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A novel procedure for routine milk fat extraction based on dichloromethane
Journal of Food Composition and Analysis 23 (2010) 852–855
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Short Communication
A novel procedure for routine milk fat extraction based on dichloromethane I. Stefanov, B. Vlaeminck, V. Fievez * Laboratory for Animal Nutrition and Animal Product Quality (Lanupro), Faculty of Bioscience Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 June 2009 Received in revised form 22 February 2010 Accepted 11 March 2010
A novel method for the extraction of milk fat using dichloromethane (CH2Cl2)–ethanol was compared to the Rose-Gottlieb (IDF Standard 1D, 1996) extraction procedure. The new extraction method consisted of a fast (approximately 30 min for 20 samples) and simple procedure involving direct mixing of raw cow milk samples with a dichloromethane–ethanol solution (2/1, v/v). The fatty acid (FA) proportions and FA groups as obtained through gas chromatography (GC) analysis of fatty acids methyl esters (FAME) showed that there was no relevant difference of the extraction procedure on the individual fatty acid proportions (%FAME). None of the main milk FA groups, such as the sum of monounsaturated (MUFA), polyunsaturated (PUFA), odd- and branched-chain (OBCFA) and saturated (SFA) FA showed any significant difference between the Rose-Gottlieb and dichloromethane methods. Total milk fat as gravimetrically determined did not differ between the two extraction procedures, although numerically a slightly lower amount (2.8% less) was extracted with the new procedure (P = 0.919). In general, the new dichloromethane extraction method was found to be much faster, less hazardous to health (shorter exposure time) and more cost efficient (lower cost based on solvent price and amount used); overall, it is much more suitable for gas chromatography analysis of milk fatty acids than the older, more widely used Rose-Gottlieb extraction method. ß 2010 Elsevier Inc. All rights reserved.
Keywords: Milk fat Extraction Dichloromethane Gas chromatography Spectrophotometry Fatty acids Rapid analysis Food analysis Food composition
1. Introduction Research at our laboratory focuses on the development and practical applications of fast gas chromatography (GC) and spectrophotometry (MIR and Raman) methods for milk fatty acid (MFA) analysis. MFA profiles are investigated either directly on raw milk or after extraction of milk fat. Several MFA of interest have concentrations in milk fat of <1.0%, and it is known that they are not accurately analysed (Soyeurt et al., 2006). Thus milk fat extraction is often required for measuring the fatty acid composition of this matrix. In milk fat extraction procedures, a typical preliminary step involves destroying the protein protection of the milk fat globules without changing the oxidation status of the fat. Theodet and Gandemer (1990) compared different methods for extraction of lipids and they recommended the method of Clark et al. (1982) for quantitative and qualitative analysis. However, this method is very time-consuming, and thus not suitable for routine analysis. The Folch and Bligh & Dyer methods are considered the classical and most reliable means for quantitatively extracting lipids from various types of animal tissues and food products. They rely on chloroform–methanol (2/1, v/v) and employ a large sample-to-solvent ratio (in some cases 1/20 respectively) (Bligh and Dyer, 1959; Folch et al., 1957). Folch and Bligh & Dyer are
* Corresponding author. Tel.: +32 9 264 90 01; fax: +32 9 264 90 99. E-mail address: [email protected] (V. Fievez). 0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2010.03.016
lengthy extraction methods. Moreover chloroform has a very low threshold limit in the workplace air, about 1–2 ppm or 5–10 mg/ m3 (International Labor Organization (ILO), 2009). Examples of milk fat extraction methods which have been extensively used and later standardized are those developed by Babcock, Gerber, Mojonnier and Rose-Gottlieb (Vaghela and Kilara, 1995). RoseGottlieb (IDF Standard 1D, 1996) is the standard milk fat extraction method used in our lab and it involves a dual organic solvent system (diethyl ether and petroleum ether) and the addition of a strong mineral alkali (ammonium hydroxide) in combination with heat to dissociate the lipid–protein complexes. All of the latter extraction methods are time-consuming, use specific equipment or, in some cases, harmful solvents; in general they are not suitable for routine applications. Therefore there is a need for a fast specialized alternative method for dairy milk that achieves the extraction of fat without chemical degradation and with the use of a safer and more user-friendly solvent. Dichloromethane is a commonly used solvent for chemical reactions, purification and isolation of intermediates or products. Dichloromethane was previously used in a reference extraction method (ruminant feed and digesta samples; Cieslak et al., 2009), due to similarity with traditional extraction protocols (chloroform and methanol 2:1, v/v; Cequier-Sanchez et al., 2008). Regarding the sample type of interest, dichloromethane was previously employed in the extraction of lyophilized milk samples (50 mg), but after hydrolysis and acidification pre-treatment steps (Czauderna and Kowalczyk, 2001). Here we present a robust,
I. Stefanov et al. / Journal of Food Composition and Analysis 23 (2010) 852–855
fast, economical (lower cost based on solvent price and volume) and more user-friendly (based on exposure time) milk fat extraction technique using dichloromethane and ethanol as solvents (referred to as the dichloromethane extraction procedure), which does not employ hydrolysis or other chemical degradation steps and can cope with the speed ability of modern fast chromatographic techniques and spectroscopic equipment for the simultaneous analysis of hundreds of samples per day. This new extraction method was compared to our standard lab procedure (referred to as the Rose-Gottlieb extraction procedure). 2. Materials and methods 2.1. Samples Bulk raw milk samples were taken from a milk tank of the Ghent University experimental dairy farm for a period of 5 weeks between 15 April 2008 and 16 September 2008. The samples were used as a part of a larger experiment, which in addition to extraction method comparison, involved the investigation of freezing at different temperatures (20 8C and 80 8C) and thawing method (room temperature vs. microwave) on the gas chromatographic fatty acid results and spectroscopic profiles. Twenty milk samples were collected each week, of which four were used for immediate fresh sample analysis, using both the reference Rose-Gottlieb (n = 2) and the new dichloromethane (n = 2) extraction procedures. The remaining 16 sample aliquots (200 mL), 8 per extraction procedure, which were performed after thawing, were brought into plastic pots with black septum sealed caps for frozen storage, half of them at 20 8C and the other half at 80 8C for the next 10 weeks. The samples were then thawed using the microwave or at room temperature and further extracted using both extraction procedures and finally analysed by GC. A total of 100 samples (50 extracted with the dichloromethane method and 50 extracted with the Rose-Gottlieb procedure) were used for comparison. 2.2. Rose-Gottlieb method The International Dairy Federation (IDF) method 1D: 1996 for lipid extraction (Rose-Gottlieb method) consisted of a fat hydrolysis in the presence of ammonia (25%), and lipids extraction with diethyl ether and petroleum ether (ISO 14156:2001). The exact application of this method in our lab is as described by Vlaeminck et al. (2005). 2.3. Dichloromethane method A quantity of fresh raw cow milk, 10 g (0.0001 g), was mixed with 16 mL of previously prepared dichloromethane–ethanol (DM–E) solution (2:1 ratio, v/v) in a 40 mL centrifuge grade glass test tube. The mixture was shaken manually with a vortex (Digital Vortex Mixer, VWR International LLC, Sacramento, CA, USA) for 90 s and then centrifuged for a minimum of 8 min (2500 g at 4 8C, Beckman J2-21, Fullerton, CA, USA). The upper aqueous phase was carefully removed with a pipette and additionally 10 mL of the DM-E solution was added to the test tube. The mixture was further vortexed and then centrifuged for a minimum of 6 min (2500 g at 4 8C). A small precipitate (containing milk protein) and an upper organic phase containing the milk fat were apparent. The latter was filtered (597, 240 mm diameter, Schleicher & Schuell, Dassel, Germany) to a suitable round bottom flask (250 mL), dichloromethane was removed by using a rotovap ¨ CHI Labor AG, Flawil, Switzerland) and samples (Bu¨chi R-104, BU were evaporated until dryness.
853
2.4. Gravimetrical assessment The total extracted milk fat from the reference Rose-Gottlieb and the dichloromethane methods was recorded gravimetrically. Of all extractions performed during 5 weeks, 47 gravimetrical determinations of each extraction procedure were recorded for this gravimetrical assessment. 2.5. Gas chromatography analysis After extraction, the lipids were resolved in 20.0 mL of diethyl ether:petroleum ether (1:1, v/v) and all samples were methylated. Briefly, the solvents in the lipid solution (25 vol) were evaporated under N2 stream (room temperature), further the lipids were dissolved in hexane (50 vol) and the fatty acids in the extracted lipids were methylated with 1.0 M NaOCH3 in methanol (1 vol), in the presence of methyl acetate (1 vol), for 10 min at room temperature (Christie, 1982). The reaction was terminated with 0.3 M C2O2(OH)2H2O in diethyl ether (1.5 vol) and CaCl2 (1.0 g) drying agent was added. The mixture was centrifuged at 3150 g for 5 min (Hermle Z383, Labnet Inc., Woodbridge, NJ, USA) and 0.5 mL of the aliquot was added to hexane (1:1, v/v) prior to gas liquid chromatography (GC) analysis. The fatty acid methyl ester (FAME) content was analysed by GC according to Vlaeminck et al. (2005), but without the use of internal standards, as the primary aim was to assess and compare the milk FA profile. Fatty acid peaks were identified based on their retention times determined by analysis of the SUPELCO external standard mix (Supelco Inc., Park Bellefonte, PA, USA) to which pure branched-chain fatty acids (iso C13:0, ante C13:0, iso C14:0, iso C15:0, ante C15:0, iso C16:0, iso C17:0 and ante C17:0) and conjugated fatty acids (cis-9, trans-11 18:2 and trans-10, cis-12 18:2) were added (Larodan Fine Chem., Malmo, Sweden). Individual trans isomers and the trans-11, cis-15 18:2 isomer were identified by cross-referencing with previously published isomeric profiles using trans-11 18:1 and cis-9, cis-12 18:2 as a landmark isomer (Shingfield et al., 2006). Identification of fatty acids was further confirmed by comparing the retention index with previously analysed samples at our laboratory both by GC–MS and GC GC. The FA profile, including 49 identified FA, were expressed in percentage of total FAME. Short-chain fatty acids were corrected for their respective theoretical relative response factors (Ackman and Sipos, 1964; Wolff et al., 1995). 2.6. Statistical analysis The FA proportions, FA groups and total FA area as obtained through GC analysis of FAME and were compared using linear mixed models in the Statistical Software Package for Windows 15.0.1 (SPSS Inc.; Chicago, IL, 1994). Fixed factors included Storage (fresh vs. frozen), Extraction (dichloromethane vs. Rose-Gottlieb) and Thawing methods (microwave vs. room temperature) nested within frozen samples. Because samples were obtained for a period of 5 weeks, week effect was included as a random factor using variance components as covariance type. Further, the mixed model was designed aiming at including all interactions of possible interest, including all two-way interactions of the fixed factors as well as the Storage or Extraction effect with Week. The statistical model is described by the following equation:
Y i jkl ¼ Si¼1;2 þ E j¼1;2 þ T k¼1;2 ðSiÞ þ W l¼1;5 þ Si E j þ E j T k ðSi Þ þ E j W l þ Si W l þ ei jkl where Si, Ej and Tk(Si) denote respectively Storage, Extraction and Thawing nested within Storage as fixed factors and Wl denotes Week as a random factor.
I. Stefanov et al. / Journal of Food Composition and Analysis 23 (2010) 852–855
854
Table 1 Effect of extraction method on estimated marginal means of individual saturated milk fatty acid proportions (g/1000 g of FAME, n = 50). FA
Extraction methods
p-Value
Rose-Gottlieb
C4:0 C6:0 C8:0 C10:0 C11:0a C12:0 C13:0a C14:0 C15:0a C16:0 C17:0a C18:0 C20:0 C22:0 iso C13:0a ante C13:0a iso C14:0a iso C15:0a ante C15:0a iso C16:0a iso C17:0a ante C17:0a OBCFA SFA a
Dichloromethane
Mean
SD
Mean
SD
35.90 22.14 13.51 30.24 2.841 34.74 0.9831 105.6 10.16 260.6 4.795 108.1 1.535 0.7291
1.618 1.129 1.081 3.738 0.275 4.537 0.1531 4.743 3.321 29.29 0.506 7.986 0.067 0.0778
35.85 22.11 13.47 30.27 2.836 34.79 0.9872 105.7 10.19 260.8 4.807 108.3 1.528 0.7494
1.773 1.105 1.075 3.700 0.273 4.527 0.1576 4.764 3.323 29.12 0.520 8.007 0.082 0.0856
0.4185 0.8764 0.9164 2.592 4.470 2.152 5.989 4.124
0.0667 0.1265 0.0732 0.2361 0.3131 0.2248 0.7032 0.3577
0.4166 0.8672 0.9191 2.599 4.466 2.156 5.996 4.123
0.0727 0.1341 0.0612 0.2358 0.3106 0.2361 0.7578 0.3408
40.32 653.3
3.024 38.57
40.37 653.9
3.010 38.36
Range Min
Max
0.701 0.464 0.048 0.088 0.816 0.117 0.410 0.207 0.006 0.418 0.696 0.030 0.603 0.417
32.95 20.50 12.25 26.51 2.521 29.33 0.7531 98.58 7.870 235.8 4.218 95.25 1.349 0.5434
39.39 24.42 15.73 37.88 3.409 43.74 1.301 114.4 17.02 318.3 5.909 122.1 1.736 0.9347
0.530 0.286 0.708 0.140 0.462 0.490 0.700 0.801
0.3162 0.6447 0.7621 2.115 4.033 1.680 4.517 3.002
0.5367 1.155 1.048 2.884 5.002 2.456 6.856 4.592
0.422 0.227
36.95 618.6
46.65 734.0
Individual FA reported are included in the sum of odd- and branched-chain fatty acids (OBCFA).
3. Results and discussion Rose-Gottlieb was used as the reference method to study the potential of the new dichloromethane procedure for the quantitative and qualitative extraction of MFA. CH2Cl2 was the solvent of choice, because it has numerous advantages over other solvents used for fat extraction, like poor miscibility with water, nonflammability, high auto-ignition temperature (556 8C) and ease of removal from products by evaporation with rotavapor and/or N2 flow. It is one of the less-harmful chlorocarbons and has a threshold limit of 50 ppm or 177 mg/m3 in the workplace (ILO, 2009). According to the European Chlorinated Solvent Association (ECSA, 1997) CH2Cl2 has been widely used in the pharmaceutical industry, paint stripping industry, manufacture of aerosols and adhesives and in other processes including metal degreasing, foam blowing, chemical processing (polyurethanes, polycarbonates) and as secondary refrigerant medium. In pharmaceuticals, CH2Cl2 has been used as solvent for chemical reactions, purification and isolation of intermediates or products. Recently Cequier-Sanchez et al. (2008) successfully applied CH2Cl2 in the lipid extraction of Echium virescens seeds and Echium acanthocarpum hairy roots. Regarding the fatty acid profile, 49 fatty acids were identified and proportionally quantified. The results of the statistical model (data for freezing and thawing effects not presented), showed that, generally (except for 5 FA), there was no effect of the extraction procedure on the individual FA proportions (%FAME). Dichloromethane method resulted in higher proportions of C15:0 (P < 0.01) and C18:0 (P < 0.05) and lower proportions of C8:0 (P < 0.05), C18:1 cis-12 (P < 0.05), C18:3 n–6 (P < 0.01) in the milk fat. The new method tended to increase C10:0 (P < 0.1) and lower C18:1 trans-6– 8 (P < 0.1). However, none of these differences are biologically meaningful (Tables 1 and 2). None of the main monounsaturated (MUFA), polyunsaturated (PUFA), odd- and branched-chain (OBCFA) and saturated (SFA) milk FA groups differed (Tables 1 and 2). Moreover, the total FA area as obtained by GC analysis did not differ; however, the total extracted milk fat from the reference RoseGottlieb and the new dichloromethane method as recorded
gravimetrically showed Rose-Gottlieb to extract slightly larger amounts, although the 2.8% difference between the means was not statistically significant (P = 0.919, Table 2). In Rose-Gottlieb method, milk samples are treated with ammonium hydroxide, which helps in denaturing the protein and disrupting the cell walls, thus allowing greater exposure of the fat to the solvents. Although milk samples in the new extraction method were not treated with an alkali solution, membrane phospholipids are assumed to be extracted through disruption of the biological membranes by the solvent effect of dichloromethane (Wackett, 1996). The slight discrepancy between the gravimetrical determination and the chromatographically determined surfaces might indicate the presence of other solvent soluble compounds with non-fatty acid structure. These compounds should not be a major problem for GC analysis of FAME, but might interfere in other analytical methods such as infrared (IR) and Raman spectrophotometry. The results of the linear mixed statistical model showed no significant two-way interactions between the storage and extraction, and thawing and extraction fixed effects, thus allowing implementation of the new procedure to extract milk fat both from fresh and frozen milk samples. The average analysis time for a batch of 20 raw milk samples using the dichloromethane method is approximately 30 min, which could be further shortened by handling more samples simultaneously. In comparison, the number of extractions, which our laboratory technicians perform using the reference procedure is at maximum 24 per day (6 h). CH2Cl2 exposure time in the new method is 6 times less compared to solvents in Rose-Gottlieb. CH2Cl2 solvent is considered not as toxic as petroleum ether and it has a threshold limit 20–25 times higher (ILO, 2009) than that of chloroform, which is used in other fat extraction procedures such as Folch and Bligh & Dyer. 4. Conclusion Based on the similarity of the proportions of the individual FA as well as the main FA groups, and taking into account the amount of solvents used, the speed and safety characteristics of the new
I. Stefanov et al. / Journal of Food Composition and Analysis 23 (2010) 852–855
855
Table 2 Effect of extraction method on estimated marginal means of individual mono- and polyunsaturated milk fatty acid proportions (g/1000 g of FAME, n = 50) as well as total gravimetrically recorded milk fat. FA
Extraction methods
p-Value
Rose-Gottlieb
Dichloromethane
Range Min
Max
1.753 10.48 17.86 1.171 2.997 3.451 2.864 4.405 20.93 5.047 237.5 12.12 3.569 1.516 6.055 1.833 1.293 322.9
Mean
SD
Mean
SD
0.6968 9.871 15.25 0.8013 2.602 2.825 2.389 3.542 15.07 4.063 215.1 9.990 3.103 1.007 5.051 1.335 1.135 293.8
0.4956 0.4304 1.304 0.2300 0.1841 0.5356 0.4503 0.7050 4.850 0.5651 26.61 1.577 0.4550 0.2654 0.8443 0.2696 0.0849 34.45
0.7006 9.861 15.29 0.8027 2.605 2.784 2.373 3.538 15.00 4.135 215.2 9.914 2.993 1.015 5.027 1.315 1.146 293.5
0.5014 0.4385 1.320 0.2271 0.1650 0.5226 0.4489 0.7145 4.814 0.6827 26.41 1.708 0.5209 0.2564 0.8261 0.2689 0.0840 34.35
0.213 0.614 0.134 0.808 0.736 0.069 0.176 0.748 0.250 0.433 0.717 0.416 0.048 0.805 0.383 0.781 0.275 0.536
0.3344 9.074 13.66 0.4296 2.294 1.661 1.379 2.076 6.897 2.119 160.3 5.519 0.9949 0.3852 3.563 0.7791 0.9791 221.1
C18:2 trans C18:2 t11,c15 C18:3 n–6 C18:3 n–3 CLA c9,t11 C20:3 n–6 C20:4 n–6 C20:5 n–3 C22:4 n–6 C22:5 n–3 PUFA
2.815 1.749 0.3733 5.519 7.908 0.9873 1.347 0.6349 1.025 0.8978 27.21
0.5871 0.6809 0.0929 0.8919 2.443 0.1317 0.1712 0.2271 0.1207 0.1090 4.790
2.809 1.747 0.3044 5.522 7.895 0.9974 1.351 0.5670 1.216 0.9211 27.25
0.5911 0.6843 0.0731 0.8800 2.445 0.1203 0.1763 0.1053 0.1653 0.1357 5.010
0.975 0.921 <0.001 0.450 0.801 0.207 0.799 0.107 0.561 0.235 0.989
1.778 0.5450 0.1751 3.907 4.114 0.8075 1.106 0.3139 0.4126 0.4076 19.41
3.753 2.504 0.6048 6.625 11.62 1.413 1.798 1.906 5.740 1.153 34.12
Total fat (%)a
4.014
0.8010
3.900
0.9750
0.919
2.321
6.519
C10:1 C14:1 cis-9 C16:1 cis-9 C16:1 trans-9 C17:1 cis-9 C18:1 trans-6-8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 trans-12 C18:1 cis-9 C18:1 cis-11 C18:1 cis-12 C18:1 cis-13 C18:1 c14,t15 C18:1 cis-15 C20:1 MUFA
a
n = 47 lower number due to omitted and/or not recorded values.
extraction procedure compared to the reference Rose-Gottlieb method, we can conclude that the extraction procedure based on the dichloromethane–ethanol solvent system is a significant achievement and a great alternative for milk fat extraction prior to GC analysis of MFA. Acknowledgements The Ph.D. research of Ivan Stefanov is supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), IWT-PhD-project: 60704. Bruno Vlaeminck is a postdoctoral fellow of the Fund for Scientific Research-Flanders (Belgium). References Ackman, R.G., Sipos, J.C., 1964. Application of specific response factors in the gas chromatographic analysis of methyl esters of fatty acids with flame ionization detectors. Journal of American Oil Chemists Society 41, 377–378. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917. Clark, R.M., Ferris, A.M., Fey, M., Brown, P.B., Hundrieser, K.E., Jensen, R.G., 1982. Changes in the lipids of human milk from 2 to 16 weeks postpartum. Journal of Pediatric Gastroenterology and Nutrition 1, 311–315. Cequier-Sanchez, E., Rodriguez, C., Ravelo, A., Zarate, R., 2008. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. Journal of Agricultural and Food Chemistry 56 (12), 4297–4303. Christie, W.W., 1982. A simple procedure for rapid transmethylation of glycerolipids and cholesteryl esters. Journal of Lipid Research 23, 1072–1075. Cieslak, A., Machmu¨ller, A., Szumacher-Strabel, M., Scheeder, M.R.L., 2009. A comparison of two extraction methods used to quantify the C18 fatty acids in feed and digesta of ruminants. Journal of Animal Feed Science 18 (2), 362–367. Czauderna, M., Kowalczyk, J., 2001. Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection. Journal of Chromatography B 760, 165–178.
European Chlorinated Solvent Association (ECSA), 1997. Risk Assessment Division Home Page: http://www.eurochlor.org/ (accessed 20.02.09). Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological. Chemistry 226, 497–509. IDF-FIL, 1996. International Standard 1D. Determination of Fat Content-Gravimetric Method (Rose-Gottlieb Reference Method) International Dairy Federation— Federation Internationale de Laiterie, Brussels, Belgium. International Labor Organization (ILO), 2009). Belgium, Ministry of Employment and Work, Annex 1, Arreˆte´ royal du 11 mars 2002 relatif a` la protection de la sante´ et de la se´curite´ des travailleurs contre les risques lie´s a` des agents chimiques sur le lieu de travail. Occupational Exposure Limits Home Page: http://www.ilo.org/ (accessed 18.02.09). ISO, 2001. Milk and Milk Products—Extraction Methods for Lipids and Liposoluble Compounds. ISO 14156:2001, ISO Publications, International Organization for Standardization, Geneva, Switzerland. Shingfield, K.J., Reynolds, C.K., Herva´s, G., Griinari, J.M., Grandison, A., Beever, D.E., 2006. Examination of the persistency of milk fatty acid composition responses to fish oil and sunflower oil in the diet of dairy cows. Journal of Dairy Science 89, 714–732. Soyeurt, H., Dardenne, P., Dehareng, F., Lognay, G., Veselko, D., Marlier, M., Bertozzi, C., 2006. Estimating fatty acid content in cow milk using mid-infrared spectrometry. Journal of Dairy Science 89, 3690–3695. Theodet, C., Gandemer, G., 1990. Comparaison de cinq mehthodes pour extraire les lipides du lactoseh rum et de ses dehrivehs. AIT 71, 41–54. Vaghela, M.N., Kilara, A., 1995. A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction. Journal of American Oil Chemists Society 72, 1117– 1121. Vlaeminck, B., Dufour, C., van Vuuren, A.M., Cabrita, A.M.R., Dewhurst, R.J., Demeyer, D., Fievez, V., 2005. Potential of odd and branched chain fatty acids as microbial markers: evaluation in rumen contents and milk. Journal of Dairy Science 88, 1031–1041. Wackett, L.P., 1996. Biodegradation of chlorinated and aliphatic compounds. In: Crawford, D.L., Crawford, R.L. (Eds.), Bioremediation: Principles and Applications. 1st ed. Cambridge University Press, Cambridge, UK, pp. 305– 306. Wolff, R.L., Bayard, C.C., Fabien, R.J., 1995. Evaluation of sequential methods for the determination of butterfat fatty acid composition with emphasis on trans-18:1 acids. Application to the study of seasonal variations in French butters. Journal of American Oil Chemists Society 72, 1471–1483.
Contents lists available at ScienceDirect
Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Short Communication
A novel procedure for routine milk fat extraction based on dichloromethane I. Stefanov, B. Vlaeminck, V. Fievez * Laboratory for Animal Nutrition and Animal Product Quality (Lanupro), Faculty of Bioscience Engineering, Ghent University, Proefhoevestraat 10, 9090 Melle, Belgium
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 June 2009 Received in revised form 22 February 2010 Accepted 11 March 2010
A novel method for the extraction of milk fat using dichloromethane (CH2Cl2)–ethanol was compared to the Rose-Gottlieb (IDF Standard 1D, 1996) extraction procedure. The new extraction method consisted of a fast (approximately 30 min for 20 samples) and simple procedure involving direct mixing of raw cow milk samples with a dichloromethane–ethanol solution (2/1, v/v). The fatty acid (FA) proportions and FA groups as obtained through gas chromatography (GC) analysis of fatty acids methyl esters (FAME) showed that there was no relevant difference of the extraction procedure on the individual fatty acid proportions (%FAME). None of the main milk FA groups, such as the sum of monounsaturated (MUFA), polyunsaturated (PUFA), odd- and branched-chain (OBCFA) and saturated (SFA) FA showed any significant difference between the Rose-Gottlieb and dichloromethane methods. Total milk fat as gravimetrically determined did not differ between the two extraction procedures, although numerically a slightly lower amount (2.8% less) was extracted with the new procedure (P = 0.919). In general, the new dichloromethane extraction method was found to be much faster, less hazardous to health (shorter exposure time) and more cost efficient (lower cost based on solvent price and amount used); overall, it is much more suitable for gas chromatography analysis of milk fatty acids than the older, more widely used Rose-Gottlieb extraction method. ß 2010 Elsevier Inc. All rights reserved.
Keywords: Milk fat Extraction Dichloromethane Gas chromatography Spectrophotometry Fatty acids Rapid analysis Food analysis Food composition
1. Introduction Research at our laboratory focuses on the development and practical applications of fast gas chromatography (GC) and spectrophotometry (MIR and Raman) methods for milk fatty acid (MFA) analysis. MFA profiles are investigated either directly on raw milk or after extraction of milk fat. Several MFA of interest have concentrations in milk fat of <1.0%, and it is known that they are not accurately analysed (Soyeurt et al., 2006). Thus milk fat extraction is often required for measuring the fatty acid composition of this matrix. In milk fat extraction procedures, a typical preliminary step involves destroying the protein protection of the milk fat globules without changing the oxidation status of the fat. Theodet and Gandemer (1990) compared different methods for extraction of lipids and they recommended the method of Clark et al. (1982) for quantitative and qualitative analysis. However, this method is very time-consuming, and thus not suitable for routine analysis. The Folch and Bligh & Dyer methods are considered the classical and most reliable means for quantitatively extracting lipids from various types of animal tissues and food products. They rely on chloroform–methanol (2/1, v/v) and employ a large sample-to-solvent ratio (in some cases 1/20 respectively) (Bligh and Dyer, 1959; Folch et al., 1957). Folch and Bligh & Dyer are
* Corresponding author. Tel.: +32 9 264 90 01; fax: +32 9 264 90 99. E-mail address: [email protected] (V. Fievez). 0889-1575/$ – see front matter ß 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2010.03.016
lengthy extraction methods. Moreover chloroform has a very low threshold limit in the workplace air, about 1–2 ppm or 5–10 mg/ m3 (International Labor Organization (ILO), 2009). Examples of milk fat extraction methods which have been extensively used and later standardized are those developed by Babcock, Gerber, Mojonnier and Rose-Gottlieb (Vaghela and Kilara, 1995). RoseGottlieb (IDF Standard 1D, 1996) is the standard milk fat extraction method used in our lab and it involves a dual organic solvent system (diethyl ether and petroleum ether) and the addition of a strong mineral alkali (ammonium hydroxide) in combination with heat to dissociate the lipid–protein complexes. All of the latter extraction methods are time-consuming, use specific equipment or, in some cases, harmful solvents; in general they are not suitable for routine applications. Therefore there is a need for a fast specialized alternative method for dairy milk that achieves the extraction of fat without chemical degradation and with the use of a safer and more user-friendly solvent. Dichloromethane is a commonly used solvent for chemical reactions, purification and isolation of intermediates or products. Dichloromethane was previously used in a reference extraction method (ruminant feed and digesta samples; Cieslak et al., 2009), due to similarity with traditional extraction protocols (chloroform and methanol 2:1, v/v; Cequier-Sanchez et al., 2008). Regarding the sample type of interest, dichloromethane was previously employed in the extraction of lyophilized milk samples (50 mg), but after hydrolysis and acidification pre-treatment steps (Czauderna and Kowalczyk, 2001). Here we present a robust,
I. Stefanov et al. / Journal of Food Composition and Analysis 23 (2010) 852–855
fast, economical (lower cost based on solvent price and volume) and more user-friendly (based on exposure time) milk fat extraction technique using dichloromethane and ethanol as solvents (referred to as the dichloromethane extraction procedure), which does not employ hydrolysis or other chemical degradation steps and can cope with the speed ability of modern fast chromatographic techniques and spectroscopic equipment for the simultaneous analysis of hundreds of samples per day. This new extraction method was compared to our standard lab procedure (referred to as the Rose-Gottlieb extraction procedure). 2. Materials and methods 2.1. Samples Bulk raw milk samples were taken from a milk tank of the Ghent University experimental dairy farm for a period of 5 weeks between 15 April 2008 and 16 September 2008. The samples were used as a part of a larger experiment, which in addition to extraction method comparison, involved the investigation of freezing at different temperatures (20 8C and 80 8C) and thawing method (room temperature vs. microwave) on the gas chromatographic fatty acid results and spectroscopic profiles. Twenty milk samples were collected each week, of which four were used for immediate fresh sample analysis, using both the reference Rose-Gottlieb (n = 2) and the new dichloromethane (n = 2) extraction procedures. The remaining 16 sample aliquots (200 mL), 8 per extraction procedure, which were performed after thawing, were brought into plastic pots with black septum sealed caps for frozen storage, half of them at 20 8C and the other half at 80 8C for the next 10 weeks. The samples were then thawed using the microwave or at room temperature and further extracted using both extraction procedures and finally analysed by GC. A total of 100 samples (50 extracted with the dichloromethane method and 50 extracted with the Rose-Gottlieb procedure) were used for comparison. 2.2. Rose-Gottlieb method The International Dairy Federation (IDF) method 1D: 1996 for lipid extraction (Rose-Gottlieb method) consisted of a fat hydrolysis in the presence of ammonia (25%), and lipids extraction with diethyl ether and petroleum ether (ISO 14156:2001). The exact application of this method in our lab is as described by Vlaeminck et al. (2005). 2.3. Dichloromethane method A quantity of fresh raw cow milk, 10 g (0.0001 g), was mixed with 16 mL of previously prepared dichloromethane–ethanol (DM–E) solution (2:1 ratio, v/v) in a 40 mL centrifuge grade glass test tube. The mixture was shaken manually with a vortex (Digital Vortex Mixer, VWR International LLC, Sacramento, CA, USA) for 90 s and then centrifuged for a minimum of 8 min (2500 g at 4 8C, Beckman J2-21, Fullerton, CA, USA). The upper aqueous phase was carefully removed with a pipette and additionally 10 mL of the DM-E solution was added to the test tube. The mixture was further vortexed and then centrifuged for a minimum of 6 min (2500 g at 4 8C). A small precipitate (containing milk protein) and an upper organic phase containing the milk fat were apparent. The latter was filtered (597, 240 mm diameter, Schleicher & Schuell, Dassel, Germany) to a suitable round bottom flask (250 mL), dichloromethane was removed by using a rotovap ¨ CHI Labor AG, Flawil, Switzerland) and samples (Bu¨chi R-104, BU were evaporated until dryness.
853
2.4. Gravimetrical assessment The total extracted milk fat from the reference Rose-Gottlieb and the dichloromethane methods was recorded gravimetrically. Of all extractions performed during 5 weeks, 47 gravimetrical determinations of each extraction procedure were recorded for this gravimetrical assessment. 2.5. Gas chromatography analysis After extraction, the lipids were resolved in 20.0 mL of diethyl ether:petroleum ether (1:1, v/v) and all samples were methylated. Briefly, the solvents in the lipid solution (25 vol) were evaporated under N2 stream (room temperature), further the lipids were dissolved in hexane (50 vol) and the fatty acids in the extracted lipids were methylated with 1.0 M NaOCH3 in methanol (1 vol), in the presence of methyl acetate (1 vol), for 10 min at room temperature (Christie, 1982). The reaction was terminated with 0.3 M C2O2(OH)2H2O in diethyl ether (1.5 vol) and CaCl2 (1.0 g) drying agent was added. The mixture was centrifuged at 3150 g for 5 min (Hermle Z383, Labnet Inc., Woodbridge, NJ, USA) and 0.5 mL of the aliquot was added to hexane (1:1, v/v) prior to gas liquid chromatography (GC) analysis. The fatty acid methyl ester (FAME) content was analysed by GC according to Vlaeminck et al. (2005), but without the use of internal standards, as the primary aim was to assess and compare the milk FA profile. Fatty acid peaks were identified based on their retention times determined by analysis of the SUPELCO external standard mix (Supelco Inc., Park Bellefonte, PA, USA) to which pure branched-chain fatty acids (iso C13:0, ante C13:0, iso C14:0, iso C15:0, ante C15:0, iso C16:0, iso C17:0 and ante C17:0) and conjugated fatty acids (cis-9, trans-11 18:2 and trans-10, cis-12 18:2) were added (Larodan Fine Chem., Malmo, Sweden). Individual trans isomers and the trans-11, cis-15 18:2 isomer were identified by cross-referencing with previously published isomeric profiles using trans-11 18:1 and cis-9, cis-12 18:2 as a landmark isomer (Shingfield et al., 2006). Identification of fatty acids was further confirmed by comparing the retention index with previously analysed samples at our laboratory both by GC–MS and GC GC. The FA profile, including 49 identified FA, were expressed in percentage of total FAME. Short-chain fatty acids were corrected for their respective theoretical relative response factors (Ackman and Sipos, 1964; Wolff et al., 1995). 2.6. Statistical analysis The FA proportions, FA groups and total FA area as obtained through GC analysis of FAME and were compared using linear mixed models in the Statistical Software Package for Windows 15.0.1 (SPSS Inc.; Chicago, IL, 1994). Fixed factors included Storage (fresh vs. frozen), Extraction (dichloromethane vs. Rose-Gottlieb) and Thawing methods (microwave vs. room temperature) nested within frozen samples. Because samples were obtained for a period of 5 weeks, week effect was included as a random factor using variance components as covariance type. Further, the mixed model was designed aiming at including all interactions of possible interest, including all two-way interactions of the fixed factors as well as the Storage or Extraction effect with Week. The statistical model is described by the following equation:
Y i jkl ¼ Si¼1;2 þ E j¼1;2 þ T k¼1;2 ðSiÞ þ W l¼1;5 þ Si E j þ E j T k ðSi Þ þ E j W l þ Si W l þ ei jkl where Si, Ej and Tk(Si) denote respectively Storage, Extraction and Thawing nested within Storage as fixed factors and Wl denotes Week as a random factor.
I. Stefanov et al. / Journal of Food Composition and Analysis 23 (2010) 852–855
854
Table 1 Effect of extraction method on estimated marginal means of individual saturated milk fatty acid proportions (g/1000 g of FAME, n = 50). FA
Extraction methods
p-Value
Rose-Gottlieb
C4:0 C6:0 C8:0 C10:0 C11:0a C12:0 C13:0a C14:0 C15:0a C16:0 C17:0a C18:0 C20:0 C22:0 iso C13:0a ante C13:0a iso C14:0a iso C15:0a ante C15:0a iso C16:0a iso C17:0a ante C17:0a OBCFA SFA a
Dichloromethane
Mean
SD
Mean
SD
35.90 22.14 13.51 30.24 2.841 34.74 0.9831 105.6 10.16 260.6 4.795 108.1 1.535 0.7291
1.618 1.129 1.081 3.738 0.275 4.537 0.1531 4.743 3.321 29.29 0.506 7.986 0.067 0.0778
35.85 22.11 13.47 30.27 2.836 34.79 0.9872 105.7 10.19 260.8 4.807 108.3 1.528 0.7494
1.773 1.105 1.075 3.700 0.273 4.527 0.1576 4.764 3.323 29.12 0.520 8.007 0.082 0.0856
0.4185 0.8764 0.9164 2.592 4.470 2.152 5.989 4.124
0.0667 0.1265 0.0732 0.2361 0.3131 0.2248 0.7032 0.3577
0.4166 0.8672 0.9191 2.599 4.466 2.156 5.996 4.123
0.0727 0.1341 0.0612 0.2358 0.3106 0.2361 0.7578 0.3408
40.32 653.3
3.024 38.57
40.37 653.9
3.010 38.36
Range Min
Max
0.701 0.464 0.048 0.088 0.816 0.117 0.410 0.207 0.006 0.418 0.696 0.030 0.603 0.417
32.95 20.50 12.25 26.51 2.521 29.33 0.7531 98.58 7.870 235.8 4.218 95.25 1.349 0.5434
39.39 24.42 15.73 37.88 3.409 43.74 1.301 114.4 17.02 318.3 5.909 122.1 1.736 0.9347
0.530 0.286 0.708 0.140 0.462 0.490 0.700 0.801
0.3162 0.6447 0.7621 2.115 4.033 1.680 4.517 3.002
0.5367 1.155 1.048 2.884 5.002 2.456 6.856 4.592
0.422 0.227
36.95 618.6
46.65 734.0
Individual FA reported are included in the sum of odd- and branched-chain fatty acids (OBCFA).
3. Results and discussion Rose-Gottlieb was used as the reference method to study the potential of the new dichloromethane procedure for the quantitative and qualitative extraction of MFA. CH2Cl2 was the solvent of choice, because it has numerous advantages over other solvents used for fat extraction, like poor miscibility with water, nonflammability, high auto-ignition temperature (556 8C) and ease of removal from products by evaporation with rotavapor and/or N2 flow. It is one of the less-harmful chlorocarbons and has a threshold limit of 50 ppm or 177 mg/m3 in the workplace (ILO, 2009). According to the European Chlorinated Solvent Association (ECSA, 1997) CH2Cl2 has been widely used in the pharmaceutical industry, paint stripping industry, manufacture of aerosols and adhesives and in other processes including metal degreasing, foam blowing, chemical processing (polyurethanes, polycarbonates) and as secondary refrigerant medium. In pharmaceuticals, CH2Cl2 has been used as solvent for chemical reactions, purification and isolation of intermediates or products. Recently Cequier-Sanchez et al. (2008) successfully applied CH2Cl2 in the lipid extraction of Echium virescens seeds and Echium acanthocarpum hairy roots. Regarding the fatty acid profile, 49 fatty acids were identified and proportionally quantified. The results of the statistical model (data for freezing and thawing effects not presented), showed that, generally (except for 5 FA), there was no effect of the extraction procedure on the individual FA proportions (%FAME). Dichloromethane method resulted in higher proportions of C15:0 (P < 0.01) and C18:0 (P < 0.05) and lower proportions of C8:0 (P < 0.05), C18:1 cis-12 (P < 0.05), C18:3 n–6 (P < 0.01) in the milk fat. The new method tended to increase C10:0 (P < 0.1) and lower C18:1 trans-6– 8 (P < 0.1). However, none of these differences are biologically meaningful (Tables 1 and 2). None of the main monounsaturated (MUFA), polyunsaturated (PUFA), odd- and branched-chain (OBCFA) and saturated (SFA) milk FA groups differed (Tables 1 and 2). Moreover, the total FA area as obtained by GC analysis did not differ; however, the total extracted milk fat from the reference RoseGottlieb and the new dichloromethane method as recorded
gravimetrically showed Rose-Gottlieb to extract slightly larger amounts, although the 2.8% difference between the means was not statistically significant (P = 0.919, Table 2). In Rose-Gottlieb method, milk samples are treated with ammonium hydroxide, which helps in denaturing the protein and disrupting the cell walls, thus allowing greater exposure of the fat to the solvents. Although milk samples in the new extraction method were not treated with an alkali solution, membrane phospholipids are assumed to be extracted through disruption of the biological membranes by the solvent effect of dichloromethane (Wackett, 1996). The slight discrepancy between the gravimetrical determination and the chromatographically determined surfaces might indicate the presence of other solvent soluble compounds with non-fatty acid structure. These compounds should not be a major problem for GC analysis of FAME, but might interfere in other analytical methods such as infrared (IR) and Raman spectrophotometry. The results of the linear mixed statistical model showed no significant two-way interactions between the storage and extraction, and thawing and extraction fixed effects, thus allowing implementation of the new procedure to extract milk fat both from fresh and frozen milk samples. The average analysis time for a batch of 20 raw milk samples using the dichloromethane method is approximately 30 min, which could be further shortened by handling more samples simultaneously. In comparison, the number of extractions, which our laboratory technicians perform using the reference procedure is at maximum 24 per day (6 h). CH2Cl2 exposure time in the new method is 6 times less compared to solvents in Rose-Gottlieb. CH2Cl2 solvent is considered not as toxic as petroleum ether and it has a threshold limit 20–25 times higher (ILO, 2009) than that of chloroform, which is used in other fat extraction procedures such as Folch and Bligh & Dyer. 4. Conclusion Based on the similarity of the proportions of the individual FA as well as the main FA groups, and taking into account the amount of solvents used, the speed and safety characteristics of the new
I. Stefanov et al. / Journal of Food Composition and Analysis 23 (2010) 852–855
855
Table 2 Effect of extraction method on estimated marginal means of individual mono- and polyunsaturated milk fatty acid proportions (g/1000 g of FAME, n = 50) as well as total gravimetrically recorded milk fat. FA
Extraction methods
p-Value
Rose-Gottlieb
Dichloromethane
Range Min
Max
1.753 10.48 17.86 1.171 2.997 3.451 2.864 4.405 20.93 5.047 237.5 12.12 3.569 1.516 6.055 1.833 1.293 322.9
Mean
SD
Mean
SD
0.6968 9.871 15.25 0.8013 2.602 2.825 2.389 3.542 15.07 4.063 215.1 9.990 3.103 1.007 5.051 1.335 1.135 293.8
0.4956 0.4304 1.304 0.2300 0.1841 0.5356 0.4503 0.7050 4.850 0.5651 26.61 1.577 0.4550 0.2654 0.8443 0.2696 0.0849 34.45
0.7006 9.861 15.29 0.8027 2.605 2.784 2.373 3.538 15.00 4.135 215.2 9.914 2.993 1.015 5.027 1.315 1.146 293.5
0.5014 0.4385 1.320 0.2271 0.1650 0.5226 0.4489 0.7145 4.814 0.6827 26.41 1.708 0.5209 0.2564 0.8261 0.2689 0.0840 34.35
0.213 0.614 0.134 0.808 0.736 0.069 0.176 0.748 0.250 0.433 0.717 0.416 0.048 0.805 0.383 0.781 0.275 0.536
0.3344 9.074 13.66 0.4296 2.294 1.661 1.379 2.076 6.897 2.119 160.3 5.519 0.9949 0.3852 3.563 0.7791 0.9791 221.1
C18:2 trans C18:2 t11,c15 C18:3 n–6 C18:3 n–3 CLA c9,t11 C20:3 n–6 C20:4 n–6 C20:5 n–3 C22:4 n–6 C22:5 n–3 PUFA
2.815 1.749 0.3733 5.519 7.908 0.9873 1.347 0.6349 1.025 0.8978 27.21
0.5871 0.6809 0.0929 0.8919 2.443 0.1317 0.1712 0.2271 0.1207 0.1090 4.790
2.809 1.747 0.3044 5.522 7.895 0.9974 1.351 0.5670 1.216 0.9211 27.25
0.5911 0.6843 0.0731 0.8800 2.445 0.1203 0.1763 0.1053 0.1653 0.1357 5.010
0.975 0.921 <0.001 0.450 0.801 0.207 0.799 0.107 0.561 0.235 0.989
1.778 0.5450 0.1751 3.907 4.114 0.8075 1.106 0.3139 0.4126 0.4076 19.41
3.753 2.504 0.6048 6.625 11.62 1.413 1.798 1.906 5.740 1.153 34.12
Total fat (%)a
4.014
0.8010
3.900
0.9750
0.919
2.321
6.519
C10:1 C14:1 cis-9 C16:1 cis-9 C16:1 trans-9 C17:1 cis-9 C18:1 trans-6-8 C18:1 trans-9 C18:1 trans-10 C18:1 trans-11 C18:1 trans-12 C18:1 cis-9 C18:1 cis-11 C18:1 cis-12 C18:1 cis-13 C18:1 c14,t15 C18:1 cis-15 C20:1 MUFA
a
n = 47 lower number due to omitted and/or not recorded values.
extraction procedure compared to the reference Rose-Gottlieb method, we can conclude that the extraction procedure based on the dichloromethane–ethanol solvent system is a significant achievement and a great alternative for milk fat extraction prior to GC analysis of MFA. Acknowledgements The Ph.D. research of Ivan Stefanov is supported by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), IWT-PhD-project: 60704. Bruno Vlaeminck is a postdoctoral fellow of the Fund for Scientific Research-Flanders (Belgium). References Ackman, R.G., Sipos, J.C., 1964. Application of specific response factors in the gas chromatographic analysis of methyl esters of fatty acids with flame ionization detectors. Journal of American Oil Chemists Society 41, 377–378. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917. Clark, R.M., Ferris, A.M., Fey, M., Brown, P.B., Hundrieser, K.E., Jensen, R.G., 1982. Changes in the lipids of human milk from 2 to 16 weeks postpartum. Journal of Pediatric Gastroenterology and Nutrition 1, 311–315. Cequier-Sanchez, E., Rodriguez, C., Ravelo, A., Zarate, R., 2008. Dichloromethane as a solvent for lipid extraction and assessment of lipid classes and fatty acids from samples of different natures. Journal of Agricultural and Food Chemistry 56 (12), 4297–4303. Christie, W.W., 1982. A simple procedure for rapid transmethylation of glycerolipids and cholesteryl esters. Journal of Lipid Research 23, 1072–1075. Cieslak, A., Machmu¨ller, A., Szumacher-Strabel, M., Scheeder, M.R.L., 2009. A comparison of two extraction methods used to quantify the C18 fatty acids in feed and digesta of ruminants. Journal of Animal Feed Science 18 (2), 362–367. Czauderna, M., Kowalczyk, J., 2001. Separation of some mono-, di- and tri-unsaturated fatty acids containing 18 carbon atoms by high-performance liquid chromatography and photodiode array detection. Journal of Chromatography B 760, 165–178.
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