Techniques - Triglycerides Profile

Individual fats (triglycerides) are generally measured by gas chromatography with mass spectrometry (GC-MS).  The profile is then compared with what is "normal" for that specific grade of edible oil or fat.

Example -  Vegetable Fats in Chocolate

Cocoa butter has unique organoleptic properties in that it melts sharply at body temperature (37degrees), which gives chocolate its pleasant mouthfeel. The legislation covering chocolate permits 5% vegetable fats, which is to standardise the properties of cocoa butter from different sources, especially for enrobing chocolate. Cocoa butter owes its melting property to its triglyceride profile.

The 3 major triglycerides, which make up nearly 80% of the triglycerides in cocoa butter are:

POP  C50 (16+18+16)                  POS C52 (16 +18 + 18)                SOS C54 (18+18+18)

 Where P= palmitic acid, O= oleic acid, S= stearic acid

The vegetable fats used are often called cocoa butter equivalents or replacers (see Table 2) because they are also tropical fats, most of which contain at least two of the three CB triglycerides POS and SOS, and palm oil mid-fraction mainly contains the triglyceride POP.

Triglyceride Distribution in Cocoa Butter (CB) and Cocoa Butter Equivalents (CBEs)

Distinguishing cocoa butter triglycerides from CBEs can be achieved by separating the principle triglycerides (C50, C52, C54) by gas chromatography (GC) . If the % C50 triglyceride is plotted against %C54, then it separates cocoa butter triglycerides from CBE triglycerides, as CBE’s have much higher C54 triglycerides.

In order to determine the amount of CBEs in CB, an improved gas chromatography method was developed (by JRC) to separate the 3 main triglycerides in CB (i.e.,C50, C52 and C54) from the other triglycerides from CBEs with the same molecular weight and making 5 groups of triglycerides in total. The abundance of the different triglycerides was determined from standard mixtures of CB and CBEs. This data was used to prepare an algorithm, which permitted individual triglycerides to be compared to the standard mixtures. The method was also calibrated with cocoa butter reference material, so that the amount of CBE could be calculated in the mixture. This improved method could detect 5% of CBE in fat extracted from dark chocolate.

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For milk chocolate the contribution of triglycerides from the milk have to be subtracted from the triglycerides of the CBE/CB mixtures.

The amount of milk fat can be calculated either by determining the butyric acid content or by the sum of %C40 + %C42 + %C44. The triglyceride composition can then be corrected for the milk fat contribution, and the CBEs calculated as before.

If a vegetarian or vegan food needs to be tested to see whether animal fats have been used then sterols, rather than fats, can be used as a marker. The main marker for animal fats is cholesterol and most animal fats apart from lard have quite high levels, the highest is in fish oil.

 Unfortunately, as this table shows, cholesterol is not unique to animal fats.  Vegetable oils and fats have low levels of cholesterol. The highest is in palm oil, but this is still a 10^th to 100^th of the level in animal fats.

When animal fats and vegetable oils and fats are refined, the sterol cholesterol is converted to a sterene, cholesta-3,5-diene. If the animal fat or vegetable fat is hydrogenated, then two different sterenes  are formed: cholest-3-ene and cholest-5-ene.

This allows the derivation of a decision boundary to diagnose the origin of the cholesterol.

Hydrogenated vegetable fats fall at the boundary edge and are difficult to differentiate from animal fats.  Fortunately, there is much less hydrogenated fats produced both in the UK and globally, as it had certain health issues with trans fats produced during hydrogenation of unsaturated triglycerides.

In order to decide whether animal fats have been used in a vegetarian food, the concentrations of cholesterol and cholesta-3,5-diene are measured by liquid chromatography, and plotted on a decision boundary diagram  If the sample fall inside the red boundary, then the fat/oil used in the sample is vegetable based and hence the food is suitable for vegetarians. If it falls outside then animal fat has been used.

Interpretation can be complex and take expertise, particularly when dealing with nuances such as correcting for the milk fat content in milk chocolate.

As with any method that compares the pattern of results from a sample with what is "normal", the key is in the construction of the reference dataset of authentic foods.  The database must be fully representative of the entire range of "genuine" samples likely to be enountered, covering all production processes and legal varieties.  This is illustrated by the case of hydrogenated vegetable fats in the "plant-based vs meat-based" decision boundary in the Vegetarian example here.