Properties,analysis and purification of milk polar lipids

The phospholipids and sphingolipids in milk are gaining interest due to their nutritional and technological qualities. Sphingolipids and their derivatives are highly bioactive compounds with anti-cancer, bacteriostatic and cholesterol-lowering properties. Several low-value process streams of the dairy industry contain considerable amounts of polar lipid and exert potential for further purification. This review deals with the structure of the main dairy polar lipids, their origin and molecular arrangement, their occurrence in raw milk and other dairy products, their methodology of analysis and purification and their nutritional and technological properties.     

The composition of bovine milk lipids: January 1995 to December 2000

Data from recent publications on bovine milk lipids are presented and discussed. This includes extraction of lipids, triacylglycerols, phospholipids, other complex lipids, sterols, isoflavones, and fatty acids. Improved gas-liquid and high performance liquid chromatography were used. Data on the trans and cis isomers of fatty acid and of conjugated linoleic acids are given, and the analyses are described. Papers about the lipids in milks and dairy products from the United States are few; where with the exception of trans-fatty acid isomers and conjugated linoleic acids, almost no research has been reported.     

Addition of milk fat globule membrane as an ingredient of infant formulas for resembling the polar lipids of human milk

Polar lipid (PL) contents in human milk (HM) from two different geographic zones in Spain (central and coastal) were determined. These PLs were also analysed in several infant formulas (IFs), three of which contained milk fat globule membrane (MFGM), an ingredient used to resemble the PL profile of HM. Total PL in HM decreased significantly (p < 0.05) from transitional milk (48.62 mg 100 mL⁻¹) to 6 months (28.66 mg 100 mL⁻¹). In HM, sphingomyelin was the most abundant PL, followed by phosphatidylethanolamine; in IFs the most abundant PL was phosphatidylethanolamine. Only IFs with MFGM (54.79–58.07 mg 100 mL⁻¹) could supply the total and individual PL content present in all lactation periods, with the exception of sphingomyelin, which did not match the content in transitional milk. PL intake by infants fed HM or IFs was determined to be 96–306 and 152–575 mg day⁻¹, respectively.     

乳脂中天然生物活性物质的存在和生化特性

在充分考虑到季节变化的影响后，总结了牛乳脂肪中各种生物活性成分的含量。研究表明，牛乳脂肪中生物活性物质总的质量分数大约为75%，但要对这些生物活性物质对人体的健康的利弊进行全面的评价是较困难的。     

Use of polar aprotic solvents to release membranes from milk lipid globules

In the search for alternatives to physical methods for release of membranes from milk lipid globules, aqueous solutions of the polar aprotic solvents dimethyl sulfoxide and dimethyl formamide were found to cause release of milk fat globule membrane. Membranes released with polar, aprotic solvents displayed some differences in polypeptide, enzymatic, and lipid composition in comparison with membranes released by churning. However, all polypeptide, enzymatic, and lipid constituents measured in membranes released by churning were also present in membranes released with dimethyl formamide and dimethyl sulfoxide. Antigenic and Concanavalin A binding activity of polypeptides was retained during exposure to dimethyl sulfoxide or dimethyl formamide. The solvent method was effective in releasing membrane from lipid globules of cow, rat, and guinea pig milks. This solvent method was rapid and minimized losses attendant with physical methods for release of milk fat globule membrane.     

Combined effects of milk fat globule membrane polar lipids and protein concentrate on the stability of oil-in-water emulsions

Milk fat globule membrane (MFGM) material isolated from reconstituted buttermilk by microfiltration (i.e., whole MFGM) contains two major fractions, namely proteins (consisting mainly of MFGM-specific proteins and serum proteins) and lipids (original membrane polar lipids and contaminating triglycerides from the globule core). In this study, MFGM proteins and polar lipid (PL) concentrate were separated from whole MFGM material using solvent extraction. The particle size distribution, stability, surface protein and polar lipid load of oil-in-water emulsions prepared with protein or PL concentrate, individually or in combination, at various concentrations were examined. At low emulsifier concentrations (<2.3%), there was an interacting effect between proteins and PLs on the droplet size. The phase separation of emulsions prepared with a combination of 0.3% proteins and 0.3% PLs was similar to that of emulsions containing 0.3% proteins. The proteins were more preferentially adsorbed at the emulsion droplet surface compared with PLs.     

Distribution and fatty acid content of phospholipids from bovine milk and bovine milk fat globule membranes

The phospholipid (PL) content was determined comparatively in the milk fat globule membrane (MFGM) and whole milk including their fatty acid profiles. The possible role of milk PLs in defence against pathogens was also addressed. The MFGM and whole milk showed a similar distribution of PL species; however, the fatty acid contents of the PL species were different. Total PL from MFGM showed a decrease in C18:0 content in parallel with an increase in C18:1 and C18:2 and very long-chain fatty acid (more than C20) content. No significant differences in the fatty acid content of phosphatidylcholine and sphingomyelin from either source were found. However, the phosphatidylethanolamine from MFGM had more C18:1 and C18:2 and less C14:0 and C16:0 than that from whole milk. A similar but less pronounced result was found for phosphatidylserine/phosphatidylinositol. Enterotoxigenic Escherichia coli strains failed to bind to PL, which had been previously separated by high-performance thin-layer chromatography.     

Buffalo vs. cow milk fat globules: Size distribution,zeta-potential,compositions in total fatty acids and in polar lipids from the milk fat globule membrane

Although buffalo milk is the second most produced milk in the world, and of primary nutritional importance in various parts of the world, few studies have focused on the physicochemical properties of buffalo milk fat globules. This study is a comparative analysis of buffalo and cow milk fat globules. The larger size of buffalo fat globules, 5 vs. 3.5 μm, was related to the higher amount of fat in the buffalo milks: 73.4 ± 9.9 vs. 41.3 ± 3.7 g/kg for cow milk. Buffalo milks contained significantly lower amount of polar lipids expressed per gram of lipids (0.26% vs. 0.36%), but significantly higher amount of polar lipids per litre of milk (+26%). Buffalo and cow milk fat globule membranes contain the same classes of polar lipids; phosphatidylethanolamine, sphingomyelin (SM) and phosphatidylcholine (PC) being the main constituents. A significant higher percentage of PC and lower percentage of SM were found for buffalo milks. The fatty acid analysis revealed that saturated fatty acids, mainly palmitic acid, trans fatty acids, linolenic acid (ω3) and conjugated linolenic acid were higher in buffalo milk than in cow milk. Such results will contribute to the improvement of the quality of buffalo milk-based dairy products.     

Changes in physical properties of bovine milk from the colostrum period to early lactation

The aim of this study was to analyze individual cows’ samples from the colostrum, postcolostrum, and early lactation periods to investigate how milk composition, physical properties, stability, and suitability for processing change throughout this period. Attention was paid to the first week postpartum in which the composition of bovine mammary secretion can change markedly. Properties including pH, titratable acidity, ethanol stability (ES), rennet clotting time, and casein micelle size were analyzed, together with some compositional factors such as fat, total protein, lactose, total and ionic calcium, magnesium, citrate, phosphorus, sodium, and potassium. Total Ca (36.2 mM) and free ionic Ca (2.58 mM), Mg (5.9 mM), P (32.2 mM), and Na (24.1 mM) appeared to be high on d 5 postpartum, having decreased substantially over the first 5 d; they gradually decreased thereafter. The average pH on d 5 was only 6.49, compared with 6.64 at 1 mo postpartum. Stability measurements showed that the average ES on d 5 was 70% and the rennet clotting time was 12.2 min, which were significantly lower than values at later stages. A number of milk properties including ES, pH, protein content, and Ca²⁺ concentration could be useful for identifying the point of transition from colostrum to the early lactation period. Knowing the composition and physical properties of colostrum and postcolostrum secretions will help establish when such milk is suitable for processing and determine the best use for that milk.     

Relationship of the estrous cycle to milk composition.

The relationship of the estrous cycle to milk composition was examined in 15 Holstein cows in first lactation which were approximately 45 days postpartum. Composite milk samples were collected twice daily and analyzed for progesterone, fat, protein, total solids, sodium, potassium, magnesium, calcium, and somatic cells. Milk progesterone was minimal (less than ng/ml) from day -1 to day 2 (day 0 = estrus) of the estrous cycle and then increased at a relatively constant rate through day 15. Milk yield and sodium and magnesium concentrations of the milk varied with days of the estrous cycle. Mean milk yield was highest and sodium and magnesium concentrations were lowest on day 1. However, sodium concentration of the milk was the only component that varied significantly during the 3 days centered on estrus (days -1, 0, and 1). No other milk component changed significantly during the estrous cycle. Although milk yield and composition varied during the estrous cycle, none of these components appeared to be a practical indicator of estrus.     

The simulated in vitro infant gastrointestinal digestion of droplets covered with milk fat globule membrane polar lipids concentrate

Milk fat globule membrane polar lipids (MPL) are increasingly used as the surface-active components for emulsions in many infant food products. However, the precise effect of the emulsifier MPL on the digestion of lipids during gastrointestinal digestion has not been elucidated. This study investigated the lipid digestion of droplets covered with MPL with different sizes in a simulated in vitro infant gastrointestinal digestion assay. The well-used surface-active component casein was used as a control. Four types of emulsions were formulated: small and large droplets covered with MPL concentrate (MPL-S and MPL-L, with volumetric means of 0.35 ± 0.01 and 4.04 ± 0.01 μm, respectively), and small and large droplets covered with casein (CN-S and CN-L, with volumetric means of 0.44 ± 0.01 and 4.09 ± 0.03 μm, respectively). The emulsions were subjected to in vitro gastrointestinal digestion using a semidynamic model mimicking infant digestion. Through the determination of particle size evolution, zeta-potential, and microstructure of emulsions, the lipid droplets covered with MPL were found to be more stable than that of the CN-S and CN-L during gastrointestinal digestion. Moreover, although CN-S and CN-L showed a higher initial lipolysis rate at the beginning of gastric digestion, droplets covered by MPL exhibited a significantly higher amount of free fatty acid release during later digestion. The amount of free fatty acid release of the emulsions in both gastric and intestinal digestion could be generally classified as MPL-S ≥ MPL-L > CN-S > CN-L. Our study highlights the crucial role of MPL in the efficient digestion of emulsions and brings new insight for the design of infant food products.     

An improved method to culture Staphylococcus aureus from bovine milk

Staphylococcus aureus is an important udder pathogen often associated with subclinical mastitis in dairy cows. Identification of Staph. aureus-positive udder quarters and cows is an important part of control programs to reduce spread of Staph. aureus within and between dairy herds. Therefore, accurate and easy-to-perform culturing methods of Staph. aureus in milk are needed. In the present study, 8 methods for isolation of Staph. aureus in bovine milk samples were investigated. The methods involved different culturing volumes, enrichment, incubation, and freezing processes as well as sedimentation and use of the Mastistrip cassette (SVA, Uppsala, Sweden). Three different sets of milk samples were collected, and 6, 5, and 4 methods were used in each subset of samples. Our results indicate an increased probability of detecting Staph. aureus in milk samples when a simple incubation step (37°C for 18 h) without additives was included before culturing. Using this incubation method, the number of Staph. aureus-positive udder quarters and cows increased by 50 and 29%, respectively, compared with using the standard method of direct culturing of 10 μL of milk. The improved method may be especially useful for detection of low concentrations of Staph. aureus in milk; for example, when screening herds for Staph. aureus.     

The behaviour of Arabian donkey milk during acidification compared to bovine milk

To better understand the fermentation kinetic of Arabian donkey milk, its physicochemical properties, conductivity and viscosity were assessed during acidification, and compared to that of the bovine milk. Donkey milk showed a shorter latency phase and slightly lower acidification rate than bovine milk. Measurement of electric conductivity during acidification showed that maximum demineralisation of casein micelles occurred at around pH_I 5.44 for donkey milk and pH_I 5.16 for bovine milk. Donkey milk was also found to be less viscous. The technological characteristics of donkey milk were different from those of bovine milk due to intrinsic physicochemical properties of both milks.     

The status of fluoride added to bovine milk

The use of bovine milk as a carrier for fluoride has been advocated as a prophylactic measure. The effect of the addition of sodium fluoride to cold milk, up to 1000 ppm, was examined by particle micro electrophoresis, centrifugation, and dialysis.
The fluoride had little effect on the electrokinetic potential, and no sedimentation calcium fluoride occurred. Even with centrifugation at 30 000 g , the bulk of the fluoride remained in solution. Dialysis showed that the fluoride could easily be removed, and that the eluent contained fluoride in the ionic (free) form.
The addition of fluoride to calcium phosphate free (CPF, milk showed that the bulk of the fluoride remained with the protein, even after addition of excess calcium chloride. Thus the fluoride does not affect the stability of milk and the element forms a reversible ionic complex. The evidence suggests that it is held in solution by the smaller milk proteins.     

Occurrence of flunixin residues in bovine milk samples from the USA

5-Hydroxy-flunixin concentrations in milk samples were quantified by two commercially available screening assays – CHARM^® and enzyme-linked immunoabsorbant assay (ELISA) – to determine whether any concentrations could be detected above the tolerance limit of 2 ng g^?1 from different regions in the United States. Milk samples came from large tanker trucks hauling milk to processing plants, and had already been screened for antibiotics. Positive results for flunixin residues based on a screening assay were confirmed by ultra-HPLC with mass spectrometric detection. Of the 500 milk samples analysed in this study, one sample was found to have a 5-hydroxy-flunixin concentration greater than the tolerance limit. The results of this study indicate that flunixin residues in milk are possible. Regulatory agencies should be aware that such residues can occur, and should consider incorporating or expanding flunixin screening tests as part of routine drug monitoring in milk. Larger studies are needed to determine the true prevalence of flunixin residues in milk from other regions in the United States as well as different countries.     

Occurrence of ivermectin in bovine milk from the Brazilian retail market

High-performance liquid chromatography (HPLC) with fluorescence detection was used for the quantification of ivermectin residues in bovine milk intended for human consumption. After liquid-liquid extraction of ivermectin and purification of the extract, the compound was derivatized with 1-methylimidazol in N,N-dimethyl formamide to form a fluorescent derivative, which was separated by HPLC, using reversed-phase C₁₈, with methanol : water (96 : 4 v/v) mobile phase at a flow rate 0.7 ml min^-1. The excitation and emission wavelengths of the fluorescence detector were adjusted at 360 and 470 nm, respectively. The linearity of the method was in the range 10-100 ng ivermectin ml^-1. Based on a sample of 5.0 ml, the limit of detection and the limit of quantification for ivermectin in milk were 0.6 and 2 ng ml^-1, respectively. The recovery rate varied from 76.4 to 87.2%, with an average of 77.9 ± 3.2%, at four fortification levels. The inter-day precision of the method was 13% (n = 5). Of 168 samples analysed, 17.8% contained ivermectin above the limit of quantification. Nevertheless, none of the samples contained ivermectin above the maximum residue limit (10 ng ml^-1) established by the Brazilian Ministry of Agriculture.     

牛乳中乳铁蛋白的纯化和抗菌活性研究

采用凝胶过滤层析和离子交换法层析分离纯化乳铁蛋白,通过SDS-PAGE和IEF-PAGE鉴定所得样品;同时利用抗菌实验(滤纸片法)进行样品的抗菌活性研究,并研究了乳铁蛋白分离纯化过程中抗菌活性的变化。结果表明,离子交换层析纯化乳铁蛋白的效果优于凝胶过滤层析,抗菌活性证实乳铁蛋白样品具有广谱抗菌作用,以蜡状芽孢杆菌为指示菌时,比活为3077.3AU/mg(试管法),纯化倍数提高了20.2倍。