar, condenced skimmed milk containing added sugar. The samples used in this method are required not to contain invert sugar.
2. Definition
Amount of saccharose in condenced milks containing added sugar is determined as explained in this method.
3. Principal
The principle of this method is based on the Clerget inversion principle. As a result of light treatment of the sample with acid, complete hydrolysis of saccharose is maintained, but lactose or other sugars are not influenced by this procedure. Amount of saccharose is obtained by determining the angular degree of solution.
Limpid filtrate of the sample is obtained by treating with ammonia without any alteration by lactose, and by proceeding neutralization and by subjecting to clarification procedure with zinc acetate and potassium hexacyanoferrate (II) solutions. The saccharose in a portion of the filtrate is hydrolyzed in a specific way.
Amount of saccharose is calculated with the rotation values before and after the inversion of filtrate.
4. Reagents
4.1. Zinc acetate solution, 1 M: 21.9 g crystallized zinc acetate dehydrate Zn(C2H3O2)2.2H2O and 3 ml of glacial acetic acid are dissolved in some amount of water, and filled up with water to 100 ml.
4.2. Potassium hexacyanoferrate-II solution, 0.25 M: 10.6 g crystallized potassium hexacyanoferrate-II-trihydrate K4[Fe(CN)6].3H2O is dissolved in some amount of water, and then filled up with water to 100 ml.
4.3. Hydrochloric acid solution, 6.35±0.2 M (20-22%) or 5.0±0.2M (16-18%)
4.4. Ammonia solution, 2±0.2M (3.5%)
4.5. Acetic acid solution, 2±0.2M (12%)
4.6. Bromothymol blue indicator, solution in 1% (mass/volume) ethanol
5. Equipment and material
5.1. Analytical balance, with the precision of 10 mg.
5.2. Polarimeter tube, exactly calibrated to a length of 1 dm.
5.3. Polarimeter or saccharimeter:
(a) Polarimeter, that has sodium light or mercury green light, readable in accuracy of minimum 0.05 angular degrees, (mercury vapor lamp with prism or special Wratten Screen No 77 A).
(b) Saccharimeter, international sugar scaled, using white light passing through a filter of 15 mm of 6 % solution of potassium bichromate, or sodium light, readable in an accuracy of minimum 0.1 international sugar scale degree.
5.4. Water bath, adjustable to 60±1 °C.
6. Procedure
6.1. Blank experiment
In order to control the procedure, reagents, tools and apparatus, the following control experiment is performed in parallel on 100 g of milk or a mixture of 110 g skimmed milk and 18 g pure saccharose. This mixture corresponds to 40.00 g of condenced milk containing 45% saccharose. Amount of sugar is calculated by the formula given in article 7. In the 1st formula m stands for amount of milk to be weighed, F amount of fat in the milk, P amount of protein in the milk. In the second formula, value of m is taken as 40.00. Calculated average value should be in the limits of 45%±0.2.
6.2. Experiment
6.2.1. About 40 g of well mixed sample is weighed in an accuracy of 10 mg into a glass beaker. 50 ml of hot water (80-90 °C) is added and mixed thoroughly.
6.2.2. The beaker is washed a few times with water in 60 °C up to a point where the mixture has a total volume in between 120 ml and 150 ml and transferred quantitatively into a 200 ml of scaled flask. It is mixed and cooled down to room temperature.
6.2.3. 5 ml of diluted ammonia solution (4.4) is added, mixed again and is waited for about 15 minutes.
6.2.4. Ammonia is neutralized by adding diluted acetic acid (4.5). By using bromtimol blue (4.6) as indicator, definite amount of diluted acetic acid in ml that is used for neutralization of ammonia solution with titration is determined.
6.2.5. Shaked flask is mixed by slightly turning and 12.5 ml of zinc acetate solution (4.1) is added.
6.2.6. 12.5 ml of potassium hexacyanoferrate-II solution is added in the same way as adding zinc acetate solution.
6.2.7. Temperature of content of the flask is raised to 20 °C and filled with water at 20 °C up to 200 ml.
Note: Up to this stage, addition of all water and reagents should be made not to cause any air bubbles. All the mixing activities should be made by circular turning, but not by shaking the flask. If the air bubbles exist, they may be eliminated by temporarily connecting the flask to a vacuum pump and by turning the flask with circular rotations, prior to completion of its volume to 200 ml.
6.2.8. The flask is closed with a dry cover and mixed by shakng violently
6.2.9. It is waited for a few minutes and filtered through a piece of dry filter paper; first 25 ml part of the filtrate is ejected.
6.2.10. Direct polarization: optical deviation of the filtrate is determined at 20 ±1 °C.
6.2.11. Inversion: 40 ml of the filtrate is transferred into a balloonjojet scaled to 50 ml. 6 ml of 6.35 M hydrochloric acid or 7.5 ml of 5 M hydrochloric acid is added. The flask waits in a water bath at 60 °C for 15 minutes, it should be ensured to immerse all of the contents of the flask. It is mixed with rotational motion during the first 5 minutes. During this period content of the flask reaches the temperature of the water bath and then cools down to 20 °C, its volume is filled up with 20 °C water, mixed and waits for an hour at this temperature.
6.2.12. Invert polarization: Optical deviation of the invert solution is determined at 20 ±0.2 °C. If the liquid temperature in the polarization tube changes more than 0.2 °C, temperature correction as indicated in Article 7.2 should be used.
7. Expression of results
7.1. Formula and method of calculation
Amount of saccharose is calculated by the following formula in terms of percentage of mass:
(i)v = (m/100) x (1.08 F + 1.55 P)
(ii) S = [(D – 1.25 I)/Q] x [(V – v)/V] x (V/L x m)
S = Amount of saccharose
m= Mass of sample, g
F= Amount of fat in the sample
P= Amount of protein in the sample (N x 6.38)
V= Filled up volume of the sample prior to filtering, ml
v= correction in the volume of solution formed during clarification, ml
D= polarimeter reading prior to inversion
I= polarimeter reading after inversion
L= Length of polarimeter tube, dm
Q= Inversion factor, values as given here below:
(a) Once exactly 40.00 g condenced milk is weighted and polarimeter with 2 dm polarimeter tube having angular degreed sodium light is used at 20.0±0.1 °C, amount of saccharose in normal condenced milk (C=9) is calculated by the following formula:
S = (D – 1.25 I) x (2.833 – 0.00612 F – 0.00878 P)
(b)If the invert polarization is measured at a temperature (T) other than 20 °C, the value of I should be multiplied by :
1 + 0.0037 (T – 20)
7.2. Values of inversion factor Q;
The following formulas give the definite values of Q together with the temperature and concentration corrections for different light sources, whenever required.
Sodium light and angular degreed polarimeter:
Q = 0.8825 + 0.0006 (C – 9) – 0.0033 (T – 20)
Mercury green light and angular degreed polarimeter:
Q = 1.0392 + 0.0007 (C – 9) – 0.0039 (T – 20)
White light with dichromate strainer and international sugar scale degreed saccharimeter:
Q = 2.549 + 0.0017 (C – 9) – 0.0095 (T – 20)
In these formulas:
C = Total sugar percentage in polarized invert solution
T = Temperature of invert solution at polarimetric reading
Note 1: The percentage of total sugar concentration (C) in invert sugar solution can be calculated by direct reading and by the change in inversion with regular method. Specific deviation values of saccharose, lactose and invert sugar are also used for this calculation.
Correction of 0.0006 (C – 9) is correct when C is equal to about 9, this correction can be neglected if C is close to 9 for normal condenced milks.
Note 2: For direct readings, 1 °C deviations from 20 °C create minor differences. However, in invert reading, deviations above 0.2 °C necessitate a correction. Correction factor – 0.0033 (T – 20) etc. is only correct between 18 – 22 °C.
7.3. Repeatability
The difference between the results of two analyses made under the same conditions, by the same analyzer, simultaneously or successively as fast for the same sample should not exceed 0.3 g of saccharose in each 100 g sample.
Method 6- Determining the amount of Lactic Acid and Lactate
1. Scope and Field of Application
This method is used for determining the amount of lactic acid and lactate in condenced skimmed milk containing added sugar, milk powder with high fat content, whole milk powder, semi skimmed milk powder, and skimmed milk powder.
2. Definition
The amount of lactic acid and lactate in milk powder; is the amount of lactic acid and lactate indicated as lactic acid determined by the given method.
3. Principle
Copper sulfate and calcium hydroxide are added into the sample. Later on, fat, protein and lactose are simultaneously removed from the solution by filtration. Lactic acid and lactates in the filtrate are converted into acetaldehyde forms by condenced sulfuric acid in the presence of copper (II) sulfate. Amount of lactic acid is determined calorimetrically by using p-hydroxydiphenyl.
Amount of lactic acid and lactates is the amount of lactic acid as mg in 100 g fat free dry substance.
4. Reagents
4.1. Copper (II) sulfate solution: 250 g copper (II) sulfate (CuSO4.5H2O) is dissolved in some amount of water and filled up to 1000 ml.
4.2. Calcium hydroxide suspension
4.2.1. 300 g of calcium hydroxide (Ca (OH) 2) is crushed with water in a mortar by using total 900 ml of water and it should be prepared as fresh.
4.2.2. 300 g of calcium hydroxide (Ca (OH) 2) is crushed with water in a mortar by using total 1400 ml of water and it should be prepared as fresh.
4.3. Sulfuric acid – copper (II) sulfate solution: 0.5 ml of copper (II) sulfate solution (4.1) is added into 300 ml of sulfuric acid (H2SO4) of 95.9 – 97 % (m/m).
4.4. p-hydroxydiphenyl (C5H6C5H4OH)solution : 0.75 g p-hydroxydiphenyl is slightly heated and dissolved by shaking in 5 ml portion of a aqueous sodium hydroxide solution which contains 5 g sodium hydroxide (NaOH) in 100 ml. It is diluted with water to 50 ml in metrical bulb. It is stored in a dark colored glass bottle in dark and cool place. It is not used further if any color change or turbidity is observed. Prepared solution withstands for 72 hours.
4.5. Standard lactic acid solution: 0.1067 g lithium lactate (CH3CHOHCOOLi) is dissolved in some amount of water, filled up to 1 L with water and prepared just before usage. 1 ml of this solution contains 0.1 mg lactic acid.
4.6. Standard reconstituted milk: By analyzing a few high quality milk powders, a sample of which ensures the lowest content of lactic acid is selected in order to prepare the calibration curve. This sample should contain maximum 30 mg lactic acid in 100 g fat free dry substance. The procedures described in Articles 6.2.1 and 6.2.2. herebelow are to be followed.
5. Equipment and materials
5.1. Analytical balance
5.2. Spectrophotometer; with a capacity of reading in 570 nm wave length
5.3. Water bath; adjustable to 30 ± 2 °C
5.4. Mortar and pestle
5.5. Filter paper; Schleicher and Schull 595, Whatman 1 or equal
5.6. Test tubes; pyrex or equal, 25 x 150 mm in size
Note: All the material should be cleaned well and used only in this determination. Glass material containing residue should be rinsed well with condenced HCl prior to washing.
6. Procedure
6.1. Blank experiment
30 ml water is put in a tube of 50 ml capacity, the procedure in steps between 6.2.4 and 6.2.11 are performed and blank experiment is completed. If the value of the blank obtained during measurement against water exceeds the amount of 20 mg equivalent lactic acid in 100 g fat free dry substance, then the solutions are checked and the ones that are not pure are replaced. Blank experiment should be completed at the same time with the sample analysis.
6.2. Experiment
Dirt contamination should be avoided particularly with sweat and spittle.
6.2.1. Fat free dry matter content of the sample is found in gram by subtracting moisture content obtained in method 2 and fat content obtained in method 4 from 100. (a)
6.2.2. The sample is weighed in an accuracy of 0.1 g as 1000 / (a – 10) g, and this is added into 100 ml of water and mixed.
6.2.3. 5 ml of the solution obtained is taken into the graduated tube of 50 ml and diluted to 30 ml with water.
6.2.4. By shaking 5 ml of copper (II) sulfate solution (4.1) is added slowly and waited for 10 minutes.
6.2.5. 5 ml of calcium hydroxide suspension (4.2.1) or 10 ml of calcium hydroxide suspension (4.2.2) is added slowly while shaking the tube.
6.2.6. It is diluted to 50 ml with water and strongly shaked, waited for 10 minutes and then filtered. First section of the filtrate is discarded.
6.2.7. 1 ml of filtrate is taken into test tube in Article 5.6.
6.2.8. 6 ml of sulfuric acid – copper (II) sulfate solution (4.3) is added into the tube by means of a burette or a pipette and mixed.
6.2.9. It is heated in a boiling water bath for 5 minutes and cooled down to room temperature under water.
6.2.10. 2 drops of p-hydroxydifenyl solution (4.4) is added and shaked strongly until a homogeneous distribution is obtained. Tube is placed in the water bath at 30±2 °C and waits there for 15 minutes by shaking occasionally.
6.2.11. Tube is held in the boiling water bath for 90 seconds. It is then cooled down to room temperature under flowing tap water.
6.2.12. Optical density is measured against blank in three hours at the wavelength designated in Article 5.2.
6.2.13. If the optical density exceeds over the highest point of the curve, the experiment is repeated by diluting the filtrate in a proper way that was obtained in Article 6.2.6.
6.3. Preparations of the standards
6.3.1. 5 ml of reconstituted milk is taken into each of 5 graduated tubes with 50 ml capacity. 0, 1, 2, 3 and 4 ml of standard solution (4.5) are added respectfully to each tube. Hence it is ensured to add 0, 20, 40, 60 and 80 mg lactic acid respectfully on to 100 g fat free dry substance of milk powder.
6.3.2. It is diluted to 30 ml with water and the procedure described between Article 6.2.4 and 6.2.11 is performed.
6.3.3. Optic densities of standards (6.3.1) are measured against blank experiment (6.1) at the specific wavelength as indicated in Article 5.2. Optical densities read against lactic acid quantities given in Article 6.3.1 that is numerically against 0 mg, 20 mg, 40 mg, 60 mg and 80 mg per 100 g fat free dry substance, are marked on the graph. Best line is drawn through the points and this line is moved down parallel to itself as to pass through the center of origin.
7. Expression of the results
7.1. Formula and method of calculation
Amount of lactic acid in the samples of 100 g fat free dry matter is calculated as mg from the optical density measurements that was indicated in Article 6.2.12 and 6.2.13 by the help of standard curve. These results are multiplied with dilution factor of filtrate diluted according to article 6.2.13.
7.2. Repeatability
The difference between the results of two analyses made under the same conditions, by the same analyzer, simultaneously or successively as fast for the same sample should not exceed 8 mg of lactic acid in 100 g fatless dry matter, for maximum value containing 80 mg. This difference for higher values should not exceed 10 % of the lowest value.
Method 7: Determining Phosphatase Activity (Modified Sanders and Sager Procedure)
1. Scope and Field of Application
This method is used in determining the phosphatase activity in milk powder with high fat content, whole milk powder, semi skimmed milk powder, skimmed milk powder.
2. Definition
Phosphatase activity in dried milks is the quantity measurement of active alkali phosphatase existing in the product. According to the procedure described here below, it is expressed as quantity of phenol as microgram liberated in 1 ml of reconstitute milk.
3. Principle
In dried milks phosphatase activity depends on the ability of phosphatase to liberate phenol from disodium phenyl phosphate. It depends on the principle of determining amount of liberated phenol by spectrophotometric measurement of color development with Gibb’s solution.
4. Solutions
4.1. Solution A:
Barium borate hydroxide buffer: pH 10.6 ± 0.1, at 20 °C: 25 g barium hydroxide (Ba(OH )2.8H2O) is dissolved in some amount of water and is filled up to 500 ml. 11 g boric acid (H3BO3)is dissolved in water and filled up to 500 ml. Both of the solutions are heated to 50 °C and mixed. The mixture is shaked and cooled down to room temperature. pH is adjusted to 10.6 ± 0.1 with barium hydroxide solution and filtered. The solution is stored in a bottle that can be tapped firmly. Prior to using, the buffer solution is diluted with equal amount of water.
4.2. Solution B:
Color developer buffer solution: 6 g sodium metaborate (NaBO2) or (12.6 g NaBO2.4H2O) and 20 g sodium chloride are dissolved in water and filled up to 1000 ml with water.
4.3. Solution C:
Buffer substrate solution:
4.3.1. 0.5 g disodium phenyl phosphate (Na2C6H5PO4.2H2O) is dissolved in 4.5 ml of solution B (4.2). 2 drops of solution E (4.5) is added and waited for 30 minutes. It is extracted with 2.5 ml of butanol (4.10). If required, the extraction process is repeated. Then separation process is performed and butanol is removed. This solution can be stored in the refrigerator for a few days. If any color development is observed in the solution, it is extracted with butanol before use.
4.3.2. 1 ml of this solution is put in 100 ml of balloonjojette and filled up to its mark line with solution A. Buffer solution is prepared fresh.
4.4. Solution D:
Precipitant: 3 g zinc sulfate (ZnSO4.7H2O) and 0.6 g copper (II) sulfate (CuSO4.5H2O) are dissolved in water and filled up to 100 ml with water.
4.5. Solution E:
Gibb’s solution: 0.040 g 2,6-dibromokynon-1,4-chloramid (O.C6H2BR2.NC1) is dissolved in 10 ml of 96% ethanol. Solution is stored in dark color bottles in the refrigerator. It is discarded when it turns out to be colorless.
4.6. Diluted color buffer: 10 ml solution B (4.2) is diluted to 100 ml with water.
4.7. Copper sulfate solution: 0.05 copper (II) sulfate (CuSO4.5H2O) is dissolved in water and filled up to 100 ml.
4.8. Standard phenol solution: 0.200±0.001 g pure phenol is dissolved in water and filled up to 100 ml inside a graduated flask. This solution can be stored in the refrigerator for a few months. 10 ml of this solution is diluted to 100 ml with water. 1 ml of this diluted solution contains 200 µg phenol and this solution can also be used while preparing further diluted solutions.
4.9. Boiling distilled water
4.10. n-butanol
5. Equipment and material
5.1. Analytical balance
5.2. Water bath; adjustable to 37 ± 1 °C
5.3. Spectrophotometer; enable to make readings in wavelength of 610 nm.
5.4. Filter paper; Schleicher and Schull 597, Whatman 42 or equivalent filter paper.
5.5. Boiling water bath
5.6. Aluminum foil
6. Procedure
Notes:
a) It should be avoided from direct sun light.
b) All glass material, carrying tools and corks should be perfectly clean. It is suggested to rinse and to boil them with water or to be traeted with steam.
c) Plastic material, for instance plastic covers should not be used, because they may contain phenol.
d) Even a little amount of spittle contamination should be avoided, because it contains phosphatase.
6.1. Preparation of the sample
6.1.1. The sample is weighed in between 0.1 g and 10 g and dissolved in 90 ml of water. The temperature should never exceed 35 °C to dissolve milk powder.
6.2. Experiment
6.2.1. 1 ml of reconstituted milk prepared as explained in Article 6.1.1. is put in each of the two test tubes.
6.2.2. One of the tubes is heated for two minutes in the boiling water bath as to keep the complete solution inside the water bath. It is cooled down to room temperature with cold water. This tube is used for blank experiment. Further steps are applied to both tubes in the same way.
6.2.3. 10 ml of solution C (4.3.2) is added, mixed and the tube is placed in the water bath at 37 °C.
6.2.4. It is left in the water bath for incubation for 60 minutes by shaking periodically.
6.2.5. Tubes are transferred right away into boiling water bath, heated for 2 minutes, cooled down to room temperature with cold water.
6.2.6. 1 ml of solution D (4.4) is added, mixed and filtered through a dry filter paper, initial filtrates are discarded until clear fluid is obtained.
6.2.7. 5 ml of each filtrate are put in the test tubes, 5 ml of solution B (4.2) and 0.1 ml of solution E (4.5) are added, mixed.
6.2.8. It is left for color development for 30 minutes in room temperature away from the direct sun light.
6.2.9. The optical density of the sample solution is measured against the blank at wavelength of 610 nm in the spectrophotometer.
6.2.10. If the optical density of the solution gets over the standard solution containing 20 µg phenol in article 7, then the process is repeated. If this limit is exceeded, reconstituted milk prepared according to 6.1.1 is diluted to a proper volume with carefully boiled milk as in 6.2.2 in order to inactivate the existing phosphate.
7. Preparing the standard curve
7.1. 1, 3, 5 and 10 ml of standard solution diluted as per article 4.8 are put into 4 ea. graduated flasks of each 100 ml by means of a pipette and filled up to its volume with water; these solutions contain 2, 6, 10 and 20 µg phenol respectively.
7.2. 1 ml water and 1 ml from each of the standard solutions (7.1) are added into the tubes by a pipette in order to obtain series of samples containing 0, 2, 6, 10 and 20 µg of phenol. The tube with 1 ml of water has the blank value.
7.3. 1 ml of copper (II) sulfate (4.7), 5 ml of diluted color buffer solution (4.6), 3 ml of water and 0.1 ml of solution E (4.5) are added to each of the test tubes and mixed.
7.4 Tubes are waited at room temperature for 30 minutes, while protecting from direct sun light.
7.5 Absorbency of the solution in each tube is measured against blank at wave length of 610 nm in the spectrophotometer.
7.6. Standard curve is formed with the µg absorbency values corresponding to the amount of phenol indicated in article 7.2.
8. Expression of results
8.1. Formula and method of calculation
8.1.1. The value obtained at the end of process in article 6.2.9 is found by the help of the standard curve.
8.1.2. Amount of phenol in 1 ml reconstitute milk in µg is calculated as its phosphatase activity by using the following formula:
Phosphatase activity = 2.4 x P
Where P = µg value of the amount of phenol obtained as per article 8
8.1.3. If the dilution process in article 6.2.10 has already been performed, the result in 8.1.2 is multiplied by dilution factor.
8.2. Repeatability
The difference between the results of two analysis made under the same conditions, by the same analyzer, simultaneously or successively as fast for the same sample should not exceed 2 µg phenol that comes out free from 1 ml of reconstitute milk.
Method 8: Determining Phosphatase Activity (Aschaffenburg and Mullen Procedure)
1. Scope and Field of application
This method is used in determining the phosphatase activity in condenced skimmed milk containing added sugar, milk powder with high fat content, whole milk powder, semi skimmed milk powder, and fatless milk powder.
2. Definition
Phosphatase activity in milk powders is the measurement of the amount of active alkali phosphatase existing in the product. Under the given conditions, it defines the amount in µg of p-nitrophenol that comes out free from 1 ml of reconstitute milk.
3. Principle
Reconstitute milk sample is diluted with substrate buffer having pH 10.2 and left in incubation at 37 °C for 2 hours. If any kind of alkali phosphatase exists in the sample, under these conditions p-nitrophenol is liberated from added disodium p-nitrophenylphosphate. Liberated p-nitrophenol is determined by using reflection light, comparing with standard colored glasses by means of a simple comparator.
4. Solutions
4.1. Sodium carbonate – bicarbonate buffer solution: 3.5 g anhydrous sodium carbonate and 1.5 g sodium bicarbonate are dissolved in water, diluted with water to 1000 ml in a graduated flask.
4.2. Buffer substrate: 1.5 g disodium p-nitrophenyl phosphate is dissolved in sodium carbonate – bicarbonate buffer solution (4.1), diluted with buffer solution (4.1) to 1000 ml. This solution is stable if stored in a refrigerator at 4 °C for 1 month. But a color control test should be carried out on such stored solutions. Precautions indicated in Article 6 should be taken into account.
4.3. Clarification solutions
4.3.1. Zinc sulfate solution: 30 g zinc sulfate (ZnSO4) is dissolved in water, diluted to 100 ml with water.
4.3.2. Potassium hexacyanoferrate-II solution: 17.2 g potassium hexacyanoferrate-II trihydrate (K4Fe(CN)6.3H2O) is dissolved in water and diluted with water to 100 ml.
5. Equipment
5.1. Analytical balance
5.2. Water bath: adjustable to 37 ± 1°C.
5.3. Comparator, having cells in size 2 x 25 mm and a special disc containing standard color glasses calibrated as to correspond amount of p-nitrophenol as µg in 1 ml milk.
6. Procedure
Notes:
a) Whenever usage is complete, the experiment tubes should be emptied, rinsed with water, washed with hot water containing an alkali detergent, then rinsed completely with clean hot tap water. Finally prior to use, they should be rinsed with water and dried.
Pipettes should be rinsed with clean cold tap water right after using, and rinsed with water and dried prior to use again.
b) Tube covers should be rinsed with hot tap water right after use and should be boiled in water for two minutes.
c) Buffer substrate solution (4.2) should stay as stable at least for a month, if it is stored in refrigerator at 4 °C or lower temperature. If it gets to yellow color, it means it is instable. In order to control buffer substrate solution; when a reading is done in 25 mm cell by means of using distilled water against boiled milk, if color reading exceeds 10 µg then the solution should not be used anymore.
d) A different pipette is used for each sample and contamination of spittle on to the pipette should be avoided.
e) Experiment should never be exposed to direct sun light.
6.1. Preparation of the sample
10 g of the milk powder is dissolved in 90 ml of water. Temperature should not exceed 35 °C to dissolve the milk powder.
6.2. Experiment
6.2.1. 15 ml of buffer substrate solution (4.2) is taken into a clean dry tube with a pipette. Then 2 ml of reconstituted milk sample (6.1) to be tested is added. Tube is closed with its cover and mixed, further placed in the water bath (5.2) at 37 °C.
6.2.2. Simultaneously, under similar conditions 15 ml of buffer substrate solution and control tube containing 2 ml of boiled reconstitute milk sample are placed in the water bath.
6.2.3. After 2 hours both tubes are taken out of the water bath. 0.5 ml of zinc sulfate solution (4.3.1) is added as precipitant, closed with its cover, shaked strongly and allowed to stand for 3 minutes. 0.5 ml of potassium hexacyanoferrate-II solution (4.3.2) is added as precipitant, mixed and filtered through folded filter paper and the clean filtrate is collected into a clean experiment tube.
6.2.4. The filtrate is transferred into 25 mm cell and compared with the boiled control sample filtrate by using special disc (5.3) in the comparator.
7. Expression of results
7.1. Formula and method of calculation
The value read in Article 6.2.4 is recorded as µg nitrophenol in each ml of the sample or direct reconstituted milk sample.
7.2. Repeatability
The difference between the results of two analysis made under the same conditions, by the same analyzer, simultaneously or successively as fast for the same sample should not exceed 2 µg p-nitrophenol that comes out free from 1 ml of reconstituted milk.