Endo-β-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N'-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid and complex N-glycans. These conjugated... more
Endo-β-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N'-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45-8.45), temperature (27.5-77.5°C), reaction time (15-475 min), and enzyme/protein ratio (1:3000-1:333) were evaluated on the release of N-glycans from bovine colostrum whey by EndoBI-1. A central composite design was used, including a two-level factorial design (2(4) ) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio and temperatures around 52 ºC resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a...
Research Interests:
Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the... more
Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N-glycans in protein functionality. Endo-β-N-acetylglucosaminidase (EndoBI-1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat stable enzyme that cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI-1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI-1 is able to cleave a high diversity of N-glycan structures. Nano-LC-Chip-Q-TOF MS data also revealed that different reaction conditions resulted in different N-gl...
Research Interests:
EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose,... more
EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2)mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans.
Research Interests:
Research Interests: Algorithms, Water, Methodology, Plant Biology, Hydroponics, and 33 moreEcology, Scanning Electron Microscopy, Multidisciplinary, Time series analysis, Phylogeny, Wavelet Analysis, Tree Physiology, Permeability, Chlorophyll, Wavelet Transform, Temperature, DNA fingerprinting, Frequency, Tree, Trees, Biological evolution, Plant Roots, Fluctuations, Germination, Forestry Sciences, Dehydration, Water Science and Technology, Pinus, Streams, Seeds, Time Factors, Geraniaceae, Sapindaceae, Species Specificity, Magnitude, Fourier Transformation, Plant Leaves, and Geranium(Ecology, Scanning Electron Microscopy, Multidisciplinary, Time series analysis, Phylogeny, Wavelet Analysis, Tree Physiology, Permeability, Chlorophyll, Wavelet Transform, Temperature, DNA fingerprinting, Frequency, Tree, Trees, Biological evolution, Plant Roots, Fluctuations, Germination, Forestry Sciences, Dehydration, Water Science and Technology, Pinus, Streams, Seeds, Time Factors, Geraniaceae, Sapindaceae, Species Specificity, Magnitude, Fourier Transformation, Plant Leaves, and Geranium)
(Ecology, Scanning Electron Microscopy, Multidisciplinary, Time series analysis, Phylogeny, Wavelet Analysis, Tree Physiology, Permeability, Chlorophyll, Wavelet Transform, Temperature, DNA fingerprinting, Frequency, Tree, Trees, Biological evolution, Plant Roots, Fluctuations, Germination, Forestry Sciences, Dehydration, Water Science and Technology, Pinus, Streams, Seeds, Time Factors, Geraniaceae, Sapindaceae, Species Specificity, Magnitude, Fourier Transformation, Plant Leaves, and Geranium)
Research Interests:
Research Interests: Multidisciplinary, Female, Animals, PLoS one, Milk, and 3 moreCattle, Dairy Products, and Oligosaccharides
Research Interests:
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)... more
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.
Research Interests:
Research Interests:
Many of milk’s functional molecules could not be discovered until the right concordance of novel separation and analytical technologies were developed and applied. Many health-promoting components still await discovery due to technical... more
Many of milk’s functional molecules could not be discovered until the right concordance of novel separation and analytical technologies were developed and applied. Many health-promoting components still await discovery due to technical challenges in their identification, isolation and testing. As new analytical technologies are assembled, new functional milk molecules will be discovered. Bovine milk is a source of a wide array of known bioactive compounds from a variety of molecular classes, including free glycans, lipids, glycolipids, peptides, proteins, glycoproteins, stem cells and microRNA. Because milk is such a complex mixture, when analyzed without fractionation or purification, many components mask the analytical signal of others, so some components cannot be detected. Modern analytics allow for the discovery and characterization of hundreds of novel milk compounds with high-resolution and highaccuracy. Liquid chromatography paired with electrospray ionization allows the sep...
ABSTRACT Milk has evolved to provide only components that will give selective advantage to the growing neonate. Oligosaccharides—short polymers made of several monosaccharides—are carbohydrates present in all mammalian milks studied so... more
ABSTRACT Milk has evolved to provide only components that will give selective advantage to the growing neonate. Oligosaccharides—short polymers made of several monosaccharides—are carbohydrates present in all mammalian milks studied so far and cannot be degraded by the infant's digestive enzymes. However, oligosaccharides selectively feed beneficial commensal bacteria. Some oligosaccharides also act as decoys for pathogenic bacteria and viruses to prevent infection in the neonate. The functional effects of human milk oligosaccharides (HMO) and the lack of commercial availability of those molecules have stimulated a search for alternative sources for clinical trials and food supplementation. Bovine milk oligosaccharides (BMO) are similar to HMO in their structural building block and complexity. BMO have been identified in the whey permeate fraction that is left over after cheese-making and whey protein recovery. The billions of gallons of whey processed yearly represent an enormous source of milk oligosaccharides for food products and supplements.
Human milk is a complete source of nourishment for the infant. Exclusive breastfeeding not only sustains the... more
Human milk is a complete source of nourishment for the infant. Exclusive breastfeeding not only sustains the infant's development but also guides the proliferation of a protective intestinal microbiota. Among the many components of milk that modulate the infant gut microbiota, the milk glycans, which comprise free oligosaccharides, glycoproteins, and glycolipids, are increasingly recognized as drivers of microbiota development and overall gut health. These glycans may display pleiotropic functions, conferring protection against infectious diseases and also acting as prebiotics, selecting for the growth of beneficial intestinal bacteria. The prebiotic effect of milk glycans has direct application to prevention of diseases such as necrotizing enterocolitis, a common and devastating disease of preterm infants. In this article, we review the impact of the human (and bovine) milk glycome on gut health through establishment of a milk-oriented microbiota in the neonate.
Research Interests:
Little is known about the digestive process in infants. In particular, the chronological activity of enzymes across the course of digestion in the infant remains largely unknown. To create a temporal picture of how milk proteins are... more
Little is known about the digestive process in infants. In particular, the chronological activity of enzymes across the course of digestion in the infant remains largely unknown. To create a temporal picture of how milk proteins are digested, enzyme activity was compared between intact human milk samples from three mothers and the gastric samples from each of their 4-12 day postpartum infants, 2 h after breast milk ingestion. The activities of 7 distinct enzymes are predicted in the infant stomach based on their observed cleavage pattern in peptidomics data. We found that the same patterns of cleavage were evident in both intact human milk and gastric milk samples, demonstrating that the enzyme activities that begin in milk persist in the infant stomach. However, the extent of enzyme activity is found to vary greatly between the intact milk and gastric samples. Overall, we observe that milk-specific proteins are cleaved at higher levels in the stomach compared to human milk. Notably, the enzymes we predict here only explain 78% of the cleavages uniquely observed in the gastric samples, highlighting that further investigation of the specific enzyme activities associated with digestion in infants is warranted.
Research Interests:
Research Interests:
Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland,... more
Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile. This study examines differences in peptides present between milk from women delivering at term and women delivering prematurely. Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d). Preterm milk peptide counts, ion abundance, and concentration were significantly ...
Research Interests:
Research Interests:
Numerous milk components, such as lactoferrin, are recognized as health-promoting compounds. A growing body of evidence suggests that glycans could mediate... more
Numerous milk components, such as lactoferrin, are recognized as health-promoting compounds. A growing body of evidence suggests that glycans could mediate lactoferrin&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s bioactivity. Goat milk lactoferrin is a candidate for infant formula supplementation because of its high homology with its human counterpart. The aim of this study was to characterize the glycosylation pattern of goat milk lactoferrin. After the protein was isolated from milk by affinity chromatography, N-glycans were enzymatically released and a complete characterization of glycan composition was carried out by advanced MS. The glycosylation of goat milk lactoferrin was compared with that of human and bovine milk glycoproteins. Nano-LC-Chip-Q-TOF MS data identified 65 structures, including high mannose, hybrid, and complex N-glycans. Among the N-glycan compositions, 37% were sialylated and 34% were fucosylated. The results demonstrated the existence of similar glycans in human and goat milk but also identified novel glycans in goat milk that were not present in human milk. These data suggest that goat milk could be a source of bioactive compounds, including lactoferrin that could be used as functional ingredients for food products beneficial to human nutrition.
Research Interests: Chemical Engineering, Analytical Chemistry, Polysaccharides, Humans, Animals, and 12 moreMs, Tandem Mass Spectrometry, Milk, High Pressure Liquid Chromatography, Goats, Cattle, Electrophoresis, Amino Acid Sequence, Glycomics, Biochemistry and cell biology, Carbohydrate Sequence, and Molecular Sequence Data
Research Interests: Obesity, Diet, Prebiotics, Lactation, Multidisciplinary, and 10 morePregnancy, Female, Animals, Male, Milk, Intestines, Rats, Dietary fiber, Microbiota, and Wistar Rats(Pregnancy, Female, Animals, Male, Milk, Intestines, Rats, Dietary fiber, Microbiota, and Wistar Rats)
(Pregnancy, Female, Animals, Male, Milk, Intestines, Rats, Dietary fiber, Microbiota, and Wistar Rats)
Research Interests:
Research Interests:
Research Interests:
Bovine milk is known to contain naturally occurring peptides, but relatively few of their sequences have been determined. Human milk contains hundreds of endogenous peptides, and the ensemble has been documented for antimicrobial actions.... more
Bovine milk is known to contain naturally occurring peptides, but relatively few of their sequences have been determined. Human milk contains hundreds of endogenous peptides, and the ensemble has been documented for antimicrobial actions. Naturally occurring peptides from bovine milk were sequenced and compared with human milk peptides. Bovine milk samples from six cows in second-stage peak lactation at 78-121 days postpartum revealed 159 peptides. Most peptides (73%) were found in all six cows sampled, demonstrating the similarity of the intramammary peptide degradation across these cows. One peptide sequence, ALPIIQKLEPQIA from bovine perilipin 2, was identical to another found in human milk. Most peptides derived from β-casein, αs1-casein, and αs2-casein. No peptides derived from abundant bovine milk proteins such as lactoferrin, β-lactoglobulin, and secretory immunoglobulin A. The enzymatic cleavage analysis revealed that milk proteins were degraded by plasmin, cathepsins B and D, and elastase in all samples.
Research Interests:
Milk oligosaccharides (OS)-free complex carbohydrates-confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as... more
Milk oligosaccharides (OS)-free complex carbohydrates-confer unique health benefits to the nursing neonate. Though human digestive enzymes cannot degrade these sugars, they provide nourishment to specific commensal microbes and act as decoys to prevent the adhesion of pathogenic micro-organisms to gastrointestinal cells. At present, the limited quantities of human milk oligosaccharides (HMO) impede research on these molecules and their potential applications in functional food formulations. Considerable progress has been made in the study of OS structures; however, the synthetic pathways leading to their synthesis in the mammary gland are poorly understood. Recent studies show that complex OS with fucose and N-acetyl neuraminic acid (key structural elements of HMO bioactivity) exist in goat milk. Polymorphisms in the CSN1S1 locus, which is responsible for synthesis of αs1-casein, affect lipid and casein micelle structure in goat milk. The present study sought to determine whether CS...
Research Interests:
Research Interests: Nutrition and Dietetics, Nutrition, Principal Component Analysis, Life Style, Lactation, and 17 moreMagnetic Resonance Spectroscopy, Animal Production, California, Prospective studies, Humans, Female, Infant, Body Mass Index, Questionnaires, Food habits, Human Milk, Food Sciences, Molecular weight, Metabolome, Logistic Models, Oligosaccharides, and Motor activity
Research Interests:
Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and... more
Milk is traditionally considered an ideal source of the basic elemental nutrients required by infants. More detailed examination is revealing that milk represents a more functional ensemble of components with benefits to both infants and mothers. A comprehensive peptidomics method was developed and used to analyze human milk yielding an extensive array of protein products present in the fluid. Over 300 milk peptides were identified originating from major and many minor protein components of milk. As expected, the majority of peptides derived from β-casein, however no peptide fragments from the major milk proteins lactoferrin, α-lactalbumin, and secretory immunoglobulin A were identified. Proteolysis in the mammary gland is selective-released peptides were drawn only from specific proteins and typically from only select parts of the parent sequence. A large number of the peptides showed significant sequence overlap with peptides with known antimicrobial or immunomodulatory functions. Antibacterial assays showed the milk peptide mixtures inhibited the growth of Escherichia coli and Staphylococcus aureus . The predigestion of milk proteins and the consequent release of antibacterial peptides may provide a selective advantage through evolution by protecting both the mother&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s mammary gland and her nursing offspring from infection.