Rafael Jiménez-Flores

Using Confocal Laser Scanning Microscopy to Probe the Milk Fat Globule Membrane and Associated Proteins

The bovine milk fat globule membrane (MFGM) is an important, biologically relevant membrane due to its functional and health properties. Its composition has been thoroughly studied, but its structure, especially the lateral organization of its components, still remains unclear. We have used confocal laser scanning microscopy (CLSM) to investigate the surface structure of the MFGM in globules with different degrees of processing using two types of fluorescently labeled phospholipid probes and a protein dye. Using this technique, we have observed heterogeneities in the distribution of MFGM lipids and proteins relating to the processing and size of the globules. The effect of pretreating the milk (centrifugation, pasteurization-homogenization and churning) was studied by double-staining the surface of the milk fat globules, followed by observation using CLSM, and by determining the phospholipid profile of raw milk, raw cream, processed milk and buttermilk powder. Our findings agree with other techniques by showing that the composition of the MFGM changes with processing through the loss of phospholipids and the adsorption of caseins and whey proteins onto the surface.

Insulin-Like Growth Factors and Insulin-Like Growth Factor Binding Proteins in Porcine Serum and Milk throughout Lactation

ABSTRACT: IGF-I, IGF-II, and IGF binding proteins (IGFBP) were characterized in porcine serum, colostrum, and milk on d 1–28 postpartum. IGF-I and -II were measured by heterologous RIA. Scrum IGFBP were characterized by Western ligand blotting and milk IGF binding activity by [125I]-IGF binding assay. IGF-II accounted for 70–85% of serum IGF and rose 2-fold between d 1 and d 28. Serum IGF-I was unaffected by duration of lactation. Milk IGF-II concentrations were higher than IGF-I concentrations on d 1–7 postpartum. After d 10, milk IGF-I and IGF-II contents were not significantly different. Serum contained IGFBP with M, of 43, 39, 34, 28, and 24 kD. Over the course of lactation, the 43− and 39-kD bands increased, the 24-kD band decreased, and the 34− and 28-kD bands were unchanged. Milk IGF binding activity increased between d 1 (28%) and d 3 (44%), then declined until d 28 (7%). Serum and milk were separated by isoelectric focusing into 20 fractions, across a gradient from pH 3 to 10, that were screened for IGFBP by Western ligand blotting. Milk contained six IGFBP of similar Mr as serum IGFBP; however, the relative amounts of the IGFBP and their apparent isoelectric points differed. In conclusion, porcine milk contains both IGF-I and -II, with IGF-II predominating. Several IGFBP with similar Mr as those found in serum are present in milk. IGF peptide concentrations were highest in prepartum secretions and colostrum, whereas IGF binding activity peaked on d 4 of lactation.

Composition and Fatty Acid Distribution of Bovine Milk Phospholipids from Processed Milk Products

The aim of this work was to assess the accuracy of different extraction methods of phospholipids and to measure the effect that processing has on phospholipid composition. Four methods of extracting phospholipids from buttermilk powder were compared to optimize recovery of sphingomyelin. Using the optimal method, the phospholipid profile of four dairy products (raw milk, raw cream, homogenized and pasteurized milk, and buttermilk powder) was determined. A total lipid extraction by the Folch method followed by a solid-phase extraction using the Bitman method was the most efficient technique to recover milk sphingomyelin. Milk processing (churning, centrifuging, homogenization, spray-drying) affected the profile of milk phospholipids, leading to a loss of sphingomyelin and phosphatidylcholine after centrifugation for cream separation. A corresponding decrease in the saturation content of the raw cream phospholipids and a loss of phosphatidylethanolamine after spray-drying to produce buttermilk powder were also observed.

Dietary Milk Fat Globule Membrane Reduces the Incidence of Aberrant Crypt Foci in Fischer-344 Rats

Milk fat globule membrane (MFGM) is a biopolymer composed primarily of membrane proteins and lipids that surround the fat globules in milk. Although it is considered to have potential as a bioactive ingredient, few feeding studies have been conducted to measure its potential benefits. The aim of this investigation was to determine if dietary MFGM confers protection against colon carcinogenesis compared to diets containing corn oil (CO) or anhydrous milk fat (AMF). Male, weanling Fischer-344 rats were randomly assigned to one of three dietary treatments that differed only in the fat source: (1) AIN-76A diet, corn oil; (2) AIN-76A diet, AMF; and (3) AIN-76A diet, 50% MFGM, 50% AMF. Each diet contained 50 g/kg diet of fat. With the exception of the fat source, diets were formulated to be identical in macro and micro nutrient content. Animals were injected with 1,2-dimethylhydrazine once per week at weeks 3 and 4, and fed experimental diets for a total of 13 weeks. Over the course of the study dietary treatment did not affect food consumption, weight gain or body composition. After 13 weeks animals were sacrificed, colons were removed and aberrant crypt foci (ACF) were counted by microscopy. Rats fed the MFGM diet (n = 16) had significantly fewer ACF (20.9 +/- 5.7) compared to rats fed corn oil (n = 17) or AMF (n = 16) diets (31.3 +/- 9.5 and 29.8 +/- 11.4 respectively; P < 0.05). Gene expression analysis of colonic mucosa did not reveal differential expression of candidate colon cancer genes, and the sphingolipid profile of the colonic mucosa was not affected by diet. While there were notable and significant differences in plasma and red blood cell lipids, there was no relationship to the cancer protection. These results support previous findings that dietary sphingolipids are protective against colon carcinogenesis yet extend this finding to MFGM, a milk fat fraction available as a food ingredient.

Membrane-rich milk fat diet provides protection against gastrointestinal leakiness in mice treated with lipopolysaccharide

Milk fat globule membrane is a protein-lipid complex that may strengthen the gut barrier. The main objective of this study was to assess the ability of a membrane-rich milk fat diet to promote the integrity of the gut barrier and to decrease systemic inflammation in lipopolysaccharide (LPS)-challenged mice. Animals were randomly assigned to one of 2 American Institute of Nutrition (AIN)-76A formulations differing only in fat source: control diet (corn oil) and milk fat diet (anhydrous milk fat with 10% milk fat globule membrane). Each diet contained 12% calories from fat. Mice were fed diets for 5 wk, then injected with vehicle or LPS (10mg/kg of BW) and gavaged with dextran-fluorescein to assess gut barrier integrity. Serum was assayed for fluorescence 24h after gavage, and 16 serum cytokines were measured to assess the inflammatory response. Gut permeability was 1.8-fold higher in LPS-challenged mice fed the control diet compared with the milk fat diet. Furthermore, mice fed the milk fat diet and injected with LPS had lower serum levels of IL-6, IL-10, IL-17, monocyte chemotactic protein (MCP)-1, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and IL-3 compared with LPS-injected mice fed the control diet. The results indicate that the membrane-rich milk fat diet decreases the inflammatory response to a systemic LPS challenge compared with corn oil, and the effect coincides with decreased gut permeability.

Cloning And Sequence Analysis Of Bovine β-Casein cDNA

Abstract A bovine β-casein cDNA clone was isolated from a cDNA library prepared from mammary gland mRNA. Sequence analysis revealed 25 nucleotides (nt) of the 5′ noncoding region, 672 nt of the complete sequence coding and a 3′ region of approximately 500 nt. When the nucleotide sequence of bovine β-casein cDNA is compared to rat β-casein cDNA (5), a high degree of homology is observed in the first 100 nt corresponding to the signal peptide of the pre-β-caseins.

Surface Characterization of Bovine Milk Phospholipid Monolayers by Langmuir Isotherms and Microscopic Techniques

Monolayers were prepared from phospholipids extracted from bovine milk and used as a model system to mimic the native milk fat globule membrane (MFGM) surface structure in various microscopic experiments. The natural complex mixtures of phospholipids were isolated from bovine raw milk, raw cream, processed whole milk, and buttermilk powder by total lipid extraction and solid-phase extraction. A Langmuir film balance mounted on an epifluorescence microscope was used to analyze the physical behavior of the monolayer films and the phase coexistence resulting from the formation of phospholipid microdomains within these films. Atomic force microscopy was used for nanometer-scale topographic resolution of the microdomains. This study allowed comparison of the behavior of phospholipid monolayers from dairy products at different stages of processing, analysis of the formation of microdomains, and the study of the effect of milk processing on lipid-lipid interactions and phase coexistence. It was observed that milk processing changes the physical behavior of phospholipid monolayers by altering the phospholipid profile and the fatty acid distribution.

Expression of a Bovine κ-CN cDNA in the Mammary Gland of Transgenic Mice Utilizing a Genomic Milk Protein Gene as an Expression Cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine κ-casein (κ-CN) cDNA under the control of the goat β-CN 5′ promoter elements and 3′ flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine κ-CN RNA to the mammary gland and secretion of bovine κ-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine κ-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine κ-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine κ-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did κ-CN from bovine milk. A high degree of variation in the production of bovine κ-CN within each of the transgenic lines was observed.

Microbial communities in pre-columbian coprolites.

The study of coprolites from earlier cultures represents a great opportunity to study an “unaltered” composition of the intestinal microbiota. To test this, pre-Columbian coprolites from two cultures, the Huecoid and Saladoid, were evaluated for the presence of DNA, proteins and lipids by cytochemical staining, human and/or dog-specific Bacteroides spp. by PCR, as well as bacteria, fungi and archaea using Terminal Restriction Fragment analyses. DNA, proteins and lipids, and human-specific Bacteroides DNA were detected in all coprolites. Multidimensional scaling analyses resulted in spatial arrangements of microbial profiles by culture, further supported by cluster analysis and ANOSIM. Differences between the microbial communities were positively correlated with culture, and SIMPER analysis indicated 68.8% dissimilarity between the Huecoid and Saladoid. Proteobacteria, Bacteroidetes and methanogens were found in all coprolite samples. Propionebacteria, Shewanella and lactic acid bacteria dominated in the Huecoid samples, while Acidobacteria, and peptococci were dominant in Saladoid samples. Yeasts, including Candida albicans and Crypotococcus spp. were found in all samples. Basidiomycetes were the most notable fungi in Huecoid samples while Ascomycetes predominated in Saladoid samples, suggesting differences in dietary habits. Our study provides an approach for the study of the microbial communities of coprolite samples from various cultures.

Characterization of Lactobacillus reuteri Interaction with Milk Fat Globule Membrane Components in Dairy Products

A set of methods has been developed to study the adhesion between four Lactobacillus reuteri strains and the milk fat globule membrane (MFGM) components in dairy products. By combining sucrose density gradient (SDG) centrifugation and bacterial DNA quantification it was found which strains of L. reuteri were more strongly associated with the dairy products, and the results were corroborated by direct binding rate and force measurements made with optical tweezers. It was determined that strong binding was associated with hydrophobicity of the bacteria and that this hydrophobicity is correlated with the presence of LiCl-extractable protein on the surface of the bacteria. Confocal laser scanning microscopy (CLSM) allowed for the visualization of interactions between bacteria and MFGM. This study demonstrates that these methods can be used in combination to characterize, both qualitatively and quantitatively, the adhesion of lactic acid bacteria strains in dairy products.