What is the phosphotase test and how is it used in the bakery

Phosphotase is an enzyme associated with animal, insect and microbial activity. The phosphotase test is used to establish whether or not an insect has been baked in a product or entered after the product has left the oven. It is valid for samples that have been stored for a long time. Even after several years' storage dead insects give a strong positive reaction. The following materials are required:

Buffer solution

Anhydrous sodium carbonate (AR grade) weighing 3.5 g and 1.5 g sodium bicarbonate (AR) per litre. (The buffer may be stored for up to three months in a tightly stoppered container of resistant glass.)


Disodium para-nitrophenol phosphate. Buffer substrate

Transfer 0.15 g of the substrate to a 100 ml measuring cylinder and make up to 100 ml with the buffer solution. This solution should not be stored for long periods, but may be kept refrigerated for a week.

250 Baking problems solved Testing procedure

Rinse the fragments with water and then crush with a glass rod. After mixing the fragments with a few drops of water, transfer to a test tube and make with rinsings to about 1 ml. Add 5 ml of buffer substrate and, after mixing, incubate at 37 °C. Simultaneously incubate a blank comprising 5 ml buffer substrate and 1 ml distilled water. After 30 min compare the colours of the tubes. Normally the presence of phosphotase is indicated by the production of a dark yellow colour within 30 min. Weakly positive tubes may require longer for the full colour to develop. A negative result suggests that the insect had been heated. The disadvantages of the test are:

• the test virtually destroys the sample;

• very small insects do not give a sufficiently strong reaction;

• false positive results may be given if the sample is heavily contaminated with microorganisms, e.g. moulds.

Continue reading here: What is meant by hydrogen ion concentration and how is the pH scale determined

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