How to Conduct Preliminary and Confirmatory Quality Tests on Milk

Quality assurance for milk and milk products require a concerted effort from all the key stake holders along the entire dairy value chain. This is because dairy products are highly susceptible to bacterial contamination and/or spoilage.

To ascertain the quality of the raw material and monitor the quality throughout the value chain, the following tests will be very instrumental.

Preliminary/Platform Quality Tests for Milk

The reception platform is the gate where you determine the quality that is fit for production purposes. Here, the quality you assign to the milk will determine the subsequent quality parameters determined at the quality assurance laboratory. These platform tests majorly serve two purposes.

• First, you can use the tests to identify if the milk is fit for production.
• Secondly, you can use the tests to monitor the quality of the milk during processing.

These tests will help you to ensure that the product is safe and of the right quality for human consumption.

Reception of Milk at the Platform

At the reception platform, the staff offload the milk from the vehicles and onto the reception platform. The quality assurance officer in charge of reception must ensure that the milk is of right quality before receiving it for further processing.

Preliminary Platform Tests

The initial platform tests are majorly organoleptic (based on the senses) and simple chemical tests. The officer knows the product to be processed from the milk; therefore, he/she ensures adherence to strict quality parameters at the platform. He/she must reject any milk that does not meet the minimum quality standards. Accepting milk of substandard quality will ruin the quality of the finished product.

The officer looks at the color of the milk, feels the temperature of the milk, and smells for an offensive odour. Tasting of raw milk is generally discouraged as it may be a conduit for distribution of zoonotic/infectious diseases. The officer must reject any milk of questionable quality based on platform tests results.

Any mistake in accepting poor quality milk will compound the problem by spoiling the whole batch. Blending a small amount of bad milk with a large volume of good quality milk does not improve its quality; it spoils the milk instead. You can never improve the quality of milk by blending proper milk with a poor-quality milk.

1. Organoleptic Tests for Milk

This involves all the preliminary quality tests conducted at the farm level, at the processing plant’s reception platform or at the collection center.

It is a simple, quick and cost effective method of checking quality of milk and allows for segregation of good quality and poor quality milk.

• The grader must have good senses of sight, smell, and touch because the results of this test is obtained instantly.
• Start testing by shaking the milk can a little bit then opening the lid and check for any aroma/flavor that emanates from the can by smelling it.
• Check for visual appearance on the milk such as colour, any other foreign objects on the surface of the milk and the sanitary condition of the milk can. The sanitation of the can indicates how the milk has been handled.
• Feel the milk can to check the milk temperature. The temperature of the milk can give more information about when the milking was done.

2. Clot on Boiling Test for Milk

This is a cheap, quick and easy to perform quality test that is mainly used to give information on the acidity of the milk. A small amount of milk is boiled in a test tube and checked for any signs of coagulation.

If the milk coagulates, it is an indication that the milk will not be able to withstand processing conditions due to high acidity.

This test is however not quantitative and does not detect any acidity level below 0.2% of lactic acid concentration. Any milk that fails this test is considered too sour for processing.

3. Alcohol Test for Milk

This is a simple and quick method used to test the acidity of milk. Mix equal amounts of alcohol (72% ethanol concentration) and milk sample and check for signs of coagulation.

The mechanism behind alcohol test.

Milk proteins remain stable due to two factors:

1. Hydration forces
2. Negative charges on the protein particles

Hydration forces keep the proteins suspended in milk owing to the hydrophilic property of ϰ-casein. Negative charges keep the proteins repellent from each other hence they do not agglomerate into lumps. Destabilizing these forces will lead to flocculation of the proteins.

Alcohol is a dehydrating agent which removes water from the system thereby affecting the hydration forces that are responsible for keeping the proteins suspended.

If such milk has elevated acidity due to microbial activity, the proteins will flocculate due to the combinative effect of hydration force destabilization and charge neutralization.

When combined with alizarin, it gives alcohol alizarin test, which is more informative than just the alcohol test. Alizarin is a dye that changes colour depending on the acidity level.

Colour	Red-brown	Yellowish-brown	Yellow	Lilac/pale red
pH	6.6 – 6.7	6.4 – 6.6	≤6.3	≥6.8
	Normal milk	Slightly acidic	Acidic (coagulates)	Alkaline

Other things t consider during platform tests

The officer must subject all the milk that pass platform tests (organoleptic) to additional confirmatory tests. These additional chemical tests further confirm that the milk is of exceptional quality.

Here, alcohol and specific gravity tests become very handy. You carry out alcohol tests by mixing equal amounts of 80% ethanol with milk using the alcohol gun. If the resultant mixture clots, the milk does not have stable proteins to withstand the heat treatment during pasteurization. Such protein instability may be due to salt imbalance (colostrum in milk or late lactation) in the milk, or the milk is already aged (high acid). Accept only the milk that does not yield a positive alcohol test.

The lactometer test is necessary to determine milk adulteration by adding water (or any other foreign products). Adulteration alters the density of milk and you can detect the difference by the using a lactometer. Normal milk has a lactometer reading ranging from 1.028 to 1.032. Any reading below this range suggests less milk solids (more water than normal) and any reading above this range suggests higher amount of milk solids than normal.

All the milk that passes these tests indicate milk with proper chemical tests. Even though you can accept the milk at this stage, some processors or milk vendors prefer to conduct resazurin test at this point just to be sure that the milk is of high quality. At this stage, you can weigh the amount of milk received for pricing purposes. After weighing, release the milk into the dump tank.

Current dump tanks come fitted with the weighing scales to ease the work at the reception platform. Again, the dump tanks have muslin clothes, which filter off foreign particles that may be present in the milk.

Confirmatory Tests for Milk & Milk Products

Once you have received the milk into the dump tank, you should take a sample of the composite to the lab for further quality assurance. Using this sample, conduct chemical tests such as titratable acidity, butter fat content, COB, etc. Please note that some milk processors will conduct resazurin tests on the platform before accepting the milk into the batch to reduce the risk of accepting bad quality milk into the batch.

Resazurin tests check the microbial load in milk; the lesser the load, the better the quality of milk. Poor resazurin results indicate the unsanitary storage conditions for the milk. It also suggests a very poor keeping quality of the milk. Titratable acidity test determines the level of natural and developed acidity in the milk. Fresh milk should have near neutral pH with little native acidity attributed to the milk proteins.

Here’s more in-depth analysis of the confirmatory tests for milk.

1. Acidity Test (Titration/pH method)

Acidity test is performed to determine the level of natural or developed acidity in milk. The level of acidity in milk can be expressed in two ways.

Titratable acidity: – shows the total acidity but does not show the strength of the acid

pH: = shows the strength of acid

Reasons for doing acidity test:

• To find out the quality of milk
• To monitor storage conditions because when dairy products are stored in favourable conditions for the growth of microorganisms, they produce acidity.
• To control manufacturing e.g. for yoghurt, fermentation is done until a desired level of acidity is achieved and then stored in a cold room.
• Cheese processing also depends on acidity

Natural acidity of freshly drawn milk is due to its constituents e.g. CO₂, albumins and globulins, casein and phosphates. These substances react in the milk and produce acidity.

Constituent	Lactic acid concentration (%)
CO2	0.01
Albumins and globulins	0.01
Casein	0.07
Citrates	0.01
Phosphates	0.03
Titratable acidity equivalent	0.13

Titratable acidity is based on the principle that an acid of a known volume and strength will completely neutralize an alkali with equal volume and strength. The alkali that is commonly used in titration of acidity in milk is 0.1N NaOH.

Titratable acidity is the measure of the amount of alkali required to shift the pH of milk of 6.6 to a determined pH based on the indicator used. When measuring the acidity of the milk, total acidity is usually used because it encompasses both natural and developed acidity.

Expression of titratable acidity:

The lactic acid concentration is measured in terms of:

• Soxhlet Henkel degree (°SH): – the volume (ml) of 0.25N NaOH used per 100ml of sample
• Thorner degree (°Th): – the volume (ml) of 0.1N NaOH used per 100ml of sample
• Domic degree (°D): – the volume (ml) of 0.1N NaOH used per 100ml of the sample

Acid determination should not be used as the sole basis of accepting or rejecting milk because milk can have acceptable acidity but suffers from flavor defects and other bacterial problems.

Relationship of acidity in different products

The acidity of milk and milk products is usually on the plasma portion of the milk. Therefore, when you want to compare two dairy products (milk and cream), which have different plasma and fat contents, the calculation of the acidity should be based on the plasma portion of the milk.

Given:

Milk with 4% fat content and 0.16% lactic acid and cream with 40% fat content, what will be the acidity of the cream?

Plasma: milk – 96%, cream – 60%.

Comparable acidity of the cream to milk

Acidity	Plasma
0.16:x	96:60
0.16/x=96/60 X=0.1% lactic acid

The same method can be used for cream and butter.

Factors affecting titratable acidity in milk:

1. Changes from calcium di-calcium phosphate to tri-calcium phosphate and then to phosphoric acid.
2. Action of calcium phosphate
3. The stage of lactation e.g. colostrum milk has high proteins hence high acidity
4. Mastitis or condition of the udder. Mastitis lowers the acidity because blood (which is alkaline) leaks into the milk.
5. Enzyme activity e.g. lipolysis of fats into fatty acids
6. The effect of the feeds

2. pH Determination

pH is the concentration of hydrogen ions in milk. It is determined using two methods

a. Calorimetric method

Different indicators that cover various pH ranges are used. The unknown substance with indicator added to it will match the colour of solutions with known pH. The colour solutions should contain the same indicators as the unknown. The accuracy of this method can be increased by using a comparator which will compensate for the turbidity of the milk.

b. Electrometric method

Uses pH meter/ potentiometer. This method involves measuring the difference in two different voltages of two electrodes in a sample. It gives direct measure of pH with direct values on the display unit.

3. Inhibitory Tests

Performed to check for antibiotics and drug/antibiotic residue in milk. When the antibiotics are present in significant amounts, they inhibit fermentation process. This makes it an important test to consider when dealing with fermented milk products.

Drug residues and the antibiotics are also a health hazard as some people are sensitive to antibiotics. The antibiotic test can be done through various methods, the most common of which are:

• Delvo test: – This is the easiest and the fastest method to use but it is very expensive to acquire and operate.
• Fermentation test: – bacteria in milk produce acid as they metabolize the milk, antibiotics and drugs inhibit growth of micro-organisms. Milk with antibiotics will take too long to ferment or fail to ferment completely.

4. Specific Gravity (Lactometer Test)

Lactometer is a hydrometer used to measure specific gravity of milk and scale is limited to the specific gravity of milk. Milk fat has a specific gravity of 0.93, milk has a specific gravity of 1.032 and the milk solids have a specific gravity of 1.036. Lactometer works on the principle of fluid displacement. The displaced fluid has equal weight as the floating body. Liquids with lower specific gravity have larger displacements than liquids with higher specific gravity.

Lactometers have temperature sensitive and are designed to be used at specific temperatures. It is used to check for adulteration of milk, either by skimming or addition of water or both. When water is added, the specific gravity is lowered towards one. The design of the lactometer allows for direct reading on the stem whose calibration ranges between 26 to 38. This is because the figure obtained from the reading are on the second and third decimal points of the specific gravity.

The first figures of the specific gravity are 1.0, which are then succeeded by the figures obtained from the lactometer reading. Any deviation in temperature from the recommended reading temperature is adjusted by either adding or subtracting accordingly.

Common methods of milk adulteration:

1. Skimming

Skimming is the removal of fat from the milk. Given that fat is the lightest portion of the milk, its removal will increase the lactometer reading. A sample of milk that has low lactometer reading has a high fat content compared to the one with a low-fat content.

Fat content	Lactometer reading	SNF content
low	high	High (skimmed)
Calculate % fat content removed		Calculate % lowering SNF

2. Watering

When adulteration is watering lactometer reading will go low and SNF will reduce as well as the fat content.

3. Skimming and watering

The percentage of fat removed and the percentage reduction of SNF are not the same when watering and skimming are done. Such samples have low fat content, low lactometer reading and low SNF than normal milk.

5. Freezing Point Test for Milk

Milk freezes at -0.55°C while pure water freezes at 0°C. A small adulteration with water will cause detectable change in freezing point of milk. This makes freezing point test the most accurate test for milk adulteration by water addition.

Freezing point depends on the soluble constituents of milk, especially lactose and chlorides. Freezing point remains the same even though these are differences in the composition of the two components. The freezing point does not depend on composition of the sample but on the concentration of dissolved salts (lactose and chloride). As milk freezes, its composition gets unbalanced; solids get concentrated on the remaining liquid part of the milk.

How to determine the amount of water in adulterated milk

Amount of added water = 100(Freezing temp of milk – Freezing temp of suspect milk)

6. Dye Reduction Test for Milk

These quality tests show comparative activity of the micro-organism in milk hence used as a rough indication of the microbial load in milk. It is based on the observation of the blue colour imparted in the milk, which will disappear with time. The length of time depends on the number of microorganisms present in the milk holding all other factors (such as nutrient content, moisture content, and temperature) constant.

The colour change is assumed to be due to two reasons:

1. Consumption of oxygen in milk by the microorganism
2. Enzyme reductase produced by the microorganisms

The time reduction indicates the possible number of microorganisms. This test has low correlation with other tests especially those that show presence of bacteria. To perform this test, take a definite amount of methylene blue and mix with 10 ml of milk. Mix uniformly and incubate at 37°C in a water bath and wait for colour change.

The longer it will take for the colour to change, the lower the microbial load. All the glassware used must be sterile. The interpretation of the results is as follows:

Table 1: Dye reduction test result intepretation of pasteurized milk

Length of time (hours)	Quality of milk	Grade of milk
5 or more	Excellent	1
3 – 5	Good	2
2 – 3	Fair	3
Less than 2 hours	Poor	4

Raw milk test has a much rapid decolouration.

7. Resazurin Test for Milk

Resazurin test can take 10 minutes for fresh milk and up to one hour for pasteurized milk. This test works on the principle of a dye indicator, which changes colour when oxidized.

Using sterile equipment; add 1 ml of the dye into 9 ml of milk sample, mix uniformly and incubate at 37°C in a water bath for 10 minutes for fresh milk and up to one hour for pasteurized milk. After the time has elapsed, observe the colour change using a lovibond comparator. The results are interpreted according to the table below

Reading	Colour	Quality of milk	Verdict
6	Blue	Excellent	Accept
5	Light blue	Very good	Accept
4	Purple	Good	Accept (conditionally)
3	Purple pink	Suspect	Separate
2	Light pink	Suspect	Separate
1	Pink	Bad	Reject
0	White	Very bad	Reject

The 10 minutes resazurin test is used at the reception to accept or reject milk due to its rapidity.

8. Butterfat Test for Milk

The most common and accurate method used for butterfat testing is the Gerber method. The method is used for determining the butterfat content in milk and milk products. Butterfat determination is important because it can be used to value the milk for payment purposes and to determine how to standardize the milk.

You will require the following chemicals and ingredients

• Amyl alcohol
• Sulphuric acid (S.G 1.820 – 1.825 or 90 -91% concentration)
• Milk

Sulphuric acid dissolves all the non-fat content of the milk. The fat content remains in a liquid form due to high heat produced by the acid-protein reaction. Amyl alcohol provides distinct layer between the two phases, which are separated due to their density differences.

Stronger/weaker concentration should be available. Stronger concentration attacks the amyl alcohol while weaker concentration reduces oxidative effect of the acid. Amyl alcohol used should be colourless – iso-amyl alcohol – an isomer of two alcohols; methyl and butanol.

Different Gerber butyrometers are used for different products depending on their different fat contents.