Effect of phospholipids on isothermal crystallisation and fractionation of milk fat

Milk fats with different concentrations of water and phospholipids (PL) were crystallised isothermally under static conditions and their crystallisation behaviour was monitored by Differential Scanning calorimetry (DSC) and pulsed nuclear magnetic resonance (pNMR). The Avrami and the Gompertz models, which were fitted by non‐linear regression, described the crystallisation process. A significant effect of phospholipid concentration was observed using both techniques (DSC and pNMR). Especially the induction time and the Avrami growth rate constant were altered: higher amounts of PL delayed the onset of static crystallisation. A similar effect of PL on the crystallisation kinetics was observed in a small‐scale fractionation. Moreover, the filtration time of the crystal suspension and melting properties of the stearin were strongly affected by the presence of higher concentrations of PL. These observations emphasise the importance of the adequate removal of PL during anhydrous milk fat production.     

The effect of phospholipids and water on the isothermal crystallisation of milk fat

Different amounts of phospholipids (0.00‐0.07%) and water (0.00‐0.70%) were added to milk fat. The mixtures were crystallised under isothermal conditions and the crystallisation was monitored by differential scanning calorimetry and pulsed nuclear magnetic resonance. The crystallisation behaviour was described with the Avrami and Gompertz model which was fitted by non‐linear regression. Variance analysis revealed significant effects, whereas especially the induction time was influenced: higher concentrations of water seemed to decrease the induction time, while higher amount of phospholipids delayed the onset of crystallisation. No interaction effects between phospholipids and water were observed. An attempt to explain the effect of phospholipids on the induction time, based on the molecular interactions between phospholipids and triglycerides is proposed. This principle can be applied for sn‐1, 2 diglycerides as well.     

The role of amino acids in the autoxidation of milk fat

The effects of amino acids and their analogs on milk fat oxidation were examined under various conditions by measuring oxygen consumption and total unsaturated fatty acids. All the amino acids tested acted as antioxidants, characteristically extending the induction period (IP). Not only primary amino groups are responsible for the antioxidative activities of amino acids, but also the side-chain groups contribute, at least partially, to the protective effects of L-cysteine, L-tryptophan and L-tyrosine. In aqueous and HCL solutions, the antioxidative effects of L-alanine were significantly reduced. The freeze-dried L-lysine-HCL and L-alanine-HCL accelerated, while the corresponding control amino acids inhibited, milk fat oxidation.     

Membrane phospholipids and sterols in microfiltered milk fat globules

Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) was separated by manual churning. After total lipid extraction and separation of polar lipids, their phospholipid (PL) and sterol composition was measured using thin‐layer chromatography, methyl ester analyses by gas chromatography, and gas chromatography coupled to mass spectrometry. The main PL species were phosphatidylethanolamine, phosphatidylcholine and sphingomyelin. The respective fatty acid composition of each PL species was measured. Many different minor bioactive sterols were detected in the MFGM, e.g. lanosterol, lathosterol, desmosterol, stigmasterol and β‐sitosterol. No significant differences in the PL and sterol profile were found between MFGM extracted from small and large milk fat globule fractions.     

Antioxidant activity and active sites of phospholipids as antioxidants

Various compounds, representative of the major functional groups in phospholipids, phosphatidylethanolamine and phosphatidylcholine, were tested for antioxidant activity (AA) in a sardine oil system to determine the relationship between molecular structure and the AA of these compounds. AA was found to be attributable not only to the side-chain amino groups but also to the cooperative effect of the hydroxy group in the side chain. Choline and ethanolamine, side-chain moieties of phospholipids, strongly inhibited increases in peroxide values in a sardine oil mixture during storage; however, phosphatidic acid derivatives and glycerol, also major functional groups of phospholipids, did not show AA. Choline and ethanolamine have hydroxy amines as functional groups; therefore, several model reagents that contained amines and alcohols were assayed to compare the activity of the amino group with that of the hydroxy group. All basic alkylamines examined had AA as decomposers of hydroperoxides. The intramolecular hydroxy group in these amines complemented AA of the amino group. Only intramolecular alcohol, which can donate a proton, showed strong synergistic activity with AAof the basic amines, while protected groups, such as methyl ether and phosphate ester, did not show this effect.     

Effect of milk fat fractions on fat bloom in dark chocolate

Anhydrous milk fat was dissolved in acetone (1∶4 wt/vol) and progressively fractionated at 5°C increments from 25 to 0°C. Six solid fractions and one 0°C liquid fraction were obtained. Melting point, melting profile, solid fat content (SFC), fatty acid and triglyceride profiles were measured for each milk fat fraction (MFF). In general, there was a trend of decreased melting point, melting profile, SFC, long-chain saturated fatty acids and large acyl carbonnumbered triglycerides with decreasing fractionation temperature. The MFFs were then added to dark chocolate at 2% (w/w) addition level. In addition, two control chocolates were made, one with 2% (w/w) full milk fat and the other with 2% (w/w) additional cocoa butter. The chocolate samples were evaluated for degree of temper, hardness and fat bloom. Fat bloom was induced with continuous temperature cycling between 26.7 and 15.7°C at 6-h intervals and monitored with a colorimeter. Chocolate hardness results showed softer chocolates with the 10°C solid fraction and low-melting fractions, and harder chocolates with high-melting fractions. Accelerated bloom tests indicated that the 10°C solid MFF and higher-melting fractions (25 to 15°C solid fractions) inhibited bloom, while the lowermelting MFFs (5 and 0°C solid fractions and 0°C liquid fraction) induced bloom compared to the control chocolates.     

Analysis of individual phospholipids by high-performance capillary electrophoresis

A method of analysis for determination of phospholipids by micellar electrokinetic capillary chromatography (MECC) has been developed. Sodium cholate (NaCh) was found suitable as the micellar phase, and 1-propanol was important in obtaining an efficient separation of individual phospholipids and as organic solvent required for acceptable solubility of the phospholipids. With equal volatility of water and organic modifier, only minor changes in effects from the modifier occur during analysis. The influence of changes in high-performance capillary electrophoresis-MECC separation conditions were evaluated in terms of migration time, relative migration time (RMT), relative normalized area (RNA), resolution and theoretical plates per meter. The method has several advantages compared to high-performance liquid chromatography: The total time of analysis is less than 25 min, and only small amounts of reagents and sample are required. Relative standard deviations were 0.26–0.48% for RMT, 1.7–2.9% for RNA, and in the linearity test correlation coefficients of 0.999 were obtained. Results from analyses of the phospholipid compositions of soybean and rapeseed lecithins are presented.     

Crystallization and pressure filtration of anhydrous milk fat: Mixing effects

Melt crystallization of anhydrous milk fat and subsequent filtration of the slurry is a common process for obtaining milk fat fractions with different physical and chemical properties. The crystallization mechanism is very complex and little is known about how the crystallizer conditions and the crystal size distribution (CSD) affect the filtration process. The objective of this study was to characterize the fractionation process and determine which geometric parameters of the crystallizer affect the filtration step. Two scales of fractionation were studied, 0.6 L and 3.6 L, with crystallization at 28°C. The slurry was pressure-filtered after 24 h at 500 kPa in a 1-L chamber. Impeller diameters and speeds were varied for both scales. Photomicroscopy and spectrophotometry were used to characterize the crystallization process, and filtration rates were measured by weighing the amount of filtrate passing through the filter. Filtration resistance values, calculated using the constant pressure filtration equation, as well as photomicroscopy results indicated that the agglomerates and crystals that formed had different morphological characteristics for the different mixing and flow regimes in the crystallizer. Crystallization conditions that provide an optimal filtration time, a solid fraction with minimal liquid entrainment, and a CSD with an intermediate range of sizes (80–500 μm) having good packing properties for filtration were found.     

Microwave roasting and phospholipids in soybeans (Glycine max. L.) at different moisture contents

The effects of microwave roasting on phospholipids in soybeans were investigated in relation to moisture. Whole soybeans at different moistures (9.6, 38.2, and 51.9%) were roasted by exposure to microwaves at a frequency of 2,450 MHz. During microwave treatments, the lower the moisture content, the higher was the internal temperature in soybeans at the end of microwave roasting. Total lipids were extracted from the beans after microwave treatment, and the phospholipids were separated with thin-layer chromatography. Phosphatidylcholine was the principal phospholipid in the extracted lipids from all unroasted and roasted bean samples. After microwave roasting, phospholipids containing an amino group, especially phosphatidylethanolamine, decreased substantially (P<0.05) in lower-moisture soybeans. However, increasing the moisture content depressed a rise in the internal temperature of soybeans and prevented a reduction in phospholipids and/or polyunsaturated fatty acids in the phospholipids. Based on the changes in the composition and fatty acid distribution of phospholipids in soybeans during microwave roasting, it is necessary to consider the moisture content in soybeans when roasting in a microwave oven.     

The antioxidant effects of phospholipids on perilla oil

Antioxidant effect of phospholipids on the oxidation of refined perilla oil (PO;α-18:3, 54.5%; 16:0, 7.2%; 18:0, 2.6%; 18:1, 18.6%; 18:2, 15.5%), tocopherol-free (POF) and tocopherol-enriched (POR) perilla oil were investigated by measuring weight-gains and by the oven test at 37°C. The oxidative stability of PO was especially increased by additions of phosphatidylethanolamine (PE) and phosphatidylserine (PS), but phosphatidylcholine (PC) scarcely showed an antioxidant effect. The oxidative stability of POF was markedly low, and none of the phospholipids (PC, PE, PS) showed an antioxidant effect on the oxidation of POF. The stability of POR was lower than that of PO regardless of its higher tocopherol contents. However, the oxidation of POR was significantly suppressed by additions of PE and PS, as was observed with PO. PC showed a small antioxidant effect on the oxidation of POR. Therefore, it seems that the antioxidant effects of phospholipids, especially of PE and PS, was due to the presence of tocopherols in the perilla oil.     

Temperature and concentration dependent effect of partial glycerides on milk fat crystallization

Two diglycerides (distearin and diolein) and two monoglycerides (monostearin and monoolein) were added to milk fat in a concentration of 0.5% and 1%. The isothermal crystallization behavior was evaluated at 22 °C, 23.5 °C, 25 °C and 26.5 °C by DSC and pNMR. The crystallization kinetic was quantified by means of two models. It was noticed that the effect of the minor components on the crystallization behavior depends on temperature and concentration. The type of esterified fatty acids and the polar head of the amphiphilic molecule determine to what extent partial glycerides influence the nucleation and crystal growth of triglycerides. Moreover the degree of insolubility of partial glycerides in the melt determines which effect (on growth or on nucleation) predominates. Stearic acid based partial glycerides enhance nucleation at low temperatures, while at higher temperatures an interaction with the crystal growth predominates. Oleic acid based partial glycerides have an effect on the nucleation process while no interaction with the crystal growth was observed.     

The effect of minor components on milk fat crystallization

Milk fat is composed of 97–98% triacylglycerols and 2–3% minor polar lipids. In this study triacylglycerols were chromatographically separated from minor components. Isolated diacylglycerols from the polar fraction were also added back to the milk fat triacylglycerols. The crystallization behaviors of native anhydrous milk fat (AMF), milk fat triacylglycerols (MF-TAG), and milk fat triacylglycerols with diacylglycerols added back (MF-DAG) were studied. Removal of minor components and addition of diacylglycerols had no effect on dropping points or equilibrium solid fat contents. Presence of the minor components, however, did delay the onset of crystallization at low degrees of supercooling. Crystallization kinetics were quantified using the Avrami model. Sharp changes in the values of the Avrami constant k and exponent n were observed for all three fats around 20.0°C. Increases in n around 20.0°C indicated a change from one-dimensional to multidimensional growth. Differences in k and n of MF-DAG from AMF and MF-TAG suggested that the presence of milk fat diacylglycerols changes the crystal growth mechanism. Apparent free energies of nucleation (ΔG_c,apparent) were determined using the Fisher-Turnbull model. (ΔG_c,apparent) for AMF was significantly greater than ΔG_c,apparent for MF-TAG, and ΔG_c,apparent for MF-DAG was significantly less than those for both AMF and MF-TAG. The microstructural networks of AMF, MF-TAG, and MF-DAG, however, were similar at both 5.0 and 25.0°C, and all three fats crystallized into the typical β′-2 polymorph. Differential scanning calorimetry in both the crystallization and melting modes revealed no differences between the heat flow properties of AMF, MF-TAG, and MF-DAG.     

Characterization of phospholipids from glyphosate-tolerant soybeans

The phospholipids from three control and two glyphosate-tolerant soybean cultivars were isolated by extraction of soy flakes with hexane and characterized after separation by high-pressure liquid chromatography. In addition, several lots of commercial fluid lecithin were analyzed and the results were compared to values published in the literature. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid were identified as major components in these cultivars and in the commercial lecithin samples. The results showed that glyphosate-tolerant soybeans yield lecithin comparable and equivalent to conventional soybean cultivars.     

Thermal analysis of isothermal crystallization kinetics in blends of cocoa butter with milk fat or milk fat fractions

The kinetics of isothermal crystallization of binary mixtures of cocoa butter with milk fat and milk fat fractions were evaluated by applying the Avrami equation. Application of the Avrami equation to isothermal crystallization of the fats and the binary fat blends revealed different nucleation and growth mechanisms for the fats, based on the Avrami exponent. The suggested mechanism for cocoa butter crystallization was heterogeneous nucleation and spherulitic growth from sporadic nuclei. For milk fat, the mechanism was instantaneous heterogeneous nucleation followed by spherulitic growth. For milk fat fractions, the mechanism was high nucleation rate at the beginning of crystallization, which decreased with time, and plate-like growth. Addition of milk fat fractions did not cause a significant change in the suggested nucleation and growth mechanism of cocoa butter.     

Application of phospholipids extracted from bovine milk to the reconstitution of cream using butter oil

Phospholipid (PL) extracted from bovine milk was tested for its emulsifying properties in conjunction with the reconstitution of cream using butter oil. PL from bovine milk dispersed in the oil phase was found to stabilize the cream, whereas PL extracted from soybean oil was found to solidify the cream. Different PL species purified from bovine milk PL were tested for their emulsifying properties. PC from bovine milk dispersed in butter oil was shown to stabilize the cream, whereas PE and sphingomyelin had no such effects. PC from soybean oil also was found to have emulsifying abilities. It was suggested that PC stabilized the reconstituted cream, regardless of its origin.     

Antioxidant properties of individual phospholipids in a salmon oil model system

The antioxidant properties of phospholipids (PL) in a refined salmon oil model system were measured by determining changes in the 2-thiobarbituric acid number and decreases in the ratio of docosahexaenoic acid (DHA)/palmitic acid (22:6/16:0) of a fish oil system incubated at 180°C for up to 3 h. The more phosphatidylcholine (PC) added to the oil system, the higher the oxidative stability obtained. The order of effectiveness of commercial phospholipids in inhibiting oxidation and the loss of polyunsaturated fatty acids was as follows: sphingomyelin (SPH)=lysophosphatidylcholine (LPC)=phosphatidylcholine (PC)=phosphatidylethanolamine (PE)>phosphatidylserine (PS)>phosphatidylinositol (PI)>phosphatidylglycerol (PG)>control salmon oil. Nitrogen containing PL, including PE, PC, LPC and SPH, were equally effective in exerting greater antioxidant properties than PS, PG and PI. The inverse relationship observed between the oxidation index (C22:6/C16:0) and color intensity for treatments following 2 h of heating suggests that Maillard-type reaction products may have contributed to the oxidative stability of PL-supplemented fish oils.     

Lipase-catalyzed synthesis of waxes from milk fat and oleyl alcohol

A screening of five lipases was carried out for the synthesis of wax esters from stoichiometric amounts of oleyl alcohol and milk fat in which long-chain fatty acid content (myristic acid, palmitic acid, stearic acid, and oleic acid) represents 70% of the total fatty acid fraction. The lipases from Alcaligenes sp. and Chromobacterium viscosum both allowed for the best ester synthesis (around 60%) within 2 and 48 h, respectively. Enzeco® Lipase Concentrate gave 30% ester yield within only 2 h. During the time period of 166 h, less than 20% ester synthesis was obtained with Lipozyme? 10,000L whereas Enzeco® Lipase XX did not catalyze the reaction. Owing to commercial availability, the food-grade Enzeco® Lipase Concentrate preparation was selected for further experiments with a view to improve wax synthesis. Wax yields were compared for three substrate molar ratios, i.e., 0.5:1, 1:1, and 1.5:1 (alcohol/fatty acid). For 0.5:1 and 1.5:1 substrate molar ratios, the addition of water increased ester yields while the effect of silica gel addition was shown to be minor. The best improvement was obtained at a substrate molar ratio of 1.5:1 with addition of water, leading to 59% wax ester synthesis.     

Differently sized native milk fat globules separated by microfiltration: fatty acid composition of the milk fat globule membrane and triglyceride core

The aim of this study was to characterize the fatty acid composition of the core and membrane of differently sized milk fat globules separated by microfiltration, which can now be used to manufacture dairy products. Native milk fat globules of various mean diameters, ranging from d₄₃ = 2.3 µm to 8.0 µm, were obtained using microfiltration of raw whole milk. After milk fat globule washing, the milk fat globule membrane (MFGM) and the triglyceride core (TC) were separated by manual churning. After total lipid extraction from each fraction, their fatty acid composition was characterized using methyl ester analysis by gas chromatography. Regardless of season, no significant differences were observed in the fatty acid composition of the MFGM phospholipids. Conversely, significant differences were found in the fatty acid composition of TC; particularly, small fat globule TC contained more medium‐chain fatty acids and less stearic acid than large fat globule TC. These results show that the previously observed differences in total fatty acid composition among differently sized milk fat globules are due to their triglyceride composition and MFGM amount rather than to the composition of the MFGM.     

Microwave roasting and positional distribution of fatty acids of phospholipids in soybeans (Glycine max L.)

Whole soybeans were exposed to microwave roasting for 6, 12, and 20 min at a frequency of 2,450 MHz and were studied not only for phospholipid composition but also for positional distribution of the fatty acids. During microwave roasting, the greatest rate of phospholipid losses (P<0.05) was observed in phosphatidylethanolamine (PE), followed by phosphatidylcholine (PC) and phosphatidylinositol (PI), respectively. Therefore, the effects of microwave roasting on the composition and positional distribution of the fatty acids are likely clearer in PE than in PC or PI. However, the principal characteristics for the positional distribution of fatty acids are still retained during microwave roasting: unsaturated fatty acids, especially linoleic, are predominantly concentrated in the 2-position, and saturated fatty acids, especially palmitic, primarily occupy the 1-position after 12 or 20 min of roasting. The results suggest that unsaturated fatty acids located in the 2-position are significantly protected from microwave roasting.     

Effect of DAG on milk fat TAG crystallization

The effect of milk fat and standard DAG on the crystallization behavior of milk fat TAG (MF-TAG) was investigated. When milk fat DAG were added to MF-TAG at the 0.1 wt% level, crystallization was delayed. Racemic purity was shown to be an important factor in the ability of DAG to influence TAG crystallization. Only sn-1,2 isomers of blends of MF-TAG with 0.1 wt% of the racemic mixtures of dipalmitin and diolein increased the activation free energy barrier to MF-TAG nucleation (ΔG _c ) and delayed the subsequent crystallization process by increasing the crystallization induction time (τ_SFC) determined from solid fat content-time measurements. Although crystallization kinetics were affected, the properties of the resulting network structures remained unchanged upon addition of milk fat minor components at the 0.1 wt% level