1. ALIZAROL TEST

1.1 OBJECTIVE OF THE ALIZAROL TEST

The Alizarol test, due to its simplicity as well as its suitability to point out  more than one defect of milk is a practical and well-suited procedure to test raw milk for acceptability, either on the farm or from the tanker at the factory.  The main objective of the test is to indicate milk in which an unaccepted level of acidity has developed.

The stability of the protein complex in milk is destabilized by acid and therefore a positive alizarol test is also an indication of the heat stability of milk (i.e. the resistance of the protein complex against the denaturation by heat).  The test is therefore also popular where the heat stability of milk proteins is of importance during processing, e.g. during the production of milk powder or UHT milk.

By adding the pH-indicator alizarine to the solution an indication of whether the milk is abnormally sour or alkaline can be obtained.

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1.2 FACTORS AFFECTING THE TEST

1.2.1 Acidity

The presence of lactic acid is the most important cause of a positive alizarol test. At the higher acid level  (lower pH )  the protein suspension in milk  will be less stable with the result that protein flocculation (coagulation) will occur  when acid milk is mixed with alcohol.

Under normal circumstances and if the concentration of the alcohol is 68% (v/v), acidity will influence the test as indicated in Table 1.

TABLE 1                       THE ALIZAROL TEST

1.2.2     Mastitis

Mastitic milk differs from normal milk as regards, amongst others, the protein and mineral composition. These differences cause the casein (protein) to flocculate more readily in mastitic milk.  As the pH of mastitic milk is normally  higher than that of  normal milk the alizarine colour will turn violet if such milk is tested.

1.2.3 Sweet curdling

Certain contaminating bacteria are capable of producing rennin like enzymes, which  increase the viscosity of the milk and even flocculate casein in the absence of acid. This phenomenon is known as sweet curdling. Such milk can be identified by  inoculating aseptically a small quantity there-off   into sterilised milk and incubating the inoculated milk at room temperature for a day or more on which the viscosity of the  milk will increase at the  normal pH of fresh milk.

Large numbers of these bacteria e,g. 800,000 /ml milk was found to create a positive reaction in previously sterilised milk.

1.2.4 Mineral imbalance

Too high or to low a concentration of minerals such as calcium, phosphate and citrate will increase the ease with which casein will flocculate in the presence of alcohol.

This aspect is complex and not well define. It is well known that the addition of very small amounts of Calcium (0.2 ml M/4 Ca-acetate / 25 ml milk = 0.6g / 1 l melk ) and Magnesium (0.1 mlM/4 MgCl₂ / _/25 mlmilk = 0.95 g / 1l melk ) will normally result in a positive test while the addition of phosphate and citrate salts may alleviate the situation. In more extreme cases excess phosphate and citrate  may however be the cause of a positive test.

Small variation e.g. 0.01% in the normal Calcium content of milk may influence the test.

It is claimed that in some instances supplementing a cows ration with mono-calcium phosphate e.g. 20 – 40 g /day / cow  may  improve the situation.

If a positive reaction is due to mineral imbalance a very slight souring of the milk, causing a portion of the colloidal fraction of the minerals to become soluble, may result in a negative test.

Heating of alizarol positive milk to 60°C for 30 minutes will cause partial precipitation of Ca₃(PO₄ ) and as result the test may turn negative.

1.2.5       Stage of laction;

The chemical composition of early and late lactation milk differs from that of normal milk

in the sense that it is characterised e,g. by higher levels of albumin, globulin and chloride.

Due to these differences the protein suspension in such milk is usually very

unstable against alcohol

Milk is normally very unstable to alcohol in early lactation, thereafter gradually becoming more stable and remaining at a fairly constant level of stability which is specific for each cow. Towards the end of lactation milks from some cows become more stable but a decrease in stability is more common.

1.2.6.   Other factors that may  cause alizarol positive reactions include;

• Irritation of the udder tisue.
• Infected uterus and vagina
• Aborsion

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1.3 PRINCIPLES OF THE TEST

The stability of the protein dispersion in milk is maintained by hydration (combining with water) and by the negative electrical charges on the protein particles. If either of these two factors under goes a change, the proteins will flocculate. Alcohol is a dehydrator and therefore destablises the protein. If the protein is already slightly unstable due to souring of the milk, dehydration with alcohol will lead to the precipitation of the protein in the form of flakes.

1.4     REAGENTS AND APPARATUS:

• Measuring equipment: E.g. 2 – 5ml pippettes, siringes or automatic measuring apparatus for measuring milk and alizarol.

• Test tubes, small containers or other apparatus for mixing milk and alizarol.

• Alizarol-solution: Either 68 % (v/v%) or other specified concentration (see Table 2) of ethanol with alizarin powder dissolved in the alcohol.

Alizarol must be kept in a brown bottle in a cool place.

Either methyl (white spirits ) or ethyl alcohol, standardized with water to specified concentration and containing 0.4g alizarin powder (1.2 – dioxy-anthrquinone – C₁₄H₈O₄, MM 240.22, colour code:  CI 58000, pH-reeks:  11.0 – 13.0).  The pH of the mixture should be neutralized to 6.7 and checked regularly.

TABLE 2              APPLICATION OF DIFFERENT ALIZAROL CONCENTRATIONS

1.5 THE PROCEDURE FOR DETERMINING PROTEIN STABILITY (ALIZAROL TEST)

(i)         Equal volumes e.g. 2 or 5 ml of milk and alizarol of prescribe concentration, e.g. 70%, is mixed in a suitable container e.g. a test tube.  (Equal volumes is critical for the accuracy of the test).

(ii)        Mix carefully by inverting the tube once of twice.

(iii)       Evaluate:                –           degree of flocculation, if any

–           colour change

(iv)      Interpretation

–          If the colour remains light purple and no flocculation occurs, accept the milk.

–          If the colour remains light purple and slight flocculation occurs, confirm acidity/ bacterial activity by performing one or more  of the following tests:

• Clot-on-boiling
• pH and/or titratable acidity
• Resazurin test

–          If the colour turns brownish-pink and flocculation occurs, reject the milk.

–          If the milk has a brownish-pink colour but no flocculation occurs, determine the pH or %TA.  If the results are unacceptable, that is, if they do not fall within the prescribed limits, reject the milk.

–          If the colour of the milk varies from brownish-yellow to yellow, reject the milk.

–          If the colour of the milk is violet but no flocculation occurs, determine the pH or %TA.  If the results are unacceptable, that is, if they do not fall within the prescribed limits, reject the milk.

–          NB!  Take care not to interpret fat granules in cold milk for protein flocculation.

For an online training course on the alizarol test that you can use forever in your dairy business to re-train and train new employees at $45 go to:

https://gum.co/alizarol