A one-year study of human milk oligosaccharide profiles in the milk of healthy UK mothers and their relationship to maternal FUT2 genotype.

Human milk oligosaccharides (HMOs) are indigestible carbohydrates with prebiotic, pathogen decoy and immunomodulatory activities that are theorized to substantially impact infant health. The objective of this study was to monitor HMO concentrations over 1 year to develop a long-term longitudinal dataset. HMO concentrations in the breast milk of healthy lactating mothers of the Cambridge Baby Growth and Breastfeeding Study (CBGS-BF) were measured at birth, 2 weeks, 6 weeks, 3 months, 6 months and 12 months postpartum. HMO quantification was conducted by high-performance anion-exchange chromatography with pulsed amperometric detection using a newly validated "dilute-and-shoot" method. This technique minimizes sample losses and expedites throughput, making it particularly suitable for the analysis of large sample sets. Varying patterns of individual HMO concentrations were observed with changes in lactation timepoint and maternal secretor status, with the most prominent temporal changes occurring during the first 3 months. These data provide valuable information for the development of human milk banks in view of targeted distribution of donor milk based on infant age. Maternal FUT2 genotype was determined based on identification at single-nucleotide polymorphism rs516246 and compared with the genotype expected based on phenotypic markers in the HMO profile. Surprisingly, two mothers genotyped as secretors produced milk that displayed very low levels of 2'-fucosylated moieties. This unexpected discrepancy between genotype and phenotype suggests that differential enzyme expression may cause substantial variation in HMO profiles between genotypically similar mothers, and current genotypic methods of secretor status determination may require validation with HMO markers from milk analysis.

A genome-wide association study reveals specific transferases as candidate loci for bovine milk oligosaccharides synthesis.

BACKGROUND: Human milk oligosaccharides (OS) play a key role in brain and gut microbiota development of the neonate, but the underlying biosynthetic steps of OS in the mammary gland are still largely unknown. As bovine milk contains OS with somewhat similar structures and functionalities there is increased interest in further understanding the genetic basis underlying the OS content of milk for eventual extraction and generation of value-added ingredients for infant formulas and nutraceuticals. The present study is the first to report on genetic parameter estimation as well as on a genome wide association study (GWAS) from the largest bovine milk OS dataset analyzed to date. RESULTS: In total 15 different bovine milk OS were monitored. Heritabilities ranged from 0 to 0.68 in Danish Holstein and from 0 to 0.92 in Danish Jersey. The GWAS identified in total 1770 SNPs (FDR < 0.10) for five different OS in Danish Holstein and 6913 SNPs (FDR < 0.10) for 11 OS in Danish Jersey. In Danish Holstein, a major overlapping QTL was identified on BTA1 for LNH and LNT explaining 24% of the variation in these OS. The most significant SNPs were associated with B3GNT5, a gene encoding a glycosyltransferase involved in glycan synthesis. In Danish Jersey, a very strong QTL was detected for the OS with composition 2 Hex 1 HexNAc (isomer 1) on BTA11. The most significant SNP had -log10(P-value) of 52.88 (BOVINEHD1100030300) and was assigned to ABO, a gene encoding ABO blood group glycosyltransferases. This SNP has been reported to be a missense mutation and explains 56% of the OS variation. Other candidate genes of interest identified for milk OS were ALG3, B3GALNT2, LOC520336, PIGV, MAN1C1, ST6GALNAC6, GLT6D1, GALNT14, GALNT17, COLGALT2, LFNG and SIGLEC. CONCLUSION: To our knowledge, this is the first study documenting a solid breeding potential for bovine milk OS and a strong indication of specific candidate genes related to OS synthesis underlying this genetic influence. This new information has the potential to guide breeding strategies to achieve production of milk with higher diversity and concentration of OS and ultimately facilitate large-scale extraction of bovine milk OS.

An improved method for the purification of milk oligosaccharides by graphitised carbon-solid phase extraction.

Milk oligosaccharides (OS) are bioactive molecules that impart a variety of health benefits to the consumer. Techniques commonly used to analyse and quantify OS require optimised extraction methods to separate the OS from more abundant milk components. Solid phase extraction (SPE) is frequently used to isolate milk OS from lactose; however, the literature contains no formal studies on its efficacy in this application. In this study, established SPE conditions were modified to improve the technique's effectiveness in purifying OS from lactose. Low concentrations of acetonitrile (ACN) and trifluoroacetic acid (TFA) were tested for solid phase washing. Lactose removal and retention of many OS were significantly improved when using 4% ACN/0.1% TFA compared with the more common water washing technique. Different behaviours between acidic and neutral OS were evident. The new SPE technique improves extraction efficiency for bovine milk OS in applications that do not require prior lactose hydrolysis.

Peptidomic screening of bitter and nonbitter casein hydrolysate fractions for insulinogenic peptides.

Sodium caseinate hydrolysates (NaCaH) contain biologically active peptides that can positively influence human health. However, their intense bitterness hinders their inclusion in food products. To our knowledge, no studies have investigated whether a correlation between bitterness and bioactivity exists in NaCaH, so it is not yet known what effect selective removal of bitterness has on NaCaH bioactivity. A deeper understanding of the physicochemical characteristics affecting both bitterness and bioactivity is therefore needed. The aim of this study was to use in silico analysis to elucidate the relationship between bitterness and bioactivity of the insulinogenic NaCaH. The NaCaH fractions were generated by membrane filtration and flash chromatography and were subsequently evaluated for bitterness by a sensory panel. In this present study, peptidomic and bioinformatic processing of these NaCaH fractions allowed for the identification of insulinogenic peptides as well as other literature-identified peptides in each of the fractions. The results showed that the most bitter fraction contained the highest abundance of insulinogenic peptides, whereas another bitter fraction contained the highest abundance of other literature-identified bioactive peptides exhibiting angiotensin-converting enzyme-inhibition activity. Although some bioactive peptides were identified in the least bitter fractions, the abundance of these peptides was very low. These observations show a correlation between bitter taste and bioactivity, highlighting potential complications in removing bitterness while maintaining bioactivity. However, as the most bitter fraction contained the highest abundance of insulinogenic peptides, there is potential for using a lower dose of this enriched bioactive fraction to exert health benefits. The second most bitter fraction contained a very low abundance of insulinogenic peptides and other bioactive peptides. Therefore, removal of this fraction could reduce the NaCaH product's bitterness without significantly altering overall bioactive potential.

Current peptidomics: applications, purification, identification, quantification, and functional analysis.

Peptidomics is an emerging field branching from proteomics that targets endogenously produced protein fragments. Endogenous peptides are often functional within the body-and can be both beneficial and detrimental. This review covers the use of peptidomics in understanding digestion, and identifying functional peptides and biomarkers. Various techniques for peptide and glycopeptide extraction, both at analytical and preparative scales, and available options for peptide detection with MS are discussed. Current algorithms for peptide sequence determination, and both analytical and computational techniques for quantification are compared. Techniques for statistical analysis, sequence mapping, enzyme prediction, and peptide function, and structure prediction are explored.

Endogenous human milk peptide release is greater after preterm birth than term birth.

BACKGROUND: 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. OBJECTIVE: This study examined differences in peptides present between milk from women delivering at term and women delivering prematurely. METHODS: 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). RESULTS: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides. CONCLUSION: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.

Investigating Milk Fat Globule Structure, Size, and Functionality after Thermal Processing and Homogenization of Human Milk.

Human milk provides bioactive compounds such as milk fat globules (MFGs), which promote brain development, modulate the immune system, and hold antimicrobial properties. To ensure microbiological safety, donor milk banks apply heat treatments. This study compares the effects of heat treatments and homogenization on MFG's physicochemical properties, bioactivity, and bioavailability. Vat pasteurization (Vat-PT), retort (RTR), and ultra-high temperature (UHT) were performed with or without homogenization. UHT, RTR, and homogenization increased the colloidal dispersion of globules, as indicated by increased zeta potential. The RTR treatment completely inactivated xanthine oxidase activity (a marker of MFG bioactivity), whereas UHT reduced its activity by 93%. Interestingly, Vat-PT resulted in less damage, with 28% activity retention. Sialic acid, an important compound for brain health, was unaffected by processing. Importantly, homogenization increased the in vitro lipolysis of MFG, suggesting that this treatment could increase the digestibility of MFG. In terms of color, homogenization led to higher L* values, indicating increased whiteness due to finer dispersion of the fat and casein micelles (and thus greater light scattering), whereas UHT and RTR increased b* values associated with Maillard reactions. This study highlights the nuanced effects of processing conditions on MFG properties, emphasizing the retention of native characteristics in Vat-PT-treated human milk.

Food glycomics: Dealing with unexpected degradation of oligosaccharides during sample preparation and analysis.

This study reveals that unexpected degradation of food oligosaccharides can occur during conventional glycomics workflows, including sample preparation and analysis by liquid chromatography-mass spectrometry (LC-MS). With the present investigation, we aim to alert the scientific community of the susceptibility of specific glycosidic linkages to degradation induced by heat and acid. Key standard oligosaccharides representing the major types found in foods (3'-sialyllactose and 6'-sialyl-N-acetyllactosamine for milk, raffinose and stachyose for legumes) were selected as model systems and underwent each of the following treatments independently: (1) labeled with the derivatizing agent 1-aminopyrene-3,6,8-trisulfonic (APTS) (followed by analysis with a capillary electrophoresis system coupled with a fluorescence detector), (2) dried from an acetonitrile-water mixture containing 0.1% trifluoroacetic acid, and (3) injected into an LC-MS system. We demonstrated that both raffinose and stachyose degraded during APTS-labeling by the acid in the labeling reagents. We also discovered that during centrifugal evaporation at 37 °C, all of the four nonderivatized oligosaccharides tested were partially degraded. Additionally, when the LC-MS eluent contained 0.1% formic acid, 3'-sialyllactose, raffinose, and stachyose underwent extensive in-source fragmentation during analysis. Lastly, we identified a simple strategy that can reduce the probability of incorrect oligosaccharide identification resulting from extensive in-source fragmentation.

Solid-Phase Extraction Approaches for Improving Oligosaccharide and Small Peptide Identification with Liquid Chromatography-High-Resolution Mass Spectrometry: A Case Study on Proteolyzed Almond Extract.

Reverse-phase solid-phase extraction (SPE) is regularly used for separating and purifying food-derived oligosaccharides and peptides prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. However, the diversity in physicochemical properties of peptides may prevent the complete separation of the two types of analytes. Peptides present in the oligosaccharide fraction not only interfere with glycomics analysis but also escape peptidomics analysis. This work evaluated different SPE approaches for improving LC-MS/MS analysis of both oligosaccharides and peptides through testing on peptide standards and a food sample of commercial interest (proteolyzed almond extract). Compared with conventional reverse-phase SPE, mixed-mode SPE (reverse-phase/strong cation exchange) was more effective in retaining small/hydrophilic peptides and capturing them in the high-organic fraction and thus allowed the identification of more oligosaccharides and dipeptides in the proteolyzed almond extract, with satisfactory MS/MS confirmation. Overall, mixed-mode SPE emerged as the ideal method for simultaneously improving the identification of food-derived oligosaccharides and small peptides using LC-MS/MS analysis.

Can cheese mites, maggots and molds enhance bioactivity? Peptidomic investigation of functional peptides in four traditional cheeses.

Aside from their amino acid content, dairy proteins are valuable for their ability to carry encrypted bioactive peptides whose activities are latent until released by digestive enzymes or endogenous enzymes within the food. Peptides can possess a wide variety of functionalities, such as antibacterial, antihypertensive, and antioxidative properties, as demonstrated by in vitro and in vivo studies. This phenomenon raises the question as to what impact various traditional cheese-making processes have on the formation of bioactive peptides in the resulting products. In this study, we have profiled the naturally-occurring peptides in two hard and two soft traditional cheeses and have identified their known bioactive sequences. While past studies have typically identified fewer than 100 peptide sequences in a single cheese, we have used modern instrumentation to identify between 2900 and 4700 sequences per cheese, an increase by a factor of about 50. We demonstrated substantial variations in proteolysis and peptide formation between the interior and rind of each cheese, which we ascribed to the differences in microbial composition between these regions. We identified a total of 111 bioactive sequences among the four cheeses, with the greatest number of sequences, 89, originating from Mimolette. The most common bioactivities identified were antimicrobial and inhibition of the angiotensin-converting enzyme. This work revealed that cheese proteolysis and the resulting peptidomes are more complex than originally thought in terms of the number of peptides released, variation in peptidome across sites within a single cheese, and variation in bioactive peptides among cheese-making techniques.

Structures and Metabolic Properties of Bovine Milk Oligosaccharides and Their Potential in the Development of Novel Therapeutics.

Among the many bioactive components in human milk, the free oligosaccharides (OS) have been intensely studied in recent decades due to their unique ability to selectively modulate the infant gut microbiota, in addition to providing numerous other health benefits. In light of the demonstrated value of these compounds, recent studies have set out to characterize the structures and properties of the similar and more widely-available OS in the dairy industry. This mini review gives a brief overview of the common analytical techniques used to characterize bovine milk OS and highlights several recent, key studies that have identified valuable physiological and metabolic effects of these molecules in vivo. Although traditionally considered indigestible by human enzymes, evidence now suggests that milk OS are partially absorbed in the intestines and likely contribute to the development of molecular structures in the brain. Furthermore, aside from their prebiotic effects, these compounds show promise as therapeutics that could alleviate numerous metabolic abnormalities, including undernutrition, obesity, and excessive intestinal permeability. The need for novel treatments to address these and related health issues is motivating the development of scalable techniques to produce large quantities of milk OS for use as food ingredients. The safety and tolerability of high dosages of bovine milk OS have been demonstrated in two independent human studies, which potentially opens the door for further research aiming to utilize these molecules to alleviate common metabolic health issues.

Peptidomic and glycomic profiling of commercial dairy products: identification, quantification and potential bioactivities.

Peptidomics and glycomics are recently established disciplines enabling researchers to characterize functional characteristics of foods at a molecular level. Milk-derived bioactive peptides and oligosaccharides have garnered both scientific and commercial interest because they possess unique functional properties, such as anti-hypertensive, immunomodulatory and prebiotic activities; therefore, the objective of this work was to employ peptidomic and glycomic tools to identify and measure relative and absolute quantities of peptides and oligosaccharides in widely consumed dairy products. Specifically, we identified up to 2117 unique peptides in 10 commercial dairy products, which together represent the most comprehensive peptidomic profiling of dairy milk in the literature to date. The quantity of peptides, measured by ion-exchange chromatography, varied between 60 and 130 mg/L among the same set of dairy products, which the majority originated from caseins, and the remaining from whey proteins. A recently published bioactive peptide database was used to identify 66 unique bioactive peptides in the dataset. In addition, 24 unique oligosaccharide compositions were identified in all the samples by nano LC Chip QTOF. Neutral oligosaccharides were the most abundant class in all samples (66-91.3%), followed by acidic (8.6-33.7%), and fucosylated oligosaccharides (0-4.6%). Variation of total oligosaccharide concentration ranged from a high of 65.78 to a low of 24.82 mg/L. Importantly, characterizing bioactive peptides and oligosaccharides in a wider number of dairy products may lead to innovations that go beyond the traditional vision of dairy components used for nutritional purposes but that will rather focus on improving human health.

Profiling of aminoxyTMT-labeled bovine milk oligosaccharides reveals substantial variation in oligosaccharide abundance between dairy cattle breeds.

Free milk oligosaccharides are bioactive molecules that function as prebiotics and prevent infections that commonly afflict developing infants. To date, few publications have examined the factors affecting bovine milk oligosaccharide production among cattle in the dairy industry. Here we have applied a high-throughput isobaric labeling technique to measure oligosaccharide abundances in milk collected from Danish Holstein-Friesian and Jersey dairy cattle by liquid chromatography-mass spectrometry. With a total of 634 milk samples, this collection represents the largest sample set used for milk oligosaccharide profiling in the current literature. This study is also the first to use isobaric labeling for the purpose of measuring free oligosaccharides in a real sample set. We have identified 13 oligosaccharides that vary significantly by breed, with most structures being more abundant in the milk of Jersey cattle. The abundances of several oligosaccharides were increased in second-parity cows, and correlations between the abundances of oligosaccharide pairs were identified, potentially indicating similarities in their synthetic pathways. Fucosylated oligosaccharide structures were widely identified among both breeds. Improving our understanding of oligosaccharide production will aid in developing strategies to recover these compounds from processing streams and may enable their use as a functional ingredient in foods for infants and adults.

Peptidomic profiling of human milk with LC-MS/MS reveals pH-specific proteolysis of milk proteins.

Human milk is a dynamic protein-protease system that delivers bioactive peptides to infants. The pH of milk changes from the mother's mammary gland to the infant's digestive tract. Although the release of human milk peptides has been studied during in vivo or in vitro digestion, these models did not explicitly vary nor observe the effect of pH. The objective of this research was to determine the effect of pH on the proteolysis of human milk. Using high-resolution accurate-mass Orbitrap mass spectrometry, profiles of endogenous human milk peptides before and after incubation at various pH levels have been mapped. Over 5000 peptides were identified. Comparative analyses classified 74 peptides that were consistently found independent of pH alterations, and 8 peptides that were released only at pH 4 or 5 (typical infant gastric pH). Results documented that the proteolysis of milk proteins, particularly ß-casein, polymeric immunoglobulin receptor, and α-lactalbumin, is pH-dependent.

Multiplexed bovine milk oligosaccharide analysis with aminoxy tandem mass tags.

Milk oligosaccharides (OS) are a key factor that influences the infant gut microbial composition, and their importance in promoting healthy infant development and disease prevention is becoming increasingly apparent. Investigating the structures, properties, and sources of these compounds requires a host of complementary analytical techniques. Relative compound quantification by mass spectral analysis of isobarically labeled samples is a relatively new technique that has been used mainly in the proteomics field. Glycomics applications have so far focused on analysis of protein-linked glycans, while analysis of free milk OS has previously been conducted only on analytical standards. In this paper, we extend the use of isobaric glycan tags to the analysis of bovine milk OS by presenting a method for separation of labeled OS on a porous graphitized carbon liquid chromatographic column with subsequent analysis by quadrupole time-of-flight tandem mass spectrometry. Abundances for 15 OS extracted from mature bovine milk were measured, with replicate injections providing coefficients of variation below 15% for most OS. Isobaric labeling improved ionization efficiency for low-abundance, high-molecular weight fucosylated OS, which are known to exist in bovine milk but have been only sporadically reported in the literature. We compared the abundances of four fucosylated OS in milk from Holstein and Jersey cattle and found that three of the compounds were more abundant in Jersey milk, which is in general agreement with a previous study. This novel method represents an advancement in our ability to characterize milk OS and provides the advantages associated with isobaric labeling, including reduced instrumental analysis time and increased analyte ionization efficiency. This improved ability to measure differences in bioactive OS abundances in large datasets will facilitate exploration of OS from all food sources for the purpose of developing health-guiding products for infants, immune-compromised elderly, and the population at large.

Milk Glycans and Their Interaction with the Infant-Gut Microbiota.

Human milk is a unique and complex fluid that provides infant nutrition and delivers an array of bioactive molecules that serve various functions. Glycans, abundant in milk, can be found as free oligosaccharides or as glycoconjugates. Milk glycans are increasingly linked to beneficial outcomes in neonates through protection from pathogens and modulation of the immune system. Indeed, these glycans influence the development of the infant and the infant-gut microbiota. Bifidobacterium species commonly are enriched in breastfed infants and are among a limited group of bacteria that readily consume human milk oligosaccharides (HMOs) and milk glycoconjugates. Given the importance of bifidobacteria in infant health, numerous studies have examined the molecular mechanisms they employ to consume HMOs and milk glycans, thus providing insight into this unique enrichment and shedding light on a range of translational opportunities to benefit at-risk infants.

Antibody-independent identification of bovine milk-derived peptides in breast-milk.

Exclusively breast-fed infants can exhibit clear signs of IgE or non IgE-mediated cow's milk allergy. However, the definite characterization of dietary cow's milk proteins (CMP) that survive the maternal digestive tract to be absorbed into the bloodstream and secreted into breast milk remains missing. Herein, we aimed at assessing possible CMP-derived peptides in breast milk. Using high performance liquid chromatography (HPLC)-high resolution mass spectrometry (MS), we compared the peptide fraction of breast milk from 12 donors, among which 6 drank a cup of milk daily and 6 were on a strict dairy-free diet. We identified two bovine ß-lactoglobulin (ß-Lg, 2 out 6 samples) and one αs1-casein (1 out 6 samples) fragments in breast milk from mothers receiving a cup of bovine milk daily. These CMP-derived fragments, namely ß-Lg (f42-54), (f42-57) and αs1-casein (f180-197), were absent in milk from mothers on dairy-free diet. In contrast, neither intact nor hydrolyzed ß-Lg was detected by western blot and competitive ELISA in any breast milk sample. Eight additional bovine milk-derived peptides identified by software-assisted MS were most likely false positive. The results of this study demonstrate that CMP-derived peptides rather than intact CMP may sensitize or elicit allergic responses in the neonate through mother's milk. Immunologically active peptides from the maternal diet could be involved in priming the newborn's immune system, driving a tolerogenic response.

Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

SCOPE: The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. METHODS AND RESULTS: Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. CONCLUSION: The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases.