Comparison of different methods to quantify fat classes in bakery products

Abstract

The definition of fat differs in different countries; thus whether fat is listed on food labels depends on the country. Some countries list crude fat content in the ‘Fat’ section on the food label, whereas other countries list total fat. In this study, three methods were used for determining fat classes and content in bakery products: the Folch method, the automated Soxhlet method, and the AOAC 996.06 method. The results using these methods were compared. Fat (crude) extracted by the Folch and Soxhlet methods was gravimetrically determined and assessed by fat class using capillary gas chromatography (GC). In most samples, fat (total) content determined by the AOAC 996.06 method was lower than the fat (crude) content determined by the Folch or automated Soxhlet methods. Furthermore, monounsaturated fat or saturated fat content determined by the AOAC 996.06 method was lowest. Almost no difference was observed between fat (crude) content determined by the Folch method and that determined by the automated Soxhlet method for nearly all samples. In three samples (wheat biscuits, butter cookies-1, and chocolate chip cookies), monounsaturated fat, saturated fat, and trans fat content obtained by the automated Soxhlet method was higher than that obtained by the Folch method. The polyunsaturated fat content obtained by the automated Soxhlet method was not higher than that obtained by the Folch method in any sample.

Introduction

Fat is important because it affects food taste and food texture and also affects human health depending on intake. Some trends have emerged in diets around the world, including excess calories, lack of essential nutrients, imbalanced nutrition, deterioration in meal quality, declines in physical activity, and increased dependence on processed foods, fast food, and instant food rather than natural foods. These dietary trends have lead to an increase in obesity, metabolic syndrome, and non-communicable disease morbidity; thus, causing heath problems. Thus, more attention should be paid to the overall quality of meals. To that end, the use of nutrition labels, which enable consumers to choose healthier foods, need to be considered. Consumers recognise the advantages of nutrition labels and also consider them important when choosing foods. The use of the Nutrition Facts Label helps consumers compare bakery products and see calories or fat content (Code of Federal Regulation, 2006). Nutritional labelling laws in many countries require all processed foods to be analysed for various kinds of saturated, mono, and polyunsaturated fatty acids and that the analytical results are reported to consumers (Federal Register, 2003). It is important to determine fatty acid profiles of food for correct nutrition labelling and also for control of labelling authenticity.

Many countries follow the Codex guidelines for nutrition labelling. However, the way they follow the guidelines differs slightly, and each nation has developed a method for quantifying nutrient content. Some countries (Korea, Japan, and Europe, etc.) list crude fat content in the ‘Fat’ section on food labels (KFDA, 2009; EEC, 1990, Pharmaceutical Society of Japan, 2010), whereas other countries (United States, Canada, and Australia, etc.) state total fat on a food label (CFIA, 2011, Hawkes, 2004). But, ‘fat’ listed on a food label is not the same as ‘total fat’. The definition of ‘total fat’, as established by the US Food and Drug Determination (FDA) in 1990 through the Nutritional Labeling and Education Act (NLEA), is ‘the sum of all fatty acids obtained in a lipid extract, expressed as triglycerides’ (Federal Register, 1993), and this definition is met by AOAC Method 996.06. However, in Hong Kong and Brazil (Brazil, 2006, Centre for food safety, 2011), the definition of total fat corresponds to that of crude fat, so gravimetric methods are accepted to determine total fat in those nations. Crude fat is a term used to refer to the crude mixture of fat-soluble material present in a food. The lipid materials may include triglycerides, diglycerides, monoglycerides, phospholipids, steroids, free fatty acids, fat soluble vitamins, carotene pigments, and chlorophylls. The definition of fat used in Europe, Korea, and Japan is that of crude fat, so fat stated on a food label is crude fat (interpreted to be food fat). The crude fat content of food has traditionally been determined by methodologies that involve extraction with organic solvents, drying of the extract, and gravimetric determination of fat.

Lipid extraction is carried out in different ways depending on the sample characteristics. Thus, some extraction methods (Weibull-Berntrop, Rose-Gottlieb, Mononnier, Folch, Werner-Schmid, and Bligh–Dyer methods) are based on hydrolysis (either acid, alkaline, or enzymatic) before solvent extraction, whereas others involve only the solvent extraction step (Soxhlet, Lickens-Nickerson) (García-Ayuso and Luque de Castro, 1999, Priego-Capote and Luque de Castro, 2005). The fat content of food has traditionally been determined by extraction with an organic solvent, followed by extract drying and gravimetric determination. Non-polar organic solvents such as chloroform and n-hexane are used for disrupting hydrophobic and ion–dipole interactions (e.g., lipid hydrophobic chain and non-polar amino acids), whereas polar organic solvents with high dielectric constants, such as methanol, are used for breaking hydrogen bonds (e.g., non-lipid compounds and lipid hydroxyl, carboxyl or amino groups) (Aued-Pimentel, Kus, Kumagai, & Ruvieri, 2010).

The Folch (Folch, Less, & Sloane, 1957) and Bligh–Dyer (Bligh & Dyer, 1959) methods are considered the classical and most reliable means for quantitatively extracting lipids from various types of animal tissues and bakery products. They rely on chloroform–methanol (2/1, v/v) and employ a large sample-to-solvent ratio (in some cases 1:20 respectively). However, the Folch and Bligh–Dyer methods are very lengthy (Stefanov, Valeminck, & Fievez, 2010). The lipid mixture obtained by solvent extraction consists of a variety of lipid classes with different polarities such as thiacylglycerols, phospholipids, free fatty acids, free sterols, and steryl esters. All of them are extracted when using a chloroform/methanol mixture. Only non-polar lipids are extracted when using petroleum ether (Petrovic, Kezic, & Bolanca, 2010).

The Soxhlet extraction technique was invented in 1879 by Franz von Soxhlet to determine fat content in milk. Then, it was generalised for extraction in agriculture before becoming the most used tool for solid–liquid extraction in many fields such as the environment, foodstuffs, and pharmaceutics. Currently, the Soxhlet apparatus is still commonplace in laboratories and has been the standard and reference method for solid–liquid extraction (Virot, Tomao, Colnagui, Visinoni, & Chemat, 2007). However, the long extraction time (16–24 h) and the high temperatures needed for Soxhlet extraction are its main shortcomings, and they might involve changes in extract composition. These alterations can influence the results of some specific types of analyses, as occurs during trans fatty acid determination due to the cis/trans configuration (Priego-Capote, Ruiz-Miménez, & Luque de Castro, 2007). Automated Soxhlet extractors compensating for the shortcomings of conventional Soxhlet extractors are often used. Automated Soxhlet extractors have various advantages, including shorter extraction time, decreased extractant volume, and simultaneous extraction of several samples (Luque de Castro & Priego-Capote, 2010).

AOAC 996.06 is a universally accepted method for determining total, saturated, polyunsaturated, and monounsaturated fats in food. AOAC 996.06 is identical to gas chromatography (GC) analysis and involves hydrolysis of a ground sample, extraction of fat into diethyl and petroleum ether solvents, evaporation of the solvents, methylation of the extracted fat, and quantification of fatty acids by GC (AOAC, 2000a, AOAC, 2000b). AOAC 996.06 is accurate and repeatable but is a laborious procedure, requiring careful attentiveness throughout the analysis (Robinson, Singh, & Kays, 2008).

The objective of this study was to evaluate methods for determining fat based on the different definitions of fat. Fat content of bakery products being sold was gravimetrically determined by the Folch, the automated Soxhlet method and also determined by AOAC 996.06 methods (acid hydrolysis-GC method), and the results were compared. In addition, a portion of the fat obtained by the gravimetric methods was taken and analysed by capillary GC to determine saturated, polyunsaturated, monounsaturated, and trans fat contents. The results were compared with those obtained by AOAC 996.06 to evaluate each method.