About the Author
Manvik Joshi is pursuing a B.Tech. in Dairy Technology from Maharana Pratap University of Agriculture and Technology (India). He has a deep interest in traditional dairy product processing technology and has written a range of papers on this topic. Manvik is also passionate about dairy microbiology, milk chemistry and ice cream technology. He writes as a freelancer for numerous food titles. He had also undergone training under Student READY (Rural Entrepreneurship Awareness Development Yojana) at KVK Chittorgarh which has allowed him to explore various aspects of dairy animal rearing and routine dairy farm operations.
COB TEST,LACTOMETER TEST,THE ALCOHOL-ALIZARIN TEST,METHYLENE BLUE REDUCTION TEST (MBRT ),THE RESAZURIN TEST.
Prologue
After accepting milk from various farmers or BMC it is crucial for all milk processing plants to ensure quality assurance of raw milk. To process dairy products within legal standards it is important to judge raw milk on various parameters via platform tests. These platform tests are the organoleptic tests which are used to determine the quality of milk in a dairy plant. These tests can be termed as 'rapid quality-control tests' which are of crucial importance.
By application of these tests the inferior quality milk (which has to be rejected) is screened out before the milk is mixed with bulk milk during milk collection and/or reception. These tests ensure that only good quality milk enters into the processing line. This is of utmost importance to focus and achieve the best quality of end products (as even a single lot of milk is of poor quality then it can necessarily spoil the rest of the milk it is mixed with). Applying a series of platform tests, not merely laboratory analysis of samples rather if there is any suspected case a sample from milk is taken to the laboratory for further inspections for quality. In case the milk does not pass the quality tests and does not comply with legal quality standards, it is rejected. The various platform tests are discussed below:
Clot-on- boiling test
( COB test)
This is a rapid test which is used to detect any increase in lactic acidity of milk. With an increase in the acidity of milk (decrease in pH) proteins being very sensitive to pH will precipitate, giving rise to clots.
However, this method is slower than alcohol test but very useful where and when an alcohol test is not available.
This test is performed by heating all amount of milk in a test tube over a flame. The result can be seen immediately. If no coagulation occurs, it indicates that milk can stand heating operations at the time of testing.
The appearance of the surface of the milk and the lid is observed and inspected instantly after removing the lid of incoming milk can or container. Any abnormal colour of the milk, visible dirt and particles, changes in viscosity etc. are also observed. Any abnormal smell can be noticed by inhalation of air standing above the milk in the upper part of the milk can.
Lactometer test
During organoleptic tests, if milk appears too thin and having its colour as 'bluish thin' then it may have added water. To check this, lactometer test is applied. This test serves as a quick method for determination of adulteration of milk by adding water. The principle behind this test is the differences between whole milk, skim milk and water from each other.
PROCEDURE
Mix the milk sample gently and pour it gently into a measuring cylinder. Let the lactometer sink slowly into the milk. Read and record the last Lactometer degree (ºL) just above the surface of the milk. If the temperature of the milk is different from the calibration temperature (Calibration temperature may be=20°C ) of the lactometer, calculate the temperature correction.
For each ºC above the calibration, temperature 0.2ºL is added and for each ºC below calibration,temperature 0.2ºL subtracted from the recorded lactometer reading. For understanding, the following table may be considered.
Alcohol test
If the the the milk sample is suspected to get a sour taste then alcohol test is applied. This test is very useful in judging the quality of raw milk which is used to make UHT milk, evaporated milk and milk powders. This test is considered e sensitive than COB test.
It is based on the fact that milk proteins will precipitate if there is any disturbance in the use of raw milk. Here, three variants of rejected milk can be seen. If milk is having too much-developed acidity or increase in the concentration of Calcium and Magnesium ions then it will be coagulated after adding alcohol. Further, if the milk contains elevated levels of albumen i.e. colostrum milk or higher levels of salt concentrates i.e. mastitis milk it will precipitate.
In dairy industries, normally three different concentrations of ethanol solution are used for this test, depending upon the usage of milk. These are 68% v/v, 65% v/v and 60% v/v. Milk which passes 68% ethanol test is considered as that of 'superior quality' and this milk can be used for processing of UHT milk and milk powders. Milk which we get in poly packs i.e. pasteurised milk must pass 60% alcohol test negative. Samples which do not even pass 60% alcohol test are rejected at the processing units.
PROCEDURE
An equal amount of milk and ethanol is mixed in a test tube. If the tested milk is of good quality,there will be no coagulation. Presence of flakes or clots indicates poor quality milk which is to be rejected.
The Alcohol-Alizarin Test
The procedure for carrying out the test remains the same as for alcohol test but this test is more Informative. 'Alizarin' is a colour indicator it means it changes colour with h change in acidity.
The Alcohol Alizarin solution can be prepared by adding 0.4 grammes alizarin powder to a litre of 61% alcohol solution.
Acidity test
Bacteria that normally proliferate in raw milk produce lactic acid by utilising milk lactose. In the acidity test, the acid is neutralised with 0.1 N Sodium hydroxide and the amount of alkaline is measured. From this, the percentage of lactic acid can be calculated. Fresh milk contains in this test also "natural acidity" which is due to the natural ability to resist pH changes. The natural acidity of milk is 0.16 - 0.18%. Figures higher than this show developed acidity in the talk.
APPARATUS:
A porcelain dish or small conical flask, 10 ml pipette, graduated 1 ml pipette, a burette, 0.1 ml graduations a glass rod for stirring the milk in the dish a 'phenolphthalein' indicator solution,0.5% in 50% Alcohol 0.1 N Sodium hydroxide solution.
PROCEDURE
9 ml of the milk measured into the porcelain dish/conical flask and 1 ml phenolphthalein is added Then slowly from the burette, 0.1 N Sodium hydroxide under continuous mixing, until a faint pink colour appears.
The number of MLS of Sodium hydroxide solution divided by 10 expresses the percentage of lactic acid.
Methylene Blue Reduction Test (MBRT )
The methylene blue reduction test is based on the fact that the colour imparted to milk by the addition of a dye 'methylene blue' will disappear more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism causes the colour to disappear. Bacteria are responsible for discolouration of milk. Generally, assumed that the greater the number of bacteria in milk, the quicker will the colour of the dye will disappear Thus, the time of reduction is taken as a measure of the number of organisms in milk.
The methylene blue reduction test has lost much of its popularity because of its low correlation with other bacterial procedures.
APPARATUS:
The necessary equipment consists of test tubes with rubber stoppers, a pipette or dipper graduated to deliver 10 ml of milk and a water bath for maintaining the samples at 35°C to 37°C.
The bath should contain a volume of water sufficient to heat the samples to 35°C within 10minutes after the tubes enter the water and should have some means of protecting the samples from light during the incubation period.
PROCEDURE:
Sterilize all glassware and rubber stoppers either in an autoclave or in boiling water. Ensure that allglassware is chemically clean.
● Measure 1 ml of the methylene blue thiocyanate solution into a test tube.
● Add 10 ml of milk into it and apply stopper.
● Tubes may be placed in the water bath immediately or may be stored in the refrigerator at 0° to 4°C for a more convenient time of incubation. When ready to perform the test, the temperature of the samples should be brought to 35°C within 10 minutes.
● When the temperature reaches 36°C, slowly invert tubes a few times to assure uniform creaming. Do not shake tubes. Record this time as the beginning of the incubation period.Cover to keep out light.
● Check samples for decolourization after 30 minutes of incubation. Make subsequent readings at an hour interval thereafter.
● After each reading, remove decolourized tubes and then slowly make one complete
inversion of remaining tubes.
● Record reduction time in whole hours between last inversion and decolourization. For example, if the sample were still blue after L 5 hours but was decolourized (white) at the2.5-hour reading, the methylene blue reduction time would be recorded as 2 hours.
Important note: decolourization is considered complete when four-fifths of the colour has disappeared.
Factors influencing MBRT:-
Since the oxygen content must be used up before the colour disappears, any manipulation that increases the oxygen significantly affects the test. Cold milk holds more oxygen than warm milk;pouring milk back and forth from one container to another increases the amount, and at milking time much oxygen may be absorbed.
The kind of organisms affects the rate of reduction. The coliforms appear to be the most rapidly reducing organisms, followed by Streptococcus lactis, some of the faecal Streptococci, and certain micrococci. Thermoduric and psychrotrophic bacteria reduce methylene blue very slowly.
A large number of leucocytes affect the reduction of time materially.
Light hastens reduction and therefore the tests should be kept covered. The concentration of the dye should be uniform as an increased concentration lengthens the time of reduction. Increasing the incubation temperature augments the activity of the bacteria and therefore shortens the
reduction time.
The creaming of the test samples causes several organisms to be removed from the body of the
milk and brought to the surface with the rising fat. This factor causes variations in the reduction
time since the bacteria are not evenly distributed. The accuracy of the test is increased, reduction
time shortened and decolourization more uniform if the samples are periodically inverted during
incubation.
Due to the above points, the MBRT test should be conducted very carefully as slight changes
from a proper experiment may give false results.
Freezing Point Determination
The freezing point of milk is regarded to be the most constant of all measurable properties of
milk. Even a small adulteration of milk with water will cause a detectable elevation of the
freezing point of milk from its normal values of -0.54ºC. Since the test is accurate and sensitive
to added water in milk, it is used to detect whether milk is of normal composition and
adulterated.
The Resazurin Test
The resazurin test is conducted similar to the methylene blue reduction test with the judgement of quality based either on the colour produced after a stated period of incubation or on the time required to reduce the dye to a given end-point. It is done to determine hygiene as well as potential keeping quality of raw milk. There are various modifications in this test. The two most commonly used are the "one-hour test" and the "triple-reading test" taken after one, two, and three hours of incubation.
The procedure for making the resazurin test is as follows: Prepare resazurin solution by dissolving one resazurin tablet (dye content/ tablet, approximately 11 mg, certified by Biological Stain Commission) in 200 ml of hot distilled water as was done in the methylene blue test. Place one ml of dye solution in a sterile test tube, then add 10 ml of sample. Stopper the tube, place in the incubator and, when the temperature reaches 36°C, invert to mix the milk and dye. Incubate at 36°C. Tubes are examined and classified at the end of an hour in the "one-hour test" or at the end of three successive hourly intervals in the "triple-reading test." The following relationships of colour and quality are generally accepted:
'Blue' (no colour change):- Excellent;
'Blue to deep mauve':- Good;
'Deep mauve to deep pink':- Fair;
'Deep pink to whitish-pink':- Poor and
'White':- Bad.
The resazurin test may be a valuable time-saving tool if properly conducted and intelligently interpreted, but should be supplemented by microscopic examination. Important note for this test is resazurin solution must not be exposed to sunlight, and it should not be used for more than
eight hours as it losses strength.
Conclusion
Quality Control remains a powerful tool to enhance production. If the raw material is of good quality then the finished product will also have good quality. It remains important for effective analysis of the lack of quality to specified standards in raw materials. It also becomes important in the context of global competitiveness. The above tests are commonly applied in the dairy industry as a platform test which detects the quality of raw milk snd and tells if the milk is ready for further processing or not.
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