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Margarine: Composition and Analysis
Margarine: Composition and Analysis MD Guille´n, ML Ibargoitia, and P Sopelana, University of the Basque Country (UPV/EHU), Vitoria, Spain ã 2016 Elsevier Ltd. All rights reserved.
Introduction Since its development as a butter substitute by the end of the nineteenth century, margarine has become a highly versatile product due to the advances in technology and the great variation in composition that it can exhibit. Margarine manufacture technology enables the production of very varied products that can satisfy the needs and requirements of different segments of the population. This has contributed to an increase in margarine consumer demand over time. Thus, in 2012, the world production of margarine amounted to 9.4 million tons.
Definition and Types of Margarines Strictly speaking, margarine is a water-in-oil (W/O) emulsion derived from vegetable and/or animal fats, with a fat content of between 80% and 90% and a milk fat content of no more than 3%. The emulsion remains solid at 20 C. Other W/O emulsions with a fat content of 39–41% may be designated as minarine or halvarine, according to the Codex Stan 256-2007. In addition to these two kinds of emulsions, there are others with fat contents outside the limits mentioned, which could be described as fat spreads. In spite of these clear differences in relation to the fat content of the different kinds of products and in order to simplify matters, this article will use the term ‘margarine’ in a general way, regardless of fat content. Table 1 shows the different kinds of margarines and related products and their denominations according to their fat content. Moreover, depending on their composition and texture, they have been described as hard, soft, and semisoft margarines.
Margarine Manufacture Process
process, which involves the addition of hydrogen gas in the presence of a suitable catalyst, raises the melting point of the oil by saturation of double bonds, providing a firmer consistency. However, partial hydrogenation can produce trans isomers of fatty acids (FAs), which have been related to harmful effects on human health. In an attempt to reduce the levels of trans isomers in margarine, other strategies and technologies, such as the introduction of some modifications in the hydrogenation process, have been developed. These include hydrogenation in critical fluids, pressure-controlled hydrogenation, the use of novel catalysts, and conditions known to suppress trans FA formation including low temperatures, high pressures, and increased catalyst loadings. Even so, it could be said that the most widely used technology to avoid the occurrence of trans isomers in margarine is interesterification, which results in an exchange of acyl groups in the glycerol backbone either of the same triglyceride (TG) or of different TGs. This does not change the unsaturation degree of the fat but causes a modification in the fat physical properties that enables the obtention of fat with suitable melting point and crystallization behavior. The reaction can be catalyzed by sodium methoxide (chemical interesterification) or by lipases (enzymatic interesterification), each process having specific technological conditions. Carrying out this type of reactions usually requires the use of fully hydrogenated oil or of another type of solid fat such as palm stearin together with an unsaturated liquid oil. Another possibility to produce the lipid-based stock for margarine manufacture is to blend different oils (hydrogenated or not), each with specific melting characteristics. However, it is sometimes necessary to include in the mixture harder components, like stearin.
Description of the process
Margarine manufacture involves the blend of a lipidic and an aqueous phase in order to obtain a W/O emulsion with a fat percentage of at least 80%. Apart from 80% of fat, the rest is usually skim milk, whey, or a water solution containing salt and flavors.
Margarine production includes the following steps:
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• Preparation of the fatty raw material One of the main requirements of the margarine lipidic fraction is that it provides an appropriate texture to the final product; this will depend both on the fat composition and on its physical structure. The type of fats used for margarine manufacture can be very varied, including vegetable oils with different unsaturation degrees, marine oils, solid fats of either vegetable or animal origin, and their mixtures. Some of these fats are usually subjected to specific treatments aimed at achieving the desired characteristics in the final product. In the beginning, partial hydrogenation of liquid vegetable oils was the technology used for margarine production. This
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Blending of the suitable fats with other oil-soluble components such as emulsifiers (lecithin, mono- and diglycerides, or a combination of them), oil-soluble vitamins, and colorants (b-carotene and annatto). Preparation of the aqueous fraction by mixing different water-soluble components. These include milk or water with salt and flavors such as diacetyl or starter distillate. Mixing of both the lipidic and the aqueous phases to form the W/O emulsion at temperatures around 50–60 C. Pasteurization of the emulsion at temperatures around 70–86 C. Chilling to 15–25 C in a scraped-surface heat exchanger while undergoing vigorous agitation and kneading (plasticizing). This makes an extremely fine dispersion of the water phase in the fat phase possible. Solidification of the margarine before extrusion to the packaging line.
Encyclopedia of Food and Health
http://dx.doi.org/10.1016/B978-0-12-384947-2.00446-3
Margarine: Composition and Analysis
Table 1 Kinds of margarines and related products according to their fat content (Council Regulation (EC) No. 1234/2007) Denomination
Fat content (%)
Margarine Fat spreads X % Three-quarter fat margarine Fat spreads X % Half-fat margarinea Fat spreads X %
80 to <90 >62 to <80 60 to 62 >41 to <60 39 to 41 <39
a
Corresponding to minarine or halvarine in Codex Stan 256-2007.
Margarine Composition Factors Influencing Margarine Composition Two main factors influence margarine composition: the raw material from which it comes from and the conditions under which its processing takes place. The raw material can vary a great deal. Vegetable oils such as soybean, sunflower, canola, palm kernel, palm, corn, cottonseed, safflower, coconut, and olive, among others, have been used for margarine production. They can be either totally or partially hydrogenated or not hydrogenated at all. This wide range of raw materials is one of the reasons for the great variability observed in margarine composition. The processing method in margarine manufacture has a decisive influence on its composition, especially on its unsaturation degree and on its content in unsaturated acyl groups with trans configuration. However, when the processing involves enzymatic interesterification instead of partial hydrogenation, the trans isomer content is considerably reduced and may even reach zero.
compositions in margarines can be observed from this table. This reflects the variety of fat sources and processing methods used for their production. Palmitic acyl groups (C16) are the most abundant of the S groups, whereas the major monounsaturated (M) groups are those with 18 carbon atoms. Of the P acyl groups, the diunsaturated are found in the highest proportions, with triunsaturated acyl group content being much lower. The widest diversity can be found in the S group, with acyl group chain lengths between 14 and 22 carbon atoms. In addition, acyl groups with fewer than 14 carbon atoms and with 24 carbon atoms have also been found in some margarines. The presence of long-chain acyl groups has been related to the use of fish oils for margarine production. As Table 2 shows, most trans unsaturation is found in the monoene fraction, especially in octadecenoic acyl groups. In general, higher trans contents have been reported for hard-type margarines than for soft margarines. Broadly speaking, a decrease in the trans content of margarine has been observed over the years.
Minor margarine components Apart from TG, other minor components can be found in margarine. Some of them are naturally present in the fats used for margarine production but their concentrations can get bigger by external addition. Others can be exclusively added during manufacture. The Codex Stan 256-2007 establishes which type of substances can be added and their maximum use level. Vitamins A, D, and E constitute an exception, since these are regulated by national legislations. The most common are the following:
• Margarine Components Triglycerides These are the main margarine components. The nature of their acyl groups and the position of these in the glyceryl backbone define the properties and physical characteristics of margarine, such as melting and crystallization temperatures, which in turn affect its textural properties. TG composition also influences margarine behavior during processing and storage, as well as its effects on nutrition and health. Many different acyl groups have been found in margarine TG, with a chain length usually ranging from eight to twenty four carbon atoms and with different unsaturation degrees. Among these, cis isomers predominate over trans isomers while acyl groups having an even number of carbon atoms predominate over odd chain acyl groups. Over the years, different compositional parameters have been described as being important for margarine quality and safety. Factors taken into account have included polyunsaturated (P), linoleic (L), saturated (S), and trans (t) acyl groups content, as well as such ratios as P/S, P/(S þ t), L/t, and omega6/omega-3 (o6/o 3). The values considered optimal for each one of these parameters have changed over time, as knowledge of their importance has advanced. Table 2 shows the ranges in percentage by weight of each one of the FAs in different studies. A wide range of acyl group
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•
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Mono- and diglycerides These components can be present in margarine derived either from the vegetable oils used in its manufacture or as a consequence of hydrolytic reactions during processing and/ or storage. They can also be added to margarine due to their emulsifying properties. Free fatty acids (FFAs) Although there is very little information about the occurrence of FFA in margarine, mean values below 0.5% have been found in margarines prepared both from hydrogenated fats and from fractionated or interesterified fats. These values are considered comparable to those usually found in refined vegetable oils. Carotenes and vitamin A (retinol) Both carotenes and vitamin A are present in many of the fats used in margarine manufacture. Moreover, in some cases, they are added for their coloring, antioxidant, nutritional, and healthy properties. Vitamin A is usually added as palmitate or acetate esters. Although there is little information about carotene and/ or vitamin A content of margarine, concentrations of carotene ranging from 0.1 to 1.3 mg 100 g1 have been reported. In margarines declared as being colored with bcarotene or carotene, the concentration of total carotenes ranges between 0.3 and 0.9 mg 100 g1. However, this value can decrease to 0.2 mg 100 g1 in margarines colored with carrot extract and in those with no colorant declared.
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Ranges, in percentage in weight of each one of the several kinds of fatty acids, found in margarine in different studies S (%)
Country Canada/the United States (1976)a Bar (hard) margarines (n ¼ 27) Tub (soft) margarines (n ¼ 58) The United States (1985)b (n ¼ 83) Canada/the United States (1991)c (n ¼ 16) Denmark (1996)d 1992 (n ¼ 40) 1995 (n ¼ 34) 1998e (n ¼ 59) Germany (2000)f 1994 (n ¼ 11) 1999 (n ¼ 9) Portugal (2002)g (n ¼ 16) Bulgaria (2002)h (n ¼ 68) Spain (2003)i (n ¼ 12) Pakistan (2006)j (n ¼ 10) 2008k (n ¼ 10) Greece (2011)l (n ¼ 31) Saudi Arabia (2013)m (n ¼ 4)
M (%)
C14
C16
C18
C20
C22
0.7–1.4 0.6–4.9
2.0–17.8 1.5–20.9 8.5–24.3 4.1–12.2
5.5–11.5 3.4–12.0 5.0–15.2 4.9–9.4
0.5–1.1 0.8–6.1
0.5–2.1
0.0–7.6 0.0–8.4 0.0–6.9
7.7–34.2 5.6–33.7 5.1–37.1
3.7–16.5 2.9–20.8 2.3–23.1
0.0–2.9 0.0–4.0 0.0–4.7
0.0–2.8 0.0–2.3 0.0–5.2
0.2–0.4 0.2–2.6 0.5–4.3 0.0–2.1 0.1–1.8 0.0–3.3 0.2–8.7 0.1–3.6 0.1–1.7
6.9–9.4 6.7–27.2 11.9–37.6 8.5–34.7 7.1–21.7 24.4–40.0 16.9–33.8 5.0–42.1 10.2–24.0
8.4–16.4 6.1–19.4 4.0–12.3 2.9–8.9 3.2–15.5 6.2–12.8 6.1–19.0 2.7–6.9 4.1–7.8
0.1–0.4 0.3–0.3
P (%)
trans (%)
C16:1
C18:1
C20:1
C18:2
C18:3
C18:1 t
11.7
32.8–81.2 18.8–80.2 21.6–41.7 17.7–68.6
2.1–6.9 1.2–12.9
1.5–51.8 5.1–66.8 6.1–46.4 12.0–57.0
0.5–6.1 0.5–6.9 0.2–3.8 0.5–6.9
3.9–30.1 0.6–14.1 (total trans)
0.0–2.2 0.0–1.7 0.0–1.1
13.3–42.2 14.8–52.9 15.3–58.4
0.0–2.5 0.0–2.2 0.0–3.2
7.3–61.1 4.2–53.8 3.8–61.2
0.0–6.0 0.0–7.3 0.0–9.1
0.0–22.3 0.0–8.2 0.0–14.2
0.2–0.7 0.3–0.7
0.1–0.3 0.1–0.2
0.1–0.4 0.2–0.2
0.3–0.5
0.2–0.7
0.0–0.1
0.1–1.3 0.3–0.7 0.4–0.8
0.0–0.8 0.1–1.3
0.0–2.3 0.1–0.6 0.1
32.5–49.2 20.6–33.4 18.2–41.2 20.7–49.9 20.3–37.0 21.9–35.8 5.7–34.8 22.8–59.7 26.6–29.8
27.0–40.8 21.7–54.5 10.6–54.0 9.3–50.5 26.1–49.5 7.0–21.0 3.8–35.4 11.5–51.2 37.0–40.6
0.2–0.6 0.2–0.5 0.1–5.2 0.0–6.4 0.2–6.1 0.0–1.5 0.0–2.0
12.9–25.9 1.8–5.6 0.1–8.2 0.0–27.2 0.1–18.6 2.5–19.1 2.2–34.7 0.0–0.4 0.0–6.9
0.0–0.7 0.0-tr 0.3–0.5
0.5–4.6
C18:2 t
0.4–2.0 0.1–0.6 0.1–2.2 0.0-tr 0.0–2.0 0.1–0.8 0.1–0.9
S, M, and P: saturated, monounsaturated, and polyunsaturated, respectively. tr, traces. a Nazir, D. J., Moorecroft, B. J. and Mishkel, M. A. (1976). Fatty acid composition of margarines. American Journal of Clinical Nutrition 29, 331–339. b Slover, H. T., Thompson Jr, R. H., Davis, C. S. and Merola, G. V. (1985). Lipids in margarines and margarine-like foods. Journal of the American Oil Chemists’ Society 62, 775–786. c De Man, L., Shen, C. F. and De Man, J. M. (1991). Composition, physical and textural characteristics of soft (tub) margarines. Journal of the American Oil Chemists’ Society 68, 70–73. d Ovesen, L., Leth, T. and Hansen, K. (1996). Fatty acid composition of Danish margarines and shortenings, with special emphasis on trans fatty acids. Lipids 31, 971–975. e Ovesen, L., Leth, T. and Hansen, K. (1998). Fatty acid composition and contents of trans monounsaturated fatty acids in frying fats, and in margarines and shortenings marketed in Denmark. Journal of the American Oil Chemists’ Society 75, 1079–1083. f Precht, D. and Molkentin, J. (2000). Recent trends in the fatty acid composition of German sunflower margarines, shortenings and cooking fats with emphasis on individual C16:1, C18:1, C18:2, C18:3 and C20:1 trans isomers. Nahrung 44, 222–228. g Torres, D., Casal, S. and Oliveira, M. P. (2002). Fatty acid composition of Portuguese spreadable fats with emphasis on trans isomers. European Food Research and Technology 214, 108–111. h Marekov, I., Tarandjiiska, R., Panayotova, S. and Nikolova, N. (2002). Comparison of fatty acid composition of domestic and imported margarines and frying fats in Bulgaria. European Journal of Lipid Science and Technology 104, 410–418. i Larque´, E., Garaulet, M., Pe´rez-Llamas, F., Zamora, S. and Tebar, F. J. (2003). Fatty acid composition and nutritional relevance of most widely consumed margarines in Spain. Grasas y Aceites 54, 65–70. j Anwar, F., Bhanger, M. I., Iqbal, S. and Sultana, B. (2006). Fatty acid composition of different margarines and butters from Pakistan with special emphasis on trans unsaturated contents. Journal of Food Quality 29, 87–96. k Kandhro, A., Sherazi, S. T. H., Mahesar, S. A., et al. (2008). GC-MS quantification of fatty acid profile including trans FA in the locally manufactured margarines of Pakistan. Food Chemistry 109, 207–211. l Kroustallaki, P., Tsimpinos, G., Vardavas, C. I. and Kafatos, A. (2011). Fatty acid composition of Greek margarines and their change in fatty acid content over the past decades. International Journal of Food Sciences and Nutrition 62, 685–691. m Bakeet, Z. A. N., Alobeidallah, F. M. and Arzoo, S. (2013). Fatty acid composition with special emphasis on unsaturated trans fatty acid content in margarines and shortenings marketed in Saudi Arabia. International Journal of Biosciences (IJB) 3, 86–93.
Margarine: Composition and Analysis
Table 2
Margarine: Composition and Analysis
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•
•
•
Tocopherols and tocotrienols Tocopherols are well-known natural antioxidants and they are present in margarine coming from the vegetable oils used in its manufacture. Of them, a-tocopherol deserves special attention from the nutritional point of view. Tocopherols can also be added externally with the aim of improving both the nutritional properties and the oxidative stability of margarine. The tocopherol content of margarine can vary widely due to the very different compositions of the fats/oils used in its manufacture. As an example, margarines produced from corn, sunflower, and cottonseed oils have been reported to have higher a-tocopherol levels than those obtained from soybean oil. Moreover, the vegetable source has also an influence on the relative proportions of the different tocopherol isomers. a-Tocopherol concentrations with values between 0.3 and 44.62 mg 100 g1 have been found in margarines from different countries. Tocotrienols, also important due to their nutritional value and to their antioxidant activity, are always in lower concentrations than tocopherols. It is worth noting that margarines made from palm oil can be expected to be among the richest in tocotrienols, since this oil is especially rich in this type of components. Vitamin D There is very little information concerning the occurrence of vitamin D in margarine. The presence of this vitamin results from its addition in the form of ergocalciferol (D2) or cholecalciferol (D3). The values reported for vitamin D3 content range from 1.08 to 3.9 IU (international units; 1 IU ¼ 25 ng) g1, exceeding the labeling claims in some cases. Vitamin K1 (phylloquinone) Some oils used for margarine manufacture, such as soybean or canola oil, contain significant amounts of vitamin K1. For this reason, it is present in certain margarines whose consumption could contribute to the dietary intake of this vitamin. Different contents of vitamin K1 have been found depending on the type of margarine, with a concentration ranging from 4 to 161 mg 100 g1. In general, hard margarines have been found to contain lower levels of vitamin K1 than soft ones, when comparing products with similar fat levels and similar oil sources. Phytosterols The phytosterols that are usually present in margarines come from the vegetable oils used in their manufacture, although in low concentrations. However, certain oils such as rapeseed or corn oils are especially rich in sterols; in fact, higher sterol contents have been observed in margarines obtained from these sources. These compounds can also be added in significant proportions (7.5–8%) to some margarines, because of their interest as functional food ingredients, which are able to lower blood cholesterol levels. In this case, they are added in the form of FA phytosteryl or phytostanyl esters, to improve their solubility. As in the case of tocopherols, the amounts of sterols in margarines can be very variable. Of these, b-sitosterol has been found to be among the most abundant, in concentrations ranging from 5.4 to 412.9 mg 100 g1, followed by campesterol (2.0–106.3 mg 100 g1) and stigmasterol (1.6–60.9 mg 100 g1).
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•
•
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Minerals There is very little information about the mineral content of margarine. Sodium and potassium appear to be the most abundant, in concentrations ranging between 13.2 and 9870 mg g1 and between 58.2 and 1140 mg g1, respectively, followed by calcium, magnesium, and iron. Some of these are important from the sensory point of view. They include sodium, responsible for a salty taste, or potassium, related to bitter notes. Others, such as iron, can favor oxidation and thus the formation of compounds responsible for both unpleasant flavors and toxic effects. Other elements such as zinc and manganese are in lower concentrations, with values between 0.38 and 2.71 mg g1 for the former and between 0.12 and 0.59 mg g1 for the latter. With regard to the elements that can be toxic in high concentrations, the small amount of existing data reveals that cadmium seems to be the most abundant, with concentrations ranging between 0.02 and 3.66 mg g1, whereas the mean contents of lead and arsenic do not exceed the maximum of 0.1 mg kg1 established for them in the Codex Alimentarius. Variable concentrations of nickel, used as catalyst in margarine manufacture, have also been observed in a few margarines, up to 1.70 mg g1. Gelatin This is sometimes used in margarines with a low fat content, to improve the stability of the emulsion. It has been observed that the addition of gelatin to the aqueous phase of margarine at 0.02% increases the firmness of the product. This enables the addition of 25% more water, when compared to margarine with no gelatin. Other components In addition to the earlier-mentioned, other components such as sugars, proteins, and different types of additives can also be found in margarine. Apart from the additives already mentioned in the text (carotenes, tocopherols, and emulsifiers such as mono- and diglycerides), acidity regulators, antifoaming agents, synthetic antioxidants such as BHA and BHT, coloring agents such as bixin-based annatto extracts, preservatives such as sorbates, stabilizers, thickeners, and flavorings can also be used. Among these latter, lactones can be cited, as they have been added to some margarines in order to give them a buttery flavor.
Composition of Margarines and Effects on Health A high content in polyunsaturated cis acyl groups and a content of both saturated and trans acyl groups as low as possible have been considered desirable from the health point of view for a long time. In fact, S acyl groups in dietary lipids have been held responsible for certain cardiovascular diseases. However, more recent studies have shown that there was insufficient scientific evidence to support this negative opinion of these groups. As far as trans groups are concerned, nutritional studies have suggested a direct relationship between this type of acyl groups and the risk of heart disease, since they increase lowdensity lipoprotein (LDL) and reduce high-density lipoprotein (HDL) cholesterol levels. In spite of this, nowadays, there are no regulations to control trans group content in margarine in a general way, although some organisms such as the World Health Organization (WHO) or the International Margarine Association of the Countries of Europe (IMACE) have given
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Margarine: Composition and Analysis
recommendations aiming at reducing the occurrence of trans isomers in margarine. Moreover, some countries have established legal limits for this type of acyl groups, resulting in an effective decrease in the trans group content of margarine. In relation to polyunsaturated acyl groups, certain values of the o6/o3 ratio have been considered optimal from the point of view of health, and great efforts have been made to produce margarines with such an acyl group profile. However, this optimal value has changed over time. It should be pointed out that there has been a recent tendency to reduce the o6 acyl group content in fat foods and to increase the content in o 3 because of the beneficial effects attributed to the latter. As a consequence of the current interest in the development of functional foods whose goal is to improve consumer health, there is a growing trend toward enriching margarine with bioactive compounds. Therefore, margarines enriched in o3 acyl groups are available on the market due to the beneficial cardiovascular effects attributed to these groups. As a result, fish oils and vegetable oils rich in o 3 acyl groups, such as camelina oil, have been used to this aim. Likewise, the enrichment of margarines with specific acyl groups considered bioactive, like pinolenic, a tri-unsaturated acyl group present in pine nut oil, has also been carried out. However, it should be noted that margarines with high levels of P acyl groups are highly susceptible to oxidation; therefore, special care should be taken in their use when thermal treatments are involved, because toxic compounds can be easily formed upon the degradation of this type of acyl groups. The effects of phytosterol and phytostanol consumption on lowering cholesterol levels seem to be well established; for this reason, margarines with significant levels of these compounds have received wide consumer acceptance. Phytostanols could have an advantage over phytosterols in view of their lesser tendency toward to oxidation. In an attempt to help prevent iodine deficiency, the possibility of obtaining iodine-fortified margarine, with a content of 1 mg iodine g1 product, from iodine-fortified sunflower oil has been studied. This could contribute in part to eradicating this deficiency, which can affect both physical development and intellectual development.
Analysis of Margarine Many efforts have been dedicated to the analysis of margarine components, especially to the nature and content of acyl groups. The methodologies and analytic techniques used have evolved over the years alongside technological improvements and the development of new and more accurate techniques.
silica gel using a mixture of petroleum ether and diethyl ether as eluent. In addition to HPLC, 13C nuclear magnetic resonance (13C NMR) has also been used for the study of the TG composition of margarines and the distribution of acyl groups in the glycerol structure.
Determination of the Different Types of Acyl Groups The most usual way to study margarine acyl groups is to perform an alkaline TG hydrolysis in methanol, either directly on the margarine sample or after solvent extraction of the lipidic fraction, followed by the transformation of FAs in the corresponding methyl esters (FAME). Once the FAME are obtained, they can be analyzed directly by gas chromatography using either flame ionization detector (GC-FID) or mass spectrometry (GC-MS) detector. Over the years, several kinds of columns have been used for this technique with the aim to achieve an optimal separation of cis and trans isomers; the best results have been obtained by using high-polar columns of great length (100 m). However, with this methodology, the separation of trans and cis C18:1 isomers cannot be totally obtained, especially when the concentration of the trans isomers is higher than the 5%. Other methodologies involve an additional isolation step by means of thin-layer chromatography (TLC) with silica gel containing silver nitrate (Ag-TLC) before the GC analysis. This technique enables the separation of FAME into fractions differing in the number and geometric configuration of the double bonds. The subsequent GC analysis of each of the fractions makes identification of the different geometric and positional isomers present in the margarine fat possible. However, this methodology is lengthy and not suitable for routine analysis. As an alternative to the combined procedure of Ag-TLC and GC, a method based on GC-FID and infrared spectrophotometry has also been used, leading to equivalent results. This latter method was recommended for samples with >5% trans unsaturation. HPLC has also been used by some authors for the determination of acyl groups in margarine. However, although some advantages over GC have been identified, HPLC has been little employed for margarine acyl group analysis. In addition to the previously mentioned techniques, 13C NMR provides information regarding the composition and nature of the acyl chains present in margarine TG. Likewise, 1 H NMR has also been proved to be very useful in determining the composition in acyl groups of margarine samples, in only a few minutes and with minimal or no sample pretreatment. The spectral signals from which the different types of acyl groups can be determined are shown in Figure 1.
Analysis of the TGs The technique used for the determination of the TG profile of margarine is high-performance liquid chromatography (HPLC) coupled either to an evaporative light-scattering detector or to an atmospheric pressure chemical ionization mass spectrometer (APCI-MS) detector. Before HPLC analysis, the TG fraction can be isolated by column chromatography on
Determination of the Unsaturation Degree The total content of double bonds in margarines has also been subject of analysis. Total double bonds can be determined by chemical methods by means of the so-called iodine index or by easier methods involving spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR) or 1H NMR.
Margarine: Composition and Analysis
651
Methylic protons SAG+Mono+Di-UAG
Sterols
1-MG 1-MG
1,2-DG 1,2-DG
Methylic protons of ω-3 AG
2-MG
Sorbic acid 5.1 5.8
Double bonds
5.5
3.9
3.8
Phytostanyl esters TG backbone
5.0
4.5
4.0
3.7
Mono-allylic protons of Mono+ Di+Tri-UAG
3.6
3.0
2.5
2.0
0.8 0.7
Sitostanol + campestanol
Bis-allylic protons of Di-+Tri-UAG
3.5
0.9
1.5
1.0
0.5
ppm
1
Figure 1 H NMR spectrum of the lipidic fraction of a margarine enriched in phytostanyl esters. AGs, acyl groups; UAGs, unsaturated acyl groups; SAGs, saturated acyl groups; MGs, monoglycerides; DGs, diglycerides.
Specific Determination of trans Unsaturation
Carotenes and Vitamin A
The most common methodology for the determination of the trans unsaturation degree of margarines is based on FTIR, it being possible either to use attenuated total reflectance or not. The advantage of this technique in relation to chromatography is that it does not require either fractionation or special sample preparation or chemical modifications of the sample. The band of trans double bonds appears near 966 cm1. This determination has also been carried out by means of optothermal window. Another spectroscopic technique used for the determination of the trans unsaturation in margarine is 13C NMR.
The analysis of these compounds has been carried out by means of different methodologies and techniques. UV spectrophotometry has been used to determine carotenes and vitamin A after previous extraction of the unsaponifiable matter, separation, isolation, and purification of both kinds of compounds. As saponification can degrade these compounds, there has been a tendency in recent years to avoid this step and carry out the determination directly in a hexane extract of the margarine sample. Carotene absorption occurs near 450 nm and that of vitamin A near 325 nm. Another method used to determine these compounds is HPLC with two UV detectors connected in series to detect both carotenes and vitamin A simultaneously. This method is faster than the previously mentioned since the isolation and purification steps are carried out by this technique automatically. With HPLC, the determination of these compounds can also be achieved directly from a margarine hexane extract, avoiding the saponification stage. The isolation step can also be carried out by gel permeation chromatography, directly on the margarine sample dissolved in methylene chloride, followed by nonaqueous reverse-phase (RP) HPLC. More recently, and with the same aim of avoiding saponification, photoacoustic spectroscopy and optothermal window, two rapid and extremely simple techniques, have also been applied to the analysis of total carotenes in margarines with satisfactory results. It should be taken into account that, with the exception of HPLC methods, these methodologies without either saponification or an isolation step only give information about total carotenes.
Mono- and Diglycerides The analysis of mono- and diglycerides in margarine has received very little attention. One of the techniques used for this purpose is HPLC. Monoglyceride determination can be carried out after derivatization with 3,5-dinitrobenzoyl chloride using a ultraviolet (UV) detector or without any sample pretreatment by using a glyceride-selective postcolumn reactor detector. Both mono- and diglycerides can also be determined by 1H NMR. This technique allows the detection and even quantification of 1- and 2-monoglycerides, as well as of 1,2- and 1,3diglycerides, simultaneously with the determination of the different kinds of acyl groups. The spectral signals from which the different types of mono- and diglycerides can be determined are shown in Figure 1.
Fatty Acids This is usually carried out by the methods used to determine this kind of compounds in other fat-containing foods such as vegetable edible oils.
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Margarine: Composition and Analysis
Tocopherols and Tocotrienols The determination of this type of components usually involves a first step of alkaline saponification. Next, the unsaponifiable material is extracted with an appropriate organic solvent such as hexane. The analysis of tocopherols can be performed either by GC, after derivatization to trimethylsilyl ethers, or by HPLC. This latter technique is the most commonly used to this aim, being the fluorescence (FL) (excitation wavelength set at 290 nm and emission wavelength at 330 nm) preferable to the UV detector. Given that tocopherols are very prone to oxidative degradation, it is recommended to perform saponification in darkness and in a nitrogen atmosphere or in the presence of an antioxidant such as ascorbic acid or pyrogallol. In order to avoid the saponification step, the determination of tocopherols has also been achieved directly from a margarine hexane extract by HPLC with FL detector. The direct determination of tocopherols from the margarine hexane extract has been also carried out by using an online coupling of both chromatographic techniques HPLC-FL and GC-FID in order to avoid interfering compounds. In addition to tocopherols, the determination of tocotrienols may also be of interest. The determination of these latter can be carried out by using some of the earlier-mentioned techniques simultaneously with that of tocopherols.
Vitamin D As in the case of other liposoluble vitamins, the methodology for vitamin D determination includes an initial alkaline saponification of the margarine sample and extraction of the unsaponifiable material with an organic solvent like hexane. HPLC is usually used for the subsequent isolation of vitamin D fraction, which is finally injected to another RP-HPLC system for the separation and quantification of vitamins D2 and D3 with a UV detector at 264/265 nm. An automatic method to simultaneously determine liposoluble vitamins (A, D3, and E) has been also used. The margarine samples were dissolved in an aqueous micellar medium and directly subjected to an online sample treatment that included alkaline hydrolysis and solid-phase extraction (SPE). This system was coupled to liquid chromatography with amperometric detection.
Vitamin K1 The methodology employed in the determination of this vitamin is based on HPLC analysis. This can be carried out after enzymatic digestion or after direct extraction with hexane and subsequent purification either by semipreparative HPLC or by SPE on a silica column. Quantification is performed by RP-HPLC equipped with a dual-electrode electrochemical detector or with FL.
Phytosterols and Phytostanols and/or Their Esters Margarine can contain phytosterols, phytostanols, and esters of these compounds. The total determination of all of them
requires alkaline hydrolysis, extraction with an organic solvent like diethyl ether, and a cleanup step using SPE or TLC. The analysis of the isolated compounds is done by GC previous derivatization to trimethylsilyl ethers. Although FID has been sometimes used, MS detectors are the most commonly used nowadays. Recently, analytic methodologies have been developed to determine phytosteryl and phytostanyl esters in enriched margarines. They are based on RP-HPLC/MS analysis with different types of ionization, such as APCI and electrospray ionization. The sample preparation steps differ from some methodologies to others, ranging from a minimal sample pretreatment to the use of SPE cartridges for the isolation of steryl esters from a hexane extract of the margarine sample. Some of the sterols present in margarine can also be determined by 1H NMR. Among these, brassicasterol, bsitosterol þcampesterol, stigmastanol, or △7-avenasterol can be mentioned. It is worth noting that this determination can be carried out simultaneously with that of the several kinds of acyl groups, mono- and diglycerides. Some of the spectral signals corresponding to phytosterols and/or phytostanols are shown in Figure 1.
Minerals The first step in determining minerals in margarine involves the release of the several elements from the fat matrix, which can be achieved by several methods. Afterward, the determination of each element can be carried out by different techniques, some of which involve sample emulsification. The most frequently used are atomic absorption spectrometry and inductively coupled plasma optical emission spectrometry. With the first technique, flame atomic absorption spectrometry is usually used to determine those elements that are in greater concentrations, whereas graphite furnace atomic absorption spectrometry is chosen for those elements whose concentrations are expected to be much lower. The determination of the isolated elements can also be achieved by ion chromatography (IC), either with a conductivity detector or with a variable-wavelength UV–vis detector, depending on the element. In addition, electrochemical methods such as differential pulse anodic (or cathodic) stripping voltammetry and galvanostatic stripping chronopotentiometry have also been used for the analysis of minerals in margarine; these have shown a good agreement with the results obtained by IC. Finally, it is worth commenting on that instrumental neutron activation analysis also enables the measurement of a large number of trace elements in margarine, with minimal sample handling and processing.
Other Components The analysis of some of the additives used in margarine manufacture, such as benzoic or sorbic acid, together with acids such as formic, lactic, citric, acetic, and butyric can be carried out by means of 1H NMR, from the margarine polar fraction. The spectral signals corresponding to sorbic acid are shown in Figure 1.
Margarine: Composition and Analysis
See also: Carotenoids: Occurrence, Properties and Determination; Cholecalciferol: Properties and Determination; Fats: Classification and Analysis; Fatty Acids: Determination and Requirements; Fatty Acids: Fatty Acids; Fatty Acids: Trans Fatty Acids; Functional Foods; Retinol: Properties and Determination; Tocopherols: Properties and Determination; Triacylglycerols: Characterization and Determination; Vegetable Oils: Composition and Analysis; Vitamin K: Properties and Determination.
Further Reading Adhikari P, Shin JA, Lee JH, et al. (2010) Production of trans-free margarine stock by enzymatic interesterification of rice bran oil, palm stearin and coconut oil. Journal of the Science of Food and Agriculture 90: 703–711. List, G. R. and Pelloso, T. (2007). Zero/low trans margarine, spreads, and shortening. In: List G, Kritchevsky D, and Ratnayake N (eds.) Trans Fats in Foods, pp. 155-176. Urbana, IL: AOCS Press.
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Miskandar MS, Man YC, Yusoff MSA, and Rahman RA (2005) Quality of margarine: fats selection and processing parameters. Asia Pacific Journal of Clinical Nutrition 14: 387–394. Schwartz H, Ollilainen V, Piironen V, and Lampi AM (2008) Tocopherol, tocotrienol and plant sterol contents of vegetable oils and industrial fats. Journal of Food Composition and Analysis 21: 152–161. Sioen I (2013) Fortified Margarine and fat spreads. In: Handbook of food fortification and health, pp. 159–171. New York: Springer. Siri-Tarino PW, Sun Q, Hu FB, and Krauss RM (2010) Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease. American Journal of Clinical Nutrition 91: 535–546. Sopelana P, Arizabaleta I, Ibargoitia ML, and Guille´n MD (2013) Characterisation of the lipidic components of margarines by 1H nuclear magnetic resonance. Food Chemistry 141: 3357–3364.
Relevant Websites http://www.imace.org/facts-on-fat/role-of-fats-in-the-diet/ – International Margarine Association of the Countries of Europe (IMACE).
Introduction Since its development as a butter substitute by the end of the nineteenth century, margarine has become a highly versatile product due to the advances in technology and the great variation in composition that it can exhibit. Margarine manufacture technology enables the production of very varied products that can satisfy the needs and requirements of different segments of the population. This has contributed to an increase in margarine consumer demand over time. Thus, in 2012, the world production of margarine amounted to 9.4 million tons.
Definition and Types of Margarines Strictly speaking, margarine is a water-in-oil (W/O) emulsion derived from vegetable and/or animal fats, with a fat content of between 80% and 90% and a milk fat content of no more than 3%. The emulsion remains solid at 20 C. Other W/O emulsions with a fat content of 39–41% may be designated as minarine or halvarine, according to the Codex Stan 256-2007. In addition to these two kinds of emulsions, there are others with fat contents outside the limits mentioned, which could be described as fat spreads. In spite of these clear differences in relation to the fat content of the different kinds of products and in order to simplify matters, this article will use the term ‘margarine’ in a general way, regardless of fat content. Table 1 shows the different kinds of margarines and related products and their denominations according to their fat content. Moreover, depending on their composition and texture, they have been described as hard, soft, and semisoft margarines.
Margarine Manufacture Process
process, which involves the addition of hydrogen gas in the presence of a suitable catalyst, raises the melting point of the oil by saturation of double bonds, providing a firmer consistency. However, partial hydrogenation can produce trans isomers of fatty acids (FAs), which have been related to harmful effects on human health. In an attempt to reduce the levels of trans isomers in margarine, other strategies and technologies, such as the introduction of some modifications in the hydrogenation process, have been developed. These include hydrogenation in critical fluids, pressure-controlled hydrogenation, the use of novel catalysts, and conditions known to suppress trans FA formation including low temperatures, high pressures, and increased catalyst loadings. Even so, it could be said that the most widely used technology to avoid the occurrence of trans isomers in margarine is interesterification, which results in an exchange of acyl groups in the glycerol backbone either of the same triglyceride (TG) or of different TGs. This does not change the unsaturation degree of the fat but causes a modification in the fat physical properties that enables the obtention of fat with suitable melting point and crystallization behavior. The reaction can be catalyzed by sodium methoxide (chemical interesterification) or by lipases (enzymatic interesterification), each process having specific technological conditions. Carrying out this type of reactions usually requires the use of fully hydrogenated oil or of another type of solid fat such as palm stearin together with an unsaturated liquid oil. Another possibility to produce the lipid-based stock for margarine manufacture is to blend different oils (hydrogenated or not), each with specific melting characteristics. However, it is sometimes necessary to include in the mixture harder components, like stearin.
Description of the process
Margarine manufacture involves the blend of a lipidic and an aqueous phase in order to obtain a W/O emulsion with a fat percentage of at least 80%. Apart from 80% of fat, the rest is usually skim milk, whey, or a water solution containing salt and flavors.
Margarine production includes the following steps:
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• Preparation of the fatty raw material One of the main requirements of the margarine lipidic fraction is that it provides an appropriate texture to the final product; this will depend both on the fat composition and on its physical structure. The type of fats used for margarine manufacture can be very varied, including vegetable oils with different unsaturation degrees, marine oils, solid fats of either vegetable or animal origin, and their mixtures. Some of these fats are usually subjected to specific treatments aimed at achieving the desired characteristics in the final product. In the beginning, partial hydrogenation of liquid vegetable oils was the technology used for margarine production. This
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Blending of the suitable fats with other oil-soluble components such as emulsifiers (lecithin, mono- and diglycerides, or a combination of them), oil-soluble vitamins, and colorants (b-carotene and annatto). Preparation of the aqueous fraction by mixing different water-soluble components. These include milk or water with salt and flavors such as diacetyl or starter distillate. Mixing of both the lipidic and the aqueous phases to form the W/O emulsion at temperatures around 50–60 C. Pasteurization of the emulsion at temperatures around 70–86 C. Chilling to 15–25 C in a scraped-surface heat exchanger while undergoing vigorous agitation and kneading (plasticizing). This makes an extremely fine dispersion of the water phase in the fat phase possible. Solidification of the margarine before extrusion to the packaging line.
Encyclopedia of Food and Health
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Margarine: Composition and Analysis
Table 1 Kinds of margarines and related products according to their fat content (Council Regulation (EC) No. 1234/2007) Denomination
Fat content (%)
Margarine Fat spreads X % Three-quarter fat margarine Fat spreads X % Half-fat margarinea Fat spreads X %
80 to <90 >62 to <80 60 to 62 >41 to <60 39 to 41 <39
a
Corresponding to minarine or halvarine in Codex Stan 256-2007.
Margarine Composition Factors Influencing Margarine Composition Two main factors influence margarine composition: the raw material from which it comes from and the conditions under which its processing takes place. The raw material can vary a great deal. Vegetable oils such as soybean, sunflower, canola, palm kernel, palm, corn, cottonseed, safflower, coconut, and olive, among others, have been used for margarine production. They can be either totally or partially hydrogenated or not hydrogenated at all. This wide range of raw materials is one of the reasons for the great variability observed in margarine composition. The processing method in margarine manufacture has a decisive influence on its composition, especially on its unsaturation degree and on its content in unsaturated acyl groups with trans configuration. However, when the processing involves enzymatic interesterification instead of partial hydrogenation, the trans isomer content is considerably reduced and may even reach zero.
compositions in margarines can be observed from this table. This reflects the variety of fat sources and processing methods used for their production. Palmitic acyl groups (C16) are the most abundant of the S groups, whereas the major monounsaturated (M) groups are those with 18 carbon atoms. Of the P acyl groups, the diunsaturated are found in the highest proportions, with triunsaturated acyl group content being much lower. The widest diversity can be found in the S group, with acyl group chain lengths between 14 and 22 carbon atoms. In addition, acyl groups with fewer than 14 carbon atoms and with 24 carbon atoms have also been found in some margarines. The presence of long-chain acyl groups has been related to the use of fish oils for margarine production. As Table 2 shows, most trans unsaturation is found in the monoene fraction, especially in octadecenoic acyl groups. In general, higher trans contents have been reported for hard-type margarines than for soft margarines. Broadly speaking, a decrease in the trans content of margarine has been observed over the years.
Minor margarine components Apart from TG, other minor components can be found in margarine. Some of them are naturally present in the fats used for margarine production but their concentrations can get bigger by external addition. Others can be exclusively added during manufacture. The Codex Stan 256-2007 establishes which type of substances can be added and their maximum use level. Vitamins A, D, and E constitute an exception, since these are regulated by national legislations. The most common are the following:
• Margarine Components Triglycerides These are the main margarine components. The nature of their acyl groups and the position of these in the glyceryl backbone define the properties and physical characteristics of margarine, such as melting and crystallization temperatures, which in turn affect its textural properties. TG composition also influences margarine behavior during processing and storage, as well as its effects on nutrition and health. Many different acyl groups have been found in margarine TG, with a chain length usually ranging from eight to twenty four carbon atoms and with different unsaturation degrees. Among these, cis isomers predominate over trans isomers while acyl groups having an even number of carbon atoms predominate over odd chain acyl groups. Over the years, different compositional parameters have been described as being important for margarine quality and safety. Factors taken into account have included polyunsaturated (P), linoleic (L), saturated (S), and trans (t) acyl groups content, as well as such ratios as P/S, P/(S þ t), L/t, and omega6/omega-3 (o6/o 3). The values considered optimal for each one of these parameters have changed over time, as knowledge of their importance has advanced. Table 2 shows the ranges in percentage by weight of each one of the FAs in different studies. A wide range of acyl group
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Mono- and diglycerides These components can be present in margarine derived either from the vegetable oils used in its manufacture or as a consequence of hydrolytic reactions during processing and/ or storage. They can also be added to margarine due to their emulsifying properties. Free fatty acids (FFAs) Although there is very little information about the occurrence of FFA in margarine, mean values below 0.5% have been found in margarines prepared both from hydrogenated fats and from fractionated or interesterified fats. These values are considered comparable to those usually found in refined vegetable oils. Carotenes and vitamin A (retinol) Both carotenes and vitamin A are present in many of the fats used in margarine manufacture. Moreover, in some cases, they are added for their coloring, antioxidant, nutritional, and healthy properties. Vitamin A is usually added as palmitate or acetate esters. Although there is little information about carotene and/ or vitamin A content of margarine, concentrations of carotene ranging from 0.1 to 1.3 mg 100 g1 have been reported. In margarines declared as being colored with bcarotene or carotene, the concentration of total carotenes ranges between 0.3 and 0.9 mg 100 g1. However, this value can decrease to 0.2 mg 100 g1 in margarines colored with carrot extract and in those with no colorant declared.
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Ranges, in percentage in weight of each one of the several kinds of fatty acids, found in margarine in different studies S (%)
Country Canada/the United States (1976)a Bar (hard) margarines (n ¼ 27) Tub (soft) margarines (n ¼ 58) The United States (1985)b (n ¼ 83) Canada/the United States (1991)c (n ¼ 16) Denmark (1996)d 1992 (n ¼ 40) 1995 (n ¼ 34) 1998e (n ¼ 59) Germany (2000)f 1994 (n ¼ 11) 1999 (n ¼ 9) Portugal (2002)g (n ¼ 16) Bulgaria (2002)h (n ¼ 68) Spain (2003)i (n ¼ 12) Pakistan (2006)j (n ¼ 10) 2008k (n ¼ 10) Greece (2011)l (n ¼ 31) Saudi Arabia (2013)m (n ¼ 4)
M (%)
C14
C16
C18
C20
C22
0.7–1.4 0.6–4.9
2.0–17.8 1.5–20.9 8.5–24.3 4.1–12.2
5.5–11.5 3.4–12.0 5.0–15.2 4.9–9.4
0.5–1.1 0.8–6.1
0.5–2.1
0.0–7.6 0.0–8.4 0.0–6.9
7.7–34.2 5.6–33.7 5.1–37.1
3.7–16.5 2.9–20.8 2.3–23.1
0.0–2.9 0.0–4.0 0.0–4.7
0.0–2.8 0.0–2.3 0.0–5.2
0.2–0.4 0.2–2.6 0.5–4.3 0.0–2.1 0.1–1.8 0.0–3.3 0.2–8.7 0.1–3.6 0.1–1.7
6.9–9.4 6.7–27.2 11.9–37.6 8.5–34.7 7.1–21.7 24.4–40.0 16.9–33.8 5.0–42.1 10.2–24.0
8.4–16.4 6.1–19.4 4.0–12.3 2.9–8.9 3.2–15.5 6.2–12.8 6.1–19.0 2.7–6.9 4.1–7.8
0.1–0.4 0.3–0.3
P (%)
trans (%)
C16:1
C18:1
C20:1
C18:2
C18:3
C18:1 t
11.7
32.8–81.2 18.8–80.2 21.6–41.7 17.7–68.6
2.1–6.9 1.2–12.9
1.5–51.8 5.1–66.8 6.1–46.4 12.0–57.0
0.5–6.1 0.5–6.9 0.2–3.8 0.5–6.9
3.9–30.1 0.6–14.1 (total trans)
0.0–2.2 0.0–1.7 0.0–1.1
13.3–42.2 14.8–52.9 15.3–58.4
0.0–2.5 0.0–2.2 0.0–3.2
7.3–61.1 4.2–53.8 3.8–61.2
0.0–6.0 0.0–7.3 0.0–9.1
0.0–22.3 0.0–8.2 0.0–14.2
0.2–0.7 0.3–0.7
0.1–0.3 0.1–0.2
0.1–0.4 0.2–0.2
0.3–0.5
0.2–0.7
0.0–0.1
0.1–1.3 0.3–0.7 0.4–0.8
0.0–0.8 0.1–1.3
0.0–2.3 0.1–0.6 0.1
32.5–49.2 20.6–33.4 18.2–41.2 20.7–49.9 20.3–37.0 21.9–35.8 5.7–34.8 22.8–59.7 26.6–29.8
27.0–40.8 21.7–54.5 10.6–54.0 9.3–50.5 26.1–49.5 7.0–21.0 3.8–35.4 11.5–51.2 37.0–40.6
0.2–0.6 0.2–0.5 0.1–5.2 0.0–6.4 0.2–6.1 0.0–1.5 0.0–2.0
12.9–25.9 1.8–5.6 0.1–8.2 0.0–27.2 0.1–18.6 2.5–19.1 2.2–34.7 0.0–0.4 0.0–6.9
0.0–0.7 0.0-tr 0.3–0.5
0.5–4.6
C18:2 t
0.4–2.0 0.1–0.6 0.1–2.2 0.0-tr 0.0–2.0 0.1–0.8 0.1–0.9
S, M, and P: saturated, monounsaturated, and polyunsaturated, respectively. tr, traces. a Nazir, D. J., Moorecroft, B. J. and Mishkel, M. A. (1976). Fatty acid composition of margarines. American Journal of Clinical Nutrition 29, 331–339. b Slover, H. T., Thompson Jr, R. H., Davis, C. S. and Merola, G. V. (1985). Lipids in margarines and margarine-like foods. Journal of the American Oil Chemists’ Society 62, 775–786. c De Man, L., Shen, C. F. and De Man, J. M. (1991). Composition, physical and textural characteristics of soft (tub) margarines. Journal of the American Oil Chemists’ Society 68, 70–73. d Ovesen, L., Leth, T. and Hansen, K. (1996). Fatty acid composition of Danish margarines and shortenings, with special emphasis on trans fatty acids. Lipids 31, 971–975. e Ovesen, L., Leth, T. and Hansen, K. (1998). Fatty acid composition and contents of trans monounsaturated fatty acids in frying fats, and in margarines and shortenings marketed in Denmark. Journal of the American Oil Chemists’ Society 75, 1079–1083. f Precht, D. and Molkentin, J. (2000). Recent trends in the fatty acid composition of German sunflower margarines, shortenings and cooking fats with emphasis on individual C16:1, C18:1, C18:2, C18:3 and C20:1 trans isomers. Nahrung 44, 222–228. g Torres, D., Casal, S. and Oliveira, M. P. (2002). Fatty acid composition of Portuguese spreadable fats with emphasis on trans isomers. European Food Research and Technology 214, 108–111. h Marekov, I., Tarandjiiska, R., Panayotova, S. and Nikolova, N. (2002). Comparison of fatty acid composition of domestic and imported margarines and frying fats in Bulgaria. European Journal of Lipid Science and Technology 104, 410–418. i Larque´, E., Garaulet, M., Pe´rez-Llamas, F., Zamora, S. and Tebar, F. J. (2003). Fatty acid composition and nutritional relevance of most widely consumed margarines in Spain. Grasas y Aceites 54, 65–70. j Anwar, F., Bhanger, M. I., Iqbal, S. and Sultana, B. (2006). Fatty acid composition of different margarines and butters from Pakistan with special emphasis on trans unsaturated contents. Journal of Food Quality 29, 87–96. k Kandhro, A., Sherazi, S. T. H., Mahesar, S. A., et al. (2008). GC-MS quantification of fatty acid profile including trans FA in the locally manufactured margarines of Pakistan. Food Chemistry 109, 207–211. l Kroustallaki, P., Tsimpinos, G., Vardavas, C. I. and Kafatos, A. (2011). Fatty acid composition of Greek margarines and their change in fatty acid content over the past decades. International Journal of Food Sciences and Nutrition 62, 685–691. m Bakeet, Z. A. N., Alobeidallah, F. M. and Arzoo, S. (2013). Fatty acid composition with special emphasis on unsaturated trans fatty acid content in margarines and shortenings marketed in Saudi Arabia. International Journal of Biosciences (IJB) 3, 86–93.
Margarine: Composition and Analysis
Table 2
Margarine: Composition and Analysis
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•
•
•
Tocopherols and tocotrienols Tocopherols are well-known natural antioxidants and they are present in margarine coming from the vegetable oils used in its manufacture. Of them, a-tocopherol deserves special attention from the nutritional point of view. Tocopherols can also be added externally with the aim of improving both the nutritional properties and the oxidative stability of margarine. The tocopherol content of margarine can vary widely due to the very different compositions of the fats/oils used in its manufacture. As an example, margarines produced from corn, sunflower, and cottonseed oils have been reported to have higher a-tocopherol levels than those obtained from soybean oil. Moreover, the vegetable source has also an influence on the relative proportions of the different tocopherol isomers. a-Tocopherol concentrations with values between 0.3 and 44.62 mg 100 g1 have been found in margarines from different countries. Tocotrienols, also important due to their nutritional value and to their antioxidant activity, are always in lower concentrations than tocopherols. It is worth noting that margarines made from palm oil can be expected to be among the richest in tocotrienols, since this oil is especially rich in this type of components. Vitamin D There is very little information concerning the occurrence of vitamin D in margarine. The presence of this vitamin results from its addition in the form of ergocalciferol (D2) or cholecalciferol (D3). The values reported for vitamin D3 content range from 1.08 to 3.9 IU (international units; 1 IU ¼ 25 ng) g1, exceeding the labeling claims in some cases. Vitamin K1 (phylloquinone) Some oils used for margarine manufacture, such as soybean or canola oil, contain significant amounts of vitamin K1. For this reason, it is present in certain margarines whose consumption could contribute to the dietary intake of this vitamin. Different contents of vitamin K1 have been found depending on the type of margarine, with a concentration ranging from 4 to 161 mg 100 g1. In general, hard margarines have been found to contain lower levels of vitamin K1 than soft ones, when comparing products with similar fat levels and similar oil sources. Phytosterols The phytosterols that are usually present in margarines come from the vegetable oils used in their manufacture, although in low concentrations. However, certain oils such as rapeseed or corn oils are especially rich in sterols; in fact, higher sterol contents have been observed in margarines obtained from these sources. These compounds can also be added in significant proportions (7.5–8%) to some margarines, because of their interest as functional food ingredients, which are able to lower blood cholesterol levels. In this case, they are added in the form of FA phytosteryl or phytostanyl esters, to improve their solubility. As in the case of tocopherols, the amounts of sterols in margarines can be very variable. Of these, b-sitosterol has been found to be among the most abundant, in concentrations ranging from 5.4 to 412.9 mg 100 g1, followed by campesterol (2.0–106.3 mg 100 g1) and stigmasterol (1.6–60.9 mg 100 g1).
•
•
•
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Minerals There is very little information about the mineral content of margarine. Sodium and potassium appear to be the most abundant, in concentrations ranging between 13.2 and 9870 mg g1 and between 58.2 and 1140 mg g1, respectively, followed by calcium, magnesium, and iron. Some of these are important from the sensory point of view. They include sodium, responsible for a salty taste, or potassium, related to bitter notes. Others, such as iron, can favor oxidation and thus the formation of compounds responsible for both unpleasant flavors and toxic effects. Other elements such as zinc and manganese are in lower concentrations, with values between 0.38 and 2.71 mg g1 for the former and between 0.12 and 0.59 mg g1 for the latter. With regard to the elements that can be toxic in high concentrations, the small amount of existing data reveals that cadmium seems to be the most abundant, with concentrations ranging between 0.02 and 3.66 mg g1, whereas the mean contents of lead and arsenic do not exceed the maximum of 0.1 mg kg1 established for them in the Codex Alimentarius. Variable concentrations of nickel, used as catalyst in margarine manufacture, have also been observed in a few margarines, up to 1.70 mg g1. Gelatin This is sometimes used in margarines with a low fat content, to improve the stability of the emulsion. It has been observed that the addition of gelatin to the aqueous phase of margarine at 0.02% increases the firmness of the product. This enables the addition of 25% more water, when compared to margarine with no gelatin. Other components In addition to the earlier-mentioned, other components such as sugars, proteins, and different types of additives can also be found in margarine. Apart from the additives already mentioned in the text (carotenes, tocopherols, and emulsifiers such as mono- and diglycerides), acidity regulators, antifoaming agents, synthetic antioxidants such as BHA and BHT, coloring agents such as bixin-based annatto extracts, preservatives such as sorbates, stabilizers, thickeners, and flavorings can also be used. Among these latter, lactones can be cited, as they have been added to some margarines in order to give them a buttery flavor.
Composition of Margarines and Effects on Health A high content in polyunsaturated cis acyl groups and a content of both saturated and trans acyl groups as low as possible have been considered desirable from the health point of view for a long time. In fact, S acyl groups in dietary lipids have been held responsible for certain cardiovascular diseases. However, more recent studies have shown that there was insufficient scientific evidence to support this negative opinion of these groups. As far as trans groups are concerned, nutritional studies have suggested a direct relationship between this type of acyl groups and the risk of heart disease, since they increase lowdensity lipoprotein (LDL) and reduce high-density lipoprotein (HDL) cholesterol levels. In spite of this, nowadays, there are no regulations to control trans group content in margarine in a general way, although some organisms such as the World Health Organization (WHO) or the International Margarine Association of the Countries of Europe (IMACE) have given
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recommendations aiming at reducing the occurrence of trans isomers in margarine. Moreover, some countries have established legal limits for this type of acyl groups, resulting in an effective decrease in the trans group content of margarine. In relation to polyunsaturated acyl groups, certain values of the o6/o3 ratio have been considered optimal from the point of view of health, and great efforts have been made to produce margarines with such an acyl group profile. However, this optimal value has changed over time. It should be pointed out that there has been a recent tendency to reduce the o6 acyl group content in fat foods and to increase the content in o 3 because of the beneficial effects attributed to the latter. As a consequence of the current interest in the development of functional foods whose goal is to improve consumer health, there is a growing trend toward enriching margarine with bioactive compounds. Therefore, margarines enriched in o3 acyl groups are available on the market due to the beneficial cardiovascular effects attributed to these groups. As a result, fish oils and vegetable oils rich in o 3 acyl groups, such as camelina oil, have been used to this aim. Likewise, the enrichment of margarines with specific acyl groups considered bioactive, like pinolenic, a tri-unsaturated acyl group present in pine nut oil, has also been carried out. However, it should be noted that margarines with high levels of P acyl groups are highly susceptible to oxidation; therefore, special care should be taken in their use when thermal treatments are involved, because toxic compounds can be easily formed upon the degradation of this type of acyl groups. The effects of phytosterol and phytostanol consumption on lowering cholesterol levels seem to be well established; for this reason, margarines with significant levels of these compounds have received wide consumer acceptance. Phytostanols could have an advantage over phytosterols in view of their lesser tendency toward to oxidation. In an attempt to help prevent iodine deficiency, the possibility of obtaining iodine-fortified margarine, with a content of 1 mg iodine g1 product, from iodine-fortified sunflower oil has been studied. This could contribute in part to eradicating this deficiency, which can affect both physical development and intellectual development.
Analysis of Margarine Many efforts have been dedicated to the analysis of margarine components, especially to the nature and content of acyl groups. The methodologies and analytic techniques used have evolved over the years alongside technological improvements and the development of new and more accurate techniques.
silica gel using a mixture of petroleum ether and diethyl ether as eluent. In addition to HPLC, 13C nuclear magnetic resonance (13C NMR) has also been used for the study of the TG composition of margarines and the distribution of acyl groups in the glycerol structure.
Determination of the Different Types of Acyl Groups The most usual way to study margarine acyl groups is to perform an alkaline TG hydrolysis in methanol, either directly on the margarine sample or after solvent extraction of the lipidic fraction, followed by the transformation of FAs in the corresponding methyl esters (FAME). Once the FAME are obtained, they can be analyzed directly by gas chromatography using either flame ionization detector (GC-FID) or mass spectrometry (GC-MS) detector. Over the years, several kinds of columns have been used for this technique with the aim to achieve an optimal separation of cis and trans isomers; the best results have been obtained by using high-polar columns of great length (100 m). However, with this methodology, the separation of trans and cis C18:1 isomers cannot be totally obtained, especially when the concentration of the trans isomers is higher than the 5%. Other methodologies involve an additional isolation step by means of thin-layer chromatography (TLC) with silica gel containing silver nitrate (Ag-TLC) before the GC analysis. This technique enables the separation of FAME into fractions differing in the number and geometric configuration of the double bonds. The subsequent GC analysis of each of the fractions makes identification of the different geometric and positional isomers present in the margarine fat possible. However, this methodology is lengthy and not suitable for routine analysis. As an alternative to the combined procedure of Ag-TLC and GC, a method based on GC-FID and infrared spectrophotometry has also been used, leading to equivalent results. This latter method was recommended for samples with >5% trans unsaturation. HPLC has also been used by some authors for the determination of acyl groups in margarine. However, although some advantages over GC have been identified, HPLC has been little employed for margarine acyl group analysis. In addition to the previously mentioned techniques, 13C NMR provides information regarding the composition and nature of the acyl chains present in margarine TG. Likewise, 1 H NMR has also been proved to be very useful in determining the composition in acyl groups of margarine samples, in only a few minutes and with minimal or no sample pretreatment. The spectral signals from which the different types of acyl groups can be determined are shown in Figure 1.
Analysis of the TGs The technique used for the determination of the TG profile of margarine is high-performance liquid chromatography (HPLC) coupled either to an evaporative light-scattering detector or to an atmospheric pressure chemical ionization mass spectrometer (APCI-MS) detector. Before HPLC analysis, the TG fraction can be isolated by column chromatography on
Determination of the Unsaturation Degree The total content of double bonds in margarines has also been subject of analysis. Total double bonds can be determined by chemical methods by means of the so-called iodine index or by easier methods involving spectroscopic techniques such as Fourier transform infrared spectroscopy (FTIR) or 1H NMR.
Margarine: Composition and Analysis
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Methylic protons SAG+Mono+Di-UAG
Sterols
1-MG 1-MG
1,2-DG 1,2-DG
Methylic protons of ω-3 AG
2-MG
Sorbic acid 5.1 5.8
Double bonds
5.5
3.9
3.8
Phytostanyl esters TG backbone
5.0
4.5
4.0
3.7
Mono-allylic protons of Mono+ Di+Tri-UAG
3.6
3.0
2.5
2.0
0.8 0.7
Sitostanol + campestanol
Bis-allylic protons of Di-+Tri-UAG
3.5
0.9
1.5
1.0
0.5
ppm
1
Figure 1 H NMR spectrum of the lipidic fraction of a margarine enriched in phytostanyl esters. AGs, acyl groups; UAGs, unsaturated acyl groups; SAGs, saturated acyl groups; MGs, monoglycerides; DGs, diglycerides.
Specific Determination of trans Unsaturation
Carotenes and Vitamin A
The most common methodology for the determination of the trans unsaturation degree of margarines is based on FTIR, it being possible either to use attenuated total reflectance or not. The advantage of this technique in relation to chromatography is that it does not require either fractionation or special sample preparation or chemical modifications of the sample. The band of trans double bonds appears near 966 cm1. This determination has also been carried out by means of optothermal window. Another spectroscopic technique used for the determination of the trans unsaturation in margarine is 13C NMR.
The analysis of these compounds has been carried out by means of different methodologies and techniques. UV spectrophotometry has been used to determine carotenes and vitamin A after previous extraction of the unsaponifiable matter, separation, isolation, and purification of both kinds of compounds. As saponification can degrade these compounds, there has been a tendency in recent years to avoid this step and carry out the determination directly in a hexane extract of the margarine sample. Carotene absorption occurs near 450 nm and that of vitamin A near 325 nm. Another method used to determine these compounds is HPLC with two UV detectors connected in series to detect both carotenes and vitamin A simultaneously. This method is faster than the previously mentioned since the isolation and purification steps are carried out by this technique automatically. With HPLC, the determination of these compounds can also be achieved directly from a margarine hexane extract, avoiding the saponification stage. The isolation step can also be carried out by gel permeation chromatography, directly on the margarine sample dissolved in methylene chloride, followed by nonaqueous reverse-phase (RP) HPLC. More recently, and with the same aim of avoiding saponification, photoacoustic spectroscopy and optothermal window, two rapid and extremely simple techniques, have also been applied to the analysis of total carotenes in margarines with satisfactory results. It should be taken into account that, with the exception of HPLC methods, these methodologies without either saponification or an isolation step only give information about total carotenes.
Mono- and Diglycerides The analysis of mono- and diglycerides in margarine has received very little attention. One of the techniques used for this purpose is HPLC. Monoglyceride determination can be carried out after derivatization with 3,5-dinitrobenzoyl chloride using a ultraviolet (UV) detector or without any sample pretreatment by using a glyceride-selective postcolumn reactor detector. Both mono- and diglycerides can also be determined by 1H NMR. This technique allows the detection and even quantification of 1- and 2-monoglycerides, as well as of 1,2- and 1,3diglycerides, simultaneously with the determination of the different kinds of acyl groups. The spectral signals from which the different types of mono- and diglycerides can be determined are shown in Figure 1.
Fatty Acids This is usually carried out by the methods used to determine this kind of compounds in other fat-containing foods such as vegetable edible oils.
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Tocopherols and Tocotrienols The determination of this type of components usually involves a first step of alkaline saponification. Next, the unsaponifiable material is extracted with an appropriate organic solvent such as hexane. The analysis of tocopherols can be performed either by GC, after derivatization to trimethylsilyl ethers, or by HPLC. This latter technique is the most commonly used to this aim, being the fluorescence (FL) (excitation wavelength set at 290 nm and emission wavelength at 330 nm) preferable to the UV detector. Given that tocopherols are very prone to oxidative degradation, it is recommended to perform saponification in darkness and in a nitrogen atmosphere or in the presence of an antioxidant such as ascorbic acid or pyrogallol. In order to avoid the saponification step, the determination of tocopherols has also been achieved directly from a margarine hexane extract by HPLC with FL detector. The direct determination of tocopherols from the margarine hexane extract has been also carried out by using an online coupling of both chromatographic techniques HPLC-FL and GC-FID in order to avoid interfering compounds. In addition to tocopherols, the determination of tocotrienols may also be of interest. The determination of these latter can be carried out by using some of the earlier-mentioned techniques simultaneously with that of tocopherols.
Vitamin D As in the case of other liposoluble vitamins, the methodology for vitamin D determination includes an initial alkaline saponification of the margarine sample and extraction of the unsaponifiable material with an organic solvent like hexane. HPLC is usually used for the subsequent isolation of vitamin D fraction, which is finally injected to another RP-HPLC system for the separation and quantification of vitamins D2 and D3 with a UV detector at 264/265 nm. An automatic method to simultaneously determine liposoluble vitamins (A, D3, and E) has been also used. The margarine samples were dissolved in an aqueous micellar medium and directly subjected to an online sample treatment that included alkaline hydrolysis and solid-phase extraction (SPE). This system was coupled to liquid chromatography with amperometric detection.
Vitamin K1 The methodology employed in the determination of this vitamin is based on HPLC analysis. This can be carried out after enzymatic digestion or after direct extraction with hexane and subsequent purification either by semipreparative HPLC or by SPE on a silica column. Quantification is performed by RP-HPLC equipped with a dual-electrode electrochemical detector or with FL.
Phytosterols and Phytostanols and/or Their Esters Margarine can contain phytosterols, phytostanols, and esters of these compounds. The total determination of all of them
requires alkaline hydrolysis, extraction with an organic solvent like diethyl ether, and a cleanup step using SPE or TLC. The analysis of the isolated compounds is done by GC previous derivatization to trimethylsilyl ethers. Although FID has been sometimes used, MS detectors are the most commonly used nowadays. Recently, analytic methodologies have been developed to determine phytosteryl and phytostanyl esters in enriched margarines. They are based on RP-HPLC/MS analysis with different types of ionization, such as APCI and electrospray ionization. The sample preparation steps differ from some methodologies to others, ranging from a minimal sample pretreatment to the use of SPE cartridges for the isolation of steryl esters from a hexane extract of the margarine sample. Some of the sterols present in margarine can also be determined by 1H NMR. Among these, brassicasterol, bsitosterol þcampesterol, stigmastanol, or △7-avenasterol can be mentioned. It is worth noting that this determination can be carried out simultaneously with that of the several kinds of acyl groups, mono- and diglycerides. Some of the spectral signals corresponding to phytosterols and/or phytostanols are shown in Figure 1.
Minerals The first step in determining minerals in margarine involves the release of the several elements from the fat matrix, which can be achieved by several methods. Afterward, the determination of each element can be carried out by different techniques, some of which involve sample emulsification. The most frequently used are atomic absorption spectrometry and inductively coupled plasma optical emission spectrometry. With the first technique, flame atomic absorption spectrometry is usually used to determine those elements that are in greater concentrations, whereas graphite furnace atomic absorption spectrometry is chosen for those elements whose concentrations are expected to be much lower. The determination of the isolated elements can also be achieved by ion chromatography (IC), either with a conductivity detector or with a variable-wavelength UV–vis detector, depending on the element. In addition, electrochemical methods such as differential pulse anodic (or cathodic) stripping voltammetry and galvanostatic stripping chronopotentiometry have also been used for the analysis of minerals in margarine; these have shown a good agreement with the results obtained by IC. Finally, it is worth commenting on that instrumental neutron activation analysis also enables the measurement of a large number of trace elements in margarine, with minimal sample handling and processing.
Other Components The analysis of some of the additives used in margarine manufacture, such as benzoic or sorbic acid, together with acids such as formic, lactic, citric, acetic, and butyric can be carried out by means of 1H NMR, from the margarine polar fraction. The spectral signals corresponding to sorbic acid are shown in Figure 1.
Margarine: Composition and Analysis
See also: Carotenoids: Occurrence, Properties and Determination; Cholecalciferol: Properties and Determination; Fats: Classification and Analysis; Fatty Acids: Determination and Requirements; Fatty Acids: Fatty Acids; Fatty Acids: Trans Fatty Acids; Functional Foods; Retinol: Properties and Determination; Tocopherols: Properties and Determination; Triacylglycerols: Characterization and Determination; Vegetable Oils: Composition and Analysis; Vitamin K: Properties and Determination.
Further Reading Adhikari P, Shin JA, Lee JH, et al. (2010) Production of trans-free margarine stock by enzymatic interesterification of rice bran oil, palm stearin and coconut oil. Journal of the Science of Food and Agriculture 90: 703–711. List, G. R. and Pelloso, T. (2007). Zero/low trans margarine, spreads, and shortening. In: List G, Kritchevsky D, and Ratnayake N (eds.) Trans Fats in Foods, pp. 155-176. Urbana, IL: AOCS Press.
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Miskandar MS, Man YC, Yusoff MSA, and Rahman RA (2005) Quality of margarine: fats selection and processing parameters. Asia Pacific Journal of Clinical Nutrition 14: 387–394. Schwartz H, Ollilainen V, Piironen V, and Lampi AM (2008) Tocopherol, tocotrienol and plant sterol contents of vegetable oils and industrial fats. Journal of Food Composition and Analysis 21: 152–161. Sioen I (2013) Fortified Margarine and fat spreads. In: Handbook of food fortification and health, pp. 159–171. New York: Springer. Siri-Tarino PW, Sun Q, Hu FB, and Krauss RM (2010) Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease. American Journal of Clinical Nutrition 91: 535–546. Sopelana P, Arizabaleta I, Ibargoitia ML, and Guille´n MD (2013) Characterisation of the lipidic components of margarines by 1H nuclear magnetic resonance. Food Chemistry 141: 3357–3364.
Relevant Websites http://www.imace.org/facts-on-fat/role-of-fats-in-the-diet/ – International Margarine Association of the Countries of Europe (IMACE).