Laddus and the science of testing ghee’s purity

Milk fat contains thousands of organic molecules called triglycerides, each structurally different, and formed with different fatty acids.

When it comes to the purity or adulteration of milk and its derivatives, the keyword is always fat. This is something that goes beyond the Telugu Desam Party’s allegations about adulterated ghee being used — or meant to be used — in making laddus for devotees at the Tirupati temple. The Supreme Court may have weighed in on that, pointing out that the party’s leaders may have jumped the gun in alleging animal fat was present in the ghee used, but it is unlikely that the last word on this has been said.

A priest performs the purification ritual to undo the alleged desecrations. (PTI)

The focus of this piece, though, is neither on politics nor laddus (nor indeed, the politics of laddus).

It is on tests for the purity of ghee.

Milk fat contains thousands of organic molecules called triglycerides, each structurally different, and formed with different fatty acids. This makes the triglyceride profile so complex that no adulterator could dream of precisely duplicating it. What analysts do, therefore, is set a standard for the mix of fatty acids that should exist in unadulterated milk fat, and examine a sample for any deviations from that standard.

What’s in ghee

Every naturally occurring fat and oil has a unique triglyceride profile. The fat in the milk from ruminants such as cows or buffalos, for example, has higher amounts of short-chain fatty acids (those with fewer carbon atoms) than medium-chain or long-chain fatty acids. Vegetable oils and animal body fats, on the other hand, mostly contain long-chain fatty acids along with medium-chain acids in smaller amounts.

Such cheaper oils and fats are well-known adulterants used in ghee. “Ghee in general is adulterated with vanaspati or margarine, vegetable oils and animal body fat. Detection of beta-sitosterol (for vegetable oil) and cholesterol (for animal fat) are indicator points for adulterations in ghee samples,” said Raj Kumar Duary, a dairy scientist and food technologist at Banaras Hindu University. He mentioned traditional methods such as the Baudouin test (for animal fats, hydrogenated fat, vanaspati and refined vegetable oil) and the furfural test (for sesame oil), both based on chemical analysis.

Advanced techniques include chromatography, which essentially means separation of components in a sample, in this case the triglycerides. When gas chromatography is used, it is sometimes combined with mass spectrometry, which examines the molecular structure of the components in order to identify them.

“The gas-liquid chromatography and gas chromatography–mass spectrometry methods analyse profiles of fatty acids along with other compounds for correlating the presence of vegetable oils and animal body fats in ghee,” Duary said.

A digression on the science of the tests may be in order

How gas chromatography works

Gas chromatography is considered the ideal technique for testing of milk fat, and has been established as the standard by the ISO and the International Dairy Federation. The process involves heating the sample into a vapour and passing it through a column, where it reacts with other chemical compounds. Every triglyceride has a different time-based way of interacting with these chemicals, and thus leaves its signature on the chromatogram, a graph that emerges as a result of chromatography.

The chromatogram appears as a line graph with a series of peaks at different times, with each peak representing one of the components of the sample. The size of the peaks gives a measure of the amount of that component, while the time of that peak indicates the kind of compound it is.

Data from the chromatogram is fed into standardised equations and formulas to determine certain values needed for the analysis. These are called S-values, a measure based on how much potassium hydroxide is required to break down how much fat.

“The S-value gives an indication of the average molecular weight of fatty acids present in the sample. If fatty acids of smaller molecular weight are present in cow ghee, the S-value should be high,” said Poonam Mishra, professor of food engineering and technology at Tezpur University.

The key, of course, is making sense of the results

Reading the results

When analysing milk fat, scientists look at more than one S-value, one for the overall content, and others as indicators of specific types of fat. This is apparent from the lab report made public by TDP leaders, showing the ghee from Tirupati temple as purportedly being adulterated with animal fat.

To be sure, the report does not establish anything about laddus given to devotees, with the Supreme Court having observed on Monday that there was no evidence that this ghee was actually used to make laddus. From the standpoint of analysis, however, the report is in a standard format that looks at S-values of different kinds of potential fat adulterants in a sample of a milk derivative.

Like the overall S-value, four other S-values are determined from standardised equations. Standard limits for these values are established for unadulterated milk fat (and by extension ghee). Any deviation from these limits could mean adulteration, with different foreign fats indicated by deviations in different S-values.

For example, one of these S-values should be in the range 95.90—104.10 for unadulterated cow milk fat. The lab report found this at 22.76, implying possible adulteration with either palm oil or beef tallow.

Mishra of Tezpur University explained why palm oil and beef tallow are indicated by deviation in the same S-value. “Palm oil contains palmitic acid (as one of the dominating fatty acids) with a carbon length of 16. Beef tallow too has palmitic acid as one of its constituents,” she said.

But which one is it?

Each S-value from an analysis of milk fat, in fact, can be indicator to the presence of more than one kind of foreign fatty acid. For example, sunflower, olive, rapeseed and linseed oil, as well as fish oil, are indicated by deviations in the same S-value.

So, once chromatography has identified a range of possible adulterants, how does one pinpoint which one or more of them are present?

“For that, gas chromatography with mass spectroscopy would be required,” Duary of BHU said.

That may well be needed to put an end to the controversy.