Milk Fat Triacylglycerols Research Articles

Regiospecific analysis of fractions of bovine milk fat triacylglycerols with the same partition number.

Bovine milk fat was fractionated using preparative reversed-phase high-performance liquid chromatography. The conditions consisted of two successive linear gradients of acetonitrile and tert-butylmethylether, followed by a final isocratic mixture of the two eluants, leading to triacylglycerols grouped by their partition number (PN). Fractions corresponding to partition numbers 32 to 50 were isolated and analyzed for fatty acid distribution between sn-1,3 and sn-2 positions by Grignard degradation. Results showed that the fatty acid distribution in milk fat triacylglycerols is nonrandom. The distribution of short-chain fatty acids, stearic (predominantly at sn-1,3 position) and palmitic (predominantly sn-2 position), did not change with triacylglycerol size. Medium-chain fatty acids were predominantly located at sn-2 position, but their proportion at this position decreased with triacylglycerol size. Oleic acid distribution was also size-dependent in that it was located in high proportions at sn-2 position in smaller triacylglycerols and vice versa. Results also showed that the sn-2 position was more unsaturated than sn-1,3 position in the PN range from 32 to 40, but it was more saturated in triacylglycerols with higher PN.

The composition of new zealand milk fat triacylglycerols by reversed‐phase high‐performance liquid chromatography

AbstractA reversed‐phase high‐performance liquid chromatography (HPLC) method was developed for the purpose of analyzing milk fat triacylglycerols by evaporative light scattering detection. With a binary solvent system, comprising dichloromethane and acetonitrile, the method allowed a separation of 61 distinct peaks, on the basis of chainlength and number of double bonds. Triacylglycerols of differing partition numbers were clearly resolved, and the resolution between peaks of the same partition number was high. An argentation thin‐layer chromatography method, separating triacylglycerols on the basis of chainlength and degree of unsaturation, provided nine band extracts that were analyzed by HPLC. By using existing fatty acid methyl ester data of these bands, an identity for each HPLC peak has been proposed.

Separation of milk fat triacylglycerols by argentation thin‐layer chromatography

AbstractArgentation thin‐layer chromatography was investigated as a method of obtaining detailed compositional information about milk fat. A modified argentation thin‐layer chromatography procedure, developed to optimize the separation of the complex mixture of total milk fat triacylglycerols, provided nine different groups of triacylglycerols, based on both the degree of unsaturation and the total length of fatty acid groups. Fatty acid methyl ester (FAME) analysis was performed to determine the composition of each band. Separation on the basis of chainlength was most pronounced among the fully saturated triacylglycerol groups, as evidenced by the high level of C4:0 and C6:0 in bands 7 and 8, respectively. For the cis‐monoenoic triacylglycerols, the separation of C4:0 and C6:0 was less distinct. The cis,cis dienes and other dienoic, trienoic, or tetraenoic species were principally observed in two bands of retention factor <0.08 on the chromatography plate. Minimal cross‐contamination of bands was observed, with the exception of the lowest of the trisaturate bands, band 7, in which trans‐monoenes were found to be present. Three samples from different points of the New Zealand dairy season were separated by argentation thin‐layer chromatography, and their FAME distributions were measured. In addition to differences in the masses of band extracts from these samples, levels of C10:0 and C12:0 in all bands, and levels of monounsaturates in the dienoic and trienoic bands, were found to differ. These changes were generally consistent with a pattern of decreasing fat hardness over the November to March period of a typical dairy season.

Identification of milk fat triacylglycerols by capillary supercritical fluid chromatography‐atmospheric pressure chemical lonization mass spectrometry

Identification of milk fat triacylglycerols was accomplished by capillary supercritical fluid chromatography (SFC) combined with atmospheric pressure chemical ionization mass spectrometry [(APCI)MS]. Supercritical carbon dioxide was the carrier fluid in SFC. Ionization was achieved by introducing vapor of ammonia in methanol into the ionization chamber which resulted in the formation of abundant [M + 18]+ and [M - RCCO]+ ions of triaclyglycerols. These ions defined both the molecular weight and the fatty acid constituents of a triacylglycerol, respectively. SFC on a nonpolar stationary phase provided an efficient separation of triacylglycerols according to the combined number of carbon atoms in the acyl chains a molecule. In addition to the identification of the major chromatographic peaks representing molecules with 26-54 acyl carbons, minor peaks representing triacylglycerols with an odd number of acyl carbons were separated and identified. Furthermore, compositional information on partially separated isobaric triacylglycerols, which differed substantially in the chain length of the fatty acyl residues, was achieved within some of the peaks. A new finding of the present study was the formation of abundant [M + 18]+ ions of saturated triacylglycerols in addition to diagnostic fragment ions,being of primary importance in structure elucidation. This extends the applicability of capillary SFC-(APCI)MS in the analysis of both saturated and unsaturated triacylglycerols.

Determination of positional distribution of butyryl groups in milkfat triacylglycerols, triacylglycerol mixtures, and isolated positional isomers of triacylglycerols by gas chromatography and 1H nuclear magnetic resonance spectroscopy.

Positional isomers (1-butyryl-2X-3Y-rac-glycerol and 2-butyryl-1X-3Y-rac-glycerol; X,Y = long-chain acyls) of saturated triacylglycerols (TAG) within 34 and 40 acyl carbons were shown to separate in two chromatographic peaks on immobilized phenyl(65%)methylsilicone column by gas-liquid chromatography, and on reversed-phase ODS-1 column by high-performance liquid chromatography. The analysis of 500-MHz 1H nuclear magnetic resonance (NMR) spectra showed distinct differences between 2-butyryl-1X-3Y-rac-glycerol and 1-butyryl-2X-3Y-rac-glycerol isomers in the resonance signals of methylene and methine protons of glycerol backbone, and carbon-2 methylene of acyl groups, and methyl protons of butyryl group. The 1H NMR spectra of three interesterified mixtures of three monacid TAG containing saturated butyrate and caproate TAG and unsaturated butyrate TAG showed that triplets of methyl protons of butyryl groups at sn-1(3)- and sn-2-positions in saturated and unsaturated TAG had similar chemical shifts and that the chemical shift of caproyl methyl protons was different from those of butyryl methyl protons. The positional distribution of butyryl groups in isolated positional isomers of butyrate TAG, interesterified TAG mixtures, and natural and interesterified butteroil can be determined by integration of these signals.

High-performance liquid chromatography with light-scattering detection and desorption chemical-ionization tandem mass spectrometry of milk fat triacylglycerols.

The utility of reverse-phase high-performance liquid chromatography (HPLC), desorption chemical-ionization mass spectrometry (DCI-MS) and tandem mass spectrometry (MS/MS) for the characterization of triacylglycerols in complex mixtures has been further explored. Triacylglycerols of anhydrous bovine milk fat were separated by using two reverse-phase C18 HPLC columns, and eluents were monitored with an evaporative light-scattering detector. Fifty-eight fractions were resolved and analyzed by positive ion isobutane DCI-MS. The formation of protonated molecules and of major fragments corresponding to the random loss of any one of the constituent fatty acids readily identified acyl carbon numbers and the number of double bonds within each fatty acid. MS/MS was only required when the original mass spectra indicated the presence of more than one triacylglycerol or of impurities in a fraction. Protonated molecules produced by DCI were fragmented using high energy collisional activation, and the resulting ions were detected by MS/MS. Odd-chain triacylglycerols were also readily distinguished using this methodology. The positive ion DCI and MS/MS techniques described here demonstrate the usefulness of this approach for the characterization of triacylglycerols in complex mixtures.

Determination of positional distribution of short‐chain fatty acids in bovine milk fat on chiral columns

AbstractThe positional distribution of acetic and butyric acids in bovine milk fat triacylglycerols was determined by chiral‐phase high‐performance liquid chromatography (HPLC) of the derived diacylglycerols. Enriched fractions of acetic and butyric acid‐containing triacylglycerols were isolated by normal‐phase thin‐layer chromatography (TLC) from a molecular distillate of butter oil, and they were fully hydrogenated. Mixedsn‐1,2(2,3)‐ andX‐1,3‐diacylglycerols of short‐ and long‐chainlength, which were generated by partial Grignard degradation of the hydrogenated triacylglycerols, were isolated by borate‐TLC. The enantiomericsn‐1,2‐andsn‐2,3‐diacylglycerols and theX‐1,3‐diacylglycerols as their 3,5‐dinitrophenylurethanes were resolved by HPLC on chiral columns. Both acetic and butyric acids were exclusively associated with thesn‐ 2,3‐ andX‐1,3‐diacylglycerols of short and long chainlength. These results establish the presence of acetic and butyric acids in thesn‐3‐position of bovine milk fat triacylglycerols. Other short‐and medium‐chainlength acids were found in progressively increasing proportions also in thesn‐1‐ andsn‐2‐positions.

Composition of Molecular Species of Triacylglycerols in Bovine Milk Fat

Triacylglycerols from bovine milk fat were fractionated by reversed-phase liquid chromatography. The fatty acid and triacylglycerol compositions of each fraction were determined by capillary gas chromatography. These data were used to determine the accurate proportions of 223 individual molecular species of even-numbered triacylglycerols, accounting for 80% of total triacylglycerols (all percentages are expressed as moles per 100mol). The three major triacylglycerols were butyroylpalmitoylacyl-glycerols, namely butyroylpalmitoyl-oleoylglycerol (4.2%), butyroyldipalm-itoylglycerol (3.2%), and butyroyl-myristoylpalmitoglycerols (3.1%)-Twenty-two triacylglycerols (>1%) contained at least two of the four major long-chain fatty acids (C14:0, C16:0, C18:0, and C18:1). Among them were eight butyroyldiacylglycerols, the proportions of which reached 19% in total but only 12% when calculated on the basis of a random distribution of the fatty acids in the triacylglycerol molecules. More generally, most of the triacylglycerols that are composed of a short-chain fatty acid (C4:0 or C6:0) and two fatty acids in the range of C12 to C18 are preferentially synthesized by the mammary gland; their proportions (36% in total) were higher than the corresponding random values (24% in total). Conversely, the total amounts of simple (.4%) and mixed (2.9%) saturated long-chain (C14:0 to C18:0) triacylglycerols were much lower than those expected from random calculation (1.9 and 6.1%, respectively).

Fatty acids generated by gastric lipase promote human milk triacylglycerol digestion by pancreatic colipase-dependent lipase

The concerted action of purified bovine gastric lipase and human pancreatic colipase-dependent lipase and colipase, or crude human pancreatic juice, in the digestion of human milk triacylglycerols was explored in vitro. Gastric lipase hydrolyzed milk triacylglycerol with an initially high rate but became severely inhibited already at low concentration of released fatty acid. In contrast, colipase-dependent lipase could not, by itself, hydrolyze milk triacylglycerol. However, a short preincubation of milk with gastric lipase, resulting in a limited lipolysis, made the milk fat triacylglycerol available for an immediate and rapid hydrolysis by pancreatic juice, and also for purified colipase-dependent lipase, provided colipase and bile salts were present. The same effect was obtained when incubation with gastric lipase was replaced by addition of long-chain fatty acid. Long-chain fatty acid increased the binding of colipase-dependent lipase to the milk fat globule. Binding was efficient only in the presence of both fatty acid and colipase. We conclude that a limited gastric lipolysis of human milk triacylglycerol, resulting in a release of a low concentration of long-chain fatty acids, is of major importance for the subsequent hydrolysis by colipase-dependent lipase in the duodenum.

Identification of the more complex triacylglycerols in bovine milk fat by gas chromatography—mass spectrometry using polar capillary columns

The fourth most volatile 2.5% molecular distillate of butteroil obtained by redistillation of the most volatile 10% cut was examined by gas chromatography on a polar capillary column (RSL-300) with electron impact and chemical ionization mass spectrometry. For this purpose the distillate was first freed from the acetyldiacylglycerols by thin-layer chromatography on plain silica gel and the remainder resolved into long and short chain length saturates, cis- and trans-monoenes, dienes and trienes by thin-layer chromatography on silver nitrate—silica gel. The order of gas chromatographic elution was established for more than 100 major and minor species making up the bulk of the molecular distillate. The results were used to derive the quantitative composition of the triacylglycerol species making up the various peaks obtained by polar capillary column gas chromatography of the total molecular distillate, which closely resembles the lower half of the molecular mass distribution of whole bovine milk fat.