Casein Hydrolysis Test: Principle, Protocol, Reagents, And Reliable Application

Casein Hydrolysis Test is the another important biochemical test that is the ability of microorganism to break down casein that is milk protein. Actually casein is a main important protein component of milk and act as a source of nutrients for microorganisms. Peptide connections between protein molecules are broken during casein hydrolysis, creating free amino acids and di- and tripeptides that help the body absorb more protein. Proteins that can be digested properly must be consumed in sufficient amounts to prevent immune system impairment and disruption of growth processes.

What is Casein Hydrolysis Test?

‘Casein Hydrolysis Test’ refers to the process of reducing milk proteins to shorter peptide sequences. This procedure, which naturally occurs in the digestive system, can be imitated in a lab setting or on a large scale.

Casein Hydrolysis Test

Principle of Casein Hydrolysis test

The primary milk protein known as casein is a macromolecule made up of amino acid subunits connected by peptide bonds. It creates the white color of milk as well as about 85% of the protein present in it. To fit inside the cell membrane, casein must be much larger. On a skim milk agar plate, an organism is grown and fed casein and food to perform this test. The plates are next examined to determine whether the casein has been digested. The absence of hydrolysis will result in the casein turning the plate white. There will be a distinct zone of hydrolysis if there is hydrolysis.

Objective of casein hydrolysis

Casein, a milk protein, is broken down into short peptides and individual amino acids by the enzyme caseinase, which is released out of the cells (an exoenzyme) into the surrounding media. These amino acids are subsequently absorbed by the organism for use as fuel or as building blocks.

skim milk agar

Reagents required

  1. Skim Milk agar
  2. inoculation loop
  3. incubator
  4. tested bacteria
  5. casein

Medium used in casein hydrolysis test

A differential medium called skim milk agar is used to assess an organism’s capacity to produce casease, an exoenzyme that hydrolyzes casein.

Protocol of Test

  1. First of the organism is inoculated on a plate either in straight or zigzag way.
  2. The plate is incubated at room temperature or 37 degree depending upon the type of bacteria.
  3. The agar plate is examined for the absence or presence of clear zone of proteolysis.
  4. Positive or negative test can be observed.

Positive Casein test

If there is hydrolysis, there will be clear zone indicates positive test for casein hydrolysis test.

Negative Casein Test

If there is no hydrolysis, casein will make the plate white, indicates negative test.

Application of casein test

It is useful to know what kind of organism produces caseinase. Determining the microorganisms that flourish in milk is assisted by the casein hydrolysis test. It aids in distinguishing between the families of Bacillaceae, Enterobacteriaceae, and other families. By using this test, it is simple to identify aerobic actinomycetes.


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