Fat Deternination

(A) Fluid Milk : Quantification of fat in milk can be done by the following dstems :

(a) Direct determination by extrction with ether as adopted in the Adam’s coil method.

(b) Separation of fat by acid/alkali treatnent and its subse-quent removal by ether. This system finds application in the Werner-Schimdt and Gottleib method.

(c) Separation of fat by chemical means followed by centri-fuging. This technique is employed in the Gerber method as well as in Lefrnann-Beam method.

  Adams’ paper coil method is the most accluate hence is employed in samples having fat percentage either of doubtful accuracy or bordering on the minimum statutory limit. This method is suitable for fresh milk only.

  Special fat-free strips of paper are needed in this method besides routine apparatus and chemicals. These strips can be prepared in the laboratory as well, from thick filter paper defatted through ether extaction and subsequenct drying.

  Method’s theory is that a fat free strip is suspended over a hot plate to dry but not scorch so that 5 ml of the sample may be spotted on this strip carefully by delivering it from a pipette. The weight of this 5 ml of milk is detemrined by weighing separately. Another similar 5 ml of the same milk sample is weighed in a platinum dish. The paper strip is dried completely lest any moisture be left behind, retained and later on carry water-soluble substances which dissolve in ether subsequently. At the same time, the thorough and/or quick drying must also be avoided lest the strip becomes flaky. This is accomplished by fitting a small fibre filter in the side tube of Soxhlet’s extractor. The dried paper strip is rolled up loosely and transferred to the Soxhlet’s extractor for extraction with ether for about four hours. The top of the condenser is plugged with cotton wool. This helps to partly feed back the escaping few vapours of ether but mostly to keep away moisture as done in non-volatile ether extract in groud spices. The rest is the same.

   The salient points of the Lefmann-Beam process are that the protein is precipitated first and dissolved later on by the acid to free fat globules. Amyl alcohol produces a great difference in surface tensions between that of fat globules and their supporting liquids, thus causing an aggregation and separation of the fat.
  Gerber’s method differs, though slightly, in approach from the Beam’s method. In  he Gerber’s method the proteins and mineral matter is dissolved in H₂SO₄  and a solution is obtained. Fat is dissolved in hot amyl alcohol. Its seperation from the heavy acid solution is effected by subjecting the mixture to centrifuging. Since this is mostly applied on a routine basis in the Indian milk plants, therefore it has been dealt here in detail.
  10 ml of conc. H₂SO₄ is run into the Gerber tube which is returned to the stand. I ml. of fat free amyl alcohol is added avoiding the threaded neck of tube.The milk sample is well mixed. 10.94 ml of this milk sample is pipetted out, the exterior of the pipette wiped clean of milk. The tip of the pipette is inserted into the slightly tilted Gerber tube without touching the neck. Rapid discharge of milk is scrupulously avoided to keep off milk which would result in obscuring the junction of the fat column with the acid after centrifuging, therefore, the milk release is controlled to keep the three liquids (acid, alcohol and milk as separate layers in the tube which is held firmly by the stem and the contents (alcohol and milk) are shaken briskly. Finally the acid lying in the stem is also mixed with the milk + alcohol solution by inverting the Gerber tube twice and brisk shaking after a few minutes. Appreciable heat is generated due to action of the acid on water, the main constituent of milk.

            The tube is cntrifuged for 3 minutes at 1000 rpm (approx). The tube is then placed in a hot water bath, keeping the stem upwards to a temperature indicated on the Gerber tube for exact volume reading, reconciling the volume changes due to glass expansion. The column of the separated fat is read off the graduated stem.

  The high rpm centrifuge drives the fat towards the narrow end of the tube. Some tips for accurate operation and result are :
    (i)     Separation by centrifugal force need to be hastened.

   (ii)     It is essential to increase the difference in the specific gravity of milk plasma                                  and its fat By increasing the sp. gravity of plasma the separation of its fat is     accelerated.

   (iii)    Casein is a disturbing factor because it appears to clasp the fat globules and unless casein is not made to divorce this characteristic, separation of fat would be slow and little.

   (iv)    Temperature rise also helps in fat separation.

   All these objectives are achieved simply by the addition of conc. H₂SO₄ which dissolves proteins to effect sp. gravity differences, brings casein solution to release fat globules. Further, during dilution of H₂SO₄ through water present in the milk, heat, sufficient for the purpose is released. By adding a trace of FeCl₃ to the H₂SO₄ used, a violet colour is produced if the milk contains formaldehyde.

            The test is simple, quick and accurate. Correctness of the results have a considerable bearing on the financial and operational administration of a milk plant where lakhs of liters of milk are handled every day. The pricing of milk supplied to the plant depends upon its fat content besides other constituents. The total fat accrued through the supply of fluid milk has to. be accounted for its distribution in the various products daily manufactured. Therefore, these precautions deserve to be always borne in mind.

            (i) Proper strength of the H₂SO₄. Too strong acid chars the reaction products  rendering difficult to distinguish the fat column from the rest of the liquids  present. Too weak acid will not dissolve all the coagulated proteins, leading to haziness and not yielding a sharp demarcation between the fat and the acid             layers. A strength between 90 to 9l% is alright. Commercial H₂SO₄(sp. gr. between 1.820 to 1.825) can be used. Generally H₂O : H₂SO₄ : : 2 : 25 gives suitable acid for this purpose.

            (ii) Graduation of the Gerber tube (0 to 8%) fat level must be accurate.

            (iii) Amyl alcohol must be free from fat and petroleum, should have specific  gravity 0.8165 at 15ºC and its boiling point should lie between 51º to 55ºC.

Fat separates out in the form of an amber coloured solution.

            Numerous reagents have been proposed for replacing the sulphuric acid used in the Gerber test by less corrosive liquids. Those which incorporate synthetic detergents appear to be the most promising. Such a method due to Macdonald (1955) appears to be convenient

            Macdonald’s neutral reagent can be prepared by using 5% w/v trisodium citrate, 5% w/v sod. salicylate, l% w/v EDTA (di-sod salt), l .l%  w/v Tween 85, 3% v/v n-butyl alcohol and 25% v/v methylated spirit (over proof 66) in water. It is inverted before use.

            Procedure : 10 ml of Macdonald’s reagent is pipetted out into a milk butyrometer (Gerber method) and 10.94 ml of milk also delivered. The contents are stoppered, inverted twice and the tube placed in water bath at 65ºC for 5 minutes. The tube is centrifuged at 1100 rpm for 5 minutes, the butyrometer tube returned to the water bath at 65ºC for atleast 3 minutes and the fat percentage read off from the scale.The tube is again returned to 65ºC water bath’ recentrifuged and read again. All determinations are required to be carried out in duplicate. The method is applicable to other dairy products also (Macdonald 1959a)

            The gravimetric Warner-Schmidt method depends upon fat separation through strong HCI which factually first precipitates the casein and then dissolves it thereby lowering the surface tension. This helps in the separation of fat and subsequent. dissolution in ether.

            A weighed quantity of milk and conc. Hydrochloric acid are heated in the Stoke’s tube for the complete precipitation and redissolution of casein followed by thorough separation of fat which is dissolved by ether in the tube itself  (total volume 40 ml). An aliquot of ether layer is pipetted out accurately in a tared flask. Ether is evaporated and fat percentage determined. The method is more suitable for sour milks.

            In the gravimetric Gottleib-Rose method., casein is precipitated by alcohol which also lowers the surface tension between fat and removes the fat as before. The method is prescribed as the reference method for determining fat in milk because it gives very accurate results provided due attention is Paid to details.

            It has been observed during court trials that after sometime say > 4/5 months, the fat content percentage in milks, preserved even with adequate dose of formalin, is found less due to hydrolysis of butyrate and caproate etc. converting them to free butyric and caproic acid respectively. These being water soluble are neither recoverable as total solids nor as fat. It is the fat in the glyceride form that on drying becomes a residue and recoverable in these aforesaid tests.

            (B)       Chhanna/Paneer  shall not contain more than 70% moisture and the milk fat content shall not be less than 50% of the dry matter.

            In several cases, the reports emanating from central Food Laboratories of the PFA deptt. mention fat content on dry weight basis.

            Same is the position in cases involving cheese hard (A. 11.02.07) and processed cheese (A. 11.02.07.01). However in processed cheese spread (A.11.02.07.02) it is on dry weight basis.

            These two positions appear to be same but in reality are not so, Determination of fat content on dry matter basis means that the material which has been dried, only that dried remanant shall be employed for determining the fat content and the calculations. on the other hand, in the event of dry weight basis two separate pieces are used, one for finding the moisture content (by any method), the other for finding the fat content and the results are calculated on

                   
                                  Wt. of fat × 100
                              ______________________    All these products are not
              fat %   =      100 – moisture percentage       

homogeneous hence the results may not be reproducible creating problems while finding the true position. Besides, during heating in the oven to drive out moisture till constant weight, many volatiles may also evaporate. This may not be the position when two separate materials are analysed separately for each test on the results calculated as above.

            It is a wrong notion that by adding water to the chhanna/parurer, the vendor can escape from the law as clear from the following example where the original coustituents of a sample are

                                                Moisture %                              Required fat %                                                                                   
                                                 40                                             = 30 but has 24

                                                Add 20 ml. water                   ie. Has 6% less fat
                                                                                               
                                                                                         

                                                                                     
                        (120-60)   =   = 50%            25%, but in a total volume of
                                                                                                100 + 20 = 120 ml. the fat                                                                                          distribution shall be

                                                                                                  i.e.  20% hence
                                                                                                has 5% less fat.
Specific Gravity

            Specific gravity in milk is determined by using specific gravity bottle which should be deemed as the reference method. The other method is by using lactometer a reliable instrument in routine testing.

            Lactometer registers sp. gravity between 1015  to 1040. The range of readings on the instrument may be from 26 to 36. Lactometers are graduated for a definite temperature [60ºF (15.6ºC)]. For temperature correction, 0.0001 is added or subtracted for every 1ºF recorded temperature above or below 60ºF respectively.