Lars Wiking

Protein denaturation of whey protein isolates (WPIs) induced by high intensity ultrasound during heat gelation

In this study, the impact of high intensity ultrasound (HIU) on proteins in whey protein isolates was examined. Effects on thermal behavior, secondary structure and nature of intra- and intermolecular bonds during heat-induced gelling were investigated. Ultrasonication (24 kHz, 300 W/cm(2), 2078 J/mL) significantly reduced denaturation enthalpies, whereas no change in secondary structure was detected by circular dichroism. The thiol-blocking agent N-ethylmaleimide was applied in order to inhibit formation of disulfide bonds during gel formation. Results showed that increased contents of α-lactalbumin (α-La) were associated with increased sensitivity to ultrasonication. The α-La:β-lactoglobulin (β-Lg) ratio greatly affected the nature of the interactions formed during gelation, where higher amounts of α-La lead to a gel more dependent on disulfide bonds. These results contribute to clarifying the mechanisms mediating the effects of HIU on whey proteins on the molecular level, thus moving further toward implementing HIU in the processing chain in the food industry.

Pellet-free isolation of human and bovine milk extracellular vesicles by size-exclusion chromatography

ABSTRACT Studies have suggested that nanoscale extracellular vesicles (EV) in human and bovine milk carry immune modulatory properties which could provide beneficial health effects to infants. In order to assess the possible health effects of milk EV, it is essential to use isolates of high purity from other more abundant milk structures with well-documented bioactive properties. Furthermore, gentle isolation procedures are important for reducing the risk of generating vesicle artefacts, particularly when EV subpopulations are investigated. In this study, we present two isolation approaches accomplished in three steps based on size-exclusion chromatography (SEC) resulting in effective and reproducible EV isolation from raw milk. The approaches do not require any EV pelleting and can be applied to both human and bovine milk. We show that SEC effectively separates phospholipid membrane vesicles from the primary casein and whey protein components in two differently obtained casein reduced milk fractions, with one of the fractions obtained without the use of ultracentrifugation. Milk EV isolates were enriched in lactadherin, CD9, CD63 and CD81 compared to minimal levels of the EV-marker proteins in other relevant milk fractions such as milk fat globules. Nanoparticle tracking analysis and electron microscopy reveals the presence of heterogeneous sized vesicle structures in milk EV isolates. Lipid analysis by thin layer chromatography shows that EV isolates are devoid of triacylglycerides and presents a phospholipid profile differing from milk fat globules surrounded by epithelial cell plasma membrane. Moreover, the milk EV fractions are enriched in RNA with distinct and diverging profiles from milk fat globules. Collectively, our data supports that successful milk EV isolation can be accomplished in few steps without the use of ultracentrifugation, as the presented isolation approaches based on SEC effectively isolates EV in both human and bovine milk.

Effects of supplemental dietary fatty acids on milk yield and fatty acid composition in high and medium yielding cows

The present study tested the hypothesis that supplemental dietary fatty acids (FA) affect the energy corrected milk yield in proportion to the milk production level of dairy cows, and increase both long chain FA proportion of milk FA and milk fat globule diameter. Sixteen Danish Holstein cows were divided into four 4x4 Latin squares with two squares of medium yielding cows (32.2 kg energy corrected milk (ECM)/d; 158 days in milk (DIM)) and two squares of high yielding cows (40.0 kg ECM/d; 74 DIM). Experimental length was 12 weeks, with three weeks for each of the four periods. The four treatments were no supplementation (17 g FA/kg dry matter (DM)) and three diets with supplemented FA (29, 40, and 52 g total FA/kg DM, respectively) obtained by substituting barley with Palm Fatty Acid Distillate (PFAD) fat. Diets were offered as total mixed rations with 63% grass/clover silage (DM basis). Dry matter intake decreased with increasing FA supplementation, but net energy intake was not affected. The general linear responses to 10 g/kg DM increase in FA level were 1.1 kg ECM (P<0.0001), 0.061 kg milk fat (P<0.0001), 0.012 kg milk protein (P=0.09) and 0.052 kg lactose (P=0.0002) per day, and linear responses in milk composition were 0.39 g fat (P=0.07), -0.71 g protein (P<0.0001) and 0.05 g lactose (P=0.3) per kg milk, and 0.092 microm (P<0.0001) in milk fat average globule diameter. Fatty acid supplementation decreased short- and medium-chain FA and C16:0 and increased C18:1 proportions of total FA in milk. Supplemental dietary FA increased ECM yield but not in proportion to production level as anticipated, and increased average FA chain length and milk fat globule diameter.

Evaluation of cooling strategies for pumping of milk – Impact of fatty acid composition on free fatty acid levels

Cooling strategies for pumping of raw milk were evaluated. Milk was pumped for 450 s at 31 degrees C, or pumped after cooling to 4 degrees C and subsequently subjected to various incubation times. Two types of milk were used; i.e. milk from cows fed a diet high in saturated fat supplements resulting in significantly larger milk fat globules than the other type of milk which comes from cows fed a low-fat diet that stimulates high de novo fat synthesis. The content of liquid fat was determined by low-field 1H NMR, which showed that milk from cows given the saturated fat diet also contained less liquid fat at both 4 degrees and 31 degrees C than the other type of milk. This can be ascribed to the differences in the fatty acid composition of the milk as a result of the fatty acid composition of the diets. After pumping of the milk at 31 degrees C, measurement of fat globule size distribution revealed a significant coalescence of milk fat globules in the milk obtained from the saturated fat diet due to pumping. Pumping at 4 degrees C or pumping the other type of milk did not result in coalescence of milk fat globules. Formation of free fatty acids increased significantly in both types of milk by pumping at 31 degrees C. Cooling the milk to 4 degrees C immediately before pumping inhibited an increased content of free fatty acids. However, when the milk was incubated at 4 degrees C for 60 min after cooling and then subjected to pumping, a significant increase in the formation of free fatty acids was observed in both types of milk. It is suggested that this increase in free fatty acids is caused by transition of polymorphic crystal forms or higher level of attached lipoprotein lipases to the milk fat globule before pumping.

Cooling causes changes in the distribution of lipoprotein lipase and milk fat globule membrane proteins between the skim milk and cream phase

Lipoprotein lipase (LPL) activity and free fatty acid levels were studied in freshly milked, uncooled milk from individual Danish Holstein or Jersey cows, or after storage for up to 24h at either a cooling temperature (4°C) or at the milking temperature (31°C). Upon cooling for up to 24h, LPL activity increased in the cream phase, whereas the activity in the skim milk was steady, as observed for Jersey cows, or increased, as seen for the Holsteins. Storage at 31°C decreased the LPL activity in both the cream phase and the skim milk phase. The increase in free fatty acid levels was found to depend on LPL activity, incubation temperature, substrate availability, and incubation time. Furthermore, the migration of milk proteins between the skim milk phase and the cream phase upon cooling of milk from Jersey cows or from Danish Holstein cows was studied using proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry. Proteins associated with the milk fat globules were isolated from all milk fractions and analyzed. Major changes in the distributions of proteins between the skim milk phase and the cream phase were observed after cooling at 4°C for 4h, where a total of 29 proteins between the 2 breeds was found to change their association with the milk fat globule membrane (MFGM) significantly. Among these, the MFGM proteins adipophilin, fatty acid-binding protein, and lactadherin, as well as the non-MFGM proteins β-casein, lactoferrin, and heat shock protein-71, were identified. Adipophilin, lactadherin, and lactoferrin were quantitatively more associated with the MFGM upon cold storage at 4°C, whereas β-casein, fatty acid-binding protein, and heat shock protein-71 were found to be less associated with the MFGM upon cold storage.

Effect of grazing fresh legumes or feeding silage on fatty acids and enzymes involved in the synthesis of milk fat in dairy cows

The impact of fresh legume types or silage on the composition of milk fatty acids and transcription of enzymes involved in the synthesis of milk fat in cows was studied. Three groups of cows grazed high proportions of white clover, red clover and lucerne, respectively. A fourth group of cows was fed maize/grass silage. The cows grazing high proportions of legumes produced significantly more 18:1 trans-11, 18:2 cis9-trans11, 18:2 trans10-cis12 and 18:3 fatty acids than cows fed silage. White clover and lucerne grazing resulted in significantly lower output of 18:1 trans9 in milk than red clover grazing and maize/grass silages. Transcription of stearoyl-CoA desaturase (SCD) in mammary tissue was significantly increased by grazing high proportions of legume whereas fatty acid synthase and acetyl-CoA carboxylase were not affected by type of feeding. Furthermore, average milk fat globule diameter was correlated to daily milk fat yield but was not affected by feeding. Although the fresh forage affected the transcription of SCD in mammary tissue, the largest effects were on the trans11-based fatty acids. It is concluded that type of forage, i.e. fresh or silage, had a greater impact on rumen fermentation pattern than on transcription of enzymes involved in the synthesis of milk fat.

Transfer of Dietary Zinc and Fat to Milk—Evaluation of Milk Fat Quality, Milk Fat Precursors, and Mastitis Indicators

The present study demonstrated that the zinc concentration in bovine milk and blood plasma is significantly affected by the intake of saturated fat supplements. Sixteen Holstein cows were used in a 4 x 4 Latin square design with 4 periods of 12 d, and 4 dietary treatments were conducted. A total mixed ration based on corn silage, grass-clover silages, and pelleted sugar beet pulp was used on all treatments. A high de novo milk fat diet was formulated by adding rapeseed meal and molasses in the total mixed ration [39 mg of Zn/kg of dry matter (DM)], and a low de novo diet by adding saturated fat, fat-rich rapeseed cake, and corn (34 mg of Zn/kg of DM). Dietary Zn levels were increased by addition of ZnO to 83 and 80 mg of Zn/kg of DM. Treatments did not affect daily DM intake, or yield of energy-corrected milk, milk fat, or milk protein. The high de novo diet significantly increased milk fat percentage and milk content of fatty acids with chain length from C6 to C16, and decreased content of C18 and C18:1. Treatments did not influence milk free fatty acids at 4 degrees C at 0 or 28 h after milking. The average diameter of milk fat globules was significantly greater in milk from cows offered low de novo diets. Furthermore, the low de novo diet significantly increased the concentration of nonesterified fatty acids and d-beta-hydroxybutyrate in blood plasma, the latter was also increased in milk. Treatments did not affect the enzyme activity of lactate dehydrogenase and N-acetyl-beta-d-glucosaminidase in milk or the activity of isocitrate dehydrogenase and malate dehydrogenase in blood plasma. The low de novo diet significantly increased plasma Zn and milk Zn content, whereas dietary Zn level did not in itself influence these parameters. This indicates that the transfer of fat from diet to milk might facilitate transfer of Zn from diet to milk.

Light sensitivity of two colorants for Cheddar cheese. Quantum yields for photodegradation in an aqueous model system in relation to light stability of cheese in illuminated display

The light stabilities of two commercial colorants for Cheddar cheese, an annatto extract and a β-carotene preparation, have been compared in a model system and tested in industrial scale production. The apparent quantum yields for photodegradation by monochromatic light were determined at 25 °C for solutions of the colorants in air-saturated 10 mm-citric acid–disodium hydrogen phosphate buffer containing 100 mg sodium caseinate/l at pH 5·2 and 5·4. Solutions of commercial β-carotene were more photostable than solutions of annatto, and although the quantum yields for photodegradation for both solutions depended significantly on both pH and irradiation wavelength, exposure to u.v. light (313 and 366 nm) caused more photobleaching than exposure to visible light (436 nm). The differences in colour stability observed in the model system were verified by tristimulus colorimetry during 9 weeks illuminated storage at 5 °C of Cheddar cheese produced from milk to which had been added (per 1000 kg) 67 g β-carotene extract or 133 ml annatto.

Seasonal variation in the composition and melting behavior of milk fat.

Dairy bulk tank milk was sampled during 1yr from 2 conventional (C1 and C2) and 1 organic dairy (O1) for studying the seasonal variation as well as the variation between dairies in the composition and properties of milk fat. The composition of fatty acids (FA) as well as triglycerides (TAG) in milk fat was analyzed, and the melting properties of milk fat were analyzed by use of differential scanning calorimetry. The main differences in fat content and composition of FA in milk fat between dairies included a higher fat content, greater proportion of C18:0, and smaller proportion of C16:0 in milk from dairy C2, which could be associated with a higher frequency of Jersey herds supplying milk to this dairy. The organic milk was characterized by a higher proportion of C18:3n-3, C18:2 cis-9,trans-11, C6 to C14, a lower proportion of C18:1 cis-9, and a higher melting point of the low-melting fraction. The TAG composition showed a greater proportion of C24 to C38 TAG in milk fat from dairy O1 and a greater proportion of C52 to C54 TAG in milk fat from dairy C2, which was in accordance with the differences in FA composition. Melting point of the low-melting fraction was higher for milk fat from dairy O1 compared with dairies C1 and C2, whereas no differences between dairies were observed with respect to melting points of the medium- and high-melting fractions. The seasonal variation in FA composition was most pronounced for dairy O1 although similar patterns were observed for all dairies. During the summer, the content of C18:0 and C18:1 cis-9 in milk fat was greater, whereas the content of C14:0 and C16:0 was lower. In addition, the content of C18:2 cis-9,trans-11 and C18:1 trans-11 increased in late summer for dairy O1. The differential scanning calorimetry thermograms of individual milk fat samples could be divided into 3 groups by principal component analysis. For dairy O1, summer samples belonged to group 1, spring and autumn samples to group 2, and winter samples to group 3. For dairy C1 winter samples (group 2), were separated from other samples (group 1), and for dairy C2 all samples were in group 1. Individual melting points were related to FA composition, and the melting point of the low-melting fraction was positively correlated to the content of C14:0 and C16:0 in milk fat and negatively correlated to the content of C18:1 cis-9 and C18:0.

Crystallization mechanisms in cream during ripening and initial butter churning

The temperature treatment of cream is the time-consuming step in butter production. A better understanding of the mechanisms leading to partial coalescence, such as fat crystallization during ripening and churning of the cream, will contribute to optimization of the production process. In this study, ripening and churning of cream were performed in a rheometer cell and the mechanisms of cream crystallization during churning of the cream, including the effect of ripening time, were investigated to understand how churning time and partial coalescence are affected. Crystallization mechanisms were studied as function of time by differential scanning calorimetry, nuclear magnetic resonance and by X-ray scattering. Microstructure formation was investigated by small deformation rheology and static light scattering. The study demonstrated that viscosity measurements can be used to detect phase inversion of the emulsion during churning of the cream in a rheometer cell. Longer ripening time (e.g., 5h vs. 0 h) resulted in larger butter grains (91 vs. 52 µm), higher viscosity (5.3 vs. 1.3 Pa · s), and solid fat content (41 vs. 13%). Both ripening and churning time had an effect on the thermal behavior of the cream. Despite the increase in solid fat content, no further changes in crystal polymorphism and in melting behavior were observed after 1h of ripening and after churning. The churning time significantly decreased after 0.5h of ripening, from 22.9 min for the cream where no ripening was applied to 16.23 min. Therefore, the crystallization state that promotes partial coalescence (i.e., aggregation of butter grains) is obtained within the first hour of cream ripening at 10 °C. The present study adds knowledge on the fundamental processes of crystallization and polymorphism of milk fat occurring during ripening and churning of cream. In addition, the dairy industry will benefit from these insights on the optimization of butter manufacturing.