- Email: [email protected]
Development of one-step sample preparation methods for fatty acid profiling of milk fat
Food Chemistry 315 (2020) 126281
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Development of one-step sample preparation methods for fatty acid profiling of milk fat
T
⁎
Zhiqian Liua, , Jianghui Wanga, Cheng Lia,b, Simone Rochforta,b a b
Agriculture Victoria Research, AgriBio, 5 Ring Road, Bundoora, Victoria 3083, Australia School of Applied Systems Biology, La Trobe University, Bundoora, Victoria 3083, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: Milk Fat Fatty acids Methylation Transesterification
Determination of the fatty acid (FA) profile of milk fat generally involves total lipid extraction from liquid milk, transesterification and GC analysis. The lipid extraction step is time consuming and often employs toxic solvents such as chloroform. Two alternative methods are presented here that skip the lipid extraction step and allow the determination of FA composition via direct transesterification of dried milk and liquid milk respectively. We have shown that dried milk can be used directly in alkaline-catalysed methylation, whereas direct transesterification of both dried milk and fresh milk is feasible with acidic methanol. Both methods generate similar results as compared to the classical two-step method (i.e. lipid extraction and FA methylation) when optimised methylation parameters (temperature, time, milk and reagent volume) are followed. By omitting the lipid extraction step, these simplified one-step methods offer a much higher throughput and a reduced cost in FA composition analysis of milk samples.
1. Introduction Lipids are one of the major components of milk solids. Fatty acid (FA) composition of milk fat is closely related to the nutritive quality of milk and physicochemical properties of dairy products (Chen et al., 2004). FA composition of milk fat is influenced by various factors such as animal genotypes, diets and physiological status of cows (Shingfield et al., 2005; Liu et al., 2017). As a result, analysis of FA composition is of great importance in lipid-related research and for dairy-based industries (Cossignani, Pollini, & Blasi, 2019). Generally, FAs are quantified as their methyl esters (FAMEs) by gas chromatography (GC) following a two-step sample preparation procedure, i.e. lipid extraction and FA methylation. For FAME preparation, both acid- and alkaline-catalysed transesterification have been widely applied so far for milk samples and review papers on this topic are also available (Jensen, 2002; Amores & Virt, 2019). The acidic protocol can convert all forms of FA into FAMEs, but the methylation process is generally slower, and could modify the profile of conjugated linoleic acids (CLA) (Kramer et al., 1997; Lee & Tweed, 2008; Liu, Ezernieks, Rochfort, & Cocks, 2018); whereas the base-catalysed reaction is fast but free FA and FA from sphingolipids cannot be methylated (Christie, 1982; Liu et al., 2018). Due to the much lower content of free FA and sphingolipids as compared to triglycerides in milk, the base-catalysed methylation is recommended for milk FA profiling (Liu et al., 2018), ⁎
which is also the official reference method for FAME preparation from milk fat (ISOIDF, 2002). Lipid extraction prior to FAME preparation is another bottleneck in FA profiling of milk and other biological samples. The classical methods for lipid extraction often involve toxic solvents such as chloroform (Folch, Lees, & Stanley, 1957; Bligh & Dyer, 1959) and are rather laborious and time-consuming. To avoid the solvent extraction step, the idea of in situ transesterification was proposed at least 30 years ago and a review paper summarising the earlier attempts in this field was also published (Carrapiso & Garcia, 2000). Since then, numerous one-step and two-step protocols have been tested for in situ transesterification of freeze-dried and fresh biological and foodstuff samples (for example, Carrapiso, Timon, Petron, Tejeda, & Garcia, 2000; O’Fallon, Busboom, Nelson & Gaskins, 2007; Abdulkadir & Tsuchiya, 2008; Araujo, Nguyen, Froyland, Wang, & Kang, 2008; Griffiths, van Hille, & Harrison, 2010; Lee, Tweed, Kim, & Scollan, 2012; Castro-Gomez, Fontecha, & Rodriguez-Alcala, 2014; Dong et al., 2015; Hidalgo, Ciudad, Schober, Mittelbach, & Navia, 2015; De Paola, Montevecchi, Masino, Antonelli, & Lo Fiego, 2017; Agnew et al., 2019). In the case of milk samples, alternative protocols for fat isolation have been proposed, such as twostep centrifugation (Feng, Lock, & Garnsworthy, 2004; Luna, Juarez, and de la Fuente, 2005) and one-step centrifugation (Liu, Moate, & Rochfort, 2019). The two-step centrifugation method appears to be reliable for fat isolation but is not easy to perform, whereas the one-step
Corresponding author. E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.foodchem.2020.126281 Received 15 August 2019; Received in revised form 10 January 2020; Accepted 21 January 2020 Available online 23 January 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
2.4. FAME preparation
centrifugation method is useful only for relative quantification of FA in raw milk samples. Compared to animal tissue samples, much less reports can be found on in situ transesterification of milk and milk products. Park and Goins (1994) reported a 2-step protocol (i.e. hydrolysis of samples by KOH in methanol, followed by acidic methylation), which appeared to be reliable for relative quantification of FA in infant formulas and human milk. There have been other attempts to achieve lipid extraction and FA methylation of milk fat with one single reagent acetyl chloride/methanol (Lepage & Roy, 1984), H2SO4/methanol (Martinez, Miranda, Franco, Cepeda, & Rodriguez, 2012 and HCl/methanol (CruzHernandez, Goeuriot, Giuffrida, Thakkar, & Destaillats, 2013). However, these protocols either employ harsh transmethylation conditions (100 °C, for example), which would degrade CLA or involve multiple steps including an over-night incubation, hence they have not found widespread acceptance. Consequently, until now none of the in-situ transesterification methods published so far has been thoroughly validated with milk samples and lipid extraction by organic solvents remains an integral step in most, if not all, studies requiring FA content determination of milk samples. Alternative or novel in situ transesterification methods that are simple (one step preferably) and most importantly reliable for milk FAME preparation are still needed. The aim of this study was to evaluate the feasibility of two novel approaches of direct transesterification of milk fat from liquid milk and dried milk by acid- and base-catalysed methylation.
Both alkaline- and acid-catalysed methylation was applied to the three types of pre-treated samples. For the alkaline-catalysed reaction, 2.5 mL of derivatization reagent (0.2 M KOH in methanol) was added to each vial that contained solvent-extracted milk fat, dried milk or liquid milk and the vial was tightly sealed with a Teflon lined cap; the methylation reaction was conducted by incubation at 50 °C for 30 min with occasional shaking. After cooling to room temperature, one mL of HCl (1 M) was added to each vial and FAMEs formed were extracted into 1 mL of hexane containing internal standard (nonadecane, 100 mg L-1) and analysed directly by GC–MS. In the case of acid-catalysed methylation, the derivatization reagent was 2.8 mL of 2% H2SO4 (in methanol) and the reaction was allowed for 2–5 h at 60 °C. Upon completion of the transesterification process, one mL of water was added before extraction of FAMEs into hexane. 2.5. GC–MS analysis of FAMEs The separation of FAMEs was achieved by a Rt-2560 column (100 m × 0.25 mm ID, 0.20 µm df, Restek) with a constant flow of 1.2 mL min−1 helium as carrier gas and the following oven temperature program: initial temperature of 100 °C and held for 4 min, increased by 6 °C min−1 to 170 °C, and then increased by 3 °C min−1 to 240 °C and held for 11 min. The injection inlet temperature was 240 °C and injection volume was 1 µL with a split ratio of 20:1. The detection was by an Agilent 7000 GC/MS Triple Quadrupole with the following settings: scanning mass range of 40–500 amu, transfer line temperature of 240 °C, source temperature of 280 °C, quad temperature of 150 °C and a solvent delay of 11.8 min. A FAME standard mix containing 37 FAMEs was used to provide absolute quantification of each FA.
2. Materials and methods 2.1. Milk sample Pasteurised and homogenised commercial milk samples were obtained from the local supermarket (Victoria, Australia); the total fat content of theses milk sample was 3.4% as shown on the label. One raw milk sample was provided by the Department of Economic Development, Jobs, Transport and Resources’ Ellinbank centre in Victoria, Australia. This sample was an aliquot from an afternoon milking of one cow; its total fat concentration was 3.3% as determined by infrared spectroscopy (Moate et al., 2013).
2.6. Statistical analysis of data All FA concentrations were expressed as µg (FA) per mL of milk. For each experiment, the mean values and the standard deviations (SD) of three technical replicates were presented either in figures or in tables. For statistical comparison between different FAME preparation methods, the FA concentration results were subjected to ANOVA (XLSTAT, Microsoft Excel); where significant differences (p < 0.05) were found between treatments by F test, a Tukey’s HSD test was applied for pairwise comparisons.
2.2. Chemicals and reagents Solvents and chemicals used for lipid extraction and FAME preparation were of chromatographic or analytical grade. Hexane, chloroform and sulfuric acid were from Ajax FineChem (Taren Point, NSW, AU); methanol was from Merck (Darmstadt, Germany); potassium hydroxide, nonadecane (used as internal standard in GC analysis) as well as the standard mix of 37 FAMEs were purchased from SigmaAldrich (Castle Hill, NSW, AU).
3. Results and discussion 3.1. Alkaline-catalysed methylation As alkaline-catalysed transesterification is a fast yet reliable way for milk FAME preparation and is used increasingly in dairy-related research (Sun et al., 2016; Yener & van Valenberg, 2019); the milder reaction conditions can also avoid isomerisation of CLA 18:2c9t11 associated with acidic methylation (Murrieta, Hess, & Rule, 2003; Lee and Tweed, 2008). So, the feasibility of directly using dried and liquid milk for FAME preparation was assessed in the first instance by alkali-based methylation method. Compared to the solvent-extracted fat (Ex-Fat), which contained only lipids, the reaction matrices were much more complex when dried milk (DM) and liquid milk (LM) were used directly for FAME preparation, as all the milk solids were present in DM and all solids and water in LM. While 20 min (at 50 °C) was adequate for methylating solvent-extracted lipids by KOH/methanol reagent (Liu et al., 2018), our preliminary tests found that the optimal methylation time for dried milk samples was 30 min using the same reagent and reaction temperature. After 30 min incubation with the methylation reagent, the reaction mixture became totally clear with the Ex-Fat sample, whereas
2.3. Sample pre-treatment Three methods of sample pre-treatment prior to transesterification reaction were compared in this study using the homogenised commercial milk samples. Method 1 (fat extraction method, Ex-fat): total lipids of the milk sample (50, 100 or 200 µL) were extracted using chloroform/methanol (2:1) by the Folch method (Folch et al., 1957); the organic phase was transferred to a 5-mL glass vial and evaporated to dryness under a stream of N2 prior to methylation. Method 2 (dried milk method, DM): fresh milk (50, 100 or 200 µL) was accurately measured into a 5-mL glass vial and then dried in a heating block (40 °C) under a stream of N2 for approximately 10, 15 and 25 min, respectively, prior to methylation. Method 3 (liquid milk method, LM): fresh milk (50, 100 or 200 µL) was subjected directly to methylation in a 5-mL glass vial without any pre-treatment. 2
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 1. GC–MS profile (total ion chromatogram) of milk FAMES with different sample pre-treatment methods prior to alkaline-catalysed methylation. A: highabundance FA; B: lower-abundance FA. IS: internal standard. Ex-Fat: milk lipids extracted by the Folch method prior to methylation; DM: methylation performed with dried milk without lipid extraction; LM: methylation performed with liquid milk without lipid extraction or drying. For all the three methods, profiles shown are from 100 µL of milk. Table 1 Major FA yield (mean ± SD in µg/mL) with different sample pre-treatment methods prior to alkaline-catalysed methylation. FA
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3
50 µL
100 µL
200 µL
Ex-Fat
DM
LM
Ex-Fat
DM
LM
Ex-Fat
DM
LM
2014 ± 64a 740 ± 15a 387 ± 4a 899 ± 11a 1104 ± 22a 3361 ± 45a 364 ± 12a 371 ± 14a 8112 ± 196a 504 ± 14a 366 ± 11a 2684 ± 43a 5112 ± 78a 494 ± 10a 211 ± 15a
2081 ± 96a 748 ± 16a 386 ± 9a 902 ± 12a 1113 ± 16a 3372 ± 31a 357 ± 7a 367 ± 3a 8231 ± 66a 497 ± 9a 361 ± 3a 2694 ± 31a 5206 ± 20a 487 ± 11a 213 ± 5a
1909 ± 35a 694 ± 5b 349 ± 6b 782 ± 6b 930 ± 15b 2907 ± 25b 300 ± 4b 304 ± 4b 7068 ± 67b 428 ± 13b 297 ± 4b 2206 ± 11b 4427 ± 33b 379 ± 8b 154 ± 4b
2142 ± 39a 753 ± 10a 412 ± 4a 987 ± 15a 1219 ± 33a 3400 ± 41a 373 ± 5a 391 ± 8a 7727 ± 131a 516 ± 14a 358 ± 11a 2813 ± 48a 5182 ± 44a 489 ± 11a 207 ± 19a
2080 ± 37a 738 ± 14a 399 ± 10a 957 ± 14a 1166 ± 27a 3301 ± 53a 362 ± 8a 376 ± 7a 7627 ± 97a 506 ± 16a 350 ± 10a 2726 ± 34a 5080 ± 63a 485 ± 6a 203 ± 8a
1719 ± 36b 619 ± 6b 327 ± 5b 751 ± 11b 902 ± 13b 2571 ± 23b 275 ± 4b 283 ± 5b 5916 ± 31b 380 ± 3b 261 ± 4b 2046 ± 20b 3917 ± 27b 355 ± 8b 152 ± 7b
2126 ± 48a 752 ± 11a 423 ± 7a 1003 ± 14a 1208 ± 16a 3460 ± 55a 375 ± 4a 391 ± 6a 7605 ± 82a 538 ± 16a 371 ± 6a 2771 ± 30a 5191 ± 75a 517 ± 9a 227 ± 4a
2088 ± 19a 740 ± 5a 422 ± 6a 990 ± 7a 1184 ± 10a 3371 ± 27a 365 ± 8a 382 ± 11a 7461 ± 58a 516 ± 10a 360 ± 8a 2705 ± 32a 5057 ± 57a 500 ± 8a 221 ± 5a
1468 ± 34b 509 ± 12b 272 ± 7b 616 ± 7b 722 ± 7b 2200 ± 26b 222 ± 3b 222 ± 4b 4966 ± 45b 311 ± 12b 217 ± 6b 1623 ± 19b 3161 ± 36b 297 ± 6b 132 ± 9b
*Within each sample size (50, 100 or 200 µL) and each FA, mean values followed by different letters are significantly different (p < 0.05). 3
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 2. Comparison of major FA yield with different methylation times. A: 50 µL liquid milk; B: 100 µL liquid milk. All methylation reactions were performed at 60 °C with 2.8 mL of reagent (2% H2SO4 in methanol). Each column represents the mean value of three technical replicates and columns with different letters are significantly different (p < 0.05). Error bars are standard deviations.
when the drying step was conducted at a mild temperature (i.e. 40 °C) and under N2. In addition, the DM method showed excellent reproducibility across replicates for all the three sample volumes, with RSD below 3% for the majority of the FA measurements; the reliability of the method was also confirmed by separate experiments. All these results suggest that using dried milk samples to prepare FAMEs without the lipid extraction step is a feasible approach. By contrast, the LM samples gave significantly reduced yield for virtually all major FA (except for C4:0 with 50 µL milk) as compared to the control method; this yield reduction in FA increased with increasing sample volume (about 15% reduction for 50 µL, about 25% for 100 µL and about 40% for 200 µL of liquid milk) (Table 1). Indeed, a slight reduction in FA level was still observed when the sample volume decreased to 25 µL (results not shown). This indicates that base-catalysed methylation is sensitive to moisture content in the reaction medium and using fresh milk to prepare FAMEs could lead to significant underestimation of milk FA. Although using liquid milk for base-catalysed methanolysis proved to be unreliable, dried milk generated reliable results as compared to the classical 2-step method. As only 10–25 min is needed for the sample drying step and both the drying and methylation steps can be performed in the same glass vial, this FAME preparation protocol is still much quicker than the classical 2-step method. It is worth mentioning that the sample drying procedure was performed with conical vials in this study, so the actual drying time required may be significantly shortened if round-bottom or flat-bottom vials are used. In addition, compared to freeze-drying samples to remove water, drying milk samples with a heating block requires no special equipment and can be performed in any laboratory. We should also point out that this sample drying approach is easily applicable to milk volume from 25 to 200 µL,
most of the milk solids still clung to the wall of the glass vial for the DM sample. In the case of the LM sample, the reaction mixture remained a cloudy suspension by the end of the methylation procedure. In all cases, upon adding hexane, phase separation was always achieved, and a clear hexane extract obtained. The overall GC–MS profile of FAMEs was found to be very similar across the three sample preparation methods for both high-abundance FA (Fig. 1A) and lower-abundance FA (Fig. 1B) (profiles of 100-µL milk samples are shown as examples). It is worth mentioning that despite the complexity of the matrix, direct methylation of dried or liquid milk did not produce any extra or interfering peaks as compared to the solventextracted “pure” lipids. This is an important finding as GC-FID is widely used in FAME quantification and any co-eluting or interfering peaks could compromise the integration accuracy of FID chromatograms. Liquid milk contains over 85% of water and dried milk contains a large proportion of non-fat components such as proteins and lactose (Cerbulis & Farrell, 1975). Despite similar GC–MS profiles shown in Fig. 1, whether water and/or other components could affect the transesterification efficiency (or FA recovery) needed to be verified. If we use the 2-step method (i.e. fat extraction by solvents and then methylation, Ex-Fat in this study) as the bench mark or control method, the yield of the DM and LM methods can be directly compared with the control method across the three sample sizes (Table 1). Close examination of the data revealed that there was no significant difference between the DM method and the control method (i.e. Ex-fat) across all the major FA (> 0.5%) including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C14:1cis-9, C15:0, C16:0, C16:1cis-9, C17:0, C18:0, C18:1cis-9, C18:2n6 and C18:3n-3), regardless of the milk volume (50, 100 or 200 µL) tested (Table 1). The fact that no significant reduction was observed in the level of C18:2/C18:3 implies that FA oxidation is minimal if any 4
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
preparation methods (Table 2, statistical annotations omitted). In addition, both the DM and the LM methods showed excellent reproducibility across replicates for both sample volumes, with RSD below 2% for most FA measurements; the reliability of the method was also confirmed by separate experiments. This means that both dried milk and liquid milk up to 100 µL can be used directly for acid-catalysed methylation with the current protocol. Clearly, acid-based transesterification shows a greater tolerance to moisture content in the samples as compared to the alkaline-based reaction, as about 3.4% of water was present in the reaction mixture that contained 100 µL of liquid milk and 2.8 mL of reagent. The presence of water is known to affect the transesterification efficiency of lipids (Carrapiso et al., 2000). However, it was reported that around 2% water in reaction mixture did not significantly affect the yield of FAMEs in acid-catalysed methylation (Ulberth & Henninger, 1992; Ichihara and Fukubayashi, 2010). Our study confirmed their finding and revealed that a longer methylation time is required when increasing the volume of fresh milk sample. By contrast, Park and Goins (1994) reported that about 8.5% (v/v) water in the total reaction mixture did not impair the in situ transesterification performed at 90 °C. Unfortunately, only the relative proportions (%) of FA were presented in their report and no information was given on the actual yield of FA. On the other hand, O’Fallon et al., 2007 demonstrated that 13% water did not affect the recovery (or yield) of FA and the percentage of each FA remained constant with up to 33% water in the reaction mixture. Whether the high water tolerance reported in these two studies is related to the 2-step protocol (hydrolysis of samples by NaOH/KOH in methanol followed by methylation with BF3/H2SO4 respectively) or simply because of different matrices (meat tissue and fish oil) involved in the 2nd study remains unclear. Acid-catalysed methylation is a slower process and a 2- to 3-h reaction time is usually needed when milder conditions are used (Agnew et al., 2019). Nevertheless, by avoiding the lipid extraction step, preparing FAME directly from liquid milk sample is still a time- and cost-saving alternative to the conventional 2-step method. It was observed that when acidic methylation was applied to LM samples, the phase separation was slow after adding hexane (to extract the FAMEs from the reaction mixture), but a brief centrifugation was enough to circumvent this drawback. It is worth noting that although the reliability of the current protocol has been confirmed for sample size up to 100 µL, using a larger sample volume is possible provided the volume of the acidic methanol reagent is proportionally increased. For example, a minimum of 5 mL of reagent is needed for methylating a 200 µL fresh milk sample.
thus meeting the requirements of quantifying both high- and lowabundance FA in milk samples. 3.2. Acid-catalysed methylation Despite being a slower process (usually a 1–4 h reaction for one-step methylation), acidic methylation is still preferred by many laboratories for milk FAME preparation (Teng, Wang, Yang, Ma, & Day, 2017). Hence, the feasibility of using dried and fresh milk was also tested with this methylation method. It is known that the efficiency of acid-catalysed transesterification depends on three parameters, acid concentration, incubation temperature and incubation time (Ichihara & Fukubayashi, 2010; Agnew et al., 2019). In order to minimise the degradation/isomerisation of conjugated linoleic acids especially C18:2c9t11, a milder methylation temperature (60 °C for example) is desirable (Liu et al., 2018). Because the feasibility of using dried milk samples for FAME preparation was already proven for alkaline-catalysed methylation, our emphasis in this experiment was to evaluate and optimise acidic methylation parameters for liquid milk samples. The optimal methylation time was first sought for 50 and 100 µL of fresh milk with fixed reaction temperature (60 °C), acid concentration (2% H2SO4) and methylation reagent volume (2.8 mL). It was found that for a sample volume of 50 µL, the yield of the major FA was rather close across three methylation times (2 h, 3 h and 4 h) (Fig. 2A). Although a slightly higher yield was observed for some FA after a 3-h as compared to a 2-h methylation, the extra gain by extending the reaction time beyond 2 h is minimal (about 3%). When the sample volume increased to 100 µL, a 2-h methylation time appears to be insufficient, whereas the difference between 3 h and 4 h/5 h, albeit significant in some cases, is actually < 5% for most FA (Fig. 2B). As a result, a 2-h and 3-h methylation time was selected for direct acidic methylation of 50 µL and 100 µL of liquid milk, respectively. Using the optimised methylation time, the profile and the yield of all major FA was compared across the three sample pre-treatment methods. As observed with FAMEs prepared by alkaline-catalysed methylation, the GC–MS profiles of FAMEs after acidic methylation again showed no visible difference between the three sample preparation procedures for both high-abundance FA and lower-abundance FA; again, no interfering or artefact peaks were found in the DM or LM samples (chromatograms not shown but are very similar to those shown in Fig. 1). The yield of the major FA was again calculated with the 2-step FAME preparation method (Ex-Fat) being used as the control for comparison. For both sample volume (50 µL and 100 µL), no significant difference was found in the yield of all major FA across the three sample
Table 2 Major FA yield (mean ± SD in µg/mL) with different sample pre-treatment methods prior to acid-catalysed methylation. FA
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3
50 µL
100 µL
Ex-Fat
DM
LM
Ex-Fat
DM
LM
2100 ± 25 588 ± 14 421 ± 5 883 ± 5 1127 ± 9 3922 ± 49 361 ± 7 369 ± 6 10239 ± 146 558 ± 8 365 ± 4 3062 ± 66 6133 ± 43 512 ± 4 218 ± 4
2087 ± 22 604 ± 6 423 ± 6 884 ± 13 1125 ± 10 3900 ± 15 350 ± 8 366 ± 5 10052 ± 44 562 ± 8 358 ± 7 2964 ± 34 6053 ± 59 510 ± 8 211 ± 3
2080 ± 72 601 ± 8 423 ± 4 886 ± 8 1108 ± 14 3859 ± 38 340 ± 13 365 ± 9 10016 ± 75 557 ± 29 363 ± 23 2940 ± 56 6024 ± 62 510 ± 3 218 ± 5
2067 ± 56 669 ± 6 422 ± 5 985 ± 13 1228 ± 16 3816 ± 37 382 ± 9 408 ± 5 9458 ± 141 561 ± 6 367 ± 13 3106 ± 55 5881 ± 46 521 ± 4 208 ± 2
2122 ± 8 675 ± 8 423 ± 3 984 ± 4 1221 ± 8 3777 ± 20 372 ± 5 406 ± 5 9328 ± 21 577 ± 18 361 ± 4 3041 ± 16 5879 ± 30 517 ± 11 207 ± 4
2148 ± 110 707 ± 20 436 ± 8 997 ± 16 1223 ± 14 3796 ± 46 376 ± 7 410 ± 5 9382 ± 134 569 ± 6 368 ± 5 3040 ± 31 5899 ± 56 520 ± 9 210 ± 2
5
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 3. Comparison of the concentration of the major FA determined by three methods. Ex-Fat (KOH): milk fat extracted by the Folch method prior to methylation with 0.2 M KOH at 50 °C for 20 min; DM (KOH): dried milk (50 µL) subjected to methylation with 0.2 M KOH at 50 °C for 30 min; LM (H2SO4): liquid milk (50 µL) subjected to methylation with 2% H2SO4 at 60 °C for 2 h. Each column represents the mean value of three technical replicates and columns with different letters are significantly different (p < 0.05). Error bars are standard deviations.
temperature, i.e. 60 °C rather than 80 or 100 °C (Liu et al., 2018). Given the relatively high abundance of this FA and the biological significance of CLA in general (Alonso, Cuesta, & Gilliland, 2004), the profile of CLA and the content of 18:2c9t11 was also examined in this raw milk experiment, which combined a milder methylation temperature (60 °C) with a lower H2SO4 concentration, i.e. 2% instead of 6% used previously (Liu et al., 2018). Fig. 4 shows that the profile of CLA obtained by acidic methylation of liquid milk is very similar to those produced by the two alkali-based methods using extracted fat and dried milk (Ex-fat and DM), with 18:2c9t11 being the single dominant CLA species whereas 18:2t9t11 being undetectable at the expected retention time (38.8 min) in all cases (Fig. 4A–C). Detailed quantification found no significant difference in 18:2c9t11 content across the three methylation regimes, indicating that direct acidic methylation of fresh milk using the current protocol is also suitable for CLA analysis in milk fat.
3.3. Verification with raw milk It is known that homogenised milk differs sharply from raw milk in terms of fat globule size and emulsion stability. Whether the above finding based on homogenised milk is valid for raw milk samples needed to be confirmed, as raw milk samples are frequently used in dairy research. Only one sample size (50 µL) of a raw milk sample was used to compare the major FA content across the three FAME preparation methods using the optimised methylation protocols, i.e. the classical 2step method with alkaline-catalysed methylation (control), drying of milk samples combined with alkaline-catalysed methylation, and direct transesterification of liquid milk sample by acidic methanolysis. No significant difference was found between the DM-KOH method and the control or between the LM-H2SO4 method and the control for most of the major FA (Fig. 3). However, a slightly higher yield was obtained with the LM-H2SO4 method as compared to the DM-KOH method for some FA; this can be explained by the fact that the former can react with all FA-containing lipids whereas the latter cannot methylate free FA and N-acyl lipids. Overall, the net difference is very small across the three methods for all the major FA. The applicability of the methods to raw milk samples was further confirmed with three more raw milk samples collected at different time (from different cows). This indicates that direct transesterification of dried milk samples by basic catalysis and direct transesterification of fresh milk samples by acidic methanolysis is equally suitable for raw milk samples. The theoretical total FA yield of about 900 µg mg−1 fat was reported for bovine milk (Moate, Chalupa, Boston, & Lean, 2007), which implies that a total of about 30 mg/mL FA would be expected for a milk sample containing 3.3% of fat. The sum of the major FA found in this experiment was very close to this level for the 2-step control method (FA yield totalling 30 mg/mL) as well as the DM (total yield of 28.8 mg/ mL) and LM (total yield of 31.5 mg/mL) methods, suggesting a satisfactory efficiency in transesterification of all the three methods. Acidic methylation could modify the profile of CLA by decreasing the level of 18:2c9t11 and a concomitant increase of 18:2t9t11; this could be mitigated to a large extent by using a lower reaction
4. Conclusions Through this systematic comparison study devoted exclusively to bovine milk, we have demonstrated that for FA composition analysis of milk (both raw and homogenised milk) skipping the lipid extraction step is possible and two new approaches have been proposed and validated, i.e. (1) using dried milk samples in combination with alkalinecatalysed transesterification, and (2) using liquid milk samples in combination with acidic methylation within a defined milk/reagent ratio. While the former requires a brief drying step but a shorter methylation reaction, the latter allows the use of fresh milk, but requires a longer methylation time. A sample size of up to 200 µL of milk is suitable for both methods, thus offering possibility of measuring both the high- and low-abundance FA. By eliminating the step of lipid extraction with organic solvents, these in situ transesterification methods are simpler and safer, and are expected to reduce the cost while increasing significantly the throughput of FA profiling of milk samples.
6
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 4. GC–MS profiles of CLA 18:2c9t11 of a raw milk sample subjected to different methylation protocols. A: milk fat extracted by the Folch method prior to methylation with 0.2 M KOH at 50 °C for 20 min; B: dried milk (50 µL) subjected to methylation with 0.2 M KOH at 50 °C for 30 min; C: liquid milk (50 µL) subjected to methylation with 2% H2SO4 at 60 °C for 2 h.
CRediT authorship contribution statement
Chen, S., Bobe, G., Zimmerman, S., Hammond, E. G., Luhman, C. M., Boylston, T. D., ... Beitz, D. C. (2004). Physical and sensory properties of dairy products from cows with various milk fatty acid compositions. Journal of Agricultural and Food Chemistry, 52, 3422–3428. Christie, W. W. (1982). Lipid analysis (2). Oxford: Pergamon Press. Cossignani, L., Pollini, L., & Blasi, F. (2019). Authentication of milk by direct and indirect analysis of triacylglycerol molecular species. Journal of Dairy Science, 2019(102), 5871–5882. Cruz-Hernandez, C., Goeuriot, S., Giuffrida, F., Thakkar, S. K., & Destaillats, F. (2013). Direct quantification of fatty acids in human milk by gas chromatography. Journal of Chromatography A, 1284, 174–179. De Paola, E. L., Montevecchi, G., Masino, F., Antonelli, A., & Lo Fiego, D. P. (2017). Single step extraction and derivatization of intramuscular lipids for fatty acid ultra-fast GC analysis: Application on pig thigh. Journal of Food Science and Technology, 54, 601–610. Dong, T., Yu, L., Gao, D., Yu, X., Miao, C., Zheng, Y., ... Chen, S. (2015). Direct quantification of fatty acids in wet microalgal and yeast biomass via a rapid in situ fatty acid methyl ester derivatization approach. Applied Microbiology and Biotechnology, 99, 10237–10247. Feng, S., Lock, A. L., & Garnsworthy, P. C. (2004). A rapid lipid separation method for determining fatty acid composition of milk. Journal of Dairy Science, 87, 3785–3788. Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497–509. Griffiths, M. J., van Hille, R. P., & Harrison, S. T. (2010). Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids, 45, 1053–1060. Hidalgo, P., Ciudad, G., Schober, S., Mittelbach, M., & Navia, R. (2015). Improving the FAME yield of in Situ transesterification from microalgal biomass through particle size reduction and cosolvent incorporation. Energy Fuels, 29, 823–832. Ichihara, K., & Fukubayashi, Y. (2010). Preparation of fatty acid methyl esters for gasliquid chromatography. Journal of Lipid Research, 51, 635–640. ISO-IDF (2002). Milk fat – Preparation of fatty acid methyl esters. International Standard ISO 15884-IDF 182:2002. International Dairy Federation, Brussels, Belgium. Jensen, R. G. (2002). The composition of bovine milk lipids: January 1995 to December 2000. Journal of Dairy Science, 85, 295–350. Kramer, J. K. G., Fellner, V., Dugan, M. E. R., Sauer, F. D., Mossoba, M. M., & Yurawecz, M. P. (1997). Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids. Lipids, 32, 1219–1228. Lee, M. R. F., & Tweed, J. K. S. (2008). Isomerisation of cis-9 trans-11 conjugated linoleic acid (CLA) to trans-9 trans-11 CLA during acidic methylation can be avoided by a rapid base catalysed methylation of milk fat. Journal of Dairy Research, 75, 354–356. Lee, M., Tweed, J., Kim, E., & Scollan, N. (2012). Beef, chicken and lamb fatty acid analysis – A simplified direct bimethylation procedure using freeze-dried material. Meat Science, 2012(92), 863–866. Lepage, G., & Roy, C. C. (1984). Improved recovery of fatty acid through direct transesterification without prior extraction or purification. Journal of Lipid Research, 25,
Zhiqian Liu: Conceptualization, Methodology, Investigation. Jianghui Wang: Investigation. Cheng Li: Investigation. Simone Rochfort: Supervision, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Abdulkadir, S., & Tsuchiya, M. (2008). One-step method for quantitative and qualitative analysis of fatty acids in marine animal samples. Journal of Experimental Marine Biology and Ecology, 354, 1–8. Agnew, M. P., Craigie, C. R., Weralupitiya, G., Reis, M. M., Johnson, P. L., & Reis, M. G. (2019). Comprehensive evaluation of parameters affecting one-step method for quantitative analysis of fatty acids in meat. Metabolites, 9, 189. https://doi.org/10. 3390/metabo9090189. Alonso, L., Cuesta, E. P., & Gilliland, S. E. (2004). Gas chromatographic method for analysis of conjugated linoleic acids isomers (c9t11, t10c12, and t9t11) in broth media as application in probiotic studies. Journal of Chromatographic Science, 42, 167–170. Amores, G., & Virt, M. (2019). Total and free fatty acids analysis in milk and dairy fat. Separation, 6, 14. https://doi.org/10.3390/separations6010014. Araujo, P., Nguyen, T. T., Froyland, L., Wang, J., & Kang, J. X. (2008). Evaluation of a rapid method for the quantitative analysis of fatty acids in various matrices. Journal of Chromatography A, 1212, 106–113. Bligh, E. G., & Dyer, W. J. (1959). A rapid method for total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Carrapiso, A. I., & Garcia, C. (2000). Development in lipid analysis: Some new extraction techniques and in situ transesterification. Lipids, 35, 1167–1177. Carrapiso, A. I., Timon, M. L., Petron, M. J., Tejeda, J. F., & Garcia, C. (2000). In situ transesterification of fatty acids from Iberian pig subcutaneous adipose tissue. Meat Science, 56, 159–164. Castro-Gomez, P., Fontecha, J., & Rodriguez-Alcala, L. M. (2014). A high-performance direct transmethylation method for total fatty acids assessment in biological and foodstuff samples. Talanta, 128, 518–523. Cerbulis, J., & Farrell, H. M. (1975). Composition of milks of dairy cattle. I. Protein, lactose, and fat contents and distribution of protein fraction. Journal of Dairy Science, 58, 817–827.
7
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis on conjugated linoleic acid. Meat Science, 65, 523–529. O’Fallon, J. V., Busboom, J. R., Nelson, M. L., & Gaskins, C. T. (2007). A direct method for fatty acid methyl ester synthesis: Application to wet meat tissues, oils, and feedstuffs. Journal of Animal Science, 85, 1511–1521. Park, P. W., & Goins, R. E. (1994). In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods. Journal of Food Science, 59, 1262–1266. Shingfield, K. J., Reynolds, C. K., Lupoli, B., Toivonen, V., Yurawecz, M. P., Delmonte, P., & Beever, D. E. (2005). Effect of forage type and proportion of concentrate in the diet on milk fatty acid composition in cows given sunflower oil and fish oil. Animal Science, 80, 225–238. Sun, C., Zou, X., Yao, Y., Jin, J., Xia, Y., Huang, J., ... Wang, X. (2016). Evaluation of fatty acid composition in commercial infant formulas on the Chinese market: a comparative study based on fat source and stage. International Dairy Journal, 63, 42–51. Teng, F., Wang, P., Yang, L., Ma, Y., & Day, L. (2017). Quantification of fatty acids in human, cow, buffalo, goat, yak, and camel milk using an improved one-step GC-FID method. Food Analytical Methods, 10, 2881–2991. Ulberth, F., & Henninger, M. (1992). One-step extraction/methylation method for determining the fatty acid composition of processed foods. Journal of the American Oil Chemists’ Society, 69, 174–177. Yener, S., & van Valenberg, H. J. F. (2019). Characterisation of triacylglycerols from bovine milk fat fractions with MALDI-TOF-MS fragmentation. Talanta, 204, 533–541.
1391–1396. Liu, Z., Ezernieks, V., Wang, J., Wanni Arachchillage, N., Garner, J. B., Wales, W. J., & Rochfort, S. (2017). Heat stress in dairy cattle alters lipid composition of milk. Scientific Reports, 7, 961. https://doi.org/10.1038/s41598-017-01120-9. Liu, Z., Ezernieks, V., Rochfort, S., & Cocks, B. (2018). Comparison of methylation methods for fatty acid analysis of milk fat. Food Chemistry, 261, 210–215. Liu, Z., Moate, P., & Rochfort, S. (2019). A simplified protocol for fatty acid profiling of milk fat without lipid extraction. International Dairy Journal, 90, 68–71. Luna, P., Juarez, M., & de la Fuente, M. A. (2005). Validation of a rapid milk fat separation method to determine the fatty acid profile by gas chromatography. Journal of Dairy Science, 88, 3377–3381. Martinez, B., Miranda, J. M., Franco, C. M., Cepeda, A., & Rodriguez, J. L. (2012). Development of a simple method for the quantitative determination of fatty acids in milk with special emphasis on long-chain fatty acids. CyTA – Journal of Food, 10, 27–35. Moate, P. J., Chalupa, W., Boston, R. C., & Lean, I. J. (2007). Milk fatty acids. I. Variation in the concentration of individual fatty acids in bovine milk. Journal of Dairy Science, 90, 4730–4739. Moate, P. J., Williams, S. R. O., Hannah, M. C., Eckard, R. J., Auldist, M. J., Ribaux, B. E., & Wales, W. J. (2013). Effects of feeding algal meal high in docosahexaenoic acid on feed intake, milk production, and methane emissions in dairy cows. Journal of Dairy Science, 96, 3177–3188. Murrieta, C. M., Hess, B. W., & Rule, D. C. (2003). Comparison of acidic and alkaline
8
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Development of one-step sample preparation methods for fatty acid profiling of milk fat
T
⁎
Zhiqian Liua, , Jianghui Wanga, Cheng Lia,b, Simone Rochforta,b a b
Agriculture Victoria Research, AgriBio, 5 Ring Road, Bundoora, Victoria 3083, Australia School of Applied Systems Biology, La Trobe University, Bundoora, Victoria 3083, Australia
A R T I C LE I N FO
A B S T R A C T
Keywords: Milk Fat Fatty acids Methylation Transesterification
Determination of the fatty acid (FA) profile of milk fat generally involves total lipid extraction from liquid milk, transesterification and GC analysis. The lipid extraction step is time consuming and often employs toxic solvents such as chloroform. Two alternative methods are presented here that skip the lipid extraction step and allow the determination of FA composition via direct transesterification of dried milk and liquid milk respectively. We have shown that dried milk can be used directly in alkaline-catalysed methylation, whereas direct transesterification of both dried milk and fresh milk is feasible with acidic methanol. Both methods generate similar results as compared to the classical two-step method (i.e. lipid extraction and FA methylation) when optimised methylation parameters (temperature, time, milk and reagent volume) are followed. By omitting the lipid extraction step, these simplified one-step methods offer a much higher throughput and a reduced cost in FA composition analysis of milk samples.
1. Introduction Lipids are one of the major components of milk solids. Fatty acid (FA) composition of milk fat is closely related to the nutritive quality of milk and physicochemical properties of dairy products (Chen et al., 2004). FA composition of milk fat is influenced by various factors such as animal genotypes, diets and physiological status of cows (Shingfield et al., 2005; Liu et al., 2017). As a result, analysis of FA composition is of great importance in lipid-related research and for dairy-based industries (Cossignani, Pollini, & Blasi, 2019). Generally, FAs are quantified as their methyl esters (FAMEs) by gas chromatography (GC) following a two-step sample preparation procedure, i.e. lipid extraction and FA methylation. For FAME preparation, both acid- and alkaline-catalysed transesterification have been widely applied so far for milk samples and review papers on this topic are also available (Jensen, 2002; Amores & Virt, 2019). The acidic protocol can convert all forms of FA into FAMEs, but the methylation process is generally slower, and could modify the profile of conjugated linoleic acids (CLA) (Kramer et al., 1997; Lee & Tweed, 2008; Liu, Ezernieks, Rochfort, & Cocks, 2018); whereas the base-catalysed reaction is fast but free FA and FA from sphingolipids cannot be methylated (Christie, 1982; Liu et al., 2018). Due to the much lower content of free FA and sphingolipids as compared to triglycerides in milk, the base-catalysed methylation is recommended for milk FA profiling (Liu et al., 2018), ⁎
which is also the official reference method for FAME preparation from milk fat (ISOIDF, 2002). Lipid extraction prior to FAME preparation is another bottleneck in FA profiling of milk and other biological samples. The classical methods for lipid extraction often involve toxic solvents such as chloroform (Folch, Lees, & Stanley, 1957; Bligh & Dyer, 1959) and are rather laborious and time-consuming. To avoid the solvent extraction step, the idea of in situ transesterification was proposed at least 30 years ago and a review paper summarising the earlier attempts in this field was also published (Carrapiso & Garcia, 2000). Since then, numerous one-step and two-step protocols have been tested for in situ transesterification of freeze-dried and fresh biological and foodstuff samples (for example, Carrapiso, Timon, Petron, Tejeda, & Garcia, 2000; O’Fallon, Busboom, Nelson & Gaskins, 2007; Abdulkadir & Tsuchiya, 2008; Araujo, Nguyen, Froyland, Wang, & Kang, 2008; Griffiths, van Hille, & Harrison, 2010; Lee, Tweed, Kim, & Scollan, 2012; Castro-Gomez, Fontecha, & Rodriguez-Alcala, 2014; Dong et al., 2015; Hidalgo, Ciudad, Schober, Mittelbach, & Navia, 2015; De Paola, Montevecchi, Masino, Antonelli, & Lo Fiego, 2017; Agnew et al., 2019). In the case of milk samples, alternative protocols for fat isolation have been proposed, such as twostep centrifugation (Feng, Lock, & Garnsworthy, 2004; Luna, Juarez, and de la Fuente, 2005) and one-step centrifugation (Liu, Moate, & Rochfort, 2019). The two-step centrifugation method appears to be reliable for fat isolation but is not easy to perform, whereas the one-step
Corresponding author. E-mail address: [email protected] (Z. Liu).
https://doi.org/10.1016/j.foodchem.2020.126281 Received 15 August 2019; Received in revised form 10 January 2020; Accepted 21 January 2020 Available online 23 January 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
2.4. FAME preparation
centrifugation method is useful only for relative quantification of FA in raw milk samples. Compared to animal tissue samples, much less reports can be found on in situ transesterification of milk and milk products. Park and Goins (1994) reported a 2-step protocol (i.e. hydrolysis of samples by KOH in methanol, followed by acidic methylation), which appeared to be reliable for relative quantification of FA in infant formulas and human milk. There have been other attempts to achieve lipid extraction and FA methylation of milk fat with one single reagent acetyl chloride/methanol (Lepage & Roy, 1984), H2SO4/methanol (Martinez, Miranda, Franco, Cepeda, & Rodriguez, 2012 and HCl/methanol (CruzHernandez, Goeuriot, Giuffrida, Thakkar, & Destaillats, 2013). However, these protocols either employ harsh transmethylation conditions (100 °C, for example), which would degrade CLA or involve multiple steps including an over-night incubation, hence they have not found widespread acceptance. Consequently, until now none of the in-situ transesterification methods published so far has been thoroughly validated with milk samples and lipid extraction by organic solvents remains an integral step in most, if not all, studies requiring FA content determination of milk samples. Alternative or novel in situ transesterification methods that are simple (one step preferably) and most importantly reliable for milk FAME preparation are still needed. The aim of this study was to evaluate the feasibility of two novel approaches of direct transesterification of milk fat from liquid milk and dried milk by acid- and base-catalysed methylation.
Both alkaline- and acid-catalysed methylation was applied to the three types of pre-treated samples. For the alkaline-catalysed reaction, 2.5 mL of derivatization reagent (0.2 M KOH in methanol) was added to each vial that contained solvent-extracted milk fat, dried milk or liquid milk and the vial was tightly sealed with a Teflon lined cap; the methylation reaction was conducted by incubation at 50 °C for 30 min with occasional shaking. After cooling to room temperature, one mL of HCl (1 M) was added to each vial and FAMEs formed were extracted into 1 mL of hexane containing internal standard (nonadecane, 100 mg L-1) and analysed directly by GC–MS. In the case of acid-catalysed methylation, the derivatization reagent was 2.8 mL of 2% H2SO4 (in methanol) and the reaction was allowed for 2–5 h at 60 °C. Upon completion of the transesterification process, one mL of water was added before extraction of FAMEs into hexane. 2.5. GC–MS analysis of FAMEs The separation of FAMEs was achieved by a Rt-2560 column (100 m × 0.25 mm ID, 0.20 µm df, Restek) with a constant flow of 1.2 mL min−1 helium as carrier gas and the following oven temperature program: initial temperature of 100 °C and held for 4 min, increased by 6 °C min−1 to 170 °C, and then increased by 3 °C min−1 to 240 °C and held for 11 min. The injection inlet temperature was 240 °C and injection volume was 1 µL with a split ratio of 20:1. The detection was by an Agilent 7000 GC/MS Triple Quadrupole with the following settings: scanning mass range of 40–500 amu, transfer line temperature of 240 °C, source temperature of 280 °C, quad temperature of 150 °C and a solvent delay of 11.8 min. A FAME standard mix containing 37 FAMEs was used to provide absolute quantification of each FA.
2. Materials and methods 2.1. Milk sample Pasteurised and homogenised commercial milk samples were obtained from the local supermarket (Victoria, Australia); the total fat content of theses milk sample was 3.4% as shown on the label. One raw milk sample was provided by the Department of Economic Development, Jobs, Transport and Resources’ Ellinbank centre in Victoria, Australia. This sample was an aliquot from an afternoon milking of one cow; its total fat concentration was 3.3% as determined by infrared spectroscopy (Moate et al., 2013).
2.6. Statistical analysis of data All FA concentrations were expressed as µg (FA) per mL of milk. For each experiment, the mean values and the standard deviations (SD) of three technical replicates were presented either in figures or in tables. For statistical comparison between different FAME preparation methods, the FA concentration results were subjected to ANOVA (XLSTAT, Microsoft Excel); where significant differences (p < 0.05) were found between treatments by F test, a Tukey’s HSD test was applied for pairwise comparisons.
2.2. Chemicals and reagents Solvents and chemicals used for lipid extraction and FAME preparation were of chromatographic or analytical grade. Hexane, chloroform and sulfuric acid were from Ajax FineChem (Taren Point, NSW, AU); methanol was from Merck (Darmstadt, Germany); potassium hydroxide, nonadecane (used as internal standard in GC analysis) as well as the standard mix of 37 FAMEs were purchased from SigmaAldrich (Castle Hill, NSW, AU).
3. Results and discussion 3.1. Alkaline-catalysed methylation As alkaline-catalysed transesterification is a fast yet reliable way for milk FAME preparation and is used increasingly in dairy-related research (Sun et al., 2016; Yener & van Valenberg, 2019); the milder reaction conditions can also avoid isomerisation of CLA 18:2c9t11 associated with acidic methylation (Murrieta, Hess, & Rule, 2003; Lee and Tweed, 2008). So, the feasibility of directly using dried and liquid milk for FAME preparation was assessed in the first instance by alkali-based methylation method. Compared to the solvent-extracted fat (Ex-Fat), which contained only lipids, the reaction matrices were much more complex when dried milk (DM) and liquid milk (LM) were used directly for FAME preparation, as all the milk solids were present in DM and all solids and water in LM. While 20 min (at 50 °C) was adequate for methylating solvent-extracted lipids by KOH/methanol reagent (Liu et al., 2018), our preliminary tests found that the optimal methylation time for dried milk samples was 30 min using the same reagent and reaction temperature. After 30 min incubation with the methylation reagent, the reaction mixture became totally clear with the Ex-Fat sample, whereas
2.3. Sample pre-treatment Three methods of sample pre-treatment prior to transesterification reaction were compared in this study using the homogenised commercial milk samples. Method 1 (fat extraction method, Ex-fat): total lipids of the milk sample (50, 100 or 200 µL) were extracted using chloroform/methanol (2:1) by the Folch method (Folch et al., 1957); the organic phase was transferred to a 5-mL glass vial and evaporated to dryness under a stream of N2 prior to methylation. Method 2 (dried milk method, DM): fresh milk (50, 100 or 200 µL) was accurately measured into a 5-mL glass vial and then dried in a heating block (40 °C) under a stream of N2 for approximately 10, 15 and 25 min, respectively, prior to methylation. Method 3 (liquid milk method, LM): fresh milk (50, 100 or 200 µL) was subjected directly to methylation in a 5-mL glass vial without any pre-treatment. 2
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 1. GC–MS profile (total ion chromatogram) of milk FAMES with different sample pre-treatment methods prior to alkaline-catalysed methylation. A: highabundance FA; B: lower-abundance FA. IS: internal standard. Ex-Fat: milk lipids extracted by the Folch method prior to methylation; DM: methylation performed with dried milk without lipid extraction; LM: methylation performed with liquid milk without lipid extraction or drying. For all the three methods, profiles shown are from 100 µL of milk. Table 1 Major FA yield (mean ± SD in µg/mL) with different sample pre-treatment methods prior to alkaline-catalysed methylation. FA
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3
50 µL
100 µL
200 µL
Ex-Fat
DM
LM
Ex-Fat
DM
LM
Ex-Fat
DM
LM
2014 ± 64a 740 ± 15a 387 ± 4a 899 ± 11a 1104 ± 22a 3361 ± 45a 364 ± 12a 371 ± 14a 8112 ± 196a 504 ± 14a 366 ± 11a 2684 ± 43a 5112 ± 78a 494 ± 10a 211 ± 15a
2081 ± 96a 748 ± 16a 386 ± 9a 902 ± 12a 1113 ± 16a 3372 ± 31a 357 ± 7a 367 ± 3a 8231 ± 66a 497 ± 9a 361 ± 3a 2694 ± 31a 5206 ± 20a 487 ± 11a 213 ± 5a
1909 ± 35a 694 ± 5b 349 ± 6b 782 ± 6b 930 ± 15b 2907 ± 25b 300 ± 4b 304 ± 4b 7068 ± 67b 428 ± 13b 297 ± 4b 2206 ± 11b 4427 ± 33b 379 ± 8b 154 ± 4b
2142 ± 39a 753 ± 10a 412 ± 4a 987 ± 15a 1219 ± 33a 3400 ± 41a 373 ± 5a 391 ± 8a 7727 ± 131a 516 ± 14a 358 ± 11a 2813 ± 48a 5182 ± 44a 489 ± 11a 207 ± 19a
2080 ± 37a 738 ± 14a 399 ± 10a 957 ± 14a 1166 ± 27a 3301 ± 53a 362 ± 8a 376 ± 7a 7627 ± 97a 506 ± 16a 350 ± 10a 2726 ± 34a 5080 ± 63a 485 ± 6a 203 ± 8a
1719 ± 36b 619 ± 6b 327 ± 5b 751 ± 11b 902 ± 13b 2571 ± 23b 275 ± 4b 283 ± 5b 5916 ± 31b 380 ± 3b 261 ± 4b 2046 ± 20b 3917 ± 27b 355 ± 8b 152 ± 7b
2126 ± 48a 752 ± 11a 423 ± 7a 1003 ± 14a 1208 ± 16a 3460 ± 55a 375 ± 4a 391 ± 6a 7605 ± 82a 538 ± 16a 371 ± 6a 2771 ± 30a 5191 ± 75a 517 ± 9a 227 ± 4a
2088 ± 19a 740 ± 5a 422 ± 6a 990 ± 7a 1184 ± 10a 3371 ± 27a 365 ± 8a 382 ± 11a 7461 ± 58a 516 ± 10a 360 ± 8a 2705 ± 32a 5057 ± 57a 500 ± 8a 221 ± 5a
1468 ± 34b 509 ± 12b 272 ± 7b 616 ± 7b 722 ± 7b 2200 ± 26b 222 ± 3b 222 ± 4b 4966 ± 45b 311 ± 12b 217 ± 6b 1623 ± 19b 3161 ± 36b 297 ± 6b 132 ± 9b
*Within each sample size (50, 100 or 200 µL) and each FA, mean values followed by different letters are significantly different (p < 0.05). 3
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 2. Comparison of major FA yield with different methylation times. A: 50 µL liquid milk; B: 100 µL liquid milk. All methylation reactions were performed at 60 °C with 2.8 mL of reagent (2% H2SO4 in methanol). Each column represents the mean value of three technical replicates and columns with different letters are significantly different (p < 0.05). Error bars are standard deviations.
when the drying step was conducted at a mild temperature (i.e. 40 °C) and under N2. In addition, the DM method showed excellent reproducibility across replicates for all the three sample volumes, with RSD below 3% for the majority of the FA measurements; the reliability of the method was also confirmed by separate experiments. All these results suggest that using dried milk samples to prepare FAMEs without the lipid extraction step is a feasible approach. By contrast, the LM samples gave significantly reduced yield for virtually all major FA (except for C4:0 with 50 µL milk) as compared to the control method; this yield reduction in FA increased with increasing sample volume (about 15% reduction for 50 µL, about 25% for 100 µL and about 40% for 200 µL of liquid milk) (Table 1). Indeed, a slight reduction in FA level was still observed when the sample volume decreased to 25 µL (results not shown). This indicates that base-catalysed methylation is sensitive to moisture content in the reaction medium and using fresh milk to prepare FAMEs could lead to significant underestimation of milk FA. Although using liquid milk for base-catalysed methanolysis proved to be unreliable, dried milk generated reliable results as compared to the classical 2-step method. As only 10–25 min is needed for the sample drying step and both the drying and methylation steps can be performed in the same glass vial, this FAME preparation protocol is still much quicker than the classical 2-step method. It is worth mentioning that the sample drying procedure was performed with conical vials in this study, so the actual drying time required may be significantly shortened if round-bottom or flat-bottom vials are used. In addition, compared to freeze-drying samples to remove water, drying milk samples with a heating block requires no special equipment and can be performed in any laboratory. We should also point out that this sample drying approach is easily applicable to milk volume from 25 to 200 µL,
most of the milk solids still clung to the wall of the glass vial for the DM sample. In the case of the LM sample, the reaction mixture remained a cloudy suspension by the end of the methylation procedure. In all cases, upon adding hexane, phase separation was always achieved, and a clear hexane extract obtained. The overall GC–MS profile of FAMEs was found to be very similar across the three sample preparation methods for both high-abundance FA (Fig. 1A) and lower-abundance FA (Fig. 1B) (profiles of 100-µL milk samples are shown as examples). It is worth mentioning that despite the complexity of the matrix, direct methylation of dried or liquid milk did not produce any extra or interfering peaks as compared to the solventextracted “pure” lipids. This is an important finding as GC-FID is widely used in FAME quantification and any co-eluting or interfering peaks could compromise the integration accuracy of FID chromatograms. Liquid milk contains over 85% of water and dried milk contains a large proportion of non-fat components such as proteins and lactose (Cerbulis & Farrell, 1975). Despite similar GC–MS profiles shown in Fig. 1, whether water and/or other components could affect the transesterification efficiency (or FA recovery) needed to be verified. If we use the 2-step method (i.e. fat extraction by solvents and then methylation, Ex-Fat in this study) as the bench mark or control method, the yield of the DM and LM methods can be directly compared with the control method across the three sample sizes (Table 1). Close examination of the data revealed that there was no significant difference between the DM method and the control method (i.e. Ex-fat) across all the major FA (> 0.5%) including C4:0, C6:0, C8:0, C10:0, C12:0, C14:0, C14:1cis-9, C15:0, C16:0, C16:1cis-9, C17:0, C18:0, C18:1cis-9, C18:2n6 and C18:3n-3), regardless of the milk volume (50, 100 or 200 µL) tested (Table 1). The fact that no significant reduction was observed in the level of C18:2/C18:3 implies that FA oxidation is minimal if any 4
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
preparation methods (Table 2, statistical annotations omitted). In addition, both the DM and the LM methods showed excellent reproducibility across replicates for both sample volumes, with RSD below 2% for most FA measurements; the reliability of the method was also confirmed by separate experiments. This means that both dried milk and liquid milk up to 100 µL can be used directly for acid-catalysed methylation with the current protocol. Clearly, acid-based transesterification shows a greater tolerance to moisture content in the samples as compared to the alkaline-based reaction, as about 3.4% of water was present in the reaction mixture that contained 100 µL of liquid milk and 2.8 mL of reagent. The presence of water is known to affect the transesterification efficiency of lipids (Carrapiso et al., 2000). However, it was reported that around 2% water in reaction mixture did not significantly affect the yield of FAMEs in acid-catalysed methylation (Ulberth & Henninger, 1992; Ichihara and Fukubayashi, 2010). Our study confirmed their finding and revealed that a longer methylation time is required when increasing the volume of fresh milk sample. By contrast, Park and Goins (1994) reported that about 8.5% (v/v) water in the total reaction mixture did not impair the in situ transesterification performed at 90 °C. Unfortunately, only the relative proportions (%) of FA were presented in their report and no information was given on the actual yield of FA. On the other hand, O’Fallon et al., 2007 demonstrated that 13% water did not affect the recovery (or yield) of FA and the percentage of each FA remained constant with up to 33% water in the reaction mixture. Whether the high water tolerance reported in these two studies is related to the 2-step protocol (hydrolysis of samples by NaOH/KOH in methanol followed by methylation with BF3/H2SO4 respectively) or simply because of different matrices (meat tissue and fish oil) involved in the 2nd study remains unclear. Acid-catalysed methylation is a slower process and a 2- to 3-h reaction time is usually needed when milder conditions are used (Agnew et al., 2019). Nevertheless, by avoiding the lipid extraction step, preparing FAME directly from liquid milk sample is still a time- and cost-saving alternative to the conventional 2-step method. It was observed that when acidic methylation was applied to LM samples, the phase separation was slow after adding hexane (to extract the FAMEs from the reaction mixture), but a brief centrifugation was enough to circumvent this drawback. It is worth noting that although the reliability of the current protocol has been confirmed for sample size up to 100 µL, using a larger sample volume is possible provided the volume of the acidic methanol reagent is proportionally increased. For example, a minimum of 5 mL of reagent is needed for methylating a 200 µL fresh milk sample.
thus meeting the requirements of quantifying both high- and lowabundance FA in milk samples. 3.2. Acid-catalysed methylation Despite being a slower process (usually a 1–4 h reaction for one-step methylation), acidic methylation is still preferred by many laboratories for milk FAME preparation (Teng, Wang, Yang, Ma, & Day, 2017). Hence, the feasibility of using dried and fresh milk was also tested with this methylation method. It is known that the efficiency of acid-catalysed transesterification depends on three parameters, acid concentration, incubation temperature and incubation time (Ichihara & Fukubayashi, 2010; Agnew et al., 2019). In order to minimise the degradation/isomerisation of conjugated linoleic acids especially C18:2c9t11, a milder methylation temperature (60 °C for example) is desirable (Liu et al., 2018). Because the feasibility of using dried milk samples for FAME preparation was already proven for alkaline-catalysed methylation, our emphasis in this experiment was to evaluate and optimise acidic methylation parameters for liquid milk samples. The optimal methylation time was first sought for 50 and 100 µL of fresh milk with fixed reaction temperature (60 °C), acid concentration (2% H2SO4) and methylation reagent volume (2.8 mL). It was found that for a sample volume of 50 µL, the yield of the major FA was rather close across three methylation times (2 h, 3 h and 4 h) (Fig. 2A). Although a slightly higher yield was observed for some FA after a 3-h as compared to a 2-h methylation, the extra gain by extending the reaction time beyond 2 h is minimal (about 3%). When the sample volume increased to 100 µL, a 2-h methylation time appears to be insufficient, whereas the difference between 3 h and 4 h/5 h, albeit significant in some cases, is actually < 5% for most FA (Fig. 2B). As a result, a 2-h and 3-h methylation time was selected for direct acidic methylation of 50 µL and 100 µL of liquid milk, respectively. Using the optimised methylation time, the profile and the yield of all major FA was compared across the three sample pre-treatment methods. As observed with FAMEs prepared by alkaline-catalysed methylation, the GC–MS profiles of FAMEs after acidic methylation again showed no visible difference between the three sample preparation procedures for both high-abundance FA and lower-abundance FA; again, no interfering or artefact peaks were found in the DM or LM samples (chromatograms not shown but are very similar to those shown in Fig. 1). The yield of the major FA was again calculated with the 2-step FAME preparation method (Ex-Fat) being used as the control for comparison. For both sample volume (50 µL and 100 µL), no significant difference was found in the yield of all major FA across the three sample
Table 2 Major FA yield (mean ± SD in µg/mL) with different sample pre-treatment methods prior to acid-catalysed methylation. FA
C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C14:1 C15:0 C16:0 C16:1 C17:0 C18:0 C18:1 C18:2 C18:3
50 µL
100 µL
Ex-Fat
DM
LM
Ex-Fat
DM
LM
2100 ± 25 588 ± 14 421 ± 5 883 ± 5 1127 ± 9 3922 ± 49 361 ± 7 369 ± 6 10239 ± 146 558 ± 8 365 ± 4 3062 ± 66 6133 ± 43 512 ± 4 218 ± 4
2087 ± 22 604 ± 6 423 ± 6 884 ± 13 1125 ± 10 3900 ± 15 350 ± 8 366 ± 5 10052 ± 44 562 ± 8 358 ± 7 2964 ± 34 6053 ± 59 510 ± 8 211 ± 3
2080 ± 72 601 ± 8 423 ± 4 886 ± 8 1108 ± 14 3859 ± 38 340 ± 13 365 ± 9 10016 ± 75 557 ± 29 363 ± 23 2940 ± 56 6024 ± 62 510 ± 3 218 ± 5
2067 ± 56 669 ± 6 422 ± 5 985 ± 13 1228 ± 16 3816 ± 37 382 ± 9 408 ± 5 9458 ± 141 561 ± 6 367 ± 13 3106 ± 55 5881 ± 46 521 ± 4 208 ± 2
2122 ± 8 675 ± 8 423 ± 3 984 ± 4 1221 ± 8 3777 ± 20 372 ± 5 406 ± 5 9328 ± 21 577 ± 18 361 ± 4 3041 ± 16 5879 ± 30 517 ± 11 207 ± 4
2148 ± 110 707 ± 20 436 ± 8 997 ± 16 1223 ± 14 3796 ± 46 376 ± 7 410 ± 5 9382 ± 134 569 ± 6 368 ± 5 3040 ± 31 5899 ± 56 520 ± 9 210 ± 2
5
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 3. Comparison of the concentration of the major FA determined by three methods. Ex-Fat (KOH): milk fat extracted by the Folch method prior to methylation with 0.2 M KOH at 50 °C for 20 min; DM (KOH): dried milk (50 µL) subjected to methylation with 0.2 M KOH at 50 °C for 30 min; LM (H2SO4): liquid milk (50 µL) subjected to methylation with 2% H2SO4 at 60 °C for 2 h. Each column represents the mean value of three technical replicates and columns with different letters are significantly different (p < 0.05). Error bars are standard deviations.
temperature, i.e. 60 °C rather than 80 or 100 °C (Liu et al., 2018). Given the relatively high abundance of this FA and the biological significance of CLA in general (Alonso, Cuesta, & Gilliland, 2004), the profile of CLA and the content of 18:2c9t11 was also examined in this raw milk experiment, which combined a milder methylation temperature (60 °C) with a lower H2SO4 concentration, i.e. 2% instead of 6% used previously (Liu et al., 2018). Fig. 4 shows that the profile of CLA obtained by acidic methylation of liquid milk is very similar to those produced by the two alkali-based methods using extracted fat and dried milk (Ex-fat and DM), with 18:2c9t11 being the single dominant CLA species whereas 18:2t9t11 being undetectable at the expected retention time (38.8 min) in all cases (Fig. 4A–C). Detailed quantification found no significant difference in 18:2c9t11 content across the three methylation regimes, indicating that direct acidic methylation of fresh milk using the current protocol is also suitable for CLA analysis in milk fat.
3.3. Verification with raw milk It is known that homogenised milk differs sharply from raw milk in terms of fat globule size and emulsion stability. Whether the above finding based on homogenised milk is valid for raw milk samples needed to be confirmed, as raw milk samples are frequently used in dairy research. Only one sample size (50 µL) of a raw milk sample was used to compare the major FA content across the three FAME preparation methods using the optimised methylation protocols, i.e. the classical 2step method with alkaline-catalysed methylation (control), drying of milk samples combined with alkaline-catalysed methylation, and direct transesterification of liquid milk sample by acidic methanolysis. No significant difference was found between the DM-KOH method and the control or between the LM-H2SO4 method and the control for most of the major FA (Fig. 3). However, a slightly higher yield was obtained with the LM-H2SO4 method as compared to the DM-KOH method for some FA; this can be explained by the fact that the former can react with all FA-containing lipids whereas the latter cannot methylate free FA and N-acyl lipids. Overall, the net difference is very small across the three methods for all the major FA. The applicability of the methods to raw milk samples was further confirmed with three more raw milk samples collected at different time (from different cows). This indicates that direct transesterification of dried milk samples by basic catalysis and direct transesterification of fresh milk samples by acidic methanolysis is equally suitable for raw milk samples. The theoretical total FA yield of about 900 µg mg−1 fat was reported for bovine milk (Moate, Chalupa, Boston, & Lean, 2007), which implies that a total of about 30 mg/mL FA would be expected for a milk sample containing 3.3% of fat. The sum of the major FA found in this experiment was very close to this level for the 2-step control method (FA yield totalling 30 mg/mL) as well as the DM (total yield of 28.8 mg/ mL) and LM (total yield of 31.5 mg/mL) methods, suggesting a satisfactory efficiency in transesterification of all the three methods. Acidic methylation could modify the profile of CLA by decreasing the level of 18:2c9t11 and a concomitant increase of 18:2t9t11; this could be mitigated to a large extent by using a lower reaction
4. Conclusions Through this systematic comparison study devoted exclusively to bovine milk, we have demonstrated that for FA composition analysis of milk (both raw and homogenised milk) skipping the lipid extraction step is possible and two new approaches have been proposed and validated, i.e. (1) using dried milk samples in combination with alkalinecatalysed transesterification, and (2) using liquid milk samples in combination with acidic methylation within a defined milk/reagent ratio. While the former requires a brief drying step but a shorter methylation reaction, the latter allows the use of fresh milk, but requires a longer methylation time. A sample size of up to 200 µL of milk is suitable for both methods, thus offering possibility of measuring both the high- and low-abundance FA. By eliminating the step of lipid extraction with organic solvents, these in situ transesterification methods are simpler and safer, and are expected to reduce the cost while increasing significantly the throughput of FA profiling of milk samples.
6
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
Fig. 4. GC–MS profiles of CLA 18:2c9t11 of a raw milk sample subjected to different methylation protocols. A: milk fat extracted by the Folch method prior to methylation with 0.2 M KOH at 50 °C for 20 min; B: dried milk (50 µL) subjected to methylation with 0.2 M KOH at 50 °C for 30 min; C: liquid milk (50 µL) subjected to methylation with 2% H2SO4 at 60 °C for 2 h.
CRediT authorship contribution statement
Chen, S., Bobe, G., Zimmerman, S., Hammond, E. G., Luhman, C. M., Boylston, T. D., ... Beitz, D. C. (2004). Physical and sensory properties of dairy products from cows with various milk fatty acid compositions. Journal of Agricultural and Food Chemistry, 52, 3422–3428. Christie, W. W. (1982). Lipid analysis (2). Oxford: Pergamon Press. Cossignani, L., Pollini, L., & Blasi, F. (2019). Authentication of milk by direct and indirect analysis of triacylglycerol molecular species. Journal of Dairy Science, 2019(102), 5871–5882. Cruz-Hernandez, C., Goeuriot, S., Giuffrida, F., Thakkar, S. K., & Destaillats, F. (2013). Direct quantification of fatty acids in human milk by gas chromatography. Journal of Chromatography A, 1284, 174–179. De Paola, E. L., Montevecchi, G., Masino, F., Antonelli, A., & Lo Fiego, D. P. (2017). Single step extraction and derivatization of intramuscular lipids for fatty acid ultra-fast GC analysis: Application on pig thigh. Journal of Food Science and Technology, 54, 601–610. Dong, T., Yu, L., Gao, D., Yu, X., Miao, C., Zheng, Y., ... Chen, S. (2015). Direct quantification of fatty acids in wet microalgal and yeast biomass via a rapid in situ fatty acid methyl ester derivatization approach. Applied Microbiology and Biotechnology, 99, 10237–10247. Feng, S., Lock, A. L., & Garnsworthy, P. C. (2004). A rapid lipid separation method for determining fatty acid composition of milk. Journal of Dairy Science, 87, 3785–3788. Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497–509. Griffiths, M. J., van Hille, R. P., & Harrison, S. T. (2010). Selection of direct transesterification as the preferred method for assay of fatty acid content of microalgae. Lipids, 45, 1053–1060. Hidalgo, P., Ciudad, G., Schober, S., Mittelbach, M., & Navia, R. (2015). Improving the FAME yield of in Situ transesterification from microalgal biomass through particle size reduction and cosolvent incorporation. Energy Fuels, 29, 823–832. Ichihara, K., & Fukubayashi, Y. (2010). Preparation of fatty acid methyl esters for gasliquid chromatography. Journal of Lipid Research, 51, 635–640. ISO-IDF (2002). Milk fat – Preparation of fatty acid methyl esters. International Standard ISO 15884-IDF 182:2002. International Dairy Federation, Brussels, Belgium. Jensen, R. G. (2002). The composition of bovine milk lipids: January 1995 to December 2000. Journal of Dairy Science, 85, 295–350. Kramer, J. K. G., Fellner, V., Dugan, M. E. R., Sauer, F. D., Mossoba, M. M., & Yurawecz, M. P. (1997). Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids. Lipids, 32, 1219–1228. Lee, M. R. F., & Tweed, J. K. S. (2008). Isomerisation of cis-9 trans-11 conjugated linoleic acid (CLA) to trans-9 trans-11 CLA during acidic methylation can be avoided by a rapid base catalysed methylation of milk fat. Journal of Dairy Research, 75, 354–356. Lee, M., Tweed, J., Kim, E., & Scollan, N. (2012). Beef, chicken and lamb fatty acid analysis – A simplified direct bimethylation procedure using freeze-dried material. Meat Science, 2012(92), 863–866. Lepage, G., & Roy, C. C. (1984). Improved recovery of fatty acid through direct transesterification without prior extraction or purification. Journal of Lipid Research, 25,
Zhiqian Liu: Conceptualization, Methodology, Investigation. Jianghui Wang: Investigation. Cheng Li: Investigation. Simone Rochfort: Supervision, Funding acquisition. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Abdulkadir, S., & Tsuchiya, M. (2008). One-step method for quantitative and qualitative analysis of fatty acids in marine animal samples. Journal of Experimental Marine Biology and Ecology, 354, 1–8. Agnew, M. P., Craigie, C. R., Weralupitiya, G., Reis, M. M., Johnson, P. L., & Reis, M. G. (2019). Comprehensive evaluation of parameters affecting one-step method for quantitative analysis of fatty acids in meat. Metabolites, 9, 189. https://doi.org/10. 3390/metabo9090189. Alonso, L., Cuesta, E. P., & Gilliland, S. E. (2004). Gas chromatographic method for analysis of conjugated linoleic acids isomers (c9t11, t10c12, and t9t11) in broth media as application in probiotic studies. Journal of Chromatographic Science, 42, 167–170. Amores, G., & Virt, M. (2019). Total and free fatty acids analysis in milk and dairy fat. Separation, 6, 14. https://doi.org/10.3390/separations6010014. Araujo, P., Nguyen, T. T., Froyland, L., Wang, J., & Kang, J. X. (2008). Evaluation of a rapid method for the quantitative analysis of fatty acids in various matrices. Journal of Chromatography A, 1212, 106–113. Bligh, E. G., & Dyer, W. J. (1959). A rapid method for total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911–917. Carrapiso, A. I., & Garcia, C. (2000). Development in lipid analysis: Some new extraction techniques and in situ transesterification. Lipids, 35, 1167–1177. Carrapiso, A. I., Timon, M. L., Petron, M. J., Tejeda, J. F., & Garcia, C. (2000). In situ transesterification of fatty acids from Iberian pig subcutaneous adipose tissue. Meat Science, 56, 159–164. Castro-Gomez, P., Fontecha, J., & Rodriguez-Alcala, L. M. (2014). A high-performance direct transmethylation method for total fatty acids assessment in biological and foodstuff samples. Talanta, 128, 518–523. Cerbulis, J., & Farrell, H. M. (1975). Composition of milks of dairy cattle. I. Protein, lactose, and fat contents and distribution of protein fraction. Journal of Dairy Science, 58, 817–827.
7
Food Chemistry 315 (2020) 126281
Z. Liu, et al.
catalysts for preparation of fatty acid methyl esters from ovine muscle with emphasis on conjugated linoleic acid. Meat Science, 65, 523–529. O’Fallon, J. V., Busboom, J. R., Nelson, M. L., & Gaskins, C. T. (2007). A direct method for fatty acid methyl ester synthesis: Application to wet meat tissues, oils, and feedstuffs. Journal of Animal Science, 85, 1511–1521. Park, P. W., & Goins, R. E. (1994). In situ preparation of fatty acid methyl esters for analysis of fatty acid composition in foods. Journal of Food Science, 59, 1262–1266. Shingfield, K. J., Reynolds, C. K., Lupoli, B., Toivonen, V., Yurawecz, M. P., Delmonte, P., & Beever, D. E. (2005). Effect of forage type and proportion of concentrate in the diet on milk fatty acid composition in cows given sunflower oil and fish oil. Animal Science, 80, 225–238. Sun, C., Zou, X., Yao, Y., Jin, J., Xia, Y., Huang, J., ... Wang, X. (2016). Evaluation of fatty acid composition in commercial infant formulas on the Chinese market: a comparative study based on fat source and stage. International Dairy Journal, 63, 42–51. Teng, F., Wang, P., Yang, L., Ma, Y., & Day, L. (2017). Quantification of fatty acids in human, cow, buffalo, goat, yak, and camel milk using an improved one-step GC-FID method. Food Analytical Methods, 10, 2881–2991. Ulberth, F., & Henninger, M. (1992). One-step extraction/methylation method for determining the fatty acid composition of processed foods. Journal of the American Oil Chemists’ Society, 69, 174–177. Yener, S., & van Valenberg, H. J. F. (2019). Characterisation of triacylglycerols from bovine milk fat fractions with MALDI-TOF-MS fragmentation. Talanta, 204, 533–541.
1391–1396. Liu, Z., Ezernieks, V., Wang, J., Wanni Arachchillage, N., Garner, J. B., Wales, W. J., & Rochfort, S. (2017). Heat stress in dairy cattle alters lipid composition of milk. Scientific Reports, 7, 961. https://doi.org/10.1038/s41598-017-01120-9. Liu, Z., Ezernieks, V., Rochfort, S., & Cocks, B. (2018). Comparison of methylation methods for fatty acid analysis of milk fat. Food Chemistry, 261, 210–215. Liu, Z., Moate, P., & Rochfort, S. (2019). A simplified protocol for fatty acid profiling of milk fat without lipid extraction. International Dairy Journal, 90, 68–71. Luna, P., Juarez, M., & de la Fuente, M. A. (2005). Validation of a rapid milk fat separation method to determine the fatty acid profile by gas chromatography. Journal of Dairy Science, 88, 3377–3381. Martinez, B., Miranda, J. M., Franco, C. M., Cepeda, A., & Rodriguez, J. L. (2012). Development of a simple method for the quantitative determination of fatty acids in milk with special emphasis on long-chain fatty acids. CyTA – Journal of Food, 10, 27–35. Moate, P. J., Chalupa, W., Boston, R. C., & Lean, I. J. (2007). Milk fatty acids. I. Variation in the concentration of individual fatty acids in bovine milk. Journal of Dairy Science, 90, 4730–4739. Moate, P. J., Williams, S. R. O., Hannah, M. C., Eckard, R. J., Auldist, M. J., Ribaux, B. E., & Wales, W. J. (2013). Effects of feeding algal meal high in docosahexaenoic acid on feed intake, milk production, and methane emissions in dairy cows. Journal of Dairy Science, 96, 3177–3188. Murrieta, C. M., Hess, B. W., & Rule, D. C. (2003). Comparison of acidic and alkaline
8