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.

Advancements in food science for Phenylketonuria (PKU) management: a comprehensive review.

This review systematically explores the pivotal role of food science and technology as a support for Phenylketonuria (PKU) dietary management. It delves into the genetic and metabolic underpinnings of PKU, highlighting the crucial need for stringent dietary regulation to manage phenylalanine levels and mitigate neurological complications. Through bibliometric analysis and current product evaluations, it identifies trends in PKU food research, emphasizing recent innovations in food formulations such as glycomacropeptide (GMP) supplements and higher appealing low-phenylalanine food products. Furthermore, it accentuates the sensory and consumer aspects of PKU dietary solutions, underscoring the importance of palatability for adherence. Notably, the review introduces 3D food printing as an emerging technology for creating personalized, nutrient-optimized, and sensory-appealing foods for PKU patients, offering a new horizon in dietary management. This comprehensive assessment underscores the dynamic interplay between nutritional science, food technology, and sensory evaluation in improving the quality of life for individuals with PKU.

Glycomacropeptide: A comprehensive understanding of its major biological characteristics and purification methodologies.

Glycomacropeptide (GMP) is a bioactive peptide derived from whey protein, consisting of 64 amino acids. It is a phenylalanine-free peptide, making it a beneficial dietary option for individuals dealing with phenylketonuria (PKU). PKU is an inherited metabolic disorder characterized by high levels of phenylalanine in the bloodstream, resulting from a deficiency of phenylalanine dehydrogenase in affected individuals. Consequently, patients with PKU require lifelong adherence to a low-phenylalanine diet, wherein a significant portion of their protein intake is typically sourced from a phenylalanine-free amino acid formula. GMP has several nutritional values, numerous bioactivity properties, and therapeutic effects in various inflammatory disorders. Despite all these features, the purification of GMP is an imperative requirement; however, there are no unique methods for achieving this goal. Traditionally, several methods have been used for GMP purification, such as thermal or acid treatment, alcoholic precipitation, ultrafiltration (UF), gel filtration, and membrane separation techniques. However, these methods have poor specificity, and the presence of large amounts of impurities can interfere with the analysis of GMP. More efficient and highly specific GMP purification methods need to be developed. In this review, we have highlighted and summarized the current research progress on the major biological features and purification methodologies associated with GMP, as well as providing an extensive overview of the recent developments in using charged UF membranes for GMP purification and the influential factors.

Glycomacropeptide-Based Protein Substitutes for Children with Phenylketonuria in Italy: A Nutritional Comparison.

Advancements in food science technology have allowed the development of new products for the therapeutic management of inherited metabolic diseases such as phenylketonuria (PKU). Glycomacropeptide (GMP), a peptide derived from casein, is naturally low in phenylalanine (Phe) and, thus, adequate for protein substitutes (PSs) for the management of PKU in children. This review aims primarily to analyse the differences in the nutritional composition of GMP-based protein substitutes in different formulations (ready to drink, powdered, and bars), and secondarily to assess the quality of these products, comparing their nutritional composition with that of standard amino acid (L-AA) mixtures. Thirty-five GMP-based PSs produced by six different companies were included in this review: twenty-one powdered PSs, eight ready to drink, and six bars. The analysis revealed great heterogeneity not only among the different formulations (powdered, ready to drink, and bars) but also within the same group, in terms of energy content and nutritional composition. GMP-based PSs were shown to have higher contents of sugars and saturated fatty acids compared to L-AA PSs, especially in ready-to-drink formulations and bars. The latter also provided the highest amounts of energy among the GMP-based products. This finding may be related to a higher risk of developing overweight and obesity. The greater palatability of these GMP-based PSs, combined with improved nutritional quality, could not only improve adherence to diet therapy but also reduce the incidence of obesity-related comorbidities in PKU.

Milk Bioactive Compounds and Gut Microbiota Modulation: The Role of Whey Proteins and Milk Oligosaccharides.

A strong correlation between the occurrence of various pathological conditions and intestinal dysbiosis is supported by a range of strong evidence. Vice versa, many pathologies have been shown, in turn, to be responsible for alterations in the gut microbiota, a condition that can worsen illness outcomes and response to therapies. For these reasons, great efforts have been made, and studies are still ongoing, to elucidate the mechanisms underlying gut microbiota alterations and to search for pharmacologic or other strategies that can effectively restore the gut microbiota. In this narrative review, we examined the most significant literature on the role of some milk bioactive compounds, such as milk oligosaccharides and whey proteins, in modulating the composition of the gut microbiota and the underlying mechanisms of action, with the aim of investigating the impact of the microbiota changes mediated by these milk bioactive molecules on human health, and their potential use as therapeutics to treat or adjuvate the treatment of gut dysbiosis and associated pathologies.

Glycomacropeptide (GMP) rescued the oxidative and inflammatory activity of free L-AAs in human Caco-2 cells: New insights that support GMP as a valid and health-promoting product for the dietary management of phenylketonuria (PKU) patients.

Phenylketonuria represents the most prevalent inborn error of amino acid metabolism. In early diagnosed patients adequate and continued dietary treatment results in a good neurologic outcome. However, due to the natural protein and phenylalanine-restricted diet, oxidative stress represents a concern in phenylketonuric patients. Clear evidences suggest that the pathophysiology of PKU is also dependent by mitochondrial impairment and oxidative stress. In this context due to the tight connection between oxidative and inflammatory stress and noncommunicable diseases (NCDs) development, it is reasonable to hypothesize that PKU patients may present a higher risk to develop NCDs during their life. Currently available protein substitutes on the market include free amino acids (L-AAs), prolonged-release protein substitute and formula containing glycomacropeptide (GMP). Our results suggest that free L-AAs significanlty worsens the intestinal hydrogen peroxide (H2O2) and lipopolysaccharides (LPS)-induced oxidative and inflammatory status in Caco-2 cells, which are significantly restored towards physiological condition by GMP alone and when present in a 1:1 mixture with free L-AAs, providing new preclinical piece of information which can shed a shadow on the mechanism of action of these products on PKU patients and their future management.

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.

Evaluation of a New Glycomacropeptide-Based Protein Substitute in Powdered and Liquid Format in Patients with PKU.

(1) Background: Good adherence to a Phe-restricted diet supplemented with an adequate amount of a protein substitute (PS) is important for good clinical outcomes in PKU. Glycomacropeptide (cGMP)-PSs are innovative, palatable alternatives to amino acid-based PSs (AA-PS). This study aimed to evaluate a new cGMP-PS in liquid and powder formats in PKU. (2) Methods: Children and adults with PKU recruited from eight centres were prescribed at least one serving/day of cGMP-PS for 7-28 days. Adherence, acceptability, and gastrointestinal tolerance were recorded at baseline and the end of the intervention. The blood Phe levels reported as part of routine care during the intervention were recorded. (3) Results: In total, 23 patients (powder group, n = 13; liquid group, n = 10) completed the study. The majority assessed the products to be palatable (77% of powder group; 100% of liquid group) and well tolerated; the adherence to the product prescription was good. A total of 14 patients provided blood Phe results during the intervention, which were within the target therapeutic range for most patients (n = 11) at baseline and during the intervention. (4) Conclusions: These new cGMP-PSs were well accepted and tolerated, and their use did not adversely affect blood Phe control.

Evaluation of a New 'Mix-In' Style Glycomacropeptide-Based Protein Substitute for Food and Drinks in Patients with Phenylketonuria and Tyrosinemia.

(1) Background: Poor palatability, large volume, and lack of variety of some liquid and powdered protein substitutes (PSs) for patients with phenylketonuria (PKU) and tyrosinemia (TYR) can result in poor adherence. This study aimed to evaluate a new unflavoured, powdered GMP-based PS designed to be mixed into drinks, foods, or with other PSs, in patients with PKU and TYR. (2) Methods: Paediatric and adult community-based patients were recruited from eight metabolic centres and prescribed ≥1 sachet/day (10 g protein equivalent (PE)) of the Mix-In-style PS over 28 days. Adherence, palatability, GI tolerance, and metabolic control were recorded at baseline and follow-up. Patients who completed at least 7 days of intervention were included in the final analysis. (3) Results: Eighteen patients (3-45 years, nine males) with PKU (n = 12) and TYR (n = 6) used the Mix-In-style PS for ≥7 days (mean 26.4 days (SD 4.6), range 11-28 days) alongside their previous PS, with a mean intake of 16.7 g (SD 7.7) PE/day. Adherence was 86% (SD 25), and GI tolerance was stable, with n = 14 experiencing no/no new symptoms and n = 3 showing improved symptoms compared to baseline. Overall palatability was rated satisfactory by 78% of patients, who successfully used the Mix-In-style PS in various foods and drinks, including smoothies, squash, and milk alternatives, as a top-up to meet their protein needs. There was no concern regarding safety/metabolic control during the intervention. (4) Conclusions: The 'Mix-In'-style PS was well adhered to, accepted, and tolerated. Collectively, these data show that providing a flexible, convenient, and novel format of PS can help with adherence and meet patients' protein needs.

Glycomacropeptide Protects against Inflammation and Oxidative Stress, and Promotes Wound Healing in an Atopic Dermatitis Model of Human Keratinocytes.

Keratinocytes are actively implicated in the physiopathology of atopic dermatitis (AD), a skin allergy condition widely distributed worldwide. Glycomacropeptide (GMP) is a milk-derived bioactive peptide generated during cheese making processes or gastric digestion. It has antiallergic and skin barrier restoring properties when it is orally administered in experimental AD. This study aimed to evaluate the effect of GMP on the inflammatory, oxidative, proliferative, and migratory responses of HaCaT keratinocytes in an in vitro AD model. GMP protected keratinocytes from death and apoptosis in a dose dependent manner. GMP at 6.3 and 25 mg/mL, respectively, reduced nitric oxide by 50% and 83.2% as well as lipid hydroperoxides by 27.5% and 45.18% in activated HaCaT cells. The gene expression of TSLP, IL33, TARC, MDC, and NGF was significantly downregulated comparably to control by GMP treatment in activated keratinocytes, while that of cGRP was enhanced. Finally, in an AD microenvironment, GMP at 25 mg/mL stimulated HaCaT cell proliferation, while concentrations of 0.01 and 0.1 mg/mL promoted the HaCaT cell migration. Therefore, we demonstrate that GMP has anti-inflammatory and antioxidative properties and stimulates wound closure on an AD model of keratinocytes, which could support its reported bioactivity in vivo.

Protective Effect of Glycomacropeptide on the Inflammatory Response of U937 Macrophages.

Macrophages play crucial roles in inflammation and oxidative stress associated with noncommunicable diseases, such as cardiovascular diseases, diabetes, and cancer. Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein that contains abundant sialic acid and has shown anti-inflammatory, antioxidative, anti-obesity, and anti-diabetic properties when is orally administered. The aim of this study was to evaluate the effect of GMP on the regulation of the inflammatory response in human macrophages and the participation of sialic acid in this activity. GMP pretreatment decreased by 35%, 35%, and 49% the production of nitrites, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α, respectively, in activated human macrophages U937. The same effect was obtained when cells were pretreated with asialo GMP, and no change on the gene expression of the lectins associated with the recognition of sialic acids, SIGLEC5, 7, and 9, was induced by GMP on macrophages, which suggests that sialic acid might not be involved in this immunoregulatory effect. Interestingly, GMP increased 8.9- and 3.5-fold the gene expression of the canonical anti-inflammatory protein SOCS3 and the antioxidant enzyme HMOX1, respectively, in U937 cells. Thus, GMP exerts anti-inflammatory and antioxidative activities on activated macrophages in a sialic acid-independent manner, which might be related to its in vivo reported bioactivity.

Functionality of glycomacropeptide glycated with lactose and maltodextrin.

The Maillard reaction (MR), under proper environmental conditions, has been used to improve protein functionality. In the present work, 2 high temperatures (50-80°C) and water activity (Aw; 0.45-0.67) were used to promote exogenous glycosylation of glycomacropeptide (GMP) while minimizing processing times (0, 8, 24, 48, and 96 h at 50°C; 0, 2, 4, 8, and 24 h at 80°C). Maltodextrin, a polysaccharide commonly used in the food industry as a functional ingredient, was used as a reducing sugar, and compared with lactose, a native milk sugar. The progression of MR was evaluated by tracking changes in molecular weight using SDS-PAGE, the formation of Amadori compounds, and browning. Aqueous glycosylated GMP solutions (5 to 20% wt/vol) were tested for solubility, rheological properties, and foam formation. As expected, MR progression was faster with Aw = 0.67 and 80°C. Glycosylated GMP powders showed no change in their solubility after MR. However, the apparent viscosity (γ˙=30s-1) of the 20% wt/vol suspensions exhibited a slight increase when GMP was glycosylated with maltodextrin for 24 h at 80°C, and a 2-log increase when GMP was glycosylated with lactose, with a high browning development in both cases. The foam expansion index of the resuspended glycosylated powders was increased by between 25 and 66% compared with the nonglycosylated powders. Better foam stability (approximately 2 h) and no browning development were observed for GMP glycosylated with maltodextrin for 2 h at Aw = 0.67 and 80°C. The results show that GMP has undergone further glycosylation by means of controlled MR, which improves viscosity and foaming index without negatively affecting solubility. These preliminary studies provide a basis for the future creation of a new ingredient with GMP and reducing sugars.

Quality of life aspects of a low protein diet using GMP in patients with phenylketonuria.

OBJECTIVE: To assess some quality of life (QOL) aspects of a low protein diet, using glycomacropeptide (GMP) as a protein substitute in patients with phenylketonuria (PKU). METHODS: This was a multicentre, prospective observational cohort, study. Metabolic control, nutritional parameters, and dietary adherence were assessed in patients with PKU before (T0), and six months after (T6) starting a low protein diet using GMP. Selected items from the PKU-QOL questionnaire were used to assess patients' acceptance of their modified diet. RESULTS: 18 patients from three Italian Centres, completed the study. With the exception of LDL-cholesterol and vitamin 25OH-D concentrations, there were no differences between T0 and T6 in metabolic or nutritional parameters. Data suggested that patients have a good acceptance of protein substitutes containing GMP, probably because of their improved palatability. CONCLUSIONS: According to our patients' responses to items related to dietary regimen, GMP based protein substitutes do not appear to significantly affect QOL.

Comparative Mass Spectrometry Analysis and Immunomodulatory Effects of Casein Glycomacropeptide O-Glycans in Bovine and Caprine Whey Powder.

Casein glycomacropeptide carries various O-glycan modifications, which, together with variations in the amino acid composition of the glycopeptide, may result in different biological activities. In this study, O-glycans of casein glycomacropeptide from bovine and caprine whey powder were qualitatively and quantitatively analyzed by LC-UV-ESI-MS/MS, and their immune activities and regulatory mechanisms were compared. O-Glycans' total content was 1.54 times higher in bovine than in caprine glycomacropeptide. The glycoform H1N1S2 (H: hexose; N: N-acetylgalactosamine; and S: N-acetylneuraminic acid) accounted for nearly 50% of total glycomacropeptide O-glycans in bovine milk but less than 20% in caprine milk. Bovine glycomacropeptide glycosylation promoted the immune activity of RAW264.7 cells, which may be linked to a higher content of disialylated O-glycans. Glycomacropeptide from both milk sources significantly upregulated the mRNA expression of IL-1α, TNF-α, and IL-10 in RAW264.7 cells and activated the MAPK immunomodulatory signaling pathway. This study demonstrates the possible use of casein glycomacropeptide as an immunomodulatory agent.

Adsorption of flexible proteins in the 'wrong side' of the isoelectric point: Casein macropeptide as a model system.

We analyze the conditions of the adsorption of a flexible peptide onto a charged substrate in the 'wrong side' of the isoelectric point (WSIP), i.e. when surface and peptide charges have the same sign. As a model system, we focus on the casein macropeptide (CMP), both in the aglycosylated (aCMP) and fully glycosydated (gCMP) forms. We model the substrate as a uniformly charged plane while CMP is treated as a bead-and-spring model including electrostatic interactions, excluded volume effects and acid/base equilibria. Adsorption coverage, aminoacid charges and concentration profiles are computed by means of Monte Carlo simulations at fixed pH and salt concentration. We conclude that for different reasons the CMP can be adsorbed to both positively and negatively charged surfaces in the WSIP. For negatively charged surfaces, WSIP adsorption is due to the patchy distribution of charges: the peptide is attached to the surface by the positively charged end of the chain, while the repulsion of the surface for the negatively charged tail is screened by the small ions of the added salt. This effect increases with salt concentration. Conversely, a positively charged substrate induces strong charge regulation of the peptide: the acidic groups are deprotonated, and the peptide becomes negatively charged. This effect is stronger at low salt concentrations and it is more intense for gCMP than for aCMP, due to the presence of the additional sialic groups in gCMP.

Glycomacropeptide Safety and Its Effect on Gut Microbiota in Patients with Phenylketonuria: A Pilot Study.

Glycomacropeptide (GMP) represents a good alternative protein source in Phenylketonuria (PKU). In a mouse model, it has been suggested to exert a prebiotic role on beneficial gut bacteria. In this study, we performed the 16S rRNA sequencing to evaluate the effect of 6 months of GMP supplementation on the gut microbiota of nine PKU patients, comparing their bacterial composition and clinical parameters before and after the intervention. GMP seems to be safe from both the microbiological and the clinical point of view. Indeed, we did not observe dramatic changes in the gut microbiota but a specific prebiotic effect on the butyrate-producer Agathobacter spp. and, to a lesser extent, of Subdoligranulum. Clinically, GMP intake did not show a significant impact on both metabolic control, as phenylalanine values were kept below the age target and nutritional parameters. On the other hand, an amelioration of calcium phosphate homeostasis was observed, with an increase in plasmatic vitamin D and a decrease in alkaline phosphatase. Our results suggest GMP as a safe alternative in the PKU diet and its possible prebiotic role on specific taxa without causing dramatic changes in the commensal microbiota.

Effects of a Formula with scGOS/lcFOS (9:1) and Glycomacropeptide (GMP) Supplementation on the Gut Microbiota of Very Preterm Infants.

Microbial colonization of very preterm (VPT) infants is detrimentally affected by the complex interplay of physiological, dietary, medical, and environmental factors. The aim of this study was to evaluate the effects of an infant formula containing the specific prebiotic mixture of scGOS/lcFOS (9:1) and glycomacropeptide (GMP) on the composition and function of VPT infants' gut microbiota. Metagenomic analysis was performed on the gut microbiota of VPT infants sampled at four time points: 24 h before the trial and 7, 14, and 28 days after the trial. Functional profiling was aggregated into gut and brain modules (GBMs) and gut metabolic modules (GMMs) based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Enterococcus faecium, Escherichia coli, Klebsiella aerogenes, and Klebsiella pneumoniae were dominant species in both the test group and the control group. After the 4-week intervention, the abundance of Bifidobacterium in the test group was significantly increased. We found two GBMs (quinolinic acid synthesis and kynurenine degradation) and four GMMs (glutamine degradation, glyoxylate bypass, dissimilatory nitrate reduction, and preparatory phase of glycolysis) were significantly enriched in the test group, respectively. The results of this study suggested that formula enriched with scGOS/lcFOS (9:1) and GPM is beneficial to the intestinal microecology of VPT infants.

Use of batch anion exchange technique for separation of κ-casein glycomacropeptide from bovine whey fraction.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide, which is considered as a health promoting compound found in cheese whey. The study described in this research communication was undertaken to determine whether GMP with undetectable level of contaminating protein or phenylalanine can be isolated from bovine whey fraction using batch anion exchange technique with chitin as an adsorbent. A soluble whey fraction (SWF) prepared from 1 g whey protein isolate (WPI) was mixed with a slurry of 1 g chitin, and the mixture was incubated at pH 3.0. After incubation, the mixture was filtered, and the residue obtained (containing chitin-GMP complex) was washed with water and eluted stepwise with 0.5 M NaCl and 2.0 M NaCl. Most of GMP (corresponding to 75.8% of total sialic acid recovered) was eluted with 0.5 M NaCl. The recovered GMP accounted for 5.4% dry weight of WPI (or 18.9% dry weight of SWF). Amino acid analysis showed that there was no detectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine in the GMP fraction. It was concluded that the batch anion exchange method with chitin developed in this study can be used for the isolation of high purity GMP from bovine SWF.

Glycomacropeptide in PKU-Does It Live Up to Its Potential?

The use of casein glycomacropeptide (CGMP) as a protein substitute in phenylketonuria (PKU) has grown in popularity. CGMP is derived from κ casein and is a sialic-rich glycophosphopeptide, formed by the action of chymosin during the production of cheese. It comprises 20-25% of total protein in whey products and has key biomodulatory properties. In PKU, the amino acid sequence of CGMP has been adapted by adding the amino acids histidine, leucine, methionine, tyrosine and tryptophan naturally low in CGMP. The use of CGMP compared to mono amino acids (L-AAs) as a protein substitute in the treatment of PKU promises several potential clinical benefits, although any advantage is supported only by evidence from non-PKU conditions or PKU animal models. This review examines if there is sufficient evidence to support the bioactive properties of CGMP leading to physiological benefits when compared to L-AAs in PKU, with a focus on blood phenylalanine control and stability, body composition, growth, bone density, breath odour and palatability.