On the utility of fluorescence-detection analytical ultracentrifugation in probing biomolecular interactions in complex solutions: a case study in milk.

ß-Lactoglobulin is the most abundant protein in the whey fraction of ruminant milks, yet is absent in human milk. It has been studied intensively due to its impact on the processing and allergenic properties of ruminant milk products. However, the physiological function of ß-lactoglobulin remains unclear. Using the fluorescence-detection system within the analytical ultracentrifuge, we observed an interaction involving fluorescently labelled ß-lactoglobulin in its native environment, i.e. cow and goat milk, for the first time. Co-elution experiments support that these ß-lactoglobulin interactions occur naturally in milk and provide evidence that the interacting partners are immunoglobulins, while further sedimentation velocity experiments confirm that an interaction occurs between these molecules. The identification of these interactions, made possible through the use of fluorescence-detected analytical ultracentrifugation, provides possible clues to the long debated physiological function of this abundant milk protein.

Supplementation with Bovine Milk or Soy Beverages Recovers Bone Mineralization in Young Growing Rats Fed an Insufficient Diet, in Contrast to an Almond Beverage.

BACKGROUND: Nondairy beverages, produced from soy, rice, oat, almond, or coconut, are increasingly being used as alternatives to dairy milk, with the perception that they are healthier and/or more sustainable products than dairy products. OBJECTIVE: The aim of this study was to compare the effects of supplementing either bovine milk, soy, or almond-based beverages to young, growing rats fed an intact-protein diet or a diet that had protein substituted with amino acids (AA-diet). METHODS: Three-week-old male Sprague-Dawley rats were randomly assigned to 5 groups (n = 10/group) and fed ad libitum for 4 wk. Two control groups were fed either standard AIN-93G food [20% casein (CN) protein] or AIN-93G with amino acids (AAs) equivalent to CN protein, and water to drink. Three treatment groups were fed AIN-93G AA and supplemented with either bovine ultra-heat treatment (UHT) milk or soy or almond UHT beverages. Rat weight gain and food intakes were recorded. During week 4, body composition was assessed using DEXA to determine lean soft tissue, fat, and bone mass. At trial end, bone biomechanical properties and blood plasma mineral concentrations were measured. RESULTS: At the end of the trial, animals supplemented with almond beverage were lightest (P > 0.05), with higher plasma calcium concentrations (P > 0.05) and lower bone mineral content (BMC) and bone density (P > 0.05) than animals supplemented with milk or soy beverage. Soy-supplemented animals had similar BMC and bone density compared with milk-supplemented animals, although the soy group gained most weight (P > 0.05) and had the highest fat:lean ratio (P > 0.05) compared with other groups. CONCLUSIONS: In the model tested, supplementing rats with bovine UHT milk and soy UHT beverage provided favorable bone health outcomes. Conversely, almond UHT beverage was not an effective supplement and could be detrimental to bone mineralization and strength outcomes.

Gastric digestion of cow and goat milk: Peptides derived from simulated conditions of infant digestion.

Infant formula products are predominantly manufactured using cow milk protein; goat milk also provides a suitable protein source. In this study, we directly compared cow and goat milk protein digestion using pH and enzyme conditions to simulate infant gastric conditions. Generated peptides, identified using liquid chromatography coupled to a mass spectrometer, show both similarities and differences in cow and goat milk post-digestion profiles. The majority of peptides were from casein proteins, 50% representing ß-casein, with many peptides unique to each species. Low or no peptides for ß-Lactoglobulin and α-Lactalbumin, respectively, suggest these proteins were highly resistant to infant gastric digestion, as reported by others. Minor milk proteins, comprising 5% of peptides, were represented by different proteins from cow and goat. Peptides with known bioactivities were also observed, both in common and unique to each species. Together these data may explain reported differences in digestion characteristics of cow and goat milk.

Nε-carboxymethyllysine in nutritional milk formulas for infants.

Production of infant formulas involves high temperature processing for microbiological safety. However, heat processes generate Advanced Glycation End-products (AGEs), including Nε-carboxymethyllysine (CML) formed between lysine and lactose. Formulas manufactured from cow or goat milk, with or without whey adjustment, or hydrolysates of cow whey proteins, were tested for CML levels using a commercially available ELISA kit. CML concentrations ranged from 2 to 210 µg/g protein in formulas containing intact proteins. Median CML concentrations were up to 3-fold greater in formulas containing 60% whey protein compared with 20% whey protein, for both cow and goat formulas. Goat milk formulas contained 7 to 12-fold less CML than cow milk formulas. Formulas made from intact proteins contained lower CML compared to formulas using whey hydrolysates. Western immunoblotting techniques detected higher CML levels in whey proteins compared with casein. This study showed whey addition to infant formula significantly contributes to CML levels.

Digestive Responses to Fortified Cow or Goat Dairy Drinks: A Randomised Controlled Trial.

Fortified milk drinks are predominantly manufactured from bovine (cow) sources. Alternative formulations include those prepared with hydrolysed bovine milk proteins or from alternate bovidae species, such as caprine (goat) milk. Currently, there is little data on protein digestive and metabolic responses following ingestion of fortified milk drinks. To examine the digestive and metabolic responses to commercially-available fortified milks, young adults (n = 15 males: 15 females), in a randomised sequence, ingested isonitrogenous quantities of whole cow-protein (WC), whole goat-protein (WG), or partially-hydrolysed whey cow-protein (HC), commercial fortified milks. Plasma amino acid (AA) and hormonal responses were measured at baseline and again at 5 h after ingestion. Paracetamol recovery, breath hydrogen, and subjective digestive responses were also measured. Postprandial plasma AA was similar between WC and WG, while AA appearance was suppressed with HC. Following HC, there was a negative incremental AUC in plasma branched-chain AAs. Further, HC had delayed gastric emptying, increased transit time, and led to exaggerated insulin and GLP-1 responses, in comparison to whole protein formulas. Overall, WC and WG had similar protein and digestive responses with no differences in digestive comfort. Contrastingly, HC led to delayed gastric emptying, attenuated AA appearance, and a heightened circulating insulin response.

The self-association and thermal denaturation of caprine and bovine ß-lactoglobulin.

Milk components, such as proteins and lipids, have different physicochemical properties depending upon the mammalian species from which they come. Understanding the different responses of these milks to digestion, processing, and differences in their immunogenicity requires detailed knowledge of these physicochemical properties. Here we report on the oligomeric state of ß-lactoglobulin from caprine milk, the most abundant protein present in the whey fraction. At pH 2.5 caprine ß-lactoglobulin is predominantly monomeric, whereas bovine ß-lactoglobulin exists in a monomer-dimer equilibrium at the same protein concentrations. This behaviour was also observed in molecular dynamics simulations and can be rationalised in terms of the amino acid substitutions present between caprine and bovine ß-lactoglobulin that result in a greater positive charge on each subunit of caprine ß-lactoglobulin at low pH. The denaturation of ß-lactoglobulin when milk is heat-treated contributes to the fouling of heat-exchange surfaces, reducing yields and increasing cleaning costs. The bovine and caprine orthologues of ß-lactoglobulin display different responses to thermal treatment, with caprine ß-lactoglobulin precipitating at higher pH values than bovine ß-lactoglobulin (pH 7.1 compared to pH 5.6) that are closer to the natural pH of these milks (pH 6.7). This property of caprine ß-lactoglobulin likely contributes to the reduced heat stability of caprine milk compared to bovine milk at its natural pH.

Effect of the dietary delivery matrix on vitamin D3 bioavailability and bone mineralisation in vitamin-D3-deficient growing male rats.

This study assessed bioavailability and utilisation of vitamin D3 in two feeding trials using young, growing Sprague-Dawley male rats. Trial one fed animals standard AIN-93G diet (casein protein) containing no vitamin D3 and goat or cow skimmed milk supplemented with vitamin D3. Trial two fed animals modified dairy-free AIN-93G diet (egg albumin) containing no vitamin D3 and goat or cow skimmed or full-fat milk supplemented with vitamin D3. Control groups received AIN-93G diets with or without vitamin D, and water. At 8 weeks of age, blood samples were collected for vitamin and mineral analysis, and femurs and spines were collected for assessment of bone mineralisation and strength. In both trials, analyses showed differences in bioavailability of vitamin D3, with ratios of serum 25-hydroxyvitamin D3 to vitamin D3 intake more than 2-fold higher in groups drinking supplemented milk compared with groups fed supplemented solid food. Bone mineralisation was higher in groups drinking supplemented milk compared with groups fed supplemented solid food, for both trials (P<0·05). There was no difference in the parameters tested between skimmed milk and full-fat milk or between cow milk and goat milk. Comparison of the two trials suggested that dietary protein source promoted bone mineralisation in a growing rat model: modified AIN-93G with egg albumin produced lower bone mineralisation compared with standard AIN-93G with casein. Overall, this study showed that effects of vitamin D3 deficiency in solid diets were reversed by offering milk supplemented with vitamin D3, and suggests that using milk as a vehicle to deliver vitamin D is advantageous.

Gastric digestion of cow and goat milk: Impact of infant and young child in vitro digestion conditions.

Many infants and young children are fed nutritional milk formulas. Although products are commonly based on cow milk, goat milk provides an alternative. We directly compared digestion of cow and goat milk proteins, varying pH, enzyme concentrations and incubation times to simulate infant and young child gastric conditions. Protein digestion and peptide formation were evaluated using electrophoresis and chromatography. Digestion of higher molecular weight whey proteins increased with decreased pH and higher enzyme concentrations of young child gastric digestion conditions compared to infant conditions. ß-lactoglobulin was poorly digested under all gastric digestion conditions. Caseins reacted to pH changes differently compared to whey proteins, with less digestion of casein at pH 3.0 than at pH 5.0. Caseins from goat milk tended to be more efficiently digested compared to caseins from cow milk and peptide profiles from goat milk were distinct from cow milk.

Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets.

BACKGROUND: Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy. METHODS: We developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions. RESULTS: A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0-32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01-4.6%) compared to subsp. longum (0.1-33.7% abundance; 21.4-100% prevalence). DISCUSSION: Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.

Comparative innate immune interactions of human and bovine secretory IgA with pathogenic and non-pathogenic bacteria.

Secretory IgA (SIgA) from milk contributes to early colonization and maintenance of commensal/symbiotic bacteria in the gut, as well as providing defence against pathogens. SIgA binds bacteria using specific antigenic sites or non-specifically via its glycans attached to α-heavy-chain and secretory component. In our study, we tested the hypothesis that human and bovine SIgA have similar innate-binding activity for bacteria. SIgAs, isolated from human and bovine milk, were incubated with a selection of commensal, pathogenic and probiotic bacteria. Using flow cytometry, we measured numbers of bacteria binding SIgA and their level of SIgA binding. The percentage of bacteria bound by human and bovine SIgA varied from 30 to 90% depending on bacterial species and strains, but was remarkably consistent between human and bovine SIgA. The level of SIgA binding per bacterial cell was lower for those bacteria that had a higher percentage of SIgA-bound bacteria, and higher for those bacteria that had lower percentage of SIgA-bound bacteria. Overall, human and bovine SIgA interacted with bacteria in a comparable way. This contributes to longer term research about the potential benefits of bovine SIgA for human consumers.

Feeding bovine milks with low or high IgA levels is associated with altered re-establishment of murine intestinal microbiota after antibiotic treatment.

Antibiotics are a vital and commonly used therapeutic tool, but their use also results in profound changes in the intestinal microbiota that can, in turn, have significant health consequences. Understanding how the microbiota recovers after antibiotic treatment will help to devise strategies for mitigating the adverse effects of antibiotics. Using a mouse model, we have characterized the changes occurring in the intestinal microbiota immediately after five days exposure to ampicillin, and then at three and fourteen days thereafter. During the fourteen day period of antibiotic recovery, groups of mice were fed either water, cows' milk containing high levels of IgA, or cows' milk containing low levels of IgA as their sole source of liquid. Effects on microbiota of feeding milks for 14 days were also assessed in groups of mice that had no ampicillin exposure. Changes in microbiota were measured by high throughput sequencing of the V4 to V6 variable regions of the 16S ribosomal RNA gene. As expected, exposure to ampicillin led to profound changes to the types and abundance of bacteria present, along with a loss of diversity. At 14 days following antibiotic exposure, mice fed water had recovered microbiota compositions similar to that prior to antibiotics. However, feeding High-IgA milk to mice that has been exposed to antibiotics was associated with altered microbiota compositions, including increased relative abundance of Lactobacillus and Barnesiella compared to the start of the study. Mice exposed to antibiotics then fed Low-IgA milk also showed increased Barnesiella at day 14. Mice without antibiotic perturbation, showed no change in their microbiota after 14 days of milk feeding. Overall, these findings add to a knowledge platform for optimizing intestinal function after treatment with antibiotics in the human population.

Immunoglobulin A in Bovine Milk: A Potential Functional Food?

Immunoglobulin A (IgA) is an anti-inflammatory antibody that plays a critical role in mucosal immunity. It is found in large quantities in human milk, but there are lower amounts in bovine milk. In humans, IgA plays a significant role in providing protection from environmental pathogens at mucosal surfaces and is a key component for the establishment and maintenance of intestinal homeostasis via innate and adaptive immune mechanisms. To date, many of the dairy-based functional foods are derived from bovine colostrum, targeting the benefits of IgG. IgA has a higher pathogenic binding capacity and greater stability against proteolytic degradation when ingested compared with IgG. This provides IgA-based products greater potential in the functional food market that has yet to be realized.

Ultra-high resolution crystal structure of recombinant caprine ß-lactoglobulin.

ß-Lactoglobulin (ßlg) is the most abundant whey protein in the milks of ruminant animals. While bovine ßlg has been subjected to a vast array of studies, little is known about the caprine ortholog. We present an ultra-high resolution crystal structure of caprine ßlg complemented by analytical ultracentrifugation and small-angle X-ray scattering data. In both solution and crystalline states caprine ßlg is dimeric (K(D)<5 µM); however, our data suggest a flexible quaternary arrangement of subunits within the dimer. These structural findings will provide insight into relationships among structural, processing, nutritional and immunological characteristics that distinguish cow's and goat's milk.

Effect of raw milk on allergic responses in a murine model of gastrointestinal allergy.

Epidemiological studies have shown an association between the consumption of raw farm milk and reduced incidence of allergy. In the present study, we fed untreated raw milk, gamma-sterilised milk, heat-treated milk or water to mice and compared their responses to allergen exposure and challenge treatment in a mouse model of gastrointestinal allergy. From weaning (3 weeks old), groups of BALB/c female mice (n 8) received raw milk, gamma-sterilised milk, heated milk or water via drink bottles, with the control group receiving water. All mice were fed a standard (dairy protein-free) rodent diet. At 6 and 8 weeks, groups were given intra-peritoneal injections with ovalbumin (OVA)/alum to sensitise them to the antigen. Controls were sham immunised. At week 10, mice were fasted and challenged four times on alternate days by intra-gastric administration with 50 mg OVA or saline. Levels of bacteria and milk proteins were assessed in milk samples. Mouse serum levels of specific IgE, IgG1 and IgG2a antibodies and mouse mast cell protease-1 (MMCP-1) were determined. Cytokine responses to 48 h activation with OVA were measured in cultured splenocytes from mice. Sterilised and heated milks contained no viable bacteria and reduced detectable levels of many milk proteins, in contrast to raw milk. Mice drinking raw milk had highest serum MMCP-1 and specific-OVA IgE responses. Cultured splenocytes from OVA-primed mice produced similar levels of IL-4 in response to the antigen; however, IL-10 levels were highest from mice drinking raw milk. Overall, the present study adds to the evidence that consuming different types of milk can affect allergic responses to a non-related dietary antigen.

Comparison of the compositions of the stool microbiotas of infants fed goat milk formula, cow milk-based formula, or breast milk.

The aim of the study was to compare the compositions of the fecal microbiotas of infants fed goat milk formula to those of infants fed cow milk formula or breast milk as the gold standard. Pyrosequencing of 16S rRNA gene sequences was used in the analysis of the microbiotas in stool samples collected from 90 Australian babies (30 in each group) at 2 months of age. Beta-diversity analysis of total microbiota sequences and Lachnospiraceae sequences revealed that they were more similar in breast milk/goat milk comparisons than in breast milk/cow milk comparisons. The Lachnospiraceae were mostly restricted to a single species (Ruminococcus gnavus) in breast milk-fed and goat milk-fed babies compared to a more diverse collection in cow milk-fed babies. Bifidobacteriaceae were abundant in the microbiotas of infants in all three groups. Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium bifidum were the most commonly detected bifidobacterial species. A semiquantitative PCR method was devised to differentiate between B. longum subsp. longum and B. longum subsp. infantis and was used to test stool samples. B. longum subsp. infantis was seldom present in stools, even of breast milk-fed babies. The presence of B. bifidum in the stools of breast milk-fed infants at abundances greater than 10% of the total microbiota was associated with the highest total abundances of Bifidobacteriaceae. When Bifidobacteriaceae abundance was low, Lachnospiraceae abundances were greater. New information about the composition of the fecal microbiota when goat milk formula is used in infant nutrition was thus obtained.

Can nutritional modulation of maternal intestinal microbiota influence the development of the infant gastrointestinal tract?

The gastrointestinal microbiota plays an important role in maintaining host health by preventing the colonization of pathogens, fermenting dietary compounds, and maintaining normal mucosal immunity. Particularly in early life, the composition of the microbiota profoundly influences the development and maturation of the gastrointestinal tract (GIT) mucosa, which may affect health in later life. Therefore, strategies to manipulate the microbiota during infancy may prevent the development of some diseases later in adult life. Earlier research suggested that term fetuses are sterile and that the initial bacterial colonization of the newborn GIT occurs only after the baby transits through the birth canal. However, recent studies have demonstrated that the colonization and/or contact of the fetus with the maternal GIT microbiota may start in utero. After vaginal birth, the colonization of the neonate GIT continues through contact with maternal feces and vaginal bacteria, leading to a relatively simple microbial community that is influenced by feeding type (breast vs. formula feeding). Maternal GIT microbiota, vaginal microbiota, and breast milk composition are influenced by maternal diet. Alterations of the maternal GIT microbiota composition via supplementation with probiotics and prebiotics have been shown; however, transfer of these benefits to the offspring remains to be demonstrated. This review focuses on the influence of maternal GIT microbiota during the pre- and postpartum periods on the colonization of the infant GIT. In particular, it examines the manipulation of the maternal GIT microbiota composition through the use of probiotics and/or prebiotics and subsequent consequences for the health of the offspring.

Changes in colon gene expression associated with increased colon inflammation in interleukin-10 gene-deficient mice inoculated with Enterococcus species.

BACKGROUND: Inappropriate responses to normal intestinal bacteria may be involved in the development of Inflammatory Bowel Diseases (IBD, e.g. Crohn's Disease (CD), Ulcerative Colitis (UC)) and variations in the host genome may mediate this process. IL-10 gene-deficient (Il10-/-) mice develop CD-like colitis mainly in the colon, in part due to inappropriate responses to normal intestinal bacteria including Enterococcus strains, and have therefore been used as an animal model of CD. Comprehensive characterization of changes in cecum gene expression levels associated with inflammation in the Il10-/- mouse model has recently been reported. Our aim was to characterize changes in colonic gene expression levels in Il10-/- and C57BL/6J (C57; control) mice resulting from oral bacterial inoculation with 12 Enterococcus faecalis and faecium (EF) strains isolated from calves or poultry, complex intestinal flora (CIF) collected from healthy control mice, or a mixture of the two (EF.CIF). We investigated two hypotheses: (1) that oral inoculation of Il10-/- mice would result in greater and more consistent intestinal inflammation than that observed in Il10-/- mice not receiving this inoculation, and (2) that this inflammation would be associated with changes in colon gene expression levels similar to those previously observed in human studies, and these mice would therefore be an appropriate model for human CD. RESULTS: At 12 weeks of age, total RNA extracted from intact colon was hybridized to Agilent 44 k mouse arrays. Differentially expressed genes were identified using linear models for microarray analysis (Bioconductor), and these genes were clustered using GeneSpring GX and Ingenuity Pathways Analysis software. Intestinal inflammation was increased in Il10-/- mice as a result of inoculation, with the strongest effect being in the EF and EF.CIF groups. Genes differentially expressed in Il10-/- mice as a result of EF or EF.CIF inoculation were associated with the following pathways: inflammatory disease (111 genes differentially expressed), immune response (209 genes), antigen presentation (11 genes, particularly major histocompatability complex Class II), fatty acid metabolism (30 genes) and detoxification (31 genes). CONCLUSIONS: Our results suggest that colonic inflammation in Il10-/- mice inoculated with solutions containing Enterococcus strains is associated with gene expression changes similar to those of human IBD, specifically CD, and that with the EF.CIF inoculum in particular this is an appropriate model to investigate food-gene interactions relevant to human CD.

Immune components of colostrum and milk--a historical perspective.

Key developments in the understanding of the immune functions of milk and colostrum are reviewed, focusing on their proteinaceous components. The topics covered include the immunoglobulins, immune cells, immunomodulatory substances, and antimicrobial proteins. The contributions of new technologies and the introduction of fresh approaches from other fields are highlighted, as are the contributions that mammary biology research has made to the development of other fields. Finally, a summary of some current outstanding questions and likely future directions of the field are given.

Production from dairy cows of semi-industrial quantities of milk-protein concentrate (MPC) containing efficacious anti-Candida albicans IgA antibodies.

Bovine milk antibodies directed against human pathogenic organisms have potential as prophylactic or therapeutic treatments of disorders affecting mucosal surfaces. The cow, however, does not naturally secrete high levels of IgA in milk, the predominant immunoglobulin of the mucosal immune system. We have patented an immunisation protocol that results in increased production of IgA in ruminant milk and in this study established that our protocol can be used on a scale sufficient to produce semi-industrial quantities of milk for processing. Cows were immunised with a common pathogenic yeast, Candida albicans and responded with high levels of antigen-specific IgA antibodies in their milk. The spray-dried milk-protein concentrate (85% protein) powder was shown to reduce adherence of Cand. albicans cells in in vitro adherence assays, demonstrating an ability to retain efficacy through the processing. These results suggest that this milk product may be of therapeutic value if the reduction in Cand. albicans adhesion observed in vitro translates to reduced colonisation in vivo.