- Email: [email protected]
Cholesterol Oxidation: Presence of 7-ketocholesterol in Different Food Products
(2000) 13, 625}631 doi:10.1006/jfca.2000.0901 Available online at http://www.idealibrary.com on
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
STUDY REVIEW Cholesterol Oxidation: Presence of 7-ketocholesterol in Different Food Products G. Lercker and M. T. Rodriguez-Estrada Universita` degli Studi di Bologna, Istituto di Industrie Agrarie, Via S. Giacomo 7, 40126 Bologna, Italy Received September 14, 1999, and in revised form March 3, 2000
Cholesterol, a monounsaturated sterol present in food of animal origin, can be oxidized during food processing and storage, thus generating a series of oxidation products, which are well known for their negative biological e!ects. The presence of 7-ketocholesterol (7-k), one of the main cholesterol oxidation products (COPs), was evaluated in biscuits, snacks, grated cheese, egg noodles, whole egg powder, whole milk powder, salami, raw and cooked meat. The content of 7-k varied depending on the type of matrix. Most food products showed a 7-k level lower than 2.0 ppm (expressed on sample basis), except for the beef meat ()3.5 ppm) and the whole egg powder ()4.6 ppm). In addition, beef meat and the meat products showed a higher percent ratio of cholesterol oxidation ()0.5%) as compared to the rest of the samples ()0.2%). 2000 Academic Press
Key =ords: cholesterol oxidation; 7-ketocholesterol; lipid oxidation; oxysterols.
INTRODUCTION Due to the presence of one double bond, cholesterol is susceptible to oxidation when exposed to light and molecular oxygen. Oxidation occurs through a free radical reaction (Nourooz-Zadeh, 1990), giving rise to more than 60 autoxidation products (Smith, 1981). Over the past few years, considerable attention has been devoted to the study of cholesterol oxidation due to the biological activities of its oxidation products (COPs) associated with human diseases. Research provides evidence that some cholesterol oxides are toxic and may facilitate development of coronary artery disease and certain cancers (Maercker, 1987; Chow, 1992; Morin and Peng, 1992; Morin et al., 1992; BoK singer et al., 1993; Haumann, 1993; Kubow, 1993). In order to avoid the formation of COPs, thermal treatments, exposure to light and oxygen should be reduced to a minimum during food processing, handling and storage. An interesting case of the e!ect of these conditions on cholesterol oxidation has been observed in fresh meat. This product is generally packaged with transparent "lms that allow a complete exposure to light and oxygen, thus favoring lipid oxidation and, in particular, cholesterol oxidation (Caboni et al., 1995); this e!ect is mainly due
To whom correspondence and reprint requests should be addressed. Tel.: #39-051-2099910. Fax: #39-051-2099911. E-mail: [email protected]. it 0889}1575/00/040625#07 $35.00/0
2000 Academic Press
626
LERCKER AND RODRIGUEZ-ESTRADA
to the presence of photocatalyzers, such as hemoglobin and hematoporphyrines. An 8-h exposure to neon light has proved to increase 10 times the initial COPs content present in the raw meat (Caboni et al., 1995). However, the intensity of oxidation directly depends on the radiation spectrum; in fact, the level of oxysterols in meat exposed to red light for 8h is only twice as much as the initial one (Tassinari, 1998). It is, therefore, important to control the quality of the raw materials, in order to evaluate the actual e!ect of processing, handling and storage on the cholesterol oxidation of the "nal product. The scope of this paper was to compare the amount of 7-ketocholesterol (7-k) found in various food products of animal origin and egg-containing foods, and to describe the di!erences among these matrices. 7-k was used as a tracer of cholesterol oxidation, because it is one of the most representative compounds among COPs from a quantitative standpoint (Park and Addis, 1985).
MATERIALS AND METHODS Grated cheese, meat and egg-containing products were purchased from a supermarket in Bologna, Italy. Whole egg powder and whole milk powder were obtained from industrial food suppliers. Salamis were provided by an Italian meat product industry. Lipid extraction from biscuits, snacks, salamis, raw and cooked meat was performed by using Folch's method (Folch et al., 1957). The powdered products and the egg noodles were extracted following the Bligh and Dyer procedure (1959). All samples were protected from light during the extraction. The determination of 7-k was performed by high-performance liquid chromatography (HPLC); the method B suggested by Penazzi et al. (1995) was utilized for this scope. Two replicates per each sample were analyzed.
RESULTS AND DISCUSSION Due to the di!erent structural characteristics of lipids in the various matrices, di!erent extraction methods were used. Since 7-k is sensitive to alkaline pH (Maerker and Unruh, 1986), a method that did not include saponi"cation was utilized for its quanti"cation; this procedure is simpler and potentially more accurate than the usual saponi"cation followed by gas chromatographic analysis. Table 1 lists the lipid content, total cholesterol content, 7-k content (expressed in lipid basis) and cholesterol oxidation percent ratio of the samples here analyzed, whereas Table 2 shows the amount of 7-k, expressed in sample basis, and the 7-k and oxysterol contents reported in the literature. As can be observed from Table 2, the 7-k content of the whole egg powder is the highest one among the analyzed food products, which is partly due to its elevated lipid percentage (44%). It is well known that spray-dried, cholesterol-containing foods are subjected to air currents and heating during processing, which particularly favors sterol oxidation during preparation and storage. In fact, our data and those found in literature con"rm this trend (Fischer and Grosch, 1985; Missler et al., 1985; Tsai and Hudson, 1985; Sander et al., 1989; Pie et al., 1990; Lai et al., 1995). However, it should be pointed out that the percent ratio of 7-k to cholesterol (i.e., percent ratio of cholesterol oxidation) shown in Table 1, is one of the lowest as compared to those detected in the other products.
627
CHOLESTEROL OXIDATION TABLE 1
Lipid content, total cholesterol content, 7-ketocholesterol content (expressed in lipid basis) and cholesterol oxidation percent ratio of some lipid-containing foods of animal origin
Food product
No. of analyzed samples
Lipids (%)
Total cholesterol (g/100 g lipids)
7-k (mg/kg lipids)
7-k/total cholesterol (%)
5 5
44 7.8}9.4
0.8}1.5
2.9}10.4 0.6}3.4
0.04}0.07
12 11 16 5 7 2 6 10
3.7}27.6 12.7}30.9 0.8}2.9 18.4}28.4 22.7 13.8 10.6}12.2 19.3}24.9
0.2}3.7 0.4}2.6 1.4}3.2 0.3}0.5 0.3}0.4 0.5 0.4}0.5 0.1}0.3
1.8}27.1 1.5}5.5 11.7}54.4 0.8}4.3 1.1}3.2 25.2 16.4}22.0 0.7}4.5
0.03}0.2 0.01}0.06 0.05}0.2 0.02}0.1 0.04}0.08 0.5 0.4}0.5 0.03}0.5
Whole egg powder Biscuits (infant formula) Biscuits prepared with eggs Snacks prepared with eggs Egg noodles Grated cheese (grana type) Whole milk powder Beef meat Raw Cooked Salami Data not available.
TABLE 2 7-ketocholesterol (expressed in sample basis) and oxysterols contents of some lipid-containing foods of animal origin 7-k Oxysterols 7-k (mg/kg sample) (mg/kg sample) (mg/kg sample) (Literature data) (Literature data)
Food product Whole egg powder Biscuits (infant formula) Biscuits prepared with eggs Snacks prepared with eggs Egg noodles Grated cheese (grana type) Whole milk powder
Beef meat
Raw Cooked
Salami
1.3}4.6
1.5}37
8}311
0.05}0.3 0.4}1.8 0.2}1.2 0.2}0.5 0.2}0.8
* * * 0.2}2.3 tr-16
* * * * 4}46
0.3}0.7
tr-3.1
tr-6.8
3.5
tr-1.1
0.5}3.4
1.7}2.5
tr-2.1
3.1}5.9
0.2}1.1
tr}2.6
tr-16.6
Reference Tsai and Hudson (1985), Sander et al. (1989), Lai et al. (1995) * * * Zunin et al. (1996) Finocchiaro et al. (1984), Sander et al. (1989) Nourooz-Zadeh and Appelqvist (1988), Angulo et al. (1997), Zunin et al. (1998) De Vore (1988), Pie et al. (1991), Hwang and Maercker (1993), Engeseth and Gray (1994) Novelli et al. (1998)
Oxysterols include 7-ketocholesterol and other oxidation products of cholesterol. tr stands for trace levels, which depend on the analytical technique used for the determination.
Considering their high oxidation susceptibility, spray-dried hen eggs should be avoided as a food ingredient, despite their excellent low-cost high-quality protein and balanced distribution of minerals and vitamins (Moreng and Avens, 1985; Shrimpton, 1987). In particular, it is not advisable to include egg powder in biscuit formulation, since these baked products are further subjected to an intense thermal treatment. Several types of biscuits (infant formula and breakfast type) were analyzed in this
628
LERCKER AND RODRIGUEZ-ESTRADA
study. The infant-formula biscuits exhibited a homogenous lipid content (Table 1) and a low 7-k level ()0.3 ppm), which somehow warrant the quality of the product from the cholesterol oxidation standpoint. Breakfast biscuits prepared with eggs, on the other hand, showed a wide range of lipid and cholesterol contents as a result of di!erent product formulations; in fact, some of these biscuits had cholesterol-containing ingredients, such as butter, milk, whole or skim milk powder. Consequently, 7-k greatly varied in these products, reaching a maximum cholesterol oxidation percent ratio equal to 0.2%. Since biscuits contained di!erent percentages of egg powder and there was no information about the initial 7-k level of these egg powders, it is di$cult to draw de"nitive conclusions with respect to the e!ect of baking on 7-k production and/or degradation. However, it should be pointed out that samples with similar formulations gave very di!erent 7-k values, which could be partly due to the type of processing technology employed. Snacks prepared with eggs, on the other hand, are also subjected to heating, but this process is milder than the one applied to biscuits; in addition, the surface-to-volume ratio exposed to oxygen is smaller in snacks than in biscuits, which makes snacks less prone to oxidation. In fact, low amounts of 7-k were detected in these products and their percent ratio of cholesterol oxidation was low as well. In any case, it should be noticed that the snacks also presented a wide range of lipid and cholesterol contents due to di!erent product formulations; besides the aforementioned ingredients used in biscuits, some snacks contained whole or skim yoghurt. Regarding egg noodles, a traditional Italian food product, the o$cial formulation claims a minimum of 4 eggs/kg of dough. Under the processing and storage conditions used for this product, a maximum of 0.5 mg/kg of 7-k in the whole sample was found; its percent ratio of cholesterol oxidation was lower than 0.2%. However, since fall 1998, the use of egg powder has been o$cially authorized, which will de"nitively a!ect its COPs content; it would be, therefore, necessary in the future to recontrol the oxysterols content of this product. Both grated cheese and milk powder have a large surface of contact with air and a low water activity, conditions that particularly enhance oxidation reactions. The quality of the raw materials, as well as the drying technology, in#uence the "nal oxidative status of these dairy products (Nourooz-Zadeh and Appelqvist, 1988). The 7-k values detected in both products are quite low (Tables 1 & 2), which might be due to the protective e!ect exerted by the fat globule membranes; these results have been con"rmed by Rose-Sallin et al. (1995). In addition, these dairy products display a low percent ratio of cholesterol oxidation (0.1%). Many papers, however, have reported much higher 7-k contents in milk powder and grated cheese, than those observed in this study. Artifact formation and co-eluting components could be the main sources of such discrepancies (Rose-Sallin et al., 1995). On the other hand, it should be pointed out that grated cheeses can be prepared from di!erent types of cheeses, so it would be necessary to have more information about origin and technology employed, in order to directly compare the results obtained from di!erent groups. Regarding meat, it is well known that, even during short storage periods after heating, lipid oxidation does occur (De Vore, 1988; Mielche and Bertelsen, 1994; Mielche and Bertelsen, 1995; Kerler and Grosch, 1997). A study was performed on the e!ect of di!erent cooking methods (oven, microwave oven, boiling, broiling, barbecue cooking, a combination of traditional oven}microwave oven) on cholesterol oxidation of a homogenous meat system (hamburger) (Rodriguez-Estrada et al., 1997). No signi"cant di!erences in the 7-k content were found among the di!erent cooking treatments, but the raw meat already presented a considerably high initial 7-k level (3.5 ppm). This result might be a direct consequence of the holding period to which meat is usually subjected; during this process, meat is often kept at 4}63C for 10}15
CHOLESTEROL OXIDATION
629
days, in order to increase the meat tenderness and to promote the #avor formation. Another point that should be noticed is that beef meat exhibits the highest percent ratio of cholesterol oxidation (0.5%) among the products analyzed here; the natural presence of oxidation catalyzers (i.e., low and high molecular weight iron) might have had an important role in this degradation process (Varman and Sutherland, 1995). On the other hand, oxidation in meat products depends on the quality of the raw materials, the amount of antioxidants added, the processing conditions and the length of ripening and storage. Meat products are usually packed with natural or arti"cial "lms, which allow a super"cial exposure to light and oxygen. Ripened meat products, such as salami, are protected, to a certain extent, against these pro-oxidant agents, due to a mold layer that grows in the surface and the catalases they release (Varman and Sutherland, 1995). In this study, the amount of 7-k found in these products was smaller than 1.1 ppm. However, some salamis presented a percent ratio of cholesterol oxidation similar to that of beef meat (0.5%); in these salamis, as well as in those meat products in which high levels of oxysterols have been detected (Novelli et al., 1998), any of the aforementioned factors could have been a determining one. In general, great discrepancies have been observed in the COPs data obtained from di!erent laboratories for the various matrices analyzed here, which may be due to di!erences in the extraction procedures or the chromatographic methods. Data could also re#ect the large quality variability of commercially available products. In any case, these large discrepancies underline, once more, the necessity of comparing the most promising methods for determination of COPs in food (Dionisi et al., 1998), which would lead to more accurate and reliable results, thus allowing the creation of a trustworthy database (Appelqvist, 1996; Dutta et al., 1998). CONCLUSIONS The presence of 7-ketocholesterol was evaluated in various food products of animal origin, as well as in egg-containing foods. The content of 7-k varied depending on the type of matrix. Most food products showed a 7-k level lower than 2.0 ppm (expressed on sample basis), except for the beef meat ()3.5 ppm) and the whole egg powder ()4.6 ppm). Due to its high 7-k content, the use of egg powder as a food ingredient would not be advisable, especially for products subjected to further heating. On the other hand, beef meat and the meat products showed a higher percent ratio of cholesterol oxidation ()0.5%) as compared to the rest of the samples ()0.2%) analyzed here; attention should be focused on the quality of the raw materials and the overall product technology, in order to reduce cholesterol oxidation in beef meat and the meat products.
REFERENCES Angulo, A. J., Romera, J. M., Ramirez, M., and Gil, A. (1997). Determination of cholesterol oxides in dairy products. E!ect of storage conditions. J. Agric. Food Chem. 45, 4318}4323. Appelqvist, L. A. (1996). Oxidized sterols. Bull. Int. Dairy Fed. 315, 52}58. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and puri"cation. Can. J. Biochem. Physiol. 37, 911}917. BoK singer, S., Luf, W. and Brandl, E. (1993). Oxysterols: their occurrence and biological e!ect. Int. Dairy J. 3, 1}33. Caboni, M. F., Fedrizzi, G., and Lercker, G. (1995). Sulla presenza di alcuni prodotti di ossidazione nella carne bovina. In Atti del =orkshop CNR-RAISA Flair Flow Europe, ;dine 5}6 dicembre 1995, pp. 143}148. Udine, Italy.
630
LERCKER AND RODRIGUEZ-ESTRADA
Chow, C. K. (1992). Biological e!ects of oxidized fatty acids. In Fatty Acids in Foods and ¹heir Health Implications (C. H. Chow, Ed.), pp. 689}705. Marcel Dekker Inc., New York. De Vore, V. R. (1988). TBA values and 7-ketocholesterol in refrigerated raw and cooked ground beef. J. Food Sci. 53, 1058}1061. Dionisi, F., Golay, P. A., Aeschlimann, J. M., and Fay, L. B. (1998). Determination of cholesterol oxidation products in milk powders: methods comparison and validation. J. Agric. Food Chem. 46, 2227}2233. Dutta, P. C., Caboni, M. F., Dionisi, F., Grandgirard, A., Guardiola, F., Kumpulainem, V., Lebovics, K., Pihlava, J.-M., Rodriguez-Estrada, M. T., and Ulberth, F. (1999). Measurements of cholesterol oxides in foods: results of an interlaboratory comparison study. In Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease (J. T. Kumpulainem and J. T. Salonen, Eds.), pp. 309}315. The Royal Society of Chemistry, Cambridge, UK. Engeseth, N. J. and Gray, J. I. (1994). Cholesterol oxidation in muscle tissue. Meat Sci. 36, 309}320. Finocchiaro, E. T., Lee, K., and Richardson, T. (1984). Identi"cation and quanti"cation of cholesterol oxides in grated cheese and bleached butteroil. J. Am. Oil Chem. Soc. 61, 877}883. Fischer, K.-H. and Grosch, W. (1985). Analysis of oxycholesterols in foods. In Proceedings of ¸ipidforum/Sik Symposium (M. Marcuse, Ed.), pp. 125}139. Scandiforum Lipid Research Technology, GoK teborg, Sweden. Folch, J., Lees, M., and Stanley, G. H. S. (1957). A simple method for the isolation and puri"cation of total lipids from animal tissues. J. Biol. Chem. 726, 497}509. Haumann, B. F. (1993). Health implications of lipid oxidation. INFORM 4, 800}810. Hwang, K. T. and Maercker, G. (1993). Quantitation of cholesterol oxidation products in unirradiated and irradiated meats. J. Am. Oil Chem. Soc. 70, 371}375. Kerler, J. and Grosch, W. (1997). Character impact odorants of boiled chicken: changes during refrigerated storage and reheating. Z. ¸ebensm. ;nters. Forsch. A 205, 232}238. Kubow, S. (1993). Lipid oxidation products in food and atherogenesis. Nutr. Rev. 51, 33}40. Lai, S.-M., Gray, J. I. and Zabik, M. E. (1995). Evaluation of solid phase extraction and gas chromatography for determination of cholesterol oxidation products in spray-dried whole egg. J. Agric. Food Chem. 43, 1122}1126. Maercker, G. (1987). Cholesterol autoxidation-current status. J. Am. Oil Chem. Soc. 64, 388}392. Maercker, G. and Unruh Jr., J. (1986). Cholesterol oxides I. Isolation and determination of some cholesterol oxidation products. J. Am. Oil Chem. Soc. 63, 767}771. Mielche, M. M. and Bertelsen, G. (1994). Approaches to prevention of warmed-over #avor. ¹rends Food Sci. ¹echnol. 5, 322}327. Mielche, M. M. and Bertelsen, G. (1995). Evaluation of a mathematical model for prediction on lipid oxidation in heat-treated beef during chill storage. Z. ¸ebensm. ;nters. Forsch. 200, 415}419. Missler, S. R., Waselchuck, B. A., and Merrit Jr., C. (1985). Separation and identi"cation of cholesterol oxidation products in dried egg preparation. J. Food Sci. 50, 595}598. Moreng, R. E. and Avens, J. S. (1985). Poultry Science and Production. Reston Publishing Company Inc., Reston, VA. Morin, R. J. and Peng, S. K. (1992). Cholesterol oxides in plasma and tissues. In Biological E+ects of Cholesterol Oxides (S. K. Peng and R. J. Morin, Eds.), pp. 89}101. CRC Press, Ann Arbor, MI. Morin, R. J., Hu, B., Peng, S. K., and Sevanian, A. (1992). Cholesterol oxidation and cancer. In Biological E+ects of Cholesterol Oxides (S. K. Peng and R. J. Morin, Eds.), pp. 191}202. CRC Press, Ann Arbor, MI. Nourooz-Zadeh, J. (1990). Determination of the autoxidation products from free or total cholesterol: a new multistep enrichment methodology including the enzymatic release of esteri"ed cholesterol. J. Agric. Food Chem. 38, 1667}1673. Nourooz-Zadeh, J. and Appelqvist, L.-A. (1988). Cholesterol oxides in Swedish foods and food ingredients: milk powder products. J. Food Sci. 53, 74}79, 87. Novelli, E., Zanardi, E., Ghiretti, G. P., Campanini, G., Dazzi, G., Madarena, G., and Chizzolini, R. (1998). Lipid and cholesterol oxidation in frozen stored pork, salame milano and mortadella. Meat Sci. 48, 29}40. Park, S. W. and Addis, P. B. (1985). HPLC determination of C-7 oxidized cholesterol derivatives in foods. J. Food Sci. 50, 1437}1441, 1444. Penazzi, G., Caboni, M. F., Zunin, P., Evangelisti, F., Tiscornia, E., Gallina Toschi, T., and Lercker, G. (1995). Routine HPLC determination of 7-ketocholesterol in some foods, by two di!erent analytical methods. J. Am. Oil Chem. Soc. 71, 1523}1527. Pie, J. E., Spahis, K., and Seillan, C. (1990). Evaluation of oxidative degradation of cholesterol in food ingredients: identi"cation and quanti"cation of cholesterol oxides. J. Agric. Food Chem. 38, 973}979.
CHOLESTEROL OXIDATION
631
Pie, J. E., Spahis, K., and Seillan, C. (1991). Cholesterol oxidation in meat products during cooking and frozen storage. J. Agric. Food Chem. 39, 250}254. Rodriguez-Estrada, M. T., Penazzi, G., Caboni, M. F., Bertacco, G., and Lercker, G. (1997). E!ect of di!erent cooking methods on some lipid and protein components of beef hamburger. Meat Sci. 45, 365}375. Rose-Sallin, C., Huggett, A., Bosset, J. O., Tabacchi, R., and Fay, L. B. (1995). Quanti"cation of cholesterol oxidation products in milk powders using [H ] cholesterol to monitor cholesterol autoxidation artifacts. J. Agric. Food Chem. 43, 935}941. Sander, B. D., Addis, P. B., Park, S. W., and Smith, D. E. (1989). Quanti"cation of cholesterol oxidation products in a variety of foods. J. Food Prot. 52, 109}114. Shrimpton, D. H. (1987). The nutritive value of eggs and their dietary signi"cance. In Egg Quality-Current Problems and Recent Advances (R. G. Wells and C. G. Belyavin, Eds.), pp. 11}25. Butterworths, London. Smith, L. L. (1981). Cholesterol Autoxidation. Plenum Press, New York/London. Tsai, L. S. and Hudson, C. A. (1985). Cholesterol oxides in commercial dry egg products: quantitation. J. Food Sci. 50, 229}231, 237. Tassinari, P. (1998). Modi,cazioni dei lipidi di carni rosse dovute a fotossidazione. Tesi di Laurea in Scienze e Tecnologie Alimentari, Universita` di Bologna, A.A. 1997}1998. Varman, A. H. and Sutherland, J. P. (1995). Meat and Meat Products. ¹echnology, Chemistry and Microbiology. Chapman & Hall, London, U.K. Zunin, P., Evangelisti, F., Calcagno, C., and Tiscornia, E. (1996). Cholesterol oxidation in dried egg pasta: detecting 7-ketocholesterol content. Cereal Chem. 73, 691}694. Zunin, P., Calcagno, C., and Evangelisti, F. (1998). Sterol oxidation in infant milk formulas and milk cereals. J. Dairy Res. 65, 591}598.
JOURNAL OF FOOD COMPOSITION AND ANALYSIS
STUDY REVIEW Cholesterol Oxidation: Presence of 7-ketocholesterol in Different Food Products G. Lercker and M. T. Rodriguez-Estrada Universita` degli Studi di Bologna, Istituto di Industrie Agrarie, Via S. Giacomo 7, 40126 Bologna, Italy Received September 14, 1999, and in revised form March 3, 2000
Cholesterol, a monounsaturated sterol present in food of animal origin, can be oxidized during food processing and storage, thus generating a series of oxidation products, which are well known for their negative biological e!ects. The presence of 7-ketocholesterol (7-k), one of the main cholesterol oxidation products (COPs), was evaluated in biscuits, snacks, grated cheese, egg noodles, whole egg powder, whole milk powder, salami, raw and cooked meat. The content of 7-k varied depending on the type of matrix. Most food products showed a 7-k level lower than 2.0 ppm (expressed on sample basis), except for the beef meat ()3.5 ppm) and the whole egg powder ()4.6 ppm). In addition, beef meat and the meat products showed a higher percent ratio of cholesterol oxidation ()0.5%) as compared to the rest of the samples ()0.2%). 2000 Academic Press
Key =ords: cholesterol oxidation; 7-ketocholesterol; lipid oxidation; oxysterols.
INTRODUCTION Due to the presence of one double bond, cholesterol is susceptible to oxidation when exposed to light and molecular oxygen. Oxidation occurs through a free radical reaction (Nourooz-Zadeh, 1990), giving rise to more than 60 autoxidation products (Smith, 1981). Over the past few years, considerable attention has been devoted to the study of cholesterol oxidation due to the biological activities of its oxidation products (COPs) associated with human diseases. Research provides evidence that some cholesterol oxides are toxic and may facilitate development of coronary artery disease and certain cancers (Maercker, 1987; Chow, 1992; Morin and Peng, 1992; Morin et al., 1992; BoK singer et al., 1993; Haumann, 1993; Kubow, 1993). In order to avoid the formation of COPs, thermal treatments, exposure to light and oxygen should be reduced to a minimum during food processing, handling and storage. An interesting case of the e!ect of these conditions on cholesterol oxidation has been observed in fresh meat. This product is generally packaged with transparent "lms that allow a complete exposure to light and oxygen, thus favoring lipid oxidation and, in particular, cholesterol oxidation (Caboni et al., 1995); this e!ect is mainly due
To whom correspondence and reprint requests should be addressed. Tel.: #39-051-2099910. Fax: #39-051-2099911. E-mail: [email protected]. it 0889}1575/00/040625#07 $35.00/0
2000 Academic Press
626
LERCKER AND RODRIGUEZ-ESTRADA
to the presence of photocatalyzers, such as hemoglobin and hematoporphyrines. An 8-h exposure to neon light has proved to increase 10 times the initial COPs content present in the raw meat (Caboni et al., 1995). However, the intensity of oxidation directly depends on the radiation spectrum; in fact, the level of oxysterols in meat exposed to red light for 8h is only twice as much as the initial one (Tassinari, 1998). It is, therefore, important to control the quality of the raw materials, in order to evaluate the actual e!ect of processing, handling and storage on the cholesterol oxidation of the "nal product. The scope of this paper was to compare the amount of 7-ketocholesterol (7-k) found in various food products of animal origin and egg-containing foods, and to describe the di!erences among these matrices. 7-k was used as a tracer of cholesterol oxidation, because it is one of the most representative compounds among COPs from a quantitative standpoint (Park and Addis, 1985).
MATERIALS AND METHODS Grated cheese, meat and egg-containing products were purchased from a supermarket in Bologna, Italy. Whole egg powder and whole milk powder were obtained from industrial food suppliers. Salamis were provided by an Italian meat product industry. Lipid extraction from biscuits, snacks, salamis, raw and cooked meat was performed by using Folch's method (Folch et al., 1957). The powdered products and the egg noodles were extracted following the Bligh and Dyer procedure (1959). All samples were protected from light during the extraction. The determination of 7-k was performed by high-performance liquid chromatography (HPLC); the method B suggested by Penazzi et al. (1995) was utilized for this scope. Two replicates per each sample were analyzed.
RESULTS AND DISCUSSION Due to the di!erent structural characteristics of lipids in the various matrices, di!erent extraction methods were used. Since 7-k is sensitive to alkaline pH (Maerker and Unruh, 1986), a method that did not include saponi"cation was utilized for its quanti"cation; this procedure is simpler and potentially more accurate than the usual saponi"cation followed by gas chromatographic analysis. Table 1 lists the lipid content, total cholesterol content, 7-k content (expressed in lipid basis) and cholesterol oxidation percent ratio of the samples here analyzed, whereas Table 2 shows the amount of 7-k, expressed in sample basis, and the 7-k and oxysterol contents reported in the literature. As can be observed from Table 2, the 7-k content of the whole egg powder is the highest one among the analyzed food products, which is partly due to its elevated lipid percentage (44%). It is well known that spray-dried, cholesterol-containing foods are subjected to air currents and heating during processing, which particularly favors sterol oxidation during preparation and storage. In fact, our data and those found in literature con"rm this trend (Fischer and Grosch, 1985; Missler et al., 1985; Tsai and Hudson, 1985; Sander et al., 1989; Pie et al., 1990; Lai et al., 1995). However, it should be pointed out that the percent ratio of 7-k to cholesterol (i.e., percent ratio of cholesterol oxidation) shown in Table 1, is one of the lowest as compared to those detected in the other products.
627
CHOLESTEROL OXIDATION TABLE 1
Lipid content, total cholesterol content, 7-ketocholesterol content (expressed in lipid basis) and cholesterol oxidation percent ratio of some lipid-containing foods of animal origin
Food product
No. of analyzed samples
Lipids (%)
Total cholesterol (g/100 g lipids)
7-k (mg/kg lipids)
7-k/total cholesterol (%)
5 5
44 7.8}9.4
0.8}1.5
2.9}10.4 0.6}3.4
0.04}0.07
12 11 16 5 7 2 6 10
3.7}27.6 12.7}30.9 0.8}2.9 18.4}28.4 22.7 13.8 10.6}12.2 19.3}24.9
0.2}3.7 0.4}2.6 1.4}3.2 0.3}0.5 0.3}0.4 0.5 0.4}0.5 0.1}0.3
1.8}27.1 1.5}5.5 11.7}54.4 0.8}4.3 1.1}3.2 25.2 16.4}22.0 0.7}4.5
0.03}0.2 0.01}0.06 0.05}0.2 0.02}0.1 0.04}0.08 0.5 0.4}0.5 0.03}0.5
Whole egg powder Biscuits (infant formula) Biscuits prepared with eggs Snacks prepared with eggs Egg noodles Grated cheese (grana type) Whole milk powder Beef meat Raw Cooked Salami Data not available.
TABLE 2 7-ketocholesterol (expressed in sample basis) and oxysterols contents of some lipid-containing foods of animal origin 7-k Oxysterols 7-k (mg/kg sample) (mg/kg sample) (mg/kg sample) (Literature data) (Literature data)
Food product Whole egg powder Biscuits (infant formula) Biscuits prepared with eggs Snacks prepared with eggs Egg noodles Grated cheese (grana type) Whole milk powder
Beef meat
Raw Cooked
Salami
1.3}4.6
1.5}37
8}311
0.05}0.3 0.4}1.8 0.2}1.2 0.2}0.5 0.2}0.8
* * * 0.2}2.3 tr-16
* * * * 4}46
0.3}0.7
tr-3.1
tr-6.8
3.5
tr-1.1
0.5}3.4
1.7}2.5
tr-2.1
3.1}5.9
0.2}1.1
tr}2.6
tr-16.6
Reference Tsai and Hudson (1985), Sander et al. (1989), Lai et al. (1995) * * * Zunin et al. (1996) Finocchiaro et al. (1984), Sander et al. (1989) Nourooz-Zadeh and Appelqvist (1988), Angulo et al. (1997), Zunin et al. (1998) De Vore (1988), Pie et al. (1991), Hwang and Maercker (1993), Engeseth and Gray (1994) Novelli et al. (1998)
Oxysterols include 7-ketocholesterol and other oxidation products of cholesterol. tr stands for trace levels, which depend on the analytical technique used for the determination.
Considering their high oxidation susceptibility, spray-dried hen eggs should be avoided as a food ingredient, despite their excellent low-cost high-quality protein and balanced distribution of minerals and vitamins (Moreng and Avens, 1985; Shrimpton, 1987). In particular, it is not advisable to include egg powder in biscuit formulation, since these baked products are further subjected to an intense thermal treatment. Several types of biscuits (infant formula and breakfast type) were analyzed in this
628
LERCKER AND RODRIGUEZ-ESTRADA
study. The infant-formula biscuits exhibited a homogenous lipid content (Table 1) and a low 7-k level ()0.3 ppm), which somehow warrant the quality of the product from the cholesterol oxidation standpoint. Breakfast biscuits prepared with eggs, on the other hand, showed a wide range of lipid and cholesterol contents as a result of di!erent product formulations; in fact, some of these biscuits had cholesterol-containing ingredients, such as butter, milk, whole or skim milk powder. Consequently, 7-k greatly varied in these products, reaching a maximum cholesterol oxidation percent ratio equal to 0.2%. Since biscuits contained di!erent percentages of egg powder and there was no information about the initial 7-k level of these egg powders, it is di$cult to draw de"nitive conclusions with respect to the e!ect of baking on 7-k production and/or degradation. However, it should be pointed out that samples with similar formulations gave very di!erent 7-k values, which could be partly due to the type of processing technology employed. Snacks prepared with eggs, on the other hand, are also subjected to heating, but this process is milder than the one applied to biscuits; in addition, the surface-to-volume ratio exposed to oxygen is smaller in snacks than in biscuits, which makes snacks less prone to oxidation. In fact, low amounts of 7-k were detected in these products and their percent ratio of cholesterol oxidation was low as well. In any case, it should be noticed that the snacks also presented a wide range of lipid and cholesterol contents due to di!erent product formulations; besides the aforementioned ingredients used in biscuits, some snacks contained whole or skim yoghurt. Regarding egg noodles, a traditional Italian food product, the o$cial formulation claims a minimum of 4 eggs/kg of dough. Under the processing and storage conditions used for this product, a maximum of 0.5 mg/kg of 7-k in the whole sample was found; its percent ratio of cholesterol oxidation was lower than 0.2%. However, since fall 1998, the use of egg powder has been o$cially authorized, which will de"nitively a!ect its COPs content; it would be, therefore, necessary in the future to recontrol the oxysterols content of this product. Both grated cheese and milk powder have a large surface of contact with air and a low water activity, conditions that particularly enhance oxidation reactions. The quality of the raw materials, as well as the drying technology, in#uence the "nal oxidative status of these dairy products (Nourooz-Zadeh and Appelqvist, 1988). The 7-k values detected in both products are quite low (Tables 1 & 2), which might be due to the protective e!ect exerted by the fat globule membranes; these results have been con"rmed by Rose-Sallin et al. (1995). In addition, these dairy products display a low percent ratio of cholesterol oxidation (0.1%). Many papers, however, have reported much higher 7-k contents in milk powder and grated cheese, than those observed in this study. Artifact formation and co-eluting components could be the main sources of such discrepancies (Rose-Sallin et al., 1995). On the other hand, it should be pointed out that grated cheeses can be prepared from di!erent types of cheeses, so it would be necessary to have more information about origin and technology employed, in order to directly compare the results obtained from di!erent groups. Regarding meat, it is well known that, even during short storage periods after heating, lipid oxidation does occur (De Vore, 1988; Mielche and Bertelsen, 1994; Mielche and Bertelsen, 1995; Kerler and Grosch, 1997). A study was performed on the e!ect of di!erent cooking methods (oven, microwave oven, boiling, broiling, barbecue cooking, a combination of traditional oven}microwave oven) on cholesterol oxidation of a homogenous meat system (hamburger) (Rodriguez-Estrada et al., 1997). No signi"cant di!erences in the 7-k content were found among the di!erent cooking treatments, but the raw meat already presented a considerably high initial 7-k level (3.5 ppm). This result might be a direct consequence of the holding period to which meat is usually subjected; during this process, meat is often kept at 4}63C for 10}15
CHOLESTEROL OXIDATION
629
days, in order to increase the meat tenderness and to promote the #avor formation. Another point that should be noticed is that beef meat exhibits the highest percent ratio of cholesterol oxidation (0.5%) among the products analyzed here; the natural presence of oxidation catalyzers (i.e., low and high molecular weight iron) might have had an important role in this degradation process (Varman and Sutherland, 1995). On the other hand, oxidation in meat products depends on the quality of the raw materials, the amount of antioxidants added, the processing conditions and the length of ripening and storage. Meat products are usually packed with natural or arti"cial "lms, which allow a super"cial exposure to light and oxygen. Ripened meat products, such as salami, are protected, to a certain extent, against these pro-oxidant agents, due to a mold layer that grows in the surface and the catalases they release (Varman and Sutherland, 1995). In this study, the amount of 7-k found in these products was smaller than 1.1 ppm. However, some salamis presented a percent ratio of cholesterol oxidation similar to that of beef meat (0.5%); in these salamis, as well as in those meat products in which high levels of oxysterols have been detected (Novelli et al., 1998), any of the aforementioned factors could have been a determining one. In general, great discrepancies have been observed in the COPs data obtained from di!erent laboratories for the various matrices analyzed here, which may be due to di!erences in the extraction procedures or the chromatographic methods. Data could also re#ect the large quality variability of commercially available products. In any case, these large discrepancies underline, once more, the necessity of comparing the most promising methods for determination of COPs in food (Dionisi et al., 1998), which would lead to more accurate and reliable results, thus allowing the creation of a trustworthy database (Appelqvist, 1996; Dutta et al., 1998). CONCLUSIONS The presence of 7-ketocholesterol was evaluated in various food products of animal origin, as well as in egg-containing foods. The content of 7-k varied depending on the type of matrix. Most food products showed a 7-k level lower than 2.0 ppm (expressed on sample basis), except for the beef meat ()3.5 ppm) and the whole egg powder ()4.6 ppm). Due to its high 7-k content, the use of egg powder as a food ingredient would not be advisable, especially for products subjected to further heating. On the other hand, beef meat and the meat products showed a higher percent ratio of cholesterol oxidation ()0.5%) as compared to the rest of the samples ()0.2%) analyzed here; attention should be focused on the quality of the raw materials and the overall product technology, in order to reduce cholesterol oxidation in beef meat and the meat products.
REFERENCES Angulo, A. J., Romera, J. M., Ramirez, M., and Gil, A. (1997). Determination of cholesterol oxides in dairy products. E!ect of storage conditions. J. Agric. Food Chem. 45, 4318}4323. Appelqvist, L. A. (1996). Oxidized sterols. Bull. Int. Dairy Fed. 315, 52}58. Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and puri"cation. Can. J. Biochem. Physiol. 37, 911}917. BoK singer, S., Luf, W. and Brandl, E. (1993). Oxysterols: their occurrence and biological e!ect. Int. Dairy J. 3, 1}33. Caboni, M. F., Fedrizzi, G., and Lercker, G. (1995). Sulla presenza di alcuni prodotti di ossidazione nella carne bovina. In Atti del =orkshop CNR-RAISA Flair Flow Europe, ;dine 5}6 dicembre 1995, pp. 143}148. Udine, Italy.
630
LERCKER AND RODRIGUEZ-ESTRADA
Chow, C. K. (1992). Biological e!ects of oxidized fatty acids. In Fatty Acids in Foods and ¹heir Health Implications (C. H. Chow, Ed.), pp. 689}705. Marcel Dekker Inc., New York. De Vore, V. R. (1988). TBA values and 7-ketocholesterol in refrigerated raw and cooked ground beef. J. Food Sci. 53, 1058}1061. Dionisi, F., Golay, P. A., Aeschlimann, J. M., and Fay, L. B. (1998). Determination of cholesterol oxidation products in milk powders: methods comparison and validation. J. Agric. Food Chem. 46, 2227}2233. Dutta, P. C., Caboni, M. F., Dionisi, F., Grandgirard, A., Guardiola, F., Kumpulainem, V., Lebovics, K., Pihlava, J.-M., Rodriguez-Estrada, M. T., and Ulberth, F. (1999). Measurements of cholesterol oxides in foods: results of an interlaboratory comparison study. In Natural Antioxidants and Anticarcinogens in Nutrition, Health and Disease (J. T. Kumpulainem and J. T. Salonen, Eds.), pp. 309}315. The Royal Society of Chemistry, Cambridge, UK. Engeseth, N. J. and Gray, J. I. (1994). Cholesterol oxidation in muscle tissue. Meat Sci. 36, 309}320. Finocchiaro, E. T., Lee, K., and Richardson, T. (1984). Identi"cation and quanti"cation of cholesterol oxides in grated cheese and bleached butteroil. J. Am. Oil Chem. Soc. 61, 877}883. Fischer, K.-H. and Grosch, W. (1985). Analysis of oxycholesterols in foods. In Proceedings of ¸ipidforum/Sik Symposium (M. Marcuse, Ed.), pp. 125}139. Scandiforum Lipid Research Technology, GoK teborg, Sweden. Folch, J., Lees, M., and Stanley, G. H. S. (1957). A simple method for the isolation and puri"cation of total lipids from animal tissues. J. Biol. Chem. 726, 497}509. Haumann, B. F. (1993). Health implications of lipid oxidation. INFORM 4, 800}810. Hwang, K. T. and Maercker, G. (1993). Quantitation of cholesterol oxidation products in unirradiated and irradiated meats. J. Am. Oil Chem. Soc. 70, 371}375. Kerler, J. and Grosch, W. (1997). Character impact odorants of boiled chicken: changes during refrigerated storage and reheating. Z. ¸ebensm. ;nters. Forsch. A 205, 232}238. Kubow, S. (1993). Lipid oxidation products in food and atherogenesis. Nutr. Rev. 51, 33}40. Lai, S.-M., Gray, J. I. and Zabik, M. E. (1995). Evaluation of solid phase extraction and gas chromatography for determination of cholesterol oxidation products in spray-dried whole egg. J. Agric. Food Chem. 43, 1122}1126. Maercker, G. (1987). Cholesterol autoxidation-current status. J. Am. Oil Chem. Soc. 64, 388}392. Maercker, G. and Unruh Jr., J. (1986). Cholesterol oxides I. Isolation and determination of some cholesterol oxidation products. J. Am. Oil Chem. Soc. 63, 767}771. Mielche, M. M. and Bertelsen, G. (1994). Approaches to prevention of warmed-over #avor. ¹rends Food Sci. ¹echnol. 5, 322}327. Mielche, M. M. and Bertelsen, G. (1995). Evaluation of a mathematical model for prediction on lipid oxidation in heat-treated beef during chill storage. Z. ¸ebensm. ;nters. Forsch. 200, 415}419. Missler, S. R., Waselchuck, B. A., and Merrit Jr., C. (1985). Separation and identi"cation of cholesterol oxidation products in dried egg preparation. J. Food Sci. 50, 595}598. Moreng, R. E. and Avens, J. S. (1985). Poultry Science and Production. Reston Publishing Company Inc., Reston, VA. Morin, R. J. and Peng, S. K. (1992). Cholesterol oxides in plasma and tissues. In Biological E+ects of Cholesterol Oxides (S. K. Peng and R. J. Morin, Eds.), pp. 89}101. CRC Press, Ann Arbor, MI. Morin, R. J., Hu, B., Peng, S. K., and Sevanian, A. (1992). Cholesterol oxidation and cancer. In Biological E+ects of Cholesterol Oxides (S. K. Peng and R. J. Morin, Eds.), pp. 191}202. CRC Press, Ann Arbor, MI. Nourooz-Zadeh, J. (1990). Determination of the autoxidation products from free or total cholesterol: a new multistep enrichment methodology including the enzymatic release of esteri"ed cholesterol. J. Agric. Food Chem. 38, 1667}1673. Nourooz-Zadeh, J. and Appelqvist, L.-A. (1988). Cholesterol oxides in Swedish foods and food ingredients: milk powder products. J. Food Sci. 53, 74}79, 87. Novelli, E., Zanardi, E., Ghiretti, G. P., Campanini, G., Dazzi, G., Madarena, G., and Chizzolini, R. (1998). Lipid and cholesterol oxidation in frozen stored pork, salame milano and mortadella. Meat Sci. 48, 29}40. Park, S. W. and Addis, P. B. (1985). HPLC determination of C-7 oxidized cholesterol derivatives in foods. J. Food Sci. 50, 1437}1441, 1444. Penazzi, G., Caboni, M. F., Zunin, P., Evangelisti, F., Tiscornia, E., Gallina Toschi, T., and Lercker, G. (1995). Routine HPLC determination of 7-ketocholesterol in some foods, by two di!erent analytical methods. J. Am. Oil Chem. Soc. 71, 1523}1527. Pie, J. E., Spahis, K., and Seillan, C. (1990). Evaluation of oxidative degradation of cholesterol in food ingredients: identi"cation and quanti"cation of cholesterol oxides. J. Agric. Food Chem. 38, 973}979.
CHOLESTEROL OXIDATION
631
Pie, J. E., Spahis, K., and Seillan, C. (1991). Cholesterol oxidation in meat products during cooking and frozen storage. J. Agric. Food Chem. 39, 250}254. Rodriguez-Estrada, M. T., Penazzi, G., Caboni, M. F., Bertacco, G., and Lercker, G. (1997). E!ect of di!erent cooking methods on some lipid and protein components of beef hamburger. Meat Sci. 45, 365}375. Rose-Sallin, C., Huggett, A., Bosset, J. O., Tabacchi, R., and Fay, L. B. (1995). Quanti"cation of cholesterol oxidation products in milk powders using [H ] cholesterol to monitor cholesterol autoxidation artifacts. J. Agric. Food Chem. 43, 935}941. Sander, B. D., Addis, P. B., Park, S. W., and Smith, D. E. (1989). Quanti"cation of cholesterol oxidation products in a variety of foods. J. Food Prot. 52, 109}114. Shrimpton, D. H. (1987). The nutritive value of eggs and their dietary signi"cance. In Egg Quality-Current Problems and Recent Advances (R. G. Wells and C. G. Belyavin, Eds.), pp. 11}25. Butterworths, London. Smith, L. L. (1981). Cholesterol Autoxidation. Plenum Press, New York/London. Tsai, L. S. and Hudson, C. A. (1985). Cholesterol oxides in commercial dry egg products: quantitation. J. Food Sci. 50, 229}231, 237. Tassinari, P. (1998). Modi,cazioni dei lipidi di carni rosse dovute a fotossidazione. Tesi di Laurea in Scienze e Tecnologie Alimentari, Universita` di Bologna, A.A. 1997}1998. Varman, A. H. and Sutherland, J. P. (1995). Meat and Meat Products. ¹echnology, Chemistry and Microbiology. Chapman & Hall, London, U.K. Zunin, P., Evangelisti, F., Calcagno, C., and Tiscornia, E. (1996). Cholesterol oxidation in dried egg pasta: detecting 7-ketocholesterol content. Cereal Chem. 73, 691}694. Zunin, P., Calcagno, C., and Evangelisti, F. (1998). Sterol oxidation in infant milk formulas and milk cereals. J. Dairy Res. 65, 591}598.