Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk

Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties.     

Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria

The bifidogenic effect of human milk oligosaccharides (HMOs) has long been known, yet the precise mechanism underlying it remains unresolved. Recent studies show that some species/subspecies of Bifidobacterium are equipped with genetic and enzymatic sets dedicated to the utilization of HMOs, and consequently they can grow on HMOs; however, the ability to metabolize HMOs has not been directly linked to the actual metabolic behavior of the bacteria. In this report, we clarify the fate of each HMO during cultivation of infant gut-associated bifidobacteria. Bifidobacterium bifidum JCM1254, Bifidobacterium longum subsp. infantis JCM1222, Bifidobacterium longum subsp. longum JCM1217, and Bifidobacterium breve JCM1192 were selected for this purpose and were grown on HMO media containing a main neutral oligosaccharide fraction. The mono- and oligosaccharides in the spent media were labeled with 2-anthranilic acid, and their concentrations were determined at various incubation times using normal phase high performance liquid chromatography. The results reflect the metabolic abilities of the respective bifidobacteria. B. bifidum used secretory glycosidases to degrade HMOs, whereas B. longum subsp. infantis assimilated all HMOs by incorporating them in their intact forms. B. longum subsp. longum and B. breve consumed lacto-N-tetraose only. Interestingly, B. bifidum left degraded HMO metabolites outside of the cell even when the cells initiate vegetative growth, which indicates that the different species/subspecies can share the produced sugars. The predominance of type 1 chains in HMOs and the preferential use of type 1 HMO by infant gut-associated bifidobacteria suggest the coevolution of the bacteria with humans.     

Studies on the Bacillus flora of milk and milk products

Bacillus licheniformis and B. cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing. Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B. cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months. Although B. licheniformis was usually isolated in larger numbers than B. cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B. cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production. Investigation of the growth kinetics of strains of both species showed that B. cereus grew faster than B. licheniformis at ambient temperatures. It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B. cereus, is not necessarily the most important source of this organism.     

Optimization of a reconstituted skim milk based medium for enhanced CLA production by bifidobacteria

Aims:  To determine the effect of a range of supplements on the bioconversion of linoleic acid to conjugated linoleic acid (CLA) by Bifidobacterium breve NCIMB 702258 in reconstituted skim milk (RSM).
Results:  Seven supplements (yeast extract, casein hydrolysate, tryptone, l -cysteine hydrochloride, sodium acetate, sodium butyrate and sodium propionate) were identified as increasing the bioconversion of linoleic acid to c9 , t 11 CLA. Using these supplements, the percentage bioconversion of linoleic acid (0·35 mg ml^−l) to the c9 , t 11 CLA isomer was elevated from 15·5 ± 1·1% in 20% RSM (w/v) to 48·1 ± 2·2% in the supplemented RSM. Through additional supplementation of 20 mg m1⁻¹ inulin and optimization of inoculum and linoleic acid concentration, the percentage bioconversion to c9 , t 11 CLA was increased to 55·0 + 3·2%.
Conclusions:  Through supplementation, the concentration of CLA produced by bifidobacteria in RSM can be increased to levels comparable to those observed in the synthetic medium cys-MRS.
Significance and Impact of the Study:  The impact of 22 supplements on the production of the c9 , t 11 CLA isomer by the strain B. breve NCIMB 702258 in milk has been determined. The results provide an understanding of the factors, which influence CLA production by bifidobacteria in RSM.     

Farm animal milk proteomics

Milk is one of the most important nutrients for humans during lifetime. Farm animal milk in all its products like cheese and other fermentation and transformation products is a widespread nutrient for the entire life of humans. Proteins are key molecules of the milk functional component repertoire and their investigation represents a major challenge. Proteins in milk, such as caseins, contribute to the formation of micelles that are different from species to species in dimension and casein-type composition; they are an integral part of the MFGM (Milk Fat Globule Membrane) that has being exhaustively studied in recent years. Milk proteins can act as enzymes or have an antimicrobial activity; they could act as hormones and, last but not least, they have a latent physiological activity encoded in their primary structure that turns active when the protein is cleaved by fermentation or digestion processes. In this review we report the last progress in proteomics, peptidomics and bioinformatics. These new approaches allow us to better characterize the milk proteome of farm animal species, to highlight specific PTMs, the peptidomic profile and even to predict the potential nutraceutical properties of the analyzed proteins.     

Temporal changes in milk proteomes reveal developing milk functions

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.     

Chemical characterization of milk oligosaccharides of a spotted hyena (Crocuta crocuta)

The Carnivora include the superfamilies Canoidea and Feloidea. In species of Canoidea other than Canidae, the milk contains only traces of lactose and much larger concentrations of oligosaccharides. In this study, the following oligosaccharides were characterized in the milk of a spotted hyena, which is a species of Feloidea species: Neu5Ac(alpha2-3)Gal(beta1-4)Glc, Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc, Gal(alpha1-3)Gal(beta1-4)Glc and Fuc(alpha1-2)Gal(beta1-4)Glc. Lactose was found to be the predominant saccharide; in this respect, the hyena milk is markedly different from the milks of most species of Canoidea species. The sole presence of 3'-SL in the spotted hyena milk is interesting, because the co-presence of 3'-SL and 6'-SL has been reported in the milk or colostrum of many mammalian species.     

The host defense proteome of human and bovine milk

Milk is the single source of nutrients for the newborn mammal. The composition of milk of different mammals has been adapted during evolution of the species to fulfill the needs of the offspring. Milk not only provides nutrients, but it also serves as a medium for transfer of host defense components to the offspring. The host defense proteins in the milk of different mammalian species are expected to reveal signatures of evolution. The aim of this study is therefore to study the difference in the host defense proteome of human and bovine milk. We analyzed human and bovine milk using a shot-gun proteomics approach focusing on host defense-related proteins. In total, 268 proteins in human milk and 269 proteins in bovine milk were identified. Of these, 44 from human milk and 51 from bovine milk are related to the host defense system. Of these proteins, 33 were found in both species but with significantly different quantities. High concentrations of proteins involved in the mucosal immune system, immunoglobulin A, CD14, lactoferrin, and lysozyme, were present in human milk. The human newborn is known to be deficient for at least two of these proteins (immunoglobulin A and CD14). On the other hand, antimicrobial proteins (5 cathelicidins and lactoperoxidase) were abundant in bovine milk. The high concentration of lactoperoxidase is probably linked to the high amount of thiocyanate in the plant-based diet of cows. This first detailed analysis of host defense proteins in human and bovine milk is an important step in understanding the function of milk in the development of the immune system of these two mammals.     

Safety of milk and milk derivatives in relation to BSE: the lactoferrin example

Bovine Spongiform Encephalopathy (BSE) belongs to Transmissible Spongiform Encephalopathies (TSEs) or Prion diseases. BSE is a feed borne infection of cattle. Epidemiological and laboratory data suggest that the BSE infectious agent is responsible for the variant form of Creutzfeldt-Jakob Disease (vCJD) and that the oral route is the most plausible way of infection. Therefore there is concern that the BSE agent can be transmitted to humans by biological materials (i.e. meat products, blood, milk) from susceptible BSE animal species (mostly cows but possibly, sheep and goats). Lactoferrin (LF) can be produced by purification from large volumes of cow's milk or whey. Therefore, a potential BSE risk for milk and milk products needs to be evaluated by risk assessment. The Committee for proprietary Medicinal Products--CPMP of the European Commission and the WHO have categorized risk tissues from TSE susceptible ruminant species in different classes in relation to the BSE risk for medicinal products. Milk, colostrum, and tissues of the mammary gland have been classified in the category of no detectable infectivity. A secondary contamination of milk can be virtually excluded (i.e. milk is taken from living animals). In the light of current scientific knowledge and irrespective of the geographical origin, milk and milk derivatives (e.g. lactoferrin, lactose) are unlikely to present any risk of TSE contamination provided that milk is sourced from healthy animals in the same conditions as milk collected from human consumption. So the risk of milk and milk derivatives in relation to BSE is negligible.     

Cellular prion protein in ovine milk

Cellular prion protein, PrP(C), is essential for the development of prion diseases where it is considered to be a substrate for the formation of the disease-associated conformer, PrP(Sc). In sheep, PrP(C) is abundant in neuronal tissue and is also found at lower concentrations in a range of non-neuronal tissues, including mammary gland. Here, we demonstrate the presence of soluble PrP(C) in the non-cellular, non-lipid fraction of clarified ovine milk. Compared with brain-derived PrP(C), ovine milk PrP(C) displays an increased electrophoretic mobility. Ovine milk PrP(C) is mainly present as three species that differ in the extent of their N-linked glycosylation, with glycoform profiles varying among animals. Similar PrP(C) species are also present in fresh and commercial homogenised/pasteurised bovine milk, with additional N-terminal PrP(C) fragments detectable in ruminant milk and commercial milk products.     

Human milk oligosaccharides promote the growth of staphylococci

Human milk oligosaccharides (HMO), which constitute a major component of human milk, promote the growth of particular bacterial species in the infant's gastrointestinal tract. We hypothesized that HMO also interact with the bacterial communities present in human milk. To test this hypothesis, two experiments were conducted. First, milk samples were collected from healthy women (n = 16); culture-independent analysis of the bacterial communities was performed, HMO content was analyzed, and the relation between these factors was investigated. A positive correlation was observed between the relative abundance of Staphylococcus and total HMO content (r = 0.66). In a follow-up study, we conducted a series of in vitro growth curve experiments utilizing Staphylococcus aureus or Staphylococcus epidermidis and HMO isolated from human milk. HMO exhibited stimulatory effects on bacterial growth under various nutritional conditions. Analysis of culture supernatants from these experiments revealed that HMO did not measurably disappear from the culture medium, indicating that the growth-enhancing effects were not a result of bacterial metabolism of the HMO. Instead, stimulation of growth caused greater utilization of amino acids in minimal medium. Collectively, the data provide evidence that HMO may promote the growth of Staphylococcus species in the lactating mammary gland.     

Potential uses of milk epithelial cells: a review

Secretions collected from the mammary gland of different species contain heterogeneous populations of cells including lymphocytes, neutrophils, macrophages and epithelial cells in different species. Several factors influence the somatic cell count in milk and the distribution of cell types, such as species, infection status, physiological status and management practices. The epithelial cells are shed into milk during the lactation process. Most of them are viable and exhibit the characteristics of fully differentiated alveolar cells. Primary cultures of epithelial cells from colostrum and milk of humans, baboons, cows and goats together with established cell lines from human and goat milk, provide a good model for the study of lactogenesis, immunity transmission, cancer research and infection by viruses. The RNA extracted from milk cells have been shown to be representative of gene expression in the mammary gland and thus provide a source of material for molecular studies of gene expression and environmental interactions.     

Glycobiology of human milk

Glycans are characteristic components of milk, and each species has unique patterns of specific carbohydrates. Human milk is unusually rich in glycans, with the major components being lactose and oligosaccharides, representing approximately 6.8 and 1% of the milk, respectively. Other sources of glycans in human milk include monosaccharides, mucins, glycosaminoglycans, glycoproteins, glycopeptides, and glycolipids. In human milk, the presence and patterns of these glycans vary depending upon the stage of lactation and the maternal genes and their genetic polymorphisms that control glycosyl transferases. The synthesis of milk glycans utilizes a significant portion of the metabolic energy that the mother expends when producing her milk, but other than lactose, these glycans contribute little to the nutritional needs of the infant. The data herein support several functions. 1) Many human milk glycans inhibit pathogens from binding to the intestinal mucosa. 2) Human milk glycans attenuate inflammation. 3) Glycans also directly stimulate the growth of beneficial (mutualist) bacteria of the microbiota (formerly considered commensal microflora of the intestine); these mutualists and their fermentation products can, in turn, (a) inhibit pathogens, (b) modulate signaling and inflammation, and (c) the fermentation products can be absorbed and utilized as a source of dietary calories. These functions can help direct and support intestinal postnatal growth, development, and ontogeny of colonization. The many functions of the milk glycans may synergistically protect infants from disease. Hence, human milk glycans and their homologs may serve as novel prophylactic or therapeutic agents for a diverse range of deleterious conditions.     

Feed intake and milk production in dairy cows fed different grass and legume species: a meta-analysis

The aim of this meta-analysis was to compare feed intake, milk production, milk composition and organic matter (OM) digestibility in dairy cows fed different grass and legume species. Data from the literature was collected and different data sets were made to compare families (grasses v. legumes, Data set 1), different legume species and grass family (Data set 2), and different grass and legume species (Data set 3+4). The first three data sets included diets where single species or family were fed as the sole forage, whereas the approach in the last data set differed by taking the proportion of single species in the forage part into account allowing diets consisting of both grasses and legumes to be included. The grass species included were perennial ryegrass, annual ryegrass, orchardgrass, timothy, meadow fescue, tall fescue and festulolium, and the legume species included were white clover, red clover, lucerne and birdsfoot trefoil. Overall, dry matter intake (DMI) and milk production were 1.3 and 1.6 kg/day higher, respectively, whereas milk protein and milk fat concentration were 0.5 and 1.4 g/kg lower, respectively, for legume-based diets compared with grass-based diets. When comparing individual legume species with grasses, only red clover resulted in a lower milk protein concentration than grasses. Cows fed white clover and birdsfoot trefoil yielded more milk than cows fed red clover and lucerne, probably caused by a higher OM digestibility of white clover and activity of condensed tannins in birdsfoot trefoil. None of the included grass species differed in DMI, milk production, milk composition or OM digestibility, indicating that different grass species have the same value for milk production, if OM digestibility is comparable. However, the comparison of different grass species relied on few observations, indicating that knowledge regarding feed intake and milk production potential of different grass species is scarce in the literature. In conclusion, different species within family similar in OM digestibility resulted in comparable DMI and milk production, but legumes increased both DMI and milk yield compared with grasses.     

耐氧短双歧杆菌的分离驯化及其应用特性的研究

从健康婴儿粪便中分离出两株厌氧无芽胞杆菌,经鉴定为短双歧杆菌。经驯化,成为在牛乳中生长良好,并使牛乳在10h内迅速凝固的菌种。它能耐受1．0％的胆盐和0．2％的苯酚,对pH3．5有较强的抵抗力,且保存性能良好。     

Diversity and temporal dynamics of primate milk microbiomes

Milk is inhabited by a community of bacteria and is one of the first postnatal sources of microbial exposure for mammalian young. Bacteria in breast milk may enhance immune development, improve intestinal health, and stimulate the gut‐brain axis for infants. Variation in milk microbiome structure (e.g., operational taxonomic unit [OTU] diversity, community composition) may lead to different infant developmental outcomes. Milk microbiome structure may depend on evolutionary processes acting at the host species level and ecological processes occurring over lactation time, among others. We quantified milk microbiomes using 16S rRNA high‐throughput sequencing for nine primate species and for six primate mothers sampled over lactation. Our data set included humans (Homo sapiens, Philippines and USA) and eight nonhuman primate species living in captivity (bonobo [Pan paniscus], chimpanzee [Pan troglodytes], western lowland gorilla [Gorilla gorilla gorilla], Bornean orangutan [Pongo pygmaeus], Sumatran orangutan [Pongo abelii], rhesus macaque [Macaca mulatta], owl monkey [Aotus nancymaae]) and in the wild (mantled howler monkey [Alouatta palliata]). For a subset of the data, we paired microbiome data with nutrient and hormone assay results to quantify the effect of milk chemistry on milk microbiomes. We detected a core primate milk microbiome of seven bacterial OTUs indicating a robust relationship between these bacteria and primate species. Milk microbiomes differed among primate species with rhesus macaques, humans and mantled howler monkeys having notably distinct milk microbiomes. Gross energy in milk from protein and fat explained some of the variations in microbiome composition among species. Microbiome composition changed in a predictable manner for three primate mothers over lactation time, suggesting that different bacterial communities may be selected for as the infant ages. Our results contribute to understanding ecological and evolutionary relationships between bacteria and primate hosts, which can have applied benefits for humans and endangered primates in our care.     

Specific detection of bifidobacterium strains in a pharmaceutical probiotic product and in human feces by polymerase chain reaction

For PCR specific detection of the strains Bifidobacterium longum Y 10, B. infantis Y 1 and B. breve Y 8 used in a new probiotic product (VSL-3), strains-specific rDNA primers have been developed. Spacer regions between the 16S and 23S rRNA genes (ITS) of the three strains were amplified by PCR with conserved primers and the nucleotide sequence of these ITSs were determined. On the basis of their comparison with the rDNA sequences retrieved from GenBank, we designed new primers which specifically recognize the species B. breve and the two strains B. infantis Y 1 and B. breve Y 8. Specificity of these primers was confirmed through the analysis of 60 bifidobacteria strains belonging to the more representative human species. The feasibility of this PCR method was investigated in commercial VSL-3 product and fecal samples collected from 4 patients affected by inflammatory bowel deseases and two healthy subjects before and after the VSL-3 administration. By PCR analysis of different VSL-3 commercial batches we were successful in differentiating and quantifying the strains B. longum Y 10, B. infantis Y 1 and B. breve Y 8. B. infantis Y 1 and B. breve Y 8 could be detected at high concentration in fecal specimens of both patients and subjects treated with the probiotic preparation, showing a different colonization behaviour. Seven days after the VSL-3 treatment suspension, no patients and subjects harbored B. infantis Y 1 and B. breve Y 8, indicating a transient presence of these exogenous strains.     

Use of the rpoB gene as an alternative to the V3 gene for the identification of spoilage and pathogenic bacteria species in milk and milk products

Aim: To evaluate the rpoB gene as an alternative to the V3 gene for the identification of bacterial species in milk and milk products. Methods and Results: DNA obtained from different bacterial species strains was amplified by PCR using rpoB primers. PCR products of each bacterial species were then separated on a DGGE gel. The molecular fingerprints of the bacterial species tested were integrated into a database. The DGGE analysis shows a single band for the rpoB gene amplicons per each bacterial species. Comparison of electrophoretic profiles obtained from V3 16S rDNA amplification with those from this study obtained with rpoB showed that for some bacterial species that co‐migrated after amplification of the V3 region, distinct bands were observed on the gel with the amplification products of the rpoB region. Conclusions: The results obtained in this study show the discriminatory power of the rpoB gene, indicating that it can be used as an alternative to the V3 16S rRNA gene for the identification of bacterial species in milk and milk products. Significance and Impact of the Study: PCR‐DGGE targeting the rpoB gene is a way of discriminating the bacterial species that co‐migrated with the amplification of the V3 gene and so avoids the sequencing of the co‐migrating bands.     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Bile salt-stimulated lipase in non-primate milk: longitudinal variation and lipase characteristics in cat and dog milk

We report the presence of bile salt-stimulated lipase in milk collected from dog and cat. This enzyme has previously been found only in the milk of human and gorilla. Bile salt-stimulated lipase activity in individual dog milk specimens (range: 4.8-107.4 U/ml; 1 U = 1 mumol [3H]oleic acid released/min) was similar, while that in cat milk specimens (range: 2.2-16.9 U/ml) was lower than in human milk (range: 10-80 U/ml). Longitudinal patterns for bile salt-stimulated lipase activity differed depending upon the enzyme source: in dog milk, lipase activity was lowest in colostrum, while in cat milk, lipase activity was highest in colostrum and decreased at mid-lactation. In human milk, bile salt-stimulated lipase activity levels remain fairly constant throughout the first 3 months of lactation. Dog, cat and human milk bile salt-stimulated lipase activity had a neutral-to-alkaline pH optimum of 7.3-8.5, was stable at low pH (above 3.0 for at least 1 h), and was inhibited 95-100% by eserine (at concentrations greater than 0.6 mM). The lipase in the milk of the three species studied had an absolute requirement for primary bile salts (tauro- and glycocholate), and was inhibited by secondary bile salts (tauro- and glycodeoxycholate). These data are the first to report bile salt-stimulated lipase activity in milk from mammals other than the highest primates. Presence of this lipase in non-primate milk will permit the study of the factors that regulate the ontogeny, synthesis and secretion of the enzyme during pregnancy and lactation as well as its function in neonatal fat digestion.