Composition of goat's milk fat triglycerides analysed by silver ion adsorption-TLC and GC–MS

International Dairy Journal 10 (2000) 119}128

Composition of goat's milk fat triglycerides analysed by silver ion adsorption-TLC and GC}MS J. Fontecha *, J.J. RmH os
, L. Lozada , M.J. Fraga, M. JuaH rez Departamento de Productos La& cteos, Instituto del Frn& o (CSIC), Ciudad Universitaria s/n, 28040 Madrid, Spain
Instituto de la Grasa (CSIC), 41012 Sevilla, Spain Departamento de Produccio& n Animal, Universidad Polite& cnica, 28040 Madrid, Spain Received 27 May 1999; accepted 23 March 2000

Abstract The triglyceride (TG) composition of goat's milk fat (from the milk of "ve herds collected monthly, from November to May) was studied using AgNO -TLC and GC}MS. Two of the four fractions obtained by TLC contained trisaturated TGs (SSS) and  represented 55% of the total TGs; they were separated by the chain length of short-chain fatty acid (FA). The TGs of the remaining fractions were identi"ed as mono- (SSM) and polyunsaturated, representing 29 and 16% of the total TGs, respectively. The distribution of two SSS fractions by carbon number (CN) was unimodal, with maxima at CN40 and CN36 respectively. The proportions of SSM and polyunsaturated TG were high between CN38}CN44 and CN46}CN54, respectively (mean values, 35 and 52%). One hundred and twenty-four peaks (some containing more that one molecular species of TGs) were detected in the chromatogram of total fat; the sum of the 30 peaks representing'1 mol% was 70% of the total TGs. One hundred and thirty-seven molecular species were identi"ed in the total goat's milk fat: 50% SSS, 30% SSM and 20% polyunsaturated. The most important in quantitative terms were medium-chain TGs containing C8, C10 or C12, and C18 : 1 as unsaturated FA.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Goat's milk fat; Triglycerides; Molecular classes; Molecular species; Silver ion adsorption-TLC; GC}MS

1. Introduction Gas chromatography (GC) with packed or short capillary columns has been used to separate milk fat triglyceride (TG) classes according to their carbon number (CN). However, to explain the physical properties (melting point, crystallization behaviour, etc.), nutritive characteristics (action of lipolytic enzymes) or biosynthesis of milk fat in the mammary gland, the composition must be known in terms of molecular species of TGs. Given the high number of fatty acids in milk fat, combined or hyphenated chromatographic techniques have been indispensable for identifying and quantifying molecular species of milk fat TGs: thin layer chromatography (TLC) or high-performance liquid chromatography (HPLC) without or with Ag> as a pre-separation step, followed by supercritical #uid chromatography (Man-

* Corresponding author. E-mail address: [email protected] (J. Fontecha).

ninen, Laakso & Kallio, 1995), GC with long capillary column (Steele & Banks, 1994; Myher, Kuksis, Marai & Sandra, 1988; Gresti, Bugaut, Maniongui & Bezard, 1993; Fraga, Fontecha, Lozada & JuaH rez, 1998), and HPLC with reverse phase and mass spectrometry (MS) as detector (Laakso & Kallio, 1993; Ruiz-Sala, Hierro, MartmH nez-Castro & Santa-MarmH a, 1996). Quite detailed information on the composition of the main TGs in cow's milk can be derived from these studies. However, there is very little information on goat's milk. The use of GC with a short column (Marai, Breckenridge & Kuksis, 1969; Parodi, 1973; Cerbulis, Parks & Farrell, 1982; Luf, Stock & Brandl, 1987; Fontecha, DmH az, Fraga & JuaH rez, 1998) showed that the occurrence of TGs in goat's milk reaches a maximum at CN38}CN42; beyond this point, the percentage of TGs decreases but not uniformly, because the values for CN48 and CN50 are close together. With regard to the fatty acid composition of TGs, in a study using TLC and GC, Dimick and Patton (1965) demonstrated that the TGs of goat's milk fat contained no more than one mole of

0958-6946/00/$ - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 0 0 ) 0 0 0 2 6 - 1

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J. Fontecha et al. / International Dairy Journal 10 (2000) 119}128

butyrate per mole, a high C18 : 0 content in the high molecular weight TGs, and a remarkable degree of uniformity in the C16 : 0 distribution. Glass, Jenness and Lohse (1969) used TLC to show that goat's milk TGs separate readily into two distinct fractions that have nearly the same C14 : 0 and C16 : 0 content, but one of them contains almost all the butyrate, and the other is much richer in C18 acids. More recently, Barron, Hierro and Santa-MarmH a (1990) and Ruiz-Sala et al. (1996) used HPLC}GC to identify respectively 116 and 181 molecular species of TGs in goat's milk fat. In both cases it was not possible to quantify the individual TGs because most of the HPLC peaks contained more than one molecular species. The purpose of this work was to study the composition of the TGs in goat's milk fat from "ve di!erent herds using a combination of AgNO -TLC and GC}MS.  2. Materials and methods 2.1. Samples and standards We used Murciana-Granadina adult female goats in "ve herds, belonging to "ve di!erent breeders in the Murcia region (Spain) and their size ranged from 98 to 125. Batches were taken once daily from storage tanks containing milk from the whole herd. Seven batches were collected for each herd during a milking period of seven months, starting in November. During the "rst 2 months after parturition (September) all the milk was used for feeding the kids. The milk fat was extracted following the procedure described by Fontecha et al. (1998). All the goat's milk fat samples from each herd were combined in equal volumes to obtain "ve mixture samples. Di!erent dilutions of the fat were prepared in hexane for chromatographic analysis. To identify the TG, a mixture of synthetic TGs (trilinolein, triolein, tristearin, tripalmitin, trimyristin, trilaurin, tricaprin, and tricaprylin) was "rst analysed to determine both the best chromatographic conditions and the retention time of these components. The other molecular species were identi"ed following previous studies carried out in similar conditions (Myher et al., 1988; Hinshaw & Seferovic, 1986; Kalo & Kemppinen, 1993) and con"rmed by MS. A reference butter oil of known TG composition, which had served as test fat in EC collaborative trials (Precht, 1991), was used to determine the response factors (RF) for quantitative studies of TG composition as described in a previous paper (Lozada, de la Fuente, Fontecha & JuaH rez, 1995). To quantify the relative percentages of TLC fractions, a solution of the TG trinanoin (C27, Sigma, Chemical CO., St. Louis, MO) was added as an internal standard to each TLC fraction scraped o! from the AgNO -TLC plate before the GC  analysis.

For the determination of fatty acid composition a BCR reference fat (CRM 164) was used. 2.2. Separation of triglycerides by AgNO3 -TLC TLC glass plates (20;20 cm) with silica gel (0.25 mm) (Merck Darmstadt, Germany) were incubated with 20% aqueous solution of AgNO (Panreac) overnight. The  plates were then activated at 1003C for 30 min and 0.5 mL of the fat solution (12 mg mL\ in chloroform, containing 0.5}0.8% ethanol) was applied to the plate. Triolein and tristearin were also applied to the edge of the plate as qualitative standards. The plate was developed twice in a saturated chamber in chloroform with 15 cm migration. After drying, the plates were sprayed with a 0.15% ethanol solution of 2,7-dichloro#uorescein and the bands were visualized under UV light. Bands were scraped o!, and 20 lL of 3.15 mg mL\ solution of C27 was added to each band as internal standard. The TGs were extracted four times with 20 mL diethyl ether protected with butylhydroxytoluene (Panreac), "ltered, and the solvent was evaporated under reduced pressure. The residue was resuspended in 140 lL hexane and the solution was used for the analysis of TGs and fatty acids. Fractionation was repeated twice, and the corresponding fractions were injected in duplicate for GC and GC}MS analysis. 2.3. Gas chromatographic analysis of triglycerides For the analysis of total TGs in goat's milk fat, 20 mg of fat was dissolved in 0.5 mL hexane. For the analysis of both goat's milk fat and fractions obtained by AgNO  TLC, 0.2 lL was injected into the gas chromatograph. The triglyceride analyses were performed on an Autosystem Gion 4072042 gas chromatograph (Perkin-Elmer, Beacons"eld, UK) equipped with an automatic injector (split/splitless) and programmed temperature. A capillary column (30 m;0.22 mm i.d.), supplied by Restek (Bellefonte, PA), Rtx-65 TG (35% dimethyl, 65% diphenyl polysiloxane) (d "0.10 lm) was used. Experi mental chromatographic conditions were as follows: the initial temperature (2203C) was raised to 3203C at a rate of 153C min\ and then to 3553C at a rate of 73C min\ and then held at this temperature for 20 min. The injector and detector temperatures were 355 and 3703C, respectively. The pressure at the top of the column was 25 psig, the split ratio was 1 : 4 and the carrier gas was helium. The calculated #ow rate was 0.8 mL min\. 2.4. Gas chromatographic analysis of fatty acids For the preparation of methyl esters of goat's milk fat, 0.1 g of fat was dissolved in 1 mL hexane and 0.05 mL of 2 M potassium hydroxide in methanol was added as described by Christopherson and Glass (1969). For the analysis of the fractions obtained by AgNO -TLC the 

J. Fontecha et al. / International Dairy Journal 10 (2000) 119}128

"nal hexane solution was methylated according to the same procedure. In both cases, 0.2 lL was injected into a gas chromatograph. A Perkin-Elmer Model 8420 gas chromatograph (Beacons"eld, UK) was used, equipped with programmed temperature vaporizer inlet, #ow splitter and hydrogen #ame ionization detector. The carrier gas was helium, the split ratio was 1 : 20 and the pressure at the top of the column was 25 psig. Calculated #ow rate was 0.7 mL min\. The column was a WCOT silica capillary (50 m;0.22 mm i.d.) containing a Silar 5CP (50% phenyl, 50% cyanopropyl) stationary phase (d "0.22 lm) (Chrompack, Middelburg, The Nether lands). Experimental chromatographic conditions were as follows: the initial temperature of 603C was maintained for 3 min, then raised to 1903C at a rate of 153C min\. The "nal temperature was maintained for 30 min. The injector and detector temperature was 2003C.

121

Fig. 1. Fractionation of triglycerides of goat's milk fat by AgNO  TLC. See Table 1 for bands identi"cation.

2.5. Gas chromatography}mass spectrometry The mass spectrometry of goat's milk fat fractions obtained by AgNO -TLC, was performed with  a Finnigan MAT95 s (Finnigan, Bremen, Germany) highresolution mass spectrometer interfaced with a HewlettPackard 5890 Series II gas chromatograph. The column and the experimental chromatographic conditions were the same as in the GC analysis of TGs. Electron impact (EI) spectra were recorded at 70 eV. Full spectra (50}1000 amu) were recorded at a scan speed of 2 s decade\ over the entire elution pro"le. Data were analysed using an ICIS II Data System from Finnigan MAT.

Table 1 Total wt% of AgNO -TLC fractions (mean values and standard devi ations of the "ve samples obtained from the corresponding herds) of goat's milk fat AgNO -TLC  fractions

Wt%

Characterization of triglycerides

A

38.5$2.62

B

16.5$1.76

C D

28.9$4.02 16.0$1.93

Saturated (C6 and longer chain fatty acids) Saturated (C4 and longer chain fatty acids) Monounsaturated Polyunsaturated

3. Results 3.1. Triglyceride separation by AgNO3 -TLC: fatty acid and triglyceride composition of the diwerent fractions Using AgNO -TLC, the TGs from the goat's milk fat  were separated into four main fractions (Fig. 1); although fractions A and D included two bands, they were too close to be individualized. Table 1 gives the relative amounts (wt%) of material recovered from the di!erent AgNO -TLC fractions (mean values and standard devi ations of the "ve goat's milk fat samples obtained from the corresponding herds) and its characterization. Table 2 shows the fatty acid composition (mol%) of TGs of the goat's milk fat and the four fractions obtained by AgNO -TLC. A large proportion of the fatty acids in  fractions A and B was saturated (95.3 and 94.3%, respectively) and hence the TGs contained in both fractions were identi"ed as trisaturates (SSS). Fraction B contained 94% of the butyric acid detected in SSS fractions, while fraction A contained larger amounts of C8, C10 and C12 fatty acids (73, 80 and 74% of the total SSS fractions, respectively).

The TGs in fraction C contained nearly one-third of the monounsaturated fatty acids (29%) and hence were identi"ed as monounsaturated (SSM). The di!erence between this percentage and the theoretical value (33.3%) could have been due to overlapping with the saturated fractions, which also contained some monounsaturated acids (0.9 and 1.5% in fractions A and B, respectively) and also due to the presence of C18 : 2 and C18 : 2U

in the SSM band (1.2 and 0.8%, respectively, Table 2). Fraction D contained 35% of monounsaturated fatty acids, mainly C18 : 1 (78% of the total), 14.6% of C18 : 2 (including 0.8% of C18 : 2U ) and 1.4% of C18 : 3. Fig. 2 shows the molar distribution of the TGs in goat's milk fat by CN, degree of unsaturation and, in the saturated TGs, length of the short fatty acid chain. The values were calculated by multiplying the molar percentage of each TG class of total goat's milk fat by the relative proportions of the four fractions obtained by AgNO  TLC in the corresponding class of TGs calculated from the GC data. Data are shown from CN28, because the molar percentages of TG classes CN22, CN24 and CN26 were low (0.09, 0.19 and 0.45% respectively).

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3.2. Composition of molecular species of main triglycerides Fig. 3 shows a chromatogram of the goat's milk fat (from the herd 1 sample), in which a total of 124 peaks were detected. Table 3 shows the relative retention times (RRT) and the molar percentages (mean values and standard deviations of the "ve samples obtained from the corresponding herds) of each peak. Also, where possible Table 2 Fatty acid molar composition (%) of TGs of goat's milk fat and of the fractions obtained by AgNO -TLC (values are means of the "ve sam ples obtained from the corresponding herds) Fatty Acid

C4 C6 C8 C10 C10 : 1 C12 C12 : 1 C14 C14 : 1# iC15 aiC15#C15 C15 : 1 iC16#C16 C16 : 1 iC17#aiC17#C17 C17 : 1 C18 C18 : 1 C18 : 2 C18 : 3 C18 : 2U

C20 C20 : 1 Others

Total fat

5.09 4.42 4.15 12.91 0.36 5.62 0.21 9.86 0.39 0.83 0.09 25.38 1.41 1.26 0.34 7.17 15.46 2.83 0.35 0.57 0.11 0.05 1.14

Mean values of two replicates.

AgNO -TLC fraction  A

B

C

D

0.56 3.83 6.70 18.57

19.26 6.87 5.81 10.79

8.72 13.54 0.85 2.18

6.96 0.05 10.01 0.73 1.67

30.54

24.26

0.88

1.05

9.57 0.90

7.17 1.47

4.43 3.94 4.57 11.74 0.60 5.21 0.41 7.95 0.69 1.06 0.37 19.64 2.79 0.66 0.60 6.58 24.24 1.19

0.06 0.18

0.12 0.42

2.92

3.37

3.01 2.50 3.98 7.75 1.84 3.80 0.40 5.15 0.86 1.08 0.53 12.30 3.98 0.98 0.73 4.47 27.35 13.80 1.44 0.76 0.28 0.18 2.83

0.76 0.11 0.03 2.43

the table indicates the tentative identi"cation of TGs contained in each peak. Data are shown from CN28, because no TG was identi"ed in TG classes CN22, CN24 and CN26. As indicated, the molecular species of TG were "rst tentatively identi"ed following previous studies carried out in similar conditions (Myher et al., 1988; Hinshaw & Seferovic, 1986; Kalo & Kemppinen, 1993). The con"rmation of identity of a TG species was made on the basis of GC and GC}MS analysis of the intact TG recovered from the four fractions obtained by AgNO  TLC. As an example, Fig. 4 shows the partial chromatograms corresponding to CN36 class TG of the total milk fat and of the four AgNO -TLC fractions. A total of eight  peaks (49}56) were recognized in the goat's milk fat, some of them containing contributions from more than one AgNO -TLC fraction. Table 4 illustrates the method  followed for the identi"cation of individual TGs in the CN36 class TG. The table indicates the composition of TG CN36 in each AgNO -TLC fraction determined by  CG}MS, the situation of each peak in relation to the RRT of total fat, and the molar percentages of the peaks for the individual AgNO -TLC fractions. The unidenti "ed peaks (NI) in each band are indicated, but only where their molar percentages multiplied by the mass proportion of the corresponding band in the total fat were '0.1%. The CG}MS analysis also provides estimations on the relative abundance of the molecular species within each peak. Thus, in Table 3, the most abundant TG in each peak is underlined. More than one species of TG was identi"ed in 37 peaks in the chromatogram in Fig. 3. Some peaks (i.e. peaks 50, 51, 54 and 55, see also Table 4 and Fig. 4) were located in more than one of the AgNO -TLC fractions. In addition,  more than one TG was identi"ed in some peaks of chromatograms from each of the AgNO -TLC fractions  (i.e. those corresponding to peaks 49 and 50 in fraction A and 50}52 in fraction C, Table 4).

Fig. 2. Distribution (mol%) of triglycerides in goat's milk fat according to carbon number and degree of unsaturation. A: saturated (C6 and longer chain fatty acids); B: saturated (C4 and longer chain fatty acids); C: monounsaturated; D: polyunsaturated.

J. Fontecha et al. / International Dairy Journal 10 (2000) 119}128

123

Fig. 3. Capillary GC pro"le of triglycerides in a goat's milk fat sample collected from herd 1.

Eleven peaks, situated at the beginning of each TG class from CN36 to CN52 (49, 50, 57, 58, 67, 75, 83, 90, 98, 105, and 112 in the chromatogram in Fig. 3), were largely located in fraction A. The largest peaks in TG classes CN28}CN38 were preferentially located in fraction B. Of these, numbers 24, 33, 42 and 51 were the most abundant in that fraction, where individual molar percentages ranged from 7 to 16%. The 10 largest peaks in the chromatogram of band C corresponded to numbers 58, 60, 68, 69, 76, 77, 84, 91, 99 and 106. However, only the last six peaks (from CN42 to CN52) were preferentially located in that band. The largest peaks in fraction D corresponded to numbers 73, 80, 85, 92, 107 and 114 (from CN40 to CN52 in the chromatogram for total fat). One hundred and thirty-seven molecular species of TGs were identi"ed: 69 (50%) saturated, 41 (30%) monoand 27 (20%) polyunsaturated. By length of chain, 59 (43%) of the molecular species identi"ed contained short-chain fatty acids (C4, C6), 101 (74%) medium-chain fatty acids (C8}C14), and 112 (82%) long-chain fatty acids (C16}C18).

4. Discussion The SSS TGs represented 55% of the total TGs (Table 1), a value higher than in cow's milk (47%, Fraga et al., 1998). All the SSSs of cow's milk fat are located in a single TLC band in most of the references consulted; however, as in the present case, Myher et al. (1988) and Fraga et al. (1998) reported that they can be separated into two bands di!erentiated mainly by the chain length of the short-chain fatty acid. The SSSs that contain butyric acid (fraction B) only represented 30% of the total SSS, which was much lower than that reported for cow's milk (46%, Fraga et al., 1998) as a consequence of the lower butyric acid content of goat's milk fat. Monounsaturated TGs of cow's milk were separated into two TLC fractions in most of the references consulted. This separation was mainly controlled by the chain length of the short-chain fatty acid (Fraga et al., 1998) or by the geometric con"guration of the unsaturated fatty acids (Parodi, 1980; Myher et al., 1988; Laakso & Kallio, 1993). However, using similar experimental conditions as

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J. Fontecha et al. / International Dairy Journal 10 (2000) 119}128

Table 3 TG composition (mol%) of goat's milk fat and identi"cation of TGs according to data of AgNO TLC-MS and the relative retention time (RRT) to C27  Peak no.

CN


RRT

mol% SD

Triglycerides identi"ed

13 14 15 16 17 18 19 20 21

C28

1.066 1.074 1.089 1.096 1.110 1.126 1.144 1.160 1.179

0.13 0.26 0.35 0.23 0.05 0.16 0.07 0.07 0.02

0.011 0.016 0.018 0.066 0.024 0.021 0.006 0.005 0.002

8,8,12/8,10,10 6,10,12 4,10,14 4,8,16

22 23 24 25 26 27 28 29 30

C30

1.205 1.216 1.234 1.242 1.253 1.275 1.286 1.300 1.319

0.36 0.39 1.13 0.13 0.25 0.17 0.13 0.10 0.05

0.028 0.009 0.077 0.026 0.017 0.022 0.014 0.009 0.017

8,10,12/10,10,10 6,10,14 4,10,16/4,12,14

31 32 33 34 35 36 37 38

C32

1.345 1.362 1.377 1.396 1.415 1.431 1.453 1.475

0.86 1.02 1.37 0.60 0.28 0.23 0.16 0.09

0.038 0.088 0.034 0.028 0.023 0.031 0.023 0.008

8,10,14/8,12,12/10,10,12 6,10,16/6,12,14 4,12,16/4,14,14/6,8,18 : 1 4,10,18 : 1

39 40 41 42 43 44 45 46 47 48

C34

1.504 1.515 1.525 1.552 1.569 1.584 1.596 1.614 1.638 1.663

0.83 0.79 1.17 2.46 0.50 0.11 0.38 0.34 0.31 0.11

0.166 0.194 0.237 0.165 0.040 0.010 0.031 0.025 0.023 0.017

8,10,16/8,12,14/10,10,14 6,10,18/6,12,16/6,14,14

49 50

C36

1.697 1.718

2.73 2.32

0.182 0.205

51

1.746

3.29

0.409

52 53 54 55 56

1.764 1.786 1.810 1.834 1.857

1.25 0.55 0.49 0.36 0.25

0.061 0.023 0.023 0.048 0.013

8,12,16/8,14,14/10,12,14 6,12,18/6,14,16/8,10,18 : 1/10, 10,16 : 1 4,14,18/4,16,16/6,12,18 : 1/6, 14,16 : 1 4,14,18 : 1/4,16,16 : 1 4,15,18/4,16,17 4,16,17/4,16 : 1,16 : 1 4,16,17/4,14 : 1,18 : 2

1.892 1.921

3.69 3.43

0.276 0.301

1.946 1.971 1.983 1.994 2.005 2.012 2.038 2.062

2.00 3.14 0.04 0.03 0.37 0.42 0.48 0.21

0.109 0.172 0.012 0.007 0.024 0.037 0.024 0.044

2.097

5.20

0.513

2.128

2.39

0.135

57 58

C38

59 60 61 62 63 64 65 66 67 68

C40

4,10,i15

4,8,18 : 1 6,10,15

Peak no.

RRT

mol% SD

Triglycerides identi"ed

2.154 2.181 2.192 2.206 2.221 2.245

2.34 0.81 0.48 0.85 0.53 0.70

0.128 0.150 0.056 0.081 0.030 0.038

4,18,18/6,16,18 : 1/8,16,16 : 1 4,18,18 : 1

2.308 2.333

4.64 2.14

0.510 0.257

2.344 2.364 2.392 2.405 2.432 2.476

1.61 1.01 0.69 0.44 0.96 0.26

0.257 0.072 0.042 0.031 0.085 0.054

10,14,18/10,16,16/12,14,16 10,14,18 : 1/10,16,16 : 1/12,14, 16 : 1 8,16,18 : 1 6,18,18 : 1 8,16 : 1,18 : 1/10,14,18 : 2 6,18 : 1,18 : 1 8,17,18 : 1/8,16 : 1,18 : 2 6,18 : 1,18 : 2

2.515

2.69

0.403

84

2.545

4.13

0.434

85 86 87 88 89

2.587 2.603 2.618 2.638 2.686

1.61 0.17 0.13 0.80 0.37

0.304 0.109 0.069 0.063 0.019

2.722 2.763

1.64 2.49

0.077 0.150

2.806 2.838 2.854 2.874 2.896 2.934

1.58 0.25 0.36 0.36 0.26 0.41

0.068 0.021 0.034 0.062 0.075 0.047

69 70 71 72 73 74 75 76

4,12,18/4,14,16 6,10,18 : 1/8,10,16 : 1 4,12,18 : 1 4,i15,16 4,ai15,16 4,15,16

8,14,16/10,12,16/10,14,14 6,14,18/6,16,16/8,12,18 : 1/ 10,10,18 : 1/10,12,16 : 1 4,16,18/6,14,18 : 1/6,16,16 : 1 4,16,18 : 1/4,18,16 : 1 4,17,18/4,16 : 1,18 : 1 4,17,18/4,16,18 : 2 4,17,18 4,16 : 1,18 : 2 8,14,18/8,16,16/10,14,16/ 12,14,14 6,16,18/10,12,18 : 1/10,14,16 : 1

Mean values of "ve herds, three replicates per herd.
Carbon number. Standard deviation. Underlined TG are the more abundant in the corresponding peak.

C42

77 78 79 80 81 82 83

4,14, ai15 4,14,15

CN


90 91

C44

C46

92 93 94 95 96 97

4,18 : 1,18 : 1 4,18,18 : 2 4,18 : 1,18 : 2

8,18,18/10,16,18/12,14,18/12, 16,16/14,14,16 8,18,18 : 1/10,16,18 : 1/12,14, 18 : 1 8,18 : 1,18 : 1/10,16 : 1,18 : 1 8,18 : 1,18 : 2 12,16,18/14,16,16 10,18,18 : 1/12,16,18 : 1/14,14, 18 : 1 10,18 : 1,18 : 1 10,18 : 1,18 : 2 14,16,17

98 99 100 101 102 103 104

C48

2.977 3.026 3.068 3.084 3.129 3.165 3.239

1.09 2.13 0.55 0.50 0.44 0.55 0.34

0.100 0.079 0.072 0.045 0.033 0.052 0.040

14,16,18/16,16,16 14,16,18 : 1 12,18 : 1,18 : 1

105 106 107 108 109 110 111

C50

3.289 3.350 3.403 3.426 3.480 3.526 3.589

0.79 2.24 0.92 0.54 0.44 0.37 0.36

0.174 0.300 0.080 0.044 0.091 0.077 0.125

16,16,18 14,18,18 : 1/16,16,18 : 1 16,16 : 1,18 : 1 14,18 : 1,18 : 1

112 113 114 115 116 117 118

C52

3.676 3.751 3.826 3.869 3.939 4.024 4.073

0.30 1.15 1.53 0.33 0.44 0.13 0.18

0.123 0.411 0.306 0.062 0.203 0.085 0.124

16,18,18 16,18,18 : 1 16,18 : 1,18 : 1

119 120 121 122 123 124

C54

4.175 4.272 4.368 4.459 4.507 4.606

0.11 0.25 0.43 0.38 0.11 0.08

0.061 0.130 0.224 0.112 0.063 0.028

16,18 : 1,18 : 2 16,18 : 2,18 : 2 18,18,18 : 1 18,18 : 1,18 : 1 18 : 1,18 : 1,18 : 1 18,18 : 1,18 : 2

J. Fontecha et al. / International Dairy Journal 10 (2000) 119}128

125

Fig. 4. Partial gas chromatograms of triglycerides (CN36 zone) of goat's milk fat (GMF) and of the AgNO -TLC fractions: A: saturated (C6 and  longer chain fatty acids); B: saturated (C4 and longer chain fatty acids); C: monounsaturated; D: polyunsaturated. Peak numbers are identi"ed in Tables 3 and 4.

Fraga et al. (1998), the SSMs in goat's milk fat were located in a single TLC fraction, probably because of the low molar percentages of butyric acid in the SSM fraction (4.4%, Table 2) in comparison with cow's milk fat. In any event, separation of the SSMs has not always been observed in cow's milk (Lund, 1988; Kemppinen & Kalo, 1993). The percentage of polyunsaturated TGs (16%) was lower than that was found in cow's milk (20%) by Fraga et al. (1998), whereas the percentage of monounsaturated TGs was similar in both the species. These results con"rm that goat's milk contains a lower concentration of total unsaturated TGs (45%) than cow's milk (52%; Fraga et al., 1998); a slightly low inter-species di!erence was reported by Ruiz-Sala et al. (1996) (51 vs. 55%). The study of the fatty acid composition of the TGs fractions indicated that the individual saturated fatty acids of goat's milk fat are distributed into saturated, mono- and polyunsaturated TGs in a very similar proportion (mean values: 66, 25 and 9%, respectively). This distribution agrees with that theoretically calculated as-

suming that the saturated fatty acids are included in the di!erent TLC fractions independent of their chain length. The saturated TGs have a unimodal molar distribution with maxima at CN40 and CN36 in fractions A and B, respectively (Fig. 2). In fraction B, the sum of TG from CN22 to CN38 made up 90% of the total fraction, as expected in a band that mainly contains butyrates. The proportion of SSSs in short-chain TGs (CN28}CN38) found in this study (60%) was higher than in cow's milk containing 49% of SSSs in short-chain TGs (Fraga et al., 1998). This di!erence links up with the need to obtain a TG composition with the appropriate melting point (Gresti et al., 1993) to allow the fat to be secreted. Thus, although the percentage of saturated TGs is higher in goat's milk than the cows milk, they have a shorter chain length. The distribution of SSMs was similar to that of total TGs in goat's milk fat. Most of the SSMs were found in TGs CN38}CN50, and only 17% were of long chain. The proportions of polyunsaturated TGs (band D) increased with the CN and represented from 39 to 89% in the long-chain TGs. This distribution also favours an

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Table 4 Location of the C36 TGs of the goat's milk fat in the AgNO -TLC fractions according to the relative retention time (RRT) to C27  Peak no.

49

CN

C36

RRT

1.697

50

1.718

51

1.746

52

1.764

53

1.786

54

1.810

55

1.834

56

1.857

AgNO -TLC fractions  A

B

8,12,16/8,14,14/10,12,14 (6.98) 6,12,18/6,14,16 (4.20)

8,12,16 (0.86) 6,14,16 (1.84) 4,14,18/4,16,16 (16.10)

NI

4,15,18/4,16,17 (1.70) 4,16,17 (Tr) 4,16,17 (0.77)

C

D

8,10,18 : 1/10,10,16 : 1 (2.24) 6,12,18 : 1/6,14,16 : 1 (1.36) 4,14,18 : 1/4,16,16 : 1 (2.49) NI

NI

4,16 : 1,16 : 1 (1.37) 4,14 : 1,18 : 2 (1.95) NI

Carbon number. NI, not identi"ed TGs (see text). In parentheses are the molar percentages in each fraction of TG identi"ed.

appropriate melting point because a higher degree of unsaturation will o!set the e!ect of a longer chain on the melting point of TGs. In qualitative terms, the chromatogram for TGs in caprine's milk fat shown in Fig. 3 is similar to that for bovine's milk published by Fraga et al. (1998) using the same experimental conditions. All the TGs listed in Table 3 have been identi"ed previously in cow's milk fat (Myher et al., 1988; Gresti et al., 1993), but a comparison with the papers cited suggests that there are considerable quantitative di!erences from one species to another. Similar "ndings were reported by Barron et al. (1990) and Ruiz-Sala et al. (1996) studying simultaneously cow's and goat's milk. Using the same procedure (HPLC and GC), the two groups of workers respectively identi"ed 116 and 181 molecular species of TG in milk from both species. Sixty three in the "rst case and 84 in the second case coincided with the ones identi"ed in the present study. Most of the TGs described by these authors and not identi"ed here are unsaturated TGs, particularly TGs containing trans-fatty acids or branched-chain TGs. As in previous studies it was not possible to quantify the TGs in terms of molecular species because, as mentioned, some peaks in chromatogram in Fig. 3 contained more than one molecular species. According to Table 3, only the three peaks (67, 75 and 84, located in TGs CN40, CN42 and CN44, respectively) individually exceeded 4% of the total content. All the TGs identi"ed in these peaks contained one of the three acids C8, C10 and C12; in all cases C18 : 1 was the unsaturated acid in the monounsaturated TGs. Mass spectrometry analysis showed that TGs 10,14,16; 10,16,16 and 10,16,18 : 1 are probably the

most abundant in goat's milk. In cow's milk, using the same experimental conditions (Fraga et al., 1998), the peaks for the corresponding RRTs made up only 3.6% of the total fat. Similarly, Barron et al. (1990) found the greatest di!erence between goat's and cow's milk in the TGs with partition numbers from 38 to 42, which contained, among others, the cited TGs. And again, when GC analyses in TG class of goat's and cow's milk fat were compared (Fontecha et al., 1998), the largest quantitative di!erences were found for TGs CN40, CN42 and CN44. There were other six peaks of over 2.5% located in the TG classes from CN36 to CN44. Four of these (49, 57, 58, and 83) contain one or more of the fatty acids C6, C8, C10 and C12. Particularly abundant are the TG species containing two identical acyl moieties (10,14,14; 6,16,16 and 14,14,16). The other two peaks (51 and 60) preferentially contain 4,16,16 and 4,16,18 : 1, respectively. In cow's milk, Gresti et al. (1993) also observed a high proportion of 6,16,16 which was higher than the corresponding random proportion; however, the high proportion of 10,14,14 was not detected in cow's milk, probably because it contained less C10. In cow's milk from the six peaks with equivalent RRT only the last two peaks which contain butyric acid (molar percentages of total fat 4.4 and 2.2, respectively) are comparable to those of goat in quantitative terms (Fraga et al., 1998). Further 21 peaks with molar percentages '1% were found, three of them (85, 92 and 114) were located preferentially in band D. The majority of these TGs contain two molecules of oleic acid; TG 16,18 : 1,18 : 1 was the most abundant polyunsaturated TG, as in cow's milk (Fraga et al., 1998). This TG is also the most abundant in

J. Fontecha et al. / International Dairy Journal 10 (2000) 119}128

human milk, where it constitutes about 10% of all TGs (Currie & Kallio, 1993). The proportions of the peaks where the TGs 16,18 : 1,18 : 1 and 14,18 : 1,18 : 1 were located (114 and 108) in the present study were about 2.5}3 times smaller than in cow's milk (Fraga et al., 1998). These TGs are negatively correlated with the butter "rmness (Bornaz, Fanni & Parmentier, 1993). Other medium-chain TGs associated with texture (10,12,18 : 1 and 8,16,18 : 1, located in peaks 68 and 77) exhibited higher proportions in goat's milk than in cow's milk. As regards the variability of TG proportions in the di!erent herds, the standard deviations for each TG class from CN28 to CN48 were low; the corresponding variation coe$cients (VC) ranged from 10 to 12%. However, TGs CN50, CN52 and CN54 showed high VC (mean values: 21, 44 and 45%, respectively) as a consequence of the high standard deviations in each peak. There has been little published on the variation in molecular species of TGs of cow's milk fat, but the data of Bornaz, Novak and Parmentier (1992) using HPLC show signi"cant regional and seasonal variations, especially for unsaturated TGs CN38, CN40, CN50 and CN52, which is consistent with our own results. Also, in a study of seasonal variation Hinrichs, Heinemann and Kessler (1992) found that most of the more-variable TGs contained C18 : 1. 5. Conclusions The distribution of TGs in goat's milk fat by CN and the degree of unsaturation can be successfully determined using a combination of the AgNO -TLC technique and  capillary GC. Fifty "ve percent of total goat's milk fat TGs are trisaturated, and a third of these contain butyric acid. The bulk of monounsaturated TGs are of medium chain length; only 17% are of long chain. The percentage of polyunsaturated TGs (16% of total TGs) increases with the CN and is high (mean value 52%) in TGs CN46}CN54. The higher proportion of short-chain TGs in the trisaturated TGs as compared to more unsaturated TGs in cow's milk fat relates to the need to obtain a TG composition with the appropriate melting point to allow the fat to be secreted. As in previous studies, it was not possible to quantify molecular species of TG since some peaks in the chromatogram contained more than one TG. However, 137 molecular species were identi"ed: 50% trisaturated, 30% mono- and 20% polyunsaturated. The most important in quantitative terms were the medium-chain TGs containing C8, C10 or C12, and the C18 : 1 as unsaturated fatty acid. Although the qualitative chromatographic pro"le of goat's milk fat TGs is similar to that of cow's milk, there are noticeable quantitative di!erences in the TGs of low or medium molecular weight associated with the short- and medium-chain fatty acids that are most abundant in either animal species.

127

Acknowledgements This work was supported by a grant from Comunidad de Madrid (Project 06G/049/96) and from FundacioH n Alfonso MartmH n Escudero.

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