High-Pressure Processing of Human Milk: A Balance between Microbial Inactivation and Bioactive Protein Preservation.

BACKGROUND: Donor human milk banks use Holder pasteurization (HoP; 62.5°C, 30 min) to reduce pathogens in donor human milk, but this process damages some bioactive milk proteins. OBJECTIVES: We aimed to determine minimal parameters for high-pressure processing (HPP) to achieve >5-log reductions of relevant bacteria in human milk and how these parameters affect an array of bioactive proteins. METHODS: Pooled raw human milk inoculated with relevant pathogens (Enterococcus faecium, Staphylococcus aureus, Listeria monocytogenes, Cronobacter sakazakii) or microbial quality indicators (Bacillus subtilis and Paenibacillus spp. spores) at 7 log CFU/mL was processed at 300-500 MPa at 16-19°C (due to adiabatic heating) for 1-9 min. Surviving microbes were enumerated using standard plate counting methods. For raw milk, and HPP-treated and HoP-treated milk, the immunoreactivity of an array of bioactive proteins was assessed via ELISA and the activity of bile salt-stimulated lipase (BSSL) was determined via a colorimetric substrate assay. RESULTS: Treatment at 500 MPa for 9 min resulted in >5-log reductions of all vegetative bacteria, but <1-log reduction in B. subtilis and Paenibacillus spores. HoP decreased immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin G, lactoferrin, elastase and polymeric immunoglobulin receptor (PIGR) concentrations, and BSSL activity. The treatment at 500 MPa for 9 min preserved more IgA, IgM, elastase, lactoferrin, PIGR, and BSSL than HoP. HoP and HPP treatments up to 500 MPa for 9 min caused no losses in osteopontin, lysozyme, α-lactalbumin and vascular endothelial growth factor. CONCLUSION: Compared with HoP, HPP at 500 MPa for 9 min provides >5-log reduction of tested vegetative neonatal pathogens with improved retention of IgA, IgM, lactoferrin, elastase, PIGR, and BSSL in human milk.

Bioactive milk peptides: an updated comprehensive overview and database.

Partial digestion of milk proteins leads to the formation of numerous bioactive peptides. Previously, our research team thoroughly examined the decades of existing literature on milk bioactive peptides across species to construct the milk bioactive peptide database (MBPDB). Herein, we provide a comprehensive update to the data within the MBPDB and a review of the current state of research for each functional category from in vitro to animal and clinical studies, including angiotensin-converting enzyme (ACE)-inhibitory, antimicrobial, antioxidant, dipeptidyl peptidase (DPP)-IV inhibitory, opioid, anti-inflammatory, immunomodulatory, calcium absorption and bone health and anticancer activity. This information will help drive future research on the bioactivities of milk peptides.

Top-Down Glycopeptidomics Reveals Intact Glycomacropeptide Is Digested to a Wide Array of Peptides in Human Jejunum.

BACKGROUND: Bovine milk κ-casein-derived caseinomacropeptide (CMP) is produced in large quantities during cheese-making and has various biological activities demonstrated via in vitro and in vivo experiments. Previous studies examined protein degradation and peptide release after casein or whey protein consumption. However, whether purified intact CMP that is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown. OBJECTIVES: The aim of this study was to determine the extent to which purified intact CMP (including glycosylated forms) is digested into peptide fragments within the jejunum of healthy human adults after consumption. METHODS: Jejunal fluids were collected from 3 adult participants (2 men and 1 woman, age: 27 ± 7 y; BMI: 23 ± 1 kg/m2) for 3 h after consuming 37.5 g of purified intact CMP. CMP and CMP-derived peptides were isolated from the collected jejunal fluids by ethanol precipitation and solid-phase extraction and identified by MS-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids. RESULTS: Intact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (<2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11 to 20 amino acids in length were predominant (accounting for 68-88% of the total peptide ion abundance) in jejunal fluids during 1-3 h post consumption. CONCLUSIONS: This study demonstrates that intact CMP (including glycosylated forms) is mostly digested in the human jejunum, releasing a wide array of CMP-derived peptide fragments. Some of the CMP-derived peptides with high homology to known bioactive peptides consistently survived across 3 h of digestion. Therefore, future research should examine the biological effects of the partially digested form-the CMP-derived fragments-rather than those of intact CMP.

Peptides Derived from In Vitro and In Vivo Digestion of Human Milk Are Immunomodulatory in THP-1 Human Macrophages.

BACKGROUND: Milk proteins contain many encrypted bioactive peptides. Whether these bioactive peptides are released in the infant intestine and exert immunomodulatory activity remains unknown. OBJECTIVE: This study examined in vitro immunomodulatory activities of peptides from in vitro- and in vivo-digested human milk. METHODS: Peptides were extracted from in vitro-digested human milk and pooled intestinal samples from 8 infants fed human milk. Peptides extracted from in vitro-digested samples were fractionated. The in vitro effects of these peptides and fractions on the secretion of TNF-α and IL-8 in LPS-treated human immune THP-1 macrophages were evaluated. The significance of differences between in vitro peptide fraction treatment and control on cytokine production was analyzed by t test. LC-MS/MS-based peptidomics was conducted to identify the peptides. The peptides were screened for potential bioactivity using a sequence homology search using the Milk Bioactive Peptide Database (MBPDB). RESULTS: Six fractions of the peptide mixture extracted from the in vitro-digested human milk significantly inhibited TNF-α production by LPS-challenged THP-1 macrophages. Fractions F4, F8, F11, F14, and F17 attenuated IL-8 secretion, and F6/7 and F18 increased IL-8 secretion. Peptides extracted from the pooled in vivo intestinal samples attenuated both TNF-α and IL-8 secretion. There were 266 and 418 peptides identified in the in vitro and in vivo samples, respectively. Among the peptides, 34 and 50 in the in vitro and in vivo samples, respectively, had >80% sequence similarity to bioactive peptides in the MBPDB. CONCLUSIONS: Peptides released by in vitro and in vivo infant digestion of human milk were immunomodulatory in human immune cells; fractions F4, F8, and F11 were anti-inflammatory; and F6/7 and F18 were proinflammatory. Thirteen peptides were present in all fractions with anti-inflammatory activity, and 38 peptides were present in all fractions with proinflammatory activity. These peptides potentially contributed to the observed immunomodulatory activity of the peptide mixtures.

Impact of pertussis-specific IgA, IgM, and IgG antibodies in mother's own breast milk and donor breast milk during preterm infant digestion.

BACKGROUND: The survival of antibody isotypes specific to pertussis toxin (PT) and filamentous hemagglutinin (FHA) from mother's own milk (MBM) and donor breast milk (DBM) during preterm infant digestion was investigated. METHODS: Feed, gastric, and stool samples were collected from 20 preterm mother-infant pairs at 8-9 days and 21-22 days postpartum. Samples were analyzed via ELISA for anti-FHA or anti-PT immunoglobulin A (IgA), IgM, and IgG. RESULTS: Anti-PT IgA, anti-FHA IgG, and anti-PT IgG were lower in MBM than DBM at 8-9 days postpartum, whereas anti-FHA IgM was higher in MBM than DBM. Anti-PT IgA, anti-PT IgG, and anti-FHA IgG in DBM decreased in gastric contents at both postpartum times but those antibodies in MBM were stable or increased during gastric digestion. Anti-FHA-specific IgA and IgM were higher in gastric contents from infants fed MBM than from infants fed DBM at 8-9 days. All pertussis antibodies were detected in infant stools at both postpartum times. CONCLUSIONS: Pertussis-specific antibodies from MBM were stable during infant digestion, whereas anti-pertussis IgA and IgG from DBM decreased in gastric contents. The constant region and variable region of antibodies and maternal immunization appear to be the critical factors for their stability to proteolytic digestion and pasteurization. IMPACT: Pertussis-specific antibodies from mother's breast milk were stable during infant digestion, whereas anti-pertussis IgA and IgG from donor breast milk decreased in gastric contents. The constant region and variable region of pertussis-specific antibodies and the maternal immunization (previous infections and vaccinations) appear to be the critical factors for their stability to proteolytic digestion and pasteurization. Pertussis-specific antibodies from either mother's breast milk or donor breast milk survived during preterm infant digestion and both types of milk will compensate for the lower IgG transplacental transfer in preterm infants compared with term infants.

Effect of digestion on stability of palivizumab IgG1 in the infant gastrointestinal tract.

BACKGROUND: Potentially, orally administered antibodies specific to enteric pathogens could be administered to infants to prevent diarrheal infections, particularly in developing countries where diarrhea is a major problem. However, to prevent infection, such antibodies would need to resist degradation within the gastrointestinal tract. METHODS: Palivizumab, a recombinant antibody specific to respiratory syncytial virus (RSV), was used in this study as a model for examining the digestion of neutralizing antibodies to enteric pathogens in infants. The survival of this recombinant IgG1 across digestion in 11 infants was assayed via an anti-idiotype ELISA and RSV F protein-specific ELISA. Concentrations were controlled for any dilution or concentration that occurred in the digestive system using mass spectrometry-based quantification of co-administered, orally supplemented, indigestible polyethylene glycol (PEG-28). RESULTS: Binding activity of Palivizumab IgG1 decreased (26-99%) across each phase of in vivo digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. CONCLUSION: Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. IMPACT: Binding activity of palivizumab IgG1 decreased (26-99%) across each phase of in vivo infant digestion as measured by both anti-idiotype and RSV F protein-specific ELISAs. Palivizumab was likely degraded by proteases and changes in pH introduced in the gut. Antibodies generated for passive protection of the infant gastrointestinal tract from pathogens will need to be more resistant to digestion than the model antibody fed to infants in this study, or provided in higher doses to be most effective. The monoclonal antibody IgG1 tested was not stable across the infant gastrointestinal tract. The observation of palivizumab reduction was unlikely due to dilution in the gastrointestinal tract. The results of this work hint that provision of antibody could be effective in preventing enteric pathogen infection in infants. Orally delivered recombinant antibodies will need to either be dosed at high levels to compensate for digestive losses or be engineered to better resist digestion. Provision of enteric pathogen-specific recombinant antibodies to at-risk infants could provide a new and previously unexplored pathway to reducing the infection in infants. The strategy of enteric recombinant antibodies deserves more investigation throughout medicine as a novel means for treatment of enteric disease targets.

Peptides from the Intestinal Tract of Breast Milk-Fed Infants Have Antimicrobial and Bifidogenic Activity.

For bioactive milk peptides to be relevant to infant health, they must be released by gastrointestinal proteolysis and resist further proteolysis until they reach their site of activity. The intestinal tract is the likeliest site for most bioactivities, but it is currently unknown whether bioactive milk peptides are present therein. The purpose of the present study was to identify antimicrobial and bifidogenic peptides in the infant intestinal tract. Milk peptides were extracted from infant intestinal samples, and the activities of the bulk peptide extracts were determined by measuring growth of Escherichia coli, Staphylococcus aureus, and Bifidobacterium longum spp. infantis after incubation with serial dilutions. The peptide profiles of active and inactive samples were determined by peptidomics analysis and compared to identify candidate peptides for bioactivity testing. We extracted peptides from 29 intestinal samples collected from 16 infants. Five samples had antimicrobial activity against S. aureus and six samples had bifidogenic activity for B. infantis. We narrowed down a list of 6645 milk peptides to 11 candidate peptides for synthesis, of which 6 fully inhibited E. coli and S. aureus growth at concentrations of 2500 and 3000 µg/mL. This study provides evidence for the potential bioactivity of milk peptides in the infant intestinal tract.

Milk Peptides Survive In Vivo Gastrointestinal Digestion and Are Excreted in the Stool of Infants.

BACKGROUND: Human milk peptides released by gastrointestinal proteases have been identified with bioactivities that can benefit the infant but must first reach their respective sites of activity. Peptides in the stool either survived to or were released inside the intestinal tract, and thus had the opportunity to exert bioactivity there. However, it is unknown whether any milk peptides, bioactive or not, can survive in the stool of infants. OBJECTIVE: The aim of this study was primarily to identify milk peptides in infant stool samples and secondarily test the hypotheses that the milk peptide profiles of stools are different between preterm infants at different days of life and between preterm and term infants. METHODS: Infant stool samples were collected from 16 preterm infants (<34 weeks gestational age) at 8 or 9 and 21 or 22 days of life (DOL), and from 10 term infants (>34 weeks gestational age) at 8 or 9 DOL. Milk peptides were isolated from the stool samples and identified using tandem MS. The peptide counts and abundances were compared between infant groups. RESULTS: In total, 118 exclusively milk-derived peptides from the caseins and α-lactalbumin were present in the stool samples, including some peptides with known or potential bioactivity. The remaining 8014 identified peptides could be derived either from milk or endogenous proteins. Although many individual milk peptides were significantly different between preterm infants at 8/9 and 21/22 DOL and between preterm and term infants, total peptide abundance and count were similar for all 3 groups. CONCLUSIONS: This is the first study to confirm the survival of milk peptides in the stool of infants. Some of the peptides had potential bioactivities that could influence infant gut development. These results are important to understand the physiological relevance of human milk peptides to the infant.

Production of functional mimics of human milk oligosaccharides by enzymatic glycosylation of bovine milk oligosaccharides.

Consumption of mothers' milk is associated with reduced incidence and severity of enteric infections, leading to reduced morbidity in breastfed infants. Fucosylated and sialylated human milk oligosaccharides (HMO) are important for both direct antimicrobial action - likely via a decoy effect - and indirect antimicrobial action through commensal growth enhancement. Bovine milk oligosaccharides (BMO) are a potential source of HMO-mimics as BMO resemble HMO; however, they have simpler and less fucosylated structures. BMO isolated at large scales from bovine whey permeate were modified by the addition of fucose and/or sialic acid to generate HMO-like glycans using high-yield and cost-effective one-pot multienzyme approaches. Quadrupole time-of-flight LC/MS analysis revealed that 22 oligosaccharides were synthesized and 9 had identical composition to known HMO. Preliminary anti-adherence activity assays indicated that fucosylated BMO decreased the uptake of enterohemorrhagic Escherichia coli O157:H7 by human intestinal epithelial Caco-2 cells more effectively than native BMO.

Cytokine Expression by Human Macrophage-Like Cells Derived from the Monocytic Cell Line THP-1 Differs Between Treatment With Milk from Preterm- and Term-Delivering Mothers and Pasteurized Donor Milk.

Immunomodulatory proteins from human milk may enhance the protection and development of the infant's gut. This study compared the immunomodulatory effects of treatment with milk from preterm-(PM) and term-delivering (TM) mothers and pasteurized donor milk (DM) on cytokine gene expression in human macrophage-like cells derived from the monocytic cell line THP-1. The gene expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-12 (p40), IL-10 and GAPDH in macrophages treated with PM, TM and DM at steady and activated (inflammatory) states were measured using real-time reverse transcription-polymerase chain reaction. TNF-α and IL-6 in macrophages (both states) with DM were higher than PM or TM. IL-10 in steady state macrophages with DM was higher than PM whereas DM increased IL-10 in activated macrophages compared with TM. TM increased IL-6 and IL-12 (p40) in steady state macrophages compared with PM. IL-12 (p40) in activated macrophages with TM was higher than PM. IL-10 in steady state macrophages with TM was higher than PM. These results suggest that DM induces higher gene expression of pro-inflammatory and anti-inflammatory cytokines in macrophages compared with PM or TM. PM reduced gene expression of pro-inflammatory cytokines compared with TM, which may decrease the development of necrotizing enterocolitis and systematic inflammation.

Peptidomics Analysis of Milk Protein-Derived Peptides Released over Time in the Preterm Infant Stomach.

Over the course of milk digestion, native milk proteases and infant digestive proteases fragment intact proteins into peptides with potential bioactivity. This study investigated the release of peptides over 3 h of gastric digestion in 14 preterm infant sample sets. The peptide content was extracted and analyzed from milk and gastric samples via Orbitrap tandem mass spectrometry. The relative ion intensity (abundance) and count of peptides in each sample were compared over time and between infants fed milk fortified with bovine milk fortifier and infants fed unfortified milk. Bioactivity of the identified peptides was predicted by sequence homology to known bioactive milk peptides. Both total and bioactive peptide abundance and count continuously increased over 3 h of gastric digestion. After accounting for infant weight, length, and postconceptual age, fortification of milk limited the release of peptides from human milk proteins. Peptides that survived further gastric digestion after their initial release were structurally more similar to bioactive peptides than nonsurviving peptides. This work is the first to provide a comprehensive profile of milk peptides released during gastric digestion over time, which is an essential step in determining which peptides are most likely to be biologically relevant in the infant. Data are available via ProteomeXchange with identifier PXD012192.

Changes in Proteases, Antiproteases, and Bioactive Proteins From Mother's Breast Milk to the Premature Infant Stomach.

OBJECTIVE: Our previous studies suggested that human milk proteases begin to hydrolyze proteins in the mammary gland and continue within the term infant' stomach. No research has measured milk protease and pepsin activity in the gastric aspirates of preterm infants after human milk feeding. This study investigated how the concentrations of human milk proteases and protease inhibitors changed in the premature infant stomach. METHODS: Human milk and infant gastric samples were collected from 18 preterm-delivering mother-infant pairs (24-32 week gestational age). Paired human milk and gastric samples were collected across postnatal age (2-47 days). Protease concentrations were determined by spectrophotometric or fluorometric assays, and the concentrations of protease inhibitors and bioactive proteins were determined by enzyme-linked immunosorbent assay. Paired t tests were applied to compare enzymes, antiproteases, and bioactive proteins between human milk and gastric samples. RESULTS: Our study reveals that although human milk proteases, including carboxypeptidase B2, kallikrein, plasmin, cathepsin D, elastase, thrombin, and cytosol aminopeptidase, are present in the preterm infant stomach, only plasmin and cathepsin D can actively hydrolyze proteins at gastric pH. Enzyme-linked immunosorbent assay and peptidomic evidence suggest that all milk antiproteases as well as lactoferrin and immunoglobulin A are partially digested in the preterm stomach. CONCLUSIONS: Most human milk proteases are active in milk but not at preterm infant gastric pH. Only cathepsin D and plasmin have potential to continue degrading milk proteins within the preterm infant stomach.

The preterm infant stomach actively degrades milk proteins with increasing breakdown across digestion time.

AIM: This study investigated the effect of time post-ingestion on gastric digestion and gastric hormones after feeding preterm infants unfortified and fortified human milk. METHODS: Human milk and infant gastric samples were collected from 14 preterm (23-32 weeks birth gestational age) mother-infant pairs within 7-98 days postnatal age. Gastric samples were collected one, two and three hours after beginning of feeding. Samples were analysed for pH, proteolysis, general protease activity and the concentrations of pepsin, gastrin and gastrin-releasing peptide (GRP). One-way ANOVA with repeated measures followed by Tukey's multiple comparisons test was used. RESULTS: Gastric pH was significantly decreased after each hour in the preterm infant stomach from one to three hours postprandial. Proteolysis increased significantly from human milk to gastric contents at one, two and three hours postprandial (by 62, 131% and 181%, p < 0.05). General protease activity increased significantly by 58% from human milk to the gastric contents at two hours postprandial. GRP was present in human milk, whereas gastrin was produced in the infant stomach. CONCLUSION: Although preterm infants may digest human milk proteins to a lesser extent than term infants, we demonstrated that the preterm infant stomach actively degrades milk proteins with increasing breakdown over digestion time.

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.

Milk Proteins Are Predigested Within the Human Mammary Gland.

Previous work demonstrates that proteases present in human milk release hundreds of peptides derived from milk proteins. However, the question of whether human milk protein digestion begins within the mammary gland remains incompletely answered. The primary objective of this study was to determine whether proteolytic degradation of human milk proteins into peptides begins within the mammary gland. The secondary objectives were to determine which milk proteases participate in the proteolysis and to predict which released peptides have bioactivity. Lactating mothers (n = 4) expressed their milk directly into a mixture of antiproteases on ice followed by immediate freezing of the milk to limit post-expression protease activity. Samples were analyzed for their peptide profiles via mass spectrometry and database searching. Peptidomics-based protease prediction and bioactivity prediction were each performed with several different approaches. The findings demonstrate that human milk contains more than 1,100 unique peptides derived from milk protein hydrolysis within the mammary gland. These peptides derived from 42 milk proteins and included 306 potential bioactive peptides. Based on the peptidomics data, plasmin was predicted to be the milk protease most active in the hydrolysis of human milk proteins within the mammary gland. Milk proteases actively cleave milk proteins within the mammary gland, initiating the release of functional peptides. Thus, the directly breastfed infant receives partially pre-digested proteins and numerous bioactive peptides.

Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown.Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age.Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age.Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL-1 · d-1 and 0.454 µg · mL-1 · d-1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples.Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not vary substantially across GA and postnatal age.

Personalizing protein nourishment.

Proteins are not equally digestible-their proteolytic susceptibility varies by their source and processing method. Incomplete digestion increases colonic microbial protein fermentation (putrefaction), which produces toxic metabolites that can induce inflammation in vitro and have been associated with inflammation in vivo. Individual humans differ in protein digestive capacity based on phenotypes, particularly disease states. To avoid putrefaction-induced intestinal inflammation, protein sources, and processing methods must be tailored to the consumer's digestive capacity. This review explores how food processing techniques alter protein digestibility and examines how physiological conditions alter digestive capacity. Possible solutions to improving digestive function or matching low digestive capacity with more digestible protein sources are explored. Beyond the ileal digestibility measurements of protein digestibility, less invasive, quicker and cheaper techniques for monitoring the extent of protein digestion and fermentation are needed to personalize protein nourishment. Biomarkers of protein digestive capacity and efficiency can be identified with the toolsets of peptidomics, metabolomics, microbial sequencing and multiplexed protein analysis of fecal and urine samples. By monitoring individual protein digestive function, the protein component of diets can be tailored via protein source and processing selection to match individual needs to minimize colonic putrefaction and, thus, optimize gut health.

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.

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.