Point-of-care human milk concentration by passive osmosis: comprehensive analysis of fresh human milk samples.

OBJECTIVE: Preterm infants need enrichment of human milk (HM) for optimal growth. This study evaluated a novel, point-of-care human milk concentration (HMC) process for water removal from fresh HM samples by passive osmotic concentration. STUDY DESIGN: Nineteen fresh HM samples were concentrated by incubation with the HMC devices for 3 h at 4 °C. Pre- and post-concentration HM samples were compared by HM properties for: pH, osmolality, macronutrients, enzyme activity, bioactive, and total cell viability. RESULTS: Passive osmotic concentration reduced HM volume by an average of 16.3% ± 3.8% without a significant effect on pH or cell viability. Ten of the 41 HM components did not differ significantly (p > 0.05) between pre- and post-concentration samples. Twenty-three increased within the expected range by volume reduction. Six increased more than expected, two less than expected, and none decreased significantly. CONCLUSION: Passive osmotic concentration of fresh HM can concentrate HM components by selective removal of water. HM osmolality and pH remained within neonatal feeding parameters.

A review of the safety evidence on recombinant human lactoferrin for use as a food ingredient.

Published studies on the glycosylation, absorption, distribution, metabolism, excretion, and safety outcomes of orally ingested recombinant human lactoferrin (rhLF) were reviewed in the context of unanswered safety questions, including alloimmunization, allergenicity, and immunotoxicity potential of rhLF during repeated exposure. The primary objective was to summarize current safety data of rhLF produced in transgenic host expression systems. Overall, results from animal and human studies showed that rhLF was well tolerated and safe. Animal data showed no significant toxicity-related outcomes among any safety or tolerability endpoints. The no observed adverse effect levels (NOAEL) were at the highest level tested in both iron-desaturated and -saturated forms of rhLF. Although one study reported outcomes of rhLF on immune parameters, no animal studies directly assessed immunogenicity or immunotoxicity from a safety perspective. Data from human studies were primarily reported as adverse events (AE). They showed no or fewer rhLF-related AE compared to control and no evidence of toxicity, dose-limiting toxicities, or changes in iron status in various subpopulations. However, no human studies evaluated the immunomodulatory potential of rhLF as a measure of safety. Following this review, a roadmap outlining preclinical and clinical studies with relevant safety endpoints was developed to address the unanswered safety questions.

Optimized Ultraviolet-C Processing Inactivates Pathogenic and Spoilage-Associated Bacteria while Preserving Bioactive Proteins, Vitamins, and Lipids in Human Milk.

Holder pasteurization (HoP) enhances donor human milk microbiological safety but damages many bioactive milk proteins. Though ultraviolet-C irradiation (UV-C) can enhance safety while better preserving some milk proteins, it has not been optimized for dose or effect on a larger array of bioactive proteins. We determined the minimal UV-C parameters that provide >5-log reductions of relevant bacteria in human milk and how these treatments affect an array of bioactive proteins, vitamin E, and lipid oxidation. Treatment at 6000 and 12 000 J/L of UV-C resulted in >5-log reductions of all vegetative bacteria and bacterial spores, respectively. Both dosages improved retention of immunoglobulin A (IgA), IgG, IgM, lactoferrin, cathepsin D, and elastase and activities of bile-salt-stimulated lipase and lysozyme compared with HoP. These UV-C doses caused minor reductions in α-tocopherol but not γ-tocopherol and no increases in lipid oxidation products. UV-C treatment is a promising approach for donor human milk processing.

Potential Prebiotic Properties of Whey Protein and Glycomacropeptide in Gut Microbiome.

Proteins in whey have prebiotic and antimicrobial properties. Whey protein comprises numerous bioactive proteins and peptides, including glycomacropeptide (GMP), a hydrophilic casein peptide that separates with the whey fraction during cheese making. GMP has traditionally been used as a protein source for individuals with phenylketonuria and also has prebiotic (supporting the growth of Bifidobacterium and lactic acid bacteria) and antimicrobial activities. GMP supplementation may help positively modulate the gut microbiome, help treat dysbiosis-related gastrointestinal disorders and improve overall health in consumers.

Corrigendum: Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization, ultra-high-temperature sterilization, freeze-thawing and homogenization.

[This corrects the article DOI: 10.3389/fnut.2022.926814.].

Prebiotic Strategies to Manage Lactose Intolerance Symptoms.

Lactose intolerance, which affects about 65-75% of the world's population, is caused by a genetic post-weaning deficiency of lactase, the enzyme required to digest the milk sugar lactose, called lactase non-persistence. Symptoms of lactose intolerance include abdominal pain, bloating and diarrhea. Genetic variations, namely lactase persistence, allow some individuals to metabolize lactose effectively post-weaning, a trait thought to be an evolutionary adaptation to dairy consumption. Although lactase non-persistence cannot be altered by diet, prebiotic strategies, including the consumption of galactooligosaccharides (GOSs) and possibly low levels of lactose itself, may shift the microbiome and mitigate symptoms of lactose consumption. This review discusses the etiology of lactose intolerance and the efficacy of prebiotic approaches like GOSs and low-dose lactose in symptom management.

Macrophage-Immunomodulatory Actions of Bovine Whey Protein Isolate, Glycomacropeptide, and Their In Vitro and In Vivo Digests.

Whey protein isolate (WPI) consists of an array of proteins and peptides obtained as a byproduct of the cheesemaking process. Research suggests that WPI, along with its peptides such as glycomacropeptide (GMP), possesses immunomodulatory properties. These properties hold potential for alleviating the adverse effects of inflammatory conditions such as inflammatory bowel disease. Although promising, the immunoregulatory properties of the digested forms of WPI and GMP-those most likely to interact with the gut immune system-remain under-investigated. To address this knowledge gap, the current study examined the effects of in vitro-digested WPI and GMP, in vivo-digested WPI, and undigested WPI and GMP on the secretion of pro-inflammatory cytokines (TNF-α and IL-1ß) in lipopolysaccharide-stimulated macrophage-like cells. Our results indicate that digested WPI and GMP reduced the expression of TNF-α and IL-1ß, two pro-inflammatory cytokines. Whole WPI had no effect on TNF-α but reduced IL-1ß levels. In contrast, in vivo-digested WPI reduced TNF-α but increased IL-1ß. Undigested GMP, on the other hand, increased the secretion of both cytokines. These results demonstrate that digestion greatly modifies the effects of WPI and GMP on macrophages and suggest that digested WPI and GMP could help mitigate gastrointestinal inflammation. Further clinical studies are necessary to determine the biological relevance of WPI and GMP digestion products within the gut and their capacity to influence gut inflammation.

Correction to: Understanding the effects of dietary components on the gut microbiome and human health.

[This corrects the article DOI: 10.1007/s10068-020-00811-w.].

Evaluating the Potential of Casein Glycomacropeptide in Adult Irritable Bowel Syndrome Management: A Pilot Study.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder that affects 10-15% of the global population and presents symptoms such as abdominal discomfort, bloating and altered bowel habits. IBS is believed to be influenced by gut microbiota alterations and low-grade inflammation. Bovine kappa-casein glycomacropeptide (GMP), a bioactive dairy-derived peptide, possesses anti-adhesive, prebiotic and immunomodulatory properties that could potentially benefit IBS patients. This pilot study investigated the effects of daily supplementation with 30 g of GMP for three weeks on gut health in five people with IBS. We assessed alterations in gut microbiota composition, fecal and blood inflammatory makers, and gut-related symptoms before, during and after the GMP feeding period. The results revealed no changes in fecal microbiota, subtle effects on systemic and intestinal immune makers, and no changes in gut-related symptoms during and after the GMP supplementation. Further research is needed to assess the potential benefits of GMP in IBS patients, including the examination of dosage and form of GMP supplementation.

The Role of Bovine Kappa-Casein Glycomacropeptide in Modulating the Microbiome and Inflammatory Responses of Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder marked by chronic abdominal pain, bloating, and irregular bowel habits. Effective treatments are still actively sought. Kappa-casein glycomacropeptide (GMP), a milk-derived peptide, holds promise because it can modulate the gut microbiome, immune responses, gut motility, and barrier functions, as well as binding toxins. These properties align with the recognized pathophysiological aspects of IBS, including gut microbiota imbalances, immune system dysregulation, and altered gut barrier functions. This review delves into GMP's role in regulating the gut microbiome, accentuating its influence on bacterial populations and its potential to promote beneficial bacteria while inhibiting pathogenic varieties. It further investigates the gut microbial shifts observed in IBS patients and contemplates GMP's potential for restoring microbial equilibrium and overall gut health. The anti-inflammatory attributes of GMP, especially its impact on vital inflammatory markers and capacity to temper the low-grade inflammation present in IBS are also discussed. In addition, this review delves into current research on GMP's effects on gut motility and barrier integrity and examines the changes in gut motility and barrier function observed in IBS sufferers. The overarching goal is to assess the potential clinical utility of GMP in IBS management.

Effects of Whey Protein Supplementation on Inflammatory Marker Concentrations in Older Adults.

Although whey protein isolate (WPI) has been shown to be immunomodulatory, its ability to modulate production of a broad array of inflammatory markers has not previously been investigated in healthy adults. We investigated the effects of daily supplementation with 35 g of WPI for 3 weeks on inflammatory marker concentrations in the blood serum and feces of 14 older adult subjects (mean age: 59). Serum was analyzed using a multiplex assay to quantify the cytokines IFN-γ, IL-1ß, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-17A and TNF-α. Fecal samples were analyzed using an ELISA for the inflammatory markers calprotectin and lactoferrin. Our results yielded high inter-subject variability and a significant proportion of cytokine concentrations that were below our method's limit of quantification. We observed decreases in serum IL-12p70 in the washout phase compared with baseline, as well as the washout stage for fecal lactoferrin relative to the intervention stage. Serum IL-13 was also significantly reduced during the intervention and washout stages. Our data suggest that whey protein supplementation did not significantly alter most inflammatory markers measured but can alter concentrations of some inflammatory markers in healthy older adults. However, our study power of 35% suggests the number of participants was too low to draw strong conclusions from our data.

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.

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.

Ecologies, synergies, and biological systems shaping human milk composition-a report from "Breastmilk Ecology: Genesis of Infant Nutrition (BEGIN)" Working Group 2.

Human milk is universally recognized as the preferred food for infants during the first 6 mo of life because it provides not only essential and conditionally essential nutrients in necessary amounts but also other biologically active components that are instrumental in protecting, communicating important information to support, and promoting optimal development and growth in infants. Despite decades of research, however, the multifaceted impacts of human milk consumption on infant health are far from understood on a biological or physiological basis. Reasons for this lack of comprehensive knowledge of human milk functions are numerous, including the fact that milk components tend to be studied in isolation, although there is reason to believe that they interact. In addition, milk composition can vary greatly within an individual as well as within and among populations. The objective of this working group within the Breastmilk Ecology: Genesis of Infant Nutrition (BEGIN) Project was to provide an overview of human milk composition, factors impacting its variation, and how its components may function to coordinately nourish, protect, and communicate complex information to the recipient infant. Moreover, we discuss the ways whereby milk components might interact such that the benefits of an intact milk matrix are greater than the sum of its parts. We then apply several examples to illustrate how milk is better thought of as a biological system rather than a more simplistic "mixture" of independent components to synergistically support optimal infant health.

Comparison of Solid-Phase Extraction Sorbents for Monitoring the In Vivo Intestinal Survival and Digestion of Kappa-Casein-Derived Caseinomacropeptide.

Kappa-casein-derived caseinomacropeptide (CMP)-a 64-amino-acid peptide-is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. It also has two major amino acid sequences with different modifications, including glycosylation, phosphorylation, and oxidation. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract, where they can exert these functions. In this study, three solid-phase extraction (SPE) methods-porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), and C18 chromatography-were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples prior to LC-MS/MS acquisition. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples.

Mass spectral profiling of caseinomacropeptide extracted from feeding material and jejunal fluid using three methods-ethanol precipitation, perchloric acid precipitation, and ultrafiltration.

The ability of bovine κ-casein-derived caseinomacropeptide (CMP) to exert bioactivity in the human gut depends on its digestive survival. Sampling from the human jejunum after feeding CMP and top-down glycopeptidomics analysis facilitates the determination of CMP survival. To reduce interference from non-target molecules in mass spectrometric analysis, CMP must be isolated from digestive fluid. To identify an optimal extraction method, this study compared the profiles of CMP extracted from feeding material (commercial CMP in water) and digestive fluid by ethanol precipitation, perchloric acid (PCA) precipitation, and ultrafiltration. Ethanol precipitation yielded the highest ion abundances for aglycosylated CMP and glycosylated CMP in both feeding material and jejunal samples. Notably, PCA precipitation yielded the highest abundance of partially digested CMP-derived fragments in jejunal samples. Overall, ethanol precipitation was the most effective among the methods tested for intact CMP extraction from jejunal fluids, whereas PCA precipitation was optimal for extraction of CMP fragments.

Structural and functional changes of bioactive proteins in donor human milk treated by vat-pasteurization, retort sterilization, ultra-high-temperature sterilization, freeze-thawing and homogenization.

Background: Donor human milk should be processed to guarantee microbiological safety prior to infant feeding, but this process can influence the structure and quantity of functional proteins. Objective: The aim of this study was to determine the effect of thawing, homogenization, vat-pasteurization (Vat-PT), retort sterilization (RTR) and ultra-high-temperature (UHT) processing on the structure of bioactive proteins in donor milk. Methods: Pooled donor milk was either not treated (Raw) or treated with an additional freeze-thaw cycle with and without homogenization, Vat-PT, RTR with and without homogenization, and UHT processing with and without homogenization. Overall protein retention was assessed via sodium-dodecyl sulfate (SDS-PAGE), and the immunoreactivity of 13 bioactive proteins were assessed via enzyme-linked immunosorbent assay (ELISA). Results: Freeze-thawing, freeze-thawing plus homogenization and Vat-PT preserved all the immunoglobulins (sIgA/IgA, IgG, IgM) in donor milk, whereas RTR and UHT degraded almost all immunoglobulins. UHT did not alter osteopontin immunoreactivity, but Vat-PT and retort decreased it by ~50 and 70%, respectively. Freeze-thawing with homogenization, Vat-PT and UHT reduced lactoferrin's immunoreactivity by 35, 65, and 84%, respectively. Lysozyme survived unaltered throughout all processing conditions. In contrast, elastase immunoreactivity was decreased by all methods except freeze-thawing. Freeze-thawing, freeze-thawing plus homogenization and Vat-PT did not alter polymeric immunoglobulin receptor (PIGR) immunoreactivity, but RTR, RTR plus homogenization and UHT increased detection. All heat processing methods increased α-lactalbumin immunoreactivity. Vat-PT preserved all the growth factors (vascular/endothelial growth factor, and transforming growth factors ß1 and ß2), and UHT treatments preserved the majority of these factors. Conclusion: Different bioactive proteins have different sensitivity to the treatments tested. Overall, Vat-PT preserved more of the bioactive proteins compared with UHT or RTR. Therefore, human milk processors should consider the impact of processing methods on key bioactive proteins in human milk.

Premature delivery impacts the concentration of plasminogen activators and a plasminogen activator inhibitor and the plasmin activity in human milk.

Background and aims: Plasmin in human milk partially hydrolyzes milk proteins within the mammary gland and may enhance the hydrolysis of milk proteins within the infant's stomach. This study examined the effects of extremely preterm (EP)-, very preterm (VP)-, and term-delivery on plasmin activity and the concentrations of plasminogen activators [urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA)], plasminogen activator inhibitor type 1 (PAI-1) and the complexes of PAI-1/uPA and PAI-1/tPA in human milk. Materials and methods: Human milk samples were collected from mothers who delivered extremely preterm infants [24-27 weeks gestational age (GA), n = 20], very preterm infants (28-32 weeks GA, n = 12), and term infants (38-39 weeks GA, n = 8) during 2-72 days postnatally. Plasmin activity was determined using fluorometric substrate assay, whereas concentrations of uPA, tPA, PAI-1, the PAI-1/uPA complex and the PAI-1/tPA complex were quantified by ELISA. Results: Plasmin activity, uPA and tPA were detected in all human milk samples, PAI-1 and the PAI-1/uPA complex were present in 42.5 and 32.5% of milk samples, respectively, and the PAI-1/tPA complex was not detected. Plasmin activity was correlated negatively with postnatal age and postmenstrual age (PMA) in the VP group and positively with postnatal age in the term group. uPA and tPA concentrations decreased with increasing postnatal age in both EP and VP groups but did not correlate in the term group. uPA concentration was correlated positively with GA in the VP group and tended to be elevated with increasing GA in the combined three groups. In contrast, tPA concentrations were correlated negatively with GA and PMA in the combined three groups (P < 0.008) and with PMA in the EP and VP groups. PAI-1 concentration tended to be correlated positively with postnatal age in the combined three groups. No correlation was detected with the PAI-1/uPA complex. Conclusion: Premature delivery impacted the plasmin activity and the concentrations of uPA, tPA, and PAI-1 in human milk. Whether these changes in milk plasminogen activators and inhibitors have a role in balancing the proteolytic digestion of premature infants remains to be investigated.

Bioavailability of Peptides Derived from the In Vitro Digestion of Human Milk Assessed by Caco-2 Cell Monolayers.

Human milk-protein-derived peptides exhibit an array of bioactivities. Certain bioactivities cannot be exerted unless the peptides are absorbed across the gastrointestinal lumen into the bloodstream. The purpose of study was to determine which peptides derived from in vitro digestion of human milk could cross human intestinal Caco-2 cell monolayers. Our results showed that the numbers of peptides absorbed by the Caco-2 cell monolayer were different at different concentrations (44 peptides out of 169 peptides detected at 10 µg/mL, 124 peptides out of 204 peptides detected at 100 µg/mL, and 175 peptides out of 236 peptides detected at 1000 µg/mL). Four peptides (NLHLPLP (ß-casein [138-144]), PLAPVHNPI (ß-casein [216-224]), PLMQQVPQPIPQ (ß-casein [148-159]), and FDPQIPK (ß-casein [126-132])) crossed to the basolateral chamber of the Caco-2 monolayer incubated with peptides at all three concentrations. Among the peptides identified in the basolateral chambers, three peptides (NLHLPLP (ß-casein [138-144]), LENLHLPLP (ß-casein [136-144]), and QVVPYPQ (ß-casein [182-188])) are known ACE-inhibitors; one peptide (LLNQELLLNPTHQIYPV (ß-casein [197-213])) is antimicrobial, and another peptide (QVVPYPQ (ß-casein [182-188])) has antioxidant activity. These findings indicate that specific milk peptides may be able to reach the bloodstream and exert bioactivity.