Healthy Fat in Chips and Sausages? A new Method for Extraction, Digestion and Analysis of Fat in Food Samples

A new method for the determination of saturated and unsaturated fatty acids in food samples such as chocolate, milk powder, sausages, and potato chips has been developed. The method consists of a fast and gentle microwave process in a closed system for sample preparation and the determination of fatty acids using gas chromatography with flame ionisation detection (GC-FID). The analytical data are compared with results of the same food samples generated on the conventional ISO procedures and advantages of the new method are explained.

Introduction

The REGULATION (EU) No 1169/ 2011 OF THE EUROPEAN PARLIAMENT requires the detailed declaration of nutrition values for food as of 2016, especially the differentiation of fat in saturated and unsaturated fatty acids [1]. This differentiation is important in order to protect consumers. For example, the American Dietetic Association (ADA), the European Food Safety Authority (EFSA) and the American ‘Academy of Nutrition and Dietetics’ recommend covering less than 35% of the body’s daily energy requirements by fat; according to ADA, less than 20% should be monounsaturated fatty acids (MUFA).
Determination of total fat content as well as unsaturated and saturated fatty acids in food is performed using methods based on ISO standards. Depending on the type of food, methods like Röse-Gottlieb or Weibull-Stoldt extraction are used for the determination of total fat content [2,3].
The following analysis of saturated and unsaturated fatty acids is done using gas chromatography (GC-FID). This analysis requires the derivatisation of fatty acids in fatty acid methyl esters (FAMEs) based on the ISO norm [4].
Unsaturated fatty acids are sensitive to oxidation processes generated through contact with air and influence of heat. That’s why a gentle microwave process has been developed which is applied in a closed system in two steps for various food compartments. This new microwave procedure is faster, cheaper and more environmentally friendly than the conventional ISO procedure.
It is assumed that the oxidation sensitive unsaturated fatty acids are treated more gently in the microwave process and oxidation is thereby minimised, resulting in a higher analytical levels result of unsaturated fatty acids.

Methods

Conventional ISO procedure:

The conventional standard procedure is based on the ISO extraction method (ISO 8262) for total fat determination in milk and milk products according to Weibull-Berntrop and the derivatisation of fatty acids in fatty acid methyl esters (FAMEs) based on the ISO method (DIN EN ISO 12966-2).
The ISO 8262 method serves as a base and correlates with the extraction method of Weibull-Stoldt for the determination of total fat content in food samples.
The ISO extraction method according to Weibull-Stoldt is based on a saponified digestion using hydrochloric acid and water, which releases fat bound to proteins in food samples. In the following hot filtration, the fat released remains in the filter. The filter including the fat is washed to neutral and then dried. Finally, the fat is extracted with a proper solvent in a Soxhlet extractor, taking several hours. As soon as the extraction is finished, the solvent is removed using a rotation evaporator and the remainder is dried. Determination of total fat is performed by weighing the dried fat.
Based on the ISO procedure on sample preparation for GC analysis, the transformation of fatty acids in fatty acids methyl esters (FAMEs) requires a sequence of alkaline and acidic derivatisation in water-free methanolic environment.
The alkaline derivatisation generates the FAMEs out of the fatty acids bound as Triglycerides and the saponification of free fatty acids. The following acidic derivatisation finally transfers the remaining saponified fatty acids in FAMEs.
The reaction equations are displayed in Figure 1 and Figure 2.

Microwave procedure:

The new microwave procedure for determination of fat has been developed for the microwave system Discover SP-X® (CEM, Kamp Lintfort). It consists of a microwave extraction method (MEM) and a microwave derivatisation method (MDM).
The MEM has been developed based on the Weibull-Stoldt method and extracts the total fat in a food sample in a closed system under defined conditions using microwave radiation simultaneously.
In a closed system (including MDM), derivatisation of the extracted fatty acids is performed under microwave radiation. Transformation of fatty acids in fatty acids methyl esters (FAMEs) also requires a sequence of alkaline and acidic derivatisation in water-free methanolic environment for subsequent GC analysis.

GC analysis:

Analysis of FAMEs of the food samples has been done using a gas chromatograph GC 2010 Plus AF with FID detector (Shimadzu Europa GmbH, Duisburg, Germany).
Influence of the method of sample preparation and derivatisation on the content of unsaturated fatty acids in the total fat content of food samples needs to be investigated. In order to compare the contents of unsaturated and saturated fatty acids in both methods, the total amounts of all fatty acid peak areas are calculated (= 100%). Peak areas of unsaturated and saturated fatty acids are then summarised, and the ratio of unsaturated vs. saturated fatty acids is calculated. This procedure has been applied for both the ISO procedure and the microwave procedure.

Experimental Part

ISO procedure:
Extraction method:
The food sample is weighed in a 600 mL glass beaker, in the expectation that a total fat weight of 2 to 3 g will be present. The sample is treated with 100 mL water and 150 mL hydrochloric acid (25 weight-%).
The glass beaker is covered with a glass lid, and the sample is
then boiled for 30 to 60 minutes. After the digestion process,
the hot sample is diluted with 100 mL of water and filtered in a
2 layer round filter which has been wetted with before hot water. Afterwards, the filters and the residue are washed with neutral with hot water. Finally, the filter paper and residue are dried in an oven for one hour at 105°C. The dried filters are then transferred in the extraction sleeve and finally positioned in a 250 mL extractor (Soxhlet-Apparatus).
The round bottom flask of the Soxhlet apparatus (250 mL) is filled with solvent at a volume corresponding to 1.5 times the volume of the Soxhlet apparatus. The temperature of the solvent petrol ether is 40 – 60°C. The reflux condenser is then set, the solvent is boiled and the fat is extracted over 3 to 6 hours. After extraction, the remaining liquid is fully evaporated in the rotation evaporator. Afterwards, the open flask is further dried for 1 hour at 105°C in order to remove the extraction liquid completely. After cooling, the flask is weighed and the total fat content determined.

ISO based fatty acid derivatisation for GC analysis:

The extracted fat is transferred in a 10 mL round flask and treated with 2 mL (0.2 molmole/L) sodium methanolate in methanol (8 g sodium hydroxide are dissolved in 1,000 mL methanol). Boiling under reflux is performed until the solution is clear (5 to 20 minutes). The boiling time depends on the chain length of the fatty acids, the longer the fatty acid, the longer the boiling takes. Afterwards the flask is removed from the heating source and two drops of phenolphthalein solution are added.
Sulphuric acid (1 mole/L) is then added in methanol solution until the solution is clear; another 0.2 mL are then added. The cooler is reinstalled, and the solution is boiled under reflux for 5 minutes. The solution is again removed from the heating source and cooled down in water. 4 mL of a saturated sodium chloride solution are now added and shaken well. Afterwards, 1 mL n-hexane is added and the flask is shaken again for 15 seconds. The solution is left to stand until both phases are separated. Saturated sodium chloride solution is again added until the aqueous phase reaches the lower level of the flask neck, approx. 2 – 3 mL. The upper phase now contains the FAMEs generated.