Demonstration of a beta-casomorphin immunoreactive material in the plasma of newborn calves after milk intake

Blood was collected from newborn calves before and after their first milk intake after birth; extracts of plasma were assayed by radioimmunoassay for the presence of beta-casomorphin-7 immunoreactive materials. No beta-casomorphin immunoreactivity was found in samples collected before milk ingestion; however, in samples collected after milk ingestion a beta-casomorphin-7 immunoreactive material was detected. Chromatographic characterization showed that this material was not identical with beta-casomorphin-7 but might rather represent a precursor thereof. The material proved resistant to enzymatic attack during a 30-min incubation period at 37 degrees C in the plasma of newborn calves, whereas beta-casomorphin-7 was degraded under these conditions. A physiological significance of beta-casomorphin-7 eventually cleaved from such a precursor material at any site in the newborn mammal is suggested.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

Crossed immunoelectrophoresis of bovine milk fat globule membrane protein solubilized with non-ionic detergent.

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg2+-ATPase and 5'-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

Erratum

Detergent solubilized bovine milk fat globule membrane material studied by crossed immunoelectrophoresis combined with histochemical techniques revealed four major protein complexes. All four were found to bind to concanavalin A and three were identified as sialoglycoproteins. Xanthine oxidase activity was associated with the non-sialoglycoprotein precipitate. Immunoabsorption with intact milk fat globules showed an internal location of the xanthine oxidase, whereas the three other main proteins plus Mg²⁺-ATPase and 5′-nucleotidase were disposed on the outer membrane surface. The major proteins from milk fat globule membrane and membrane material isolated from skim milk showed immunochemical identity.     

Plasma membrane fragments in bovine and caprine skim milks

By either differential or linear gradient ultracentrifugation of bovine or caprine skim milks it was possible to obtain fractions which contained 45–75% of the lipid phosphorus and unesterified cholesterol of the skim milk. Electron microscopy of these fractions revealed the presence of numerous membrane-bound vesicles, microvilli and membrane fragments. Assay of the fractions for certain membrane-bound enzymes; i.e. 5′-nucleotide pyrophosphatase, alkaline phosphatase and ATPases, established the presence of all but the latter in the membrane-rich fractions. The distributions of the enzymes in the various fractions were correlated with their lipid phosphorus and cholesterol contents.Compositions of the phospholipids from skim milk membranes, milk fat globule membranes and the plasma membrane of the lactating mammary cell were observed to be similar and unique for having a relatively high level (20–25%) of sphingomyelin. By virtue of secretory processes, all of these membranes appear to be interrelated with each other and with Golgi vesicle membranes. It is concluded that the membrane material in the skim milk originates primarily from plasma membrane of the lactating cell. The possibiltiy that Golgi vesicle membranes form a substantial part of this material is not precluded by the results of this study.Separation of bovine skim milk on a Sepharose 4B gel column demonstrated that virtually all of the 5′-nucleotidase and lipid phosphorus are recovered together in the void volume of the column. Considering the particle size discriminating characteristics of this gel, the skim milk membrane material appears to be constituted of relatively large structures rather than of discrete subunits.     

L-ascorbic acid microencapsulated with polyacylglycerol monostearate for milk fortification

Efficiency was examined of microencapsulating L-ascorbic acid by polyglycerol monostearate (PGMS), and changes in the chemical and sensorial aspects of L-ascorbic acid and/or iron-fortified milk during storage were evaluated. The selected core materials were ferric ammonium sulfate and L-ascorbic acid. The highest efficiency (94.2%) of microencapsulation was found with the ratio of 5:1 as the coating to core material. The release of ascorbic acid from the microcapsules increased sharply from 1.6 to 6.7% up to 5 d of storage. The TBA value was the lowest in the milk sample with added encapsulated iron and unencapsulated L-ascorbic acid up to 5 d of storage in comparison with the other treated samples. A sensory analysis showed that most aspects were not significantly different between the control and fortified samples encapsulated with ascorbic acid after 5 d of storage. The results indicate that L-ascorbic acid microencapsulated with PGMS can be applied to fortify milk and acceptable milk products can be prepared with microencapsulated L-ascorbic acid and iron.     

Identification of inflammatory cells in bovine milk by flow cytometry

Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.     

Milk composition of captive vervet monkey (Chlorocebus pygerythrus) and rhesus macaque (Macaca mulatta) with observations on gorilla (Gorilla gorilla gorilla) and white handed gibbon (Hylobates lar)

The nutrient content and fatty acid composition of vervet monkey milk has been determined and is compared with rhesus macaque, and two hominoid apes, the white handed gibbon and gorilla. With 15.7 ± 4.1 g protein, 33.1 ± 9.4 g fat, and 85.1 ± 7.5 g lactose per kg milk, vervet monkey milk does not differ from that of rhesus macaque, and is within the range of other primates. Small amounts (> 1 g kg^− 1) of oligosaccharides, glucose, galactose and fucose were noted. In comparison, gorilla milk has a low fat content of 13.8 g kg^− 1, but contains high levels of oligosaccharides at 7.0 g kg^− 1 milk. The hominoid partner, the white handed gibbon, contains no oligosaccharides and a milk fat content similar to other hominoid species. Differences between vervet monkey and rhesus macaque milks were observed in the electrophoretic pattern of the milk proteins, mainly amongst the κ- and γ-caseins, which also differ from that of the hominids. The fatty acid contents of these milks differ from studies where a natural diet of leafy material was available in that a low content of α-linolenic acid (18:3n−3) was noted. A phylogenetic effect is observed for the content of 8:0, 10:0 fatty acids between the Cercopithecidae and Hominoidea, and a further phylogenetic effect suggested between the Hylobatidae and Hominidae.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

The lipid isolated from the fat globule membrane of milk was quickly autoxidized. The development of off-flavor like fishy flavor and brown color took place simultaneously. The browning material seemed to decompose fat peroxide. The addition of α-, γ- and δ-tocopherol into the membrane lipid inhibited the formation of fat peroxide and off-flavor and decreased the browning degree. The addition of the membrane lipid prolonged the induction period of the oxidation of the milk fat obtained by churning. The antioxidant activity of aα-, γ- and δ-tocopherols added into the churned milk fat containing 1% of the membrane lipid was higher than that of the tocopherols added into the churned milk fat containing no membrane lipid.     

Casein as a necessary factor in the production of stimulatory material for associative growth of lactic streptococci

Strains of Streptococcus cremoris KH and HC produced material that was stimulatory for S. cremoris R₆ in milk and in the dialyzable fraction of milk, but not in the dialysate fraction of milk, lactic acid whey, or lactose broth. The addition of casein to these latter media permitted the production of this stimulatory material to occur. Tryptone, peptone, and yeast extract could not be substituted for casein in producing the stimulatory material or in initiating associative growth in the lactic acid whey. The minimum concentration of casein required appeared to be from 2.0 to 2.5%.     

根系分泌物对紫云英油菜间作的响应

根系分泌物是植物与土壤进行物质交换和信息传递的重要载体,也是间作体系中作物-土壤-微生物互作的重要调控者。为进一步揭示间作体系中作物之间的互作机制,本研究通过紫云英单作、油菜单作和紫云英油菜间作,重点分析了紫云英油菜间作下根系分泌物的响应特征。结果表明: 共检测到紫云英和油菜根系分泌物391种,定性93种,包括了9种代谢物类型,其中有机氧化物含量最高,主要是以核糖醇的形式存在。不同种植模式中,紫云英、油菜的根系分泌物含量差异显著,紫云英油菜间作时根系分泌物特征与油菜单作相似,与紫云英单作差异较大。不同种植模式的差异根系分泌物中,仅9-芴酮1与其他差异分泌物间呈负相关关系。不同种植模式的差异根系分泌物主要为苯系物、脂类和类脂分子、有机酸及其衍生物、有机氧化物等,其中苯系物、脂类和类脂分子是表征紫云英、油菜根系分泌物变化的重要类型。可见,紫云英油菜间作改变了作物的根系分泌物特征,其变化特征与苯系物、脂类和类脂分子关系密切。     

Yb-TCPP metal–organic framework as fluorescence sensor for detecting tetracycline in milk

Developing effective means for detecting contamination in milk during production, processing, and storage is both important and challenging. Tetracycline (TC), due to its use in treating animal infections, is among the most prevalent organic pollutants in milk, posing potential and significant threats to human health. However, efficient and in situ monitoring of TC remains lacking. Nevertheless, we have successfully developed a highly sensitive and selective fluorescence method for detecting TC in milk using a metal–organic framework material made from Yb-TCPP (ytterbium-tetra(4-carboxyphenyl)porphyrin). The calculated Stern–Volmer constant (K_SV) was 12,310.88 M⁻¹, and the detection limit was 2.44 × 10⁻⁶ M, surpassing previous reports. Crucially, Yb-TCPP fluoresces in the near-infrared region, promising its development into a specific fluorescence detection product for practical TC detection in milk, offering potential application value.     

Enzyme activities and electronmicroscopic structure of rat milk fractions obtained after centrifugation

Summary LDH and MDH activities were found to increase after freeze-thawing of the cream and a non-fat fraction of rat milk isolated by centrifugation.Electronmicroscopy of these fractions revealed that cellular components occurred especially in association with milk fat globules but also in combination with secretion granules.The fat globule fraction represented only 20% of the milk volume but accounted for more than 50% of the LDH and 75% of the MDH activities in milk.The results show that in the rat mammary gland an apocrine secretion type occurs. The reason why the LDH and MDH activities increase in the milk during the lactation of the rat must be that increasing amounts of cellular material is passed into milk at secretion, containing increasing activities of LDH and MDH.This investigation was supported by grants from the Foundation of Magnus Bergwall and the Swedish Natural Science Research Council.The skillful technical assistance of Mrs Anna-Greta Petersen and Mrs Marie Adler-Maihofer and the secretarial help of Miss Marianne Andersson are gratefully acknowledged.     

Antagonism of Lactic Streptococci Toward Staphylococcus aureus in Associative Milk Cultures

The inhibition of growth of Staphylococcus aureus by lactic streptococci in associative cultures in milk was not due to hydrogen peroxide produced by the streptococci. Dialyzed whey from the milk culture of lactic streptococci was more inhibitory than dialyzed whey from milk acidified with lactic acid, indicating that material other than lactate was also involved. Analyses of cation and anion exchange fractions from the dialyzed whey showed that only the neutral fraction was inhibitory.     

Coupling flash liquid chromatography with mass spectrometry for enrichment and isolation of milk oligosaccharides for functional studies

Mass spectrometry has been coupled with flash liquid chromatography to yield new capabilities for isolating nonchromophoric material from complicated biological mixtures. A flash liquid chromatography/tandem mass spectrometry (LC/MS/MS) method enabled fraction collection of milk oligosaccharides from biological mixtures based on composition and structure. The method is compatible with traditional gas pressure-driven flow flash chromatography widely employed in organic chemistry laboratories. The online mass detector enabled real-time optimization of chromatographic parameters to favor separation of oligosaccharides that would otherwise be indistinguishable from coeluting components with a nonspecific detector. Unlike previously described preparative LC/MS techniques, we have employed a dynamic flow connection that permits any flow rate from the flash system to be delivered from 1 to 200 ml/min without affecting the ionization conditions of the mass spectrometer. A new way of packing large amounts of graphitized carbon allowed the enrichment and separation of milligram quantities of structurally heterogeneous mixtures of human milk oligosaccharides (HMOs) and bovine milk oligosaccharides (BMOs). Abundant saccharide components in milk, such as lactose and lacto-N-tetraose, were separated from the rarer and less abundant oligosaccharides that have greater structural diversity and biological functionality. Neutral and acidic HMOs and BMOs were largely separated and enriched with a dual binary solvent system.     

Collaborative study on the isolation of salmonella from artificially contaminated milk powder

The suitability of artificially contaminated milk powder as a substrate for salmonella reference samples and its stability under different storage conditions were studied. The need for a reconstitution step in the standard isolation method for salmonellas from milk powders was also investigated. When milk powder was examined in this way with a reconstitution step, differences in laboratory methods and/or storage times had no significant effect on the results after storage at 4 degrees C. With powder stored at room temperature there was a systematic decrease in the number of samples positive as the storage time increased. It is concluded therefore that milk powder contaminated with salmonellas should be stored at 4 degrees C. Examination of such milk powder with a reconstitution step yielded better results than without it and this step is therefore necessary for improving the reproducibility of the method. No significant differences were encountered between the standard isolation method and that used in the authors' laboratories. The results of this study indicate that milk powder is suitable as basic material for reference samples and that a reconstitution step should be included in the standard salmonella isolation procedure.     

A comparison of the properties of membranes isolated from bovine skim milk and cream

Membrane material was isolated from skim milk and cream using the same samples of whole milk and similar purification techniques. The membranes from these two sources were characterised and compared by lipid, carbohydrate, enzymatic and electrophoretic analyses. The skim milk membranes contained higher levels of cholesterol, phospholipid and carbohydrate per mg of protein than the cream membranes. In general, the specific activities of the enzymes tested were also higher in the skim milk membranes, nucleotide pyrophosphatase, γ-glutamyltranspeptidase and sulphydryl oxidase being particularly active in these membranes. The major protein of the skim milk material had a molecular weight of approx. 85 000 and constituted 32% of the total protein. This particular protein band was almost absent in the cream membranes (only 3% of the total protein) where the major protein had a molecular weight of approx. 70 000 and constituted 34% of the total protein. Glycoprotein bands were also located in both membrane preparations but the position of these bands did not correspond with the areas which were stained with the protein staining reagent Coomassie blue. The major glycoprotein in both skim milk and cream membranes had an apparent molecular weight of 115 000. The biochemical and compositional differences between these membranes in milk provide further evidence for the skim milk membranes being more closely related to secretory cell plasma membrane than to the cream membranes. The data also lend support to the hypothesis of Keenan et al. ((1970) J. Cell Biol. 44, 80) that the cream membrane undergoes morphological and structural changes while evolving from the plasma membrane of the secretory cell.     

The survival of Staphylococcus aureus in raw milk with different cell count

The antibacterial activity of the individual udder quarter milk sample cannot be evaluated by the number of somatic cells. Milk with the highest number of cells is not always the most bactericidal. However, when the data were obtained from a material large enough for statistical calculations, negative and highly significant correlation coefficient (-0.465, P less than or equal to 0.01) between number of somatic cells in milk and number of bacteria surviving in it, and negative and significant correlation coefficient (-0.203, P 0.05) between number of somatic cells in milk and number of bacteria surviving in whey, were found. Results of the analysis of variance demonstrated highly significant differences as regards number of cells and bacteria surviving in milk and in whey between cows, quarters and stimulation time. Nevertheless, among ten investigated cows, three cows with high cell count and high antibacterial activity in milk and whey, highly reacting to stimulant, were found.