Exploring bioactive compounds in chickpea and bean aquafaba: Insights from glycomics and peptidomics analyses.

The objective of this study was to identify bioactive oligosaccharides and peptides in the cooking water of chickpeas and common beans, known as aquafaba. The oligosaccharides stachyose, raffinose and verbascose were quantified by high-performance anion-exchange chromatography; 78 and 67 additional oligosaccharides were identified in chickpea and common bean aquafaba, respectively, by LC-MS/MS. Chickpea aquafaba uniquely harbored ciceritol and other methyl-inositol-containing oligosaccharides. In prebiotic growth assays, chickpea aquafaba oligosaccharides were differentially utilized, promoting growth of Limosilactobacillus reuteri DSM 20016 and Bifidobacterium longum subsp. infantis ATCC 15697, but not Lacticaseibacillus rhamnosus GG. Dimethyl labeling, along with LC-MS/MS, effectively differentiated α- and γ-glutamyl peptides, revealing the presence of several γ-glutamyl peptides known to possess kokumi and anti-inflammatory activities, including γ-Glu-Phe and γ-Glu-Tyr in chickpeas aquafaba and γ-Glu-S-methyl-Cys and γ-Glu-Leu in beans aquafaba. This work uncovered unique bioactive peptides and oligosaccharides in aquafaba, helping promote its valorization, food system sustainability, and future health-promoting claims.

A multi-centre peptidomics investigation of food digesta: current state of the art in mass spectrometry analysis and data visualisation.

Mass spectrometry has become the technique of choice for the assessment of a high variety of molecules in complex food matrices. It is best suited for monitoring the evolution of digestive processes in vivo and in vitro. However, considering the variety of equipment available in different laboratories and the diversity of sample preparation methods, instrumental settings for data acquisition, statistical evaluations, and interpretations of results, it is difficult to predict a priori the ideal parameters for optimal results. The present work addressed this uncertainty by executing an inter-laboratory study with samples collected during in vitro digestion and presenting an overview of the state-of-the-art mass spectrometry applications and analytical capabilities available for studying food digestion. Three representative high-protein foods - skim milk powder (SMP), cooked chicken breast and tofu - were digested according to the static INFOGEST protocol with sample collection at five different time points during gastric and intestinal digestion. Ten laboratories analysed all digesta with their in-house equipment and applying theirconventional workflow. The compiled results demonstrate in general, that soy proteins had a slower gastric digestion and the presence of longer peptide sequences in the intestinal phase compared to SMP or chicken proteins, suggesting a higher resistance to the digestion of soy proteins. Differences in results among the various laboratories were attributed more to the peptide selection criteria than to the individual analytical platforms. Overall, the combination of mass spectrometry techniques with suitable methodological and statistical approaches is adequate for contributing to the characterisation of the recently defined digestome.

A complete workflow for discovering small bioactive peptides in foods by LC-MS/MS: A case study on almonds.

Identification of bioactive peptides is an increasingly important target for food chemists, particularly in consideration of the widespread application of proteolytic enzymes in food processing. Because the characterization of small peptides by LC-MS/MS is challenging, we optimized a dimethyl labeling technique to facilitate small peptide identification, using almond proteins as a model. The method was validated by comparing the MS/MS spectra of standards and almond-derived peptides in their nonderivatized and derivatized forms. Signal enhancement of a1 ions was proved to effectively aid in the full-length sequencing of small peptides. We further validated this method using two industrially-relevant protein-rich extracts from almond flour: 1737 medium-sized peptides (5-39 amino acids) and 843 small peptides (2-4 amino acids) were identified. The use of an online bioactive peptide database, complemented by the existing literature, allowed the discovery of 208 small bioactive peptides, whereas for medium-sized peptides, only one was reported being bioactive.

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.

Indole-3-lactic acid associated with Bifidobacterium-dominated microbiota significantly decreases inflammation in intestinal epithelial cells.

BACKGROUND: Bifidobacterium longum subsp. infantis (B. infantis) is a commensal bacterium that colonizes the gastrointestinal tract of breast-fed infants. B. infantis can efficiently utilize the abundant supply of oligosaccharides found in human milk (HMO) to help establish residence. We hypothesized that metabolites from B. infantis grown on HMO produce a beneficial effect on the host. RESULTS: In a previous study, we demonstrated that B. infantis routinely dominated the fecal microbiota of a breast fed Bangladeshi infant cohort (1). Characterization of the fecal metabolome of binned samples representing high and low B. infantis populations from this cohort revealed higher amounts of the tryptophan metabolite indole-3-lactic acid (ILA) in feces with high levels of B. infantis. Further in vitro analysis confirmed that B. infantis produced significantly greater quantities of the ILA when grown on HMO versus lactose, suggesting a growth substrate relationship to ILA production. The direct effects of ILA were assessed in a macrophage cell line and intestinal epithelial cell lines. ILA (1-10 mM) significantly attenuated lipopolysaccharide (LPS)-induced activation of NF-kB in macrophages. ILA significantly attenuated TNF-α- and LPS-induced increase in the pro-inflammatory cytokine IL-8 in intestinal epithelial cells. ILA increased mRNA expression of the aryl hydrogen receptor (AhR)-target gene CYP1A1 and nuclear factor erythroid 2-related factor 2 (Nrf2)-targeted genes glutathione reductase 2 (GPX2), superoxide dismutase 2 (SOD2), and NAD(P) H dehydrogenase (NQO1). Pretreatment with either the AhR antagonist or Nrf-2 antagonist inhibited the response of ILA on downstream effectors. CONCLUSIONS: These findings suggest that ILA, a predominant metabolite from B. infantis grown on HMO and elevated in infant stool high in B. infantis, and protects gut epithelial cells in culture via activation of the AhR and Nrf2 pathway.

Interactions between whey proteins and cranberry juice after thermal or non-thermal processing during in vitro gastrointestinal digestion.

The objective of this study was to understand the possible interactions between whey protein and cranberry juice after processing that could impact either the protein digestibility or the bioaccessibility of cranberry antioxidants using an in vitro gastrointestinal digestion model. Whey protein isolate (27 or 54 mg of protein per mL) was dissolved in either cranberry juice or water and used as a model beverage system. Beverages were either non-processed or underwent thermal (low: 85 °C for 1 min, medium: 99 °C for 10 s and long: 99 °C for 5 min) or high-pressure processing (600 MPa for 4 min). After processing, beverages underwent oral (30 s), gastric (2 h) and small intestinal (2 h) digestion. During in vitro digestion, protein hydrolysis was monitored by the o-phthalaldehyde (OPA) assay, SDS-PAGE, soluble amino acid content, and pepidomic profiling using Orbitrap mass-spectrometry. Antioxidant capacity was measured with Ferric Reducing Antioxidant Power (FRAP) and 2,2-azinobis (3-ethlybenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays before and during in vitro digestion. Whey protein isolate dissolved in water had a significantly higher (p < 0.05) degree of hydrolysis and soluble amino acid content during small intestinal digestion compared to protein dissolved in cranberry juice, suggesting that cranberry juice had an effect on how protein was hydrolyzed during digestion. In all processing treatments except for long thermal processing, water and cranberry juice protein solutions had similar ß-lactoglobulin digestibility (p > 0.05), suggesting that the cranberry juice interactions with the protein do not significantly decrease ß-lactoglobulin resistance to hydrolysis by pepsin. Peptide formation also differed between whey protein dissolved in either water or juice. Cranberry juice protein solutions showed a slightly lower peptide count compared with whey protein isolate dissolved in water. Antioxidant bioaccessibility by FRAP during gastric digestion significantly increased in cranberry juice with addition of whey protein isolate. This trend might indicate a protective effect of whey protein isolate to cranberry antioxidant compounds.

The effect of synbiotics Bifidobacterium infantis and milk oligosaccharides on shaping gut microbiota community structure and NASH treatment.

Probiotic Bifidobacterium longum subspecies infantis (Bifidobacterium infantis) consumes human milk oligosaccharides (MO) and protects intestinal permeability thereby having anti-inflammatory effects (Underwood et al., 2015; Bode, 2006; Asakuma et al., 2011) [1-3]. Via the gut-liver axis, gut barrier disruption and dysbiosis lead to hepatic inflammation (Sheng et al., 2017; Jena et al., 2017) [4,5,6]. Our published data revealed that butyrate, as well as synbiotics of B. infantis in combination with MO, had protective effects against cancer-prone non-alcoholic steatohepatitis (NASH) mouse models, i.e., Western diet (WD)-fed bile acid receptor FXR (farnesoid x receptor) knockout (KO) mice (Jena et al., 2018) [6,7]. In addition, MO was particularly effective in increasing the blooming of butyrate-generating bacteria (Jena et al., 2018) [7]. In the present study, we further showed that the reduced ileal short chain fatty acid (SCFA) signaling found in WD-fed FXR KO mice could be reversed by B. infantis and/or MO treatment. Moreover, ileal mRNA levels of SCFA receptors i.e. Gpr41 (Ffar3), Gpr109 (Hcar2), and Gpr43 (Ffar2) were increased in B. infantis and/or MO-treated mice suggesting increased SCFA signaling (Fig. 1). Further, nuclear magnetic resonance (NMR) data revealed that MO and B. Infantis plus MO increased intestinal acetate, propionate, butyrate, and valerate levels (Fig. 2). In addition, B. infantis and/or MO reduced the abundance of genus Bilophila and the relative copy number of bacterial genes including dissimilatory sulfite reductase (dsrA) and methyl coenzyme M reductase A (mcrA), which were all increased in cancer-prone FXR KO mice (Fig. 3).

Synbiotics Bifidobacterium infantis and milk oligosaccharides are effective in reversing cancer-prone nonalcoholic steatohepatitis using western diet-fed FXR knockout mouse models.

Milk oligosaccharides (MO) selectively increase the growth of Bifidobacterium infantis (B. infantis). This study examines the effects of bovine MO and B. infantis in preventing nonalcoholic steatohepatitis (NASH) in Western diet (WD)-fed bile acid (BA) receptor FXR (farnesoid x receptor) knockout (KO) mice. WD-fed FXR KO mice have cancer-prone NASH and reduced B. infantis. MO and/or B. infantis supplementation improved their insulin sensitivity and reduced hepatic inflammation. Additionally, B. infantis, but not MO, decreased hepatic triglyceride and cholesterol. A combination of both further reduced hepatic cholesterol, the precursor of BAs. All three treatments modulated serum and hepatic BA profile. Moreover, B. infantis and MO decreased hepatic CYP7A1 and induced Sult2a1, Sult2a2, and Sult2a3 suggesting reduced BA synthesis and increased detoxification. Furthermore, B. infantis and MO increased ileal BA membrane receptor TGR5-regulated signaling. Together, via BA-regulated signaling, synbiotics B. infantis and MO have their unique and combined effects in reversing NASH.

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.

Validation of a paper-disk approach to facilitate the sensory evaluation of bitterness in dairy protein hydrolysates from a newly developed food-grade fractionation system.

Casein-hydrolysates (NaCaH) are desirable functional ingredients, but their bitterness impedes usage in foods. This study sought to validate a paper-disk approach to help evaluate bitterness in NaCaHs and to develop a food-grade approach to separate a NaCaH into distinct fractions, which could be evaluated by a sensory panel. Membrane filtration generated <0.2-µm and <3-kDa permeates. Further fractionation of the <3-kDa permeate by flash-chromatography generated four fractions using ethanol (EtOH) concentrations of 5, 10, 30 and 50%. As some fractions were poorly soluble in water, the fractions were resolubilzed in EtOH and impregnated into paper-disks for sensory evaluation. Bitterness differences observed in the membrane fractions using this sensory evaluation approach reflected those observed for the same fractions presented as a liquid. The flash-chromatography fractions increased in bitterness with an increase in hydrophobicity, except for the 50% EtOH fraction which had little bitterness. Amino acid analysis of the fractions showed enrichment of different essential amino acids in both the bitter and less bitter fractions. Practical Applications: The developed food-grade fractionation system, allowed for a simple and reasonably scaled approach to separating a NaCaH, into physicochemically different fractions that could be evaluated by a sensory panel. The method of sensory evaluation used in this study, in which NaCaH samples are impregnated into paper-disks, provided potential solutions for issues such as sample insolubility and limited quantities of sample. As the impregnated paper-disk samples were dehydrated, their long storage life could also be suitable for sensory evaluations distributed by mail for large consumer studies. The research, in this study, allowed for a greater understanding of the physicochemical basis for bitterness in this NaCaH. As some essential amino acids were enriched in the less bitter fractions, selective removal of bitter fractions could allow for the incorporation of the less bitter NaCaH fractions into food products for added nutritional value, without negatively impacting sensory properties. There is potential for this approach to be applied to other food ingredients with undesirable tastes, such as polyphenols.

Bovine milk oligosaccharides decrease gut permeability and improve inflammation and microbial dysbiosis in diet-induced obese mice.

Obesity is characterized by altered gut homeostasis, including dysbiosis and increased gut permeability closely linked to the development of metabolic disorders. Milk oligosaccharides are complex sugars that selectively enhance the growth of specific beneficial bacteria in the gastrointestinal tract and could be used as prebiotics. The aim of the study was to demonstrate the effects of bovine milk oligosaccharides (BMO) and Bifidobacterium longum ssp. infantis (B. infantis) on restoring diet-induced obesity intestinal microbiota and barrier function defects in mice. Male C57/BL6 mice were fed a Western diet (WD, 40% fat/kcal) or normal chow (C, 14% fat/kcal) for 7 wk. During the final 2 wk of the study, the diet of a subgroup of WD-fed mice was supplemented with BMO (7% wt/wt). Weekly gavage of B. infantis was performed in all mice starting at wk 3, yet B. infantis could not be detected in any luminal contents when mice were killed. Supplementation of the WD with BMO normalized the cecal and colonic microbiota with increased abundance of Lactobacillus compared with both WD and C mice and restoration of Allobaculum and Ruminococcus levels to that of C mice. The BMO supplementation reduced WD-induced increase in paracellular and transcellular flux in the large intestine as well as mRNA levels of the inflammatory marker tumor necrosis factor α. In conclusion, BMO are promising prebiotics to modulate gut microbiota and intestinal barrier function for enhanced health.

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.

Oligosaccharides Released from Milk Glycoproteins Are Selective Growth Substrates for Infant-Associated Bifidobacteria.

UNLABELLED: Milk, in addition to nourishing the neonate, provides a range of complex glycans whose construction ensures a specific enrichment of key members of the gut microbiota in the nursing infant, a consortium known as the milk-oriented microbiome. Milk glycoproteins are thought to function similarly, as specific growth substrates for bifidobacteria common to the breast-fed infant gut. Recently, a cell wall-associated endo-ß-N-acetylglucosaminidase (EndoBI-1) found in various infant-borne bifidobacteria was shown to remove a range of intact N-linked glycans. We hypothesized that these released oligosaccharide structures can serve as a sole source for the selective growth of bifidobacteria. We demonstrated that EndoBI-1 released N-glycans from concentrated bovine colostrum at the pilot scale. EndoBI-1-released N-glycans supported the rapid growth of Bifidobacterium longum subsp. infantis (B. infantis), a species that grows well on human milk oligosaccharides, but did not support growth of Bifidobacterium animalis subsp. lactis (B. lactis), a species which does not. Conversely, B. infantis ATCC 15697 did not grow on the deglycosylated milk protein fraction, clearly demonstrating that the glycan portion of milk glycoproteins provided the key substrate for growth. Mass spectrometry-based profiling revealed that B. infantis consumed 73% of neutral and 92% of sialylated N-glycans, while B. lactis degraded only 11% of neutral and virtually no (<1%) sialylated N-glycans. These results provide mechanistic support that N-linked glycoproteins from milk serve as selective substrates for the enrichment of infant-associated bifidobacteria capable of carrying out the initial deglycosylation. Moreover, released N-glycans were better growth substrates than the intact milk glycoproteins, suggesting that EndoBI-1 cleavage is a key initial step in consumption of glycoproteins. Finally, the variety of N-glycans released from bovine milk glycoproteins suggests that they may serve as novel prebiotic substrates with selective properties similar to those of human milk oligosaccharides. IMPORTANCE: It has been previously shown that glycoproteins serve as growth substrates for bifidobacteria. However, which part of a glycoprotein (glycans or polypeptides) is responsible for this function was not known. In this study, we used a novel enzyme to cleave conjugated N-glycans from milk glycoproteins and tested their consumption by various bifidobacteria. The results showed that the glycans selectively stimulated the growth of B. infantis, which is a key infant gut microbe. The selectivity of consumption of individual N-glycans was determined using advanced mass spectrometry (nano-liquid chromatography chip-quadrupole time of flight mass spectrometry [nano-LC-Chip-Q-TOF MS]) to reveal that B. infantis can consume the range of glycan structures released from whey protein concentrate.

Milk proteins, peptides, and oligosaccharides: effects against the 21st century disorders.

Milk is the most complete food for mammals, as it supplies all the energy and nutrients needed for the proper growth and development of the neonate. Milk is a source of many bioactive components, which not only help meeting the nutritional requirements of the consumers, but also play a relevant role in preventing various disorders. Milk-derived proteins and peptides have the potential to act as coadjuvants in conventional therapies, addressing cardiovascular diseases, metabolic disorders, intestinal health, and chemopreventive properties. In addition to being a source of proteins and peptides, milk contains complex oligosaccharides that possess important functions related to the newborn's development and health. Some of the health benefits attributed to milk oligosaccharides include prebiotic probifidogenic effects, antiadherence of pathogenic bacteria, and immunomodulation. This review focuses on recent findings demonstrating the biological activities of milk peptides, proteins, and oligosaccharides towards the prevention of diseases of the 21st century. Processing challenges hindering large-scale production and commercialization of those bioactive compounds have been also addressed.

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.

Top-down analysis of highly post-translationally modified peptides by Fourier transform ion cyclotron resonance mass spectrometry.

Bovine κ-caseinoglycomacropeptide (GMP) is a highly modified peptide from κ-casein produced during the cheese making process. The chemical nature of GMP makes analysis by traditional proteomic approaches difficult, as the peptide bears a strong net negative charge and a variety of post-translational modifications. In this work, we describe the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FT-ICR MS) for the top-down analysis of GMP. The method allows the simultaneous detection of different GMP forms that result from the combination of amino acid genetic variations and post-translational modifications, specifically phosphorylation and O-glycosylation. The different GMP forms were identified by high resolution mass spectrometry in both negative and positive mode and confirmation was achieved by tandem MS. The results showed the predominance of two genetic variants of GMP that occur as either mono- or bi-phosphorylated species. Additionally, these four forms can be modified with up to two O-glycans generally sialylated. The results demonstrate the presence of glycosylated, bi-phosphorylated forms of GMP never described before.

Following the digestion of milk proteins from mother to baby.

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.

Mechanistic peptidomics: factors that dictate specificity in the formation of endogenous peptides in human milk.

An extensive mass spectrometry analysis of the human milk peptidome has revealed almost 700 endogenous peptides from 30 different proteins. Two in-house computational tools were created and used to visualize and interpret the data through both alignment of the peptide quasi-molecular ion intensities and estimation of the differential enzyme participation. These results reveal that the endogenous proteolytic activity in the mammary gland is remarkably specific and well conserved. Certain proteins-not necessarily the most abundant ones-are digested by the proteases present in milk, yielding endogenous peptides from selected regions. Our results strongly suggest that factors such as the presence of specific proteases, the position and concentration of cleavage sites, and, more important, the intrinsic disorder of segments of the protein drive this proteolytic specificity in the mammary gland. As a consequence of this selective hydrolysis, proteins that typically need to be cleaved at specific positions in order to exert their activity are properly digested, and bioactive peptides encoded in certain protein sequences are released. Proteins that must remain intact in order to maintain their activity in the mammary gland or in the neonatal gastrointestinal tract are unaffected by the hydrolytic environment present in milk. These results provide insight into the intrinsic structural mechanisms that facilitate the selectivity of the endogenous milk protease activity and might be useful to those studying the peptidomes of other biofluids.

A peptidomic analysis of human milk digestion in the infant stomach reveals protein-specific degradation patterns.

In vitro digestion of isolated milk proteins results in milk peptides with a variety of actions. However, it remains unclear to what degree protein degradation occurs in vivo in the infant stomach and whether peptides previously annotated for bioactivity are released. This study combined nanospray LC separation with time-of-flight mass spectrometry, comprehensive structural libraries, and informatics to analyze milk from 3 human mothers and the gastric aspirates from their 4- to 12-d-old postpartum infants. Milk from the mothers contained almost 200 distinct peptides, demonstrating enzymatic degradation of milk proteins beginning either during lactation or between milk collection and feeding. In the gastric samples, 649 milk peptides were identified, demonstrating that digestion continues in the infant stomach. Most peptides in both the intact milk and gastric samples were derived from ß-casein. The numbers of peptides from ß-casein, lactoferrin, α-lactalbumin, lactadherin, κ-casein, serum albumin, bile salt-associated lipase, and xanthine dehydrogenase/oxidase were significantly higher in the gastric samples than in the milk samples (P < 0.05). A total of 603 peptides differed significantly in abundance between milk and gastric samples (P < 0.05). Most of the identified peptides have previously identified biologic activity. Gastric proteolysis occurs in the term infant in the first 2 wk of life, releasing biologically active milk peptides with immunomodulatory and antibacterial properties of clinical relevance to the proximal intestinal tract. Data are available via ProteomeXchange (identifier PXD000688).

Peptidomic profile of milk of Holstein cows at peak lactation.

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 ß-casein, αs1-casein, and αs2-casein. No peptides derived from abundant bovine milk proteins such as lactoferrin, ß-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.