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.

Evaluation of a casein glycomacropeptide-based protein substitute, in the dietary management of NTBC-induced tyrosinaemia in patients with alkaptonuria: A prospective open-label study.

BACKGROUND: 2-(2-Nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) treatment of alkaptonuria (AKU) leads to increased blood tyrosine levels, causing skin issues and potentially sight-threatening corneal keratopathy. Adherence to dietary management of NTBC-induced tyrosinemia, a low-protein diet with or without protein substitutes, can be difficult for patients. This 28-day interventional study evaluated a low tyrosine casein glycomacropeptide (cGMP) protein substitute (TYR sphere)®, a 20 g protein equivalent, cGMP-based protein substitute, in terms of adherence, palatability, usability, comparison to amino acid (AA)-based protein substitutes, gastrointestinal tolerance and metabolic control in adults with NTBC-induced tyrosinaemia. METHODS: Four adults (mean 61.1 years, range 53.3-69.3 years) with AKU and NTBC-induced tyrosinaemia were recruited from the United Kingdom National Alkaptonuria Centre (NAC). The cGMP protein substitute was prescribed based on individual nutritional requirements, replacing ≥1 AA-based protein substitute. Participants recorded product-related data in study diaries, using five-point Likert scales and daily and weekly logs. To determine metabolic control, prestudy blood tyrosine levels were compared to weekly blood spot tests during the study. RESULTS: Median cGMP protein substitute adherence was 98%. Most participants rated palatability and usability positively, and preferred cGMP protein substitute to AA-based products. There were no notable gastrointestinal changes, and metabolic control was maintained. CONCLUSIONS: cGMP protein substitute is a palatable and well-tolerated option in the dietary management of AKU patients with NTBC-induced tyrosinaemia.

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.

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.

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.

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.

Dietary supplementation with casein glycomacropeptide, leucine and tryptophan reduces plasma amino acid levels in men.

BACKGROUND: The treatment of mania in bipolar disorders needs to be more efficient, as the manic condition creates severe problems for the patient when it comes to work, finances, relationships and health. This proof-of-concept study examines to what extent casein glycomacropeptide (CGMP) may reduce the precursors of dopamine, phenylalanine and tyrosine, in plasma, and therefore be a potential new intervention to treat acute manic episodes. METHOD: The study was designed as a double-blind randomised dose-response study of CGMP (with added leucine and tryptophan) in 15 healthy men, receiving 3 different doses of CGMP with an interval of at least 14 days. RESULTS: Administration of CGMP produced a dose-dependent depletion of plasma aromatic amino acids. The total area under the curve of plasma ratios of phenylalanine-tyrosine compared to the level of leucine-isoleucine-valine--tryptophan was CGMP (20 g): 3.648 [SE:0.3281]; CGMP (40 g): 2.368 [SE:0.1858]; and CGMP (60 g)1.887 [SE:0.2591]. A comparison of the groups showed a dose-dependent statistical difference, with a one-way ANOVA summary (Dunnett) F = 11.87, p = 0.0003, CGMP 20 g versus CGMP 40 g, p = 0.0042, CGMP 20 g versus CGMP 60 g, p = 0.0002. No significant side effects were observed. CONCLUSIONS: This study demonstrate CGMP is a well-tolerated and effective mixture, and that 60 g of CGMP produced the highest depletion of plasma aromatic amino acids (phenylalanine and tyrosine). The effect seems to be highest after 3-4 h. We therefore conclude that this dose should be the one considered for future studies involving CGMP in humans.

Casein glycomacropeptide is well tolerated in healthy adults and changes neither high-sensitive C-reactive protein, gut microbiota nor faecal butyrate: a restricted randomised trial.

Casein glycomacropeptide (CGMP) is a bioactive milk-derived peptide with potential anti-inflammatory effects. Animal studies suggest that CGMP may work by altering gut microbiota composition and enhancing butyrate production. Its effects on intestinal homoeostasis, microbiota and metabolites in humans are unknown. The aim of the present study was to assess both the intestinal and systemic immunomodulatory effects of orally ingested CGMP. We hypothesised that daily oral CGMP intake would reduce high-sensitive C-reactive protein (hsCRP) in healthy adults. In a single-centre limited but randomised, double-blinded, reference-controlled study, we compared the effects of a 4-week intervention of either 25 g of oral powder-based chocolate-flavoured CGMP or a reference drink. We included twenty-four healthy adults who all completed the study. CGMP had no systemic or intestinal immunomodulatory effects compared with a reference drink, with regard to either hsCRP or faecal calprotectin level, faecal microbiota composition or faecal SCFA content. CGMP ingestion did not affect satiety or body weight, and it caused no severe adverse events. The palatability of CGMP was acceptable, and adherence was high. CGMP did not induce or change gastrointestinal symptoms. In conclusion, we found no immunomodulatory effects of CGMP in healthy adults. In a minor group of healthy adults, oral ingestion of 25 g of CGMP during 4 weeks was safe, well tolerated, had acceptable palatability and was without any effects on body weight.

Large-scale preparation and glycan characterization of sialylglycopeptide from bovine milk glycomacropeptide and its bifidogenic properties.

Bovine glycomacropeptide (GMP) is a 7,000-Da glycopolypeptide released from κ-casein during cheese making. The O-glycan chains linked to GMP have many biological activities, but their utilization for nutraceutical products is limited due to their low content. To concentrate the functional glycan chains of GMP, we prepared sialylglycopeptide concentrate (SGC) from GMP-containing whey protein concentrate via proteolytic digestion of peptide chains and concentration of sialylglycopeptide by ultrafiltration using membranes with a molecular weight cut-off of 1,000 Da. The abundant saccharides detected in the prepared SGC were N-acetylneuraminic acid (Neu5Ac: 32.3% wt/wt), N-acetylgalactosamine (11.3%), and galactose (10.2%), which constitute O-glycans attached to GMP. The Neu5Ac content in SGC was found concentrated at approximately 4.8-fold of its content in GMP-containing whey protein concentrate (6.8%). Structural analysis of O-glycopeptides by liquid chromatography tandem mass spectrometry identified 88 O-glycopeptides. Moreover, O-acetylated or O-diacetylated Neu5Ac was detected in addition to the previously characterized O-glycans of GMP. Quantitative analysis of O-glycan in SGC by fluorescence labeling of chemically released O-glycan revealed that a disialylated tetrasaccharide was the most abundant glycan (76.6% of the total O-glycan). We further examined bifidogenic properties of SGC in vitro, which revealed that SGC served as a more potent carbon source than GMP and contributes to the growth-promoting effects on certain species of bifidobacteria. Overall, our study findings indicate that SGC contains abundant O-glycans and has a bifidogenic activity. Moreover, the protocol for the preparation of SGC described herein is relatively simple, providing a high yield of glycan, and can be used for large-scale preparation.

Unveiling the Metabolic Effects of Glycomacropeptide.

For many years, the main nitrogen source for patients with phenylketonuria (PKU) was phenylalanine-free amino acid supplements. Recently, casein glycomacropeptide (GMP) supplements have been prescribed due to its functional and sensorial properties. Nevertheless, many doubts still persist about the metabolic effects of GMP compared to free amino acids (fAA) and intact proteins such as casein (CAS). We endeavour to compare, in rats, the metabolic effects of different nitrogen sources. Twenty-four male Wistar rats were fed equal energy density diets plus CAS (control, n = 8), fAA (n = 8) or GMP (n = 8) for 8 weeks. Food, liquid intake and body weight were measured weekly. Blood biochemical parameters and markers of glycidic metabolism were assessed. Glucagon-like peptide-1 (GLP-1) was analysed by ELISA and immunohistochemistry. Food intake was higher in rats fed CAS compared to fAA or GMP throughout the treatment period. Fluid intake was similar between rats fed fAA and GMP. Body weight was systematically lower in rats fed fAA and GMP compared to those fed CAS, and still, from week 4 onwards, there were differences between fAA and GMP. None of the treatments appeared to induce consistent changes in glycaemia, while insulin levels were significantly higher in GMP. Likewise, the production of GLP-1 was higher in rats fed GMP when compared to fAA. Decreased urea, total protein and triglycerides were seen both in fAA and GMP related to CAS. GMP also reduced albumin and triglycerides in comparison to CAS and fAA, respectively. The chronic consumption of the diets triggers different metabolic responses which may provide clues to further study potential underlying mechanisms.

The phenylketonuria patient: A recent dietetic therapeutic approach.

Phenylalanine hydroxylase (PAH) deficiency, commonly named phenylketonuria (PKU) is a disorder of phenylalanine (Phe) metabolism inherited with an autosomal recessive trait. It is characterized by high blood and cerebral Phe levels, resulting in intellectual disabilities, seizures, etc. Early diagnosis and treatment of the patients prevent major neuro-cognitive deficits. Treatment consists of a lifelong restriction of Phe intake, combined with the supplementation of special medical foods, such as Amino Acid medical food (AA-mf), enriched in tyrosine (Tyr) and other amino acids and nutrients to avoid nutritional deficits. Developmental and neurocognitive outcomes for patients, however, remain suboptimal, especially when adherence to the demanding diet is poor. Additions to treatment include new, more palatable foods, based on Glycomacropeptide that contains limited amounts of Phe, the administration of large neutral amino acids to prevent phenylalanine entry into the brain and tetrahydrobiopterin cofactor capable of increasing residual PAH activity. Moreover, further efforts are underway to develop an oral therapy containing phenylalanine ammonia-lyase. Nutritional support of PKU future mothers (maternal PKU) is also discussed. This review aims to summarize the current literature on new PKU treatment strategies.

Isolation of κ-casein glycomacropeptide from bovine whey fraction using food grade anion exchange resin and chitin as an adsorbent.

Bovine κ-casein glycomacropeptide (GMP) found in cheese whey is a sialylated phosphorylated peptide which is thought to be a potential ingredient for functional food as well as dietetic food. This study was undertaken to determine whether high purity GMP can be isolated from soluble whey fraction (SWF) using column chromatography on food grade anion exchange resin and chitin as an adsorbent. Samples of commercially available anion exchange resin (resin A, resin B and resin C) and those of chitin (chitin A, chitin B and chitin C) were examined in this experiment. The GMP fraction obtained from each column was analyzed for amino acid composition which reflects the purity of the peptide. Major findings for commercial anion exchange resin were that: (1) the proportion of GMP monitored as sialic acid in total recovered sialic acid was similar among the three samples of resin accounting for average 78% of recovered sialic acid; (2) the GMP fraction from resin A or resin B contained undetectable level of contaminating amino acids including phenylalanine, histidine, arginine and tyrosine; (3) the GMP fraction from resin C contained small amounts (<1 mol%) of contaminating amino acids, arginine, phenylalanine and tyrosine; and (4) the GMP binding capacity expressed as mg/100 mg dry weight of resin was more than 2.5 times higher in resin C (average 22.9) than in resin A or resin B with no difference between resin A and resin B averaging 8.7. Results obtained for chitin A, chitin B and chitin C were in general similar to those found with resin A and resin B. Since chitin has a remarkable GMP binding capacity averaging 8.6 mg/100 mg dry weight of chitin, it may be a useful adsorbent for whey fractionation. Further research is needed to develop an efficient inexpensive method to purify GMP.

Production of sialic acid rich glycopeptide from bovine κ-casein glycomacropeptide by hydrolyzing with papain.

Bovine κ-casein glycomacropeptide (GMP) is a sialic acid containing glycopeptide having many biological activities. The study described in this research communication was undertaken to determine whether sialic acid rich glycopeptide can be produced from GMP by proteinase treatment. A sample of GMP was hydrolyzed with papain, and the obtained hydrolysate was chromatographed on a column of diethylaminoethyl-Sephacel to obtain a glycopeptide fraction (GPF). This product accounted for average 48.1% dry weight of GMP or 81.1% total recovered sialic acid from GMP. The content of sialic acid (expressed as % dry weight) was 1.7 times higher in GPF (22.6) than in unhydrolyzed GMP (13.4). Major differences in amino acid composition between GPF and GMP were found in the contents (mol%) of: lysine (<1 and 4.5, respectively), serine (20.3 and 10.3, approximately twice higher in GPF), asparagine/aspartic acid and isoleucine. The contents of the last two amino acids were approximately twice lower in GPF. On gel filtration chromatography with Sephacryl S-100, GMP was eluted as a single peak with elution volume similar to that of dimeric ß-lactoglobulin (36.6 kDa) whereas GPF was eluted in two peaks both with elution volumes greater than that of α-lactalbumin (14.2 kDa). These peak fractions containing high (fraction I) and low (fraction II) molecular size glycopeptides gave different sialic acid to peptide ratio, which was 1.7 times higher in fraction I than in fraction II. Results of size exclusion HPLC on Superdex-75 were consistent with those of gel filtlation chromatography. On cellulose acetate electrophoresis, the mobility of GPF relative to that of GMP as 1.0 was found to average 1.2, suggesting a higher negative charge density in GPF than in GMP. It was concluded that papain digestion of GMP is an efficient method to produce glycopeptide with high sialic acid content.

Glycomacropeptide Ameliorates Indomethacin-Induced Enteropathy in Rats by Modifying Intestinal Inflammation and Oxidative Stress.

Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy is considered a serious and increasing clinical problem without available treatment. Glycomacropeptide (GMP) is a 64-amino acid peptide derived from milk κ-casein with numerous biological activities. The aim of this study was to investigate the protective effect of GMP on NSAID enteropathy in rats. Enteropathy was induced by seven days oral indomethacin administration. Rats were orally GMP treated from seven days previous and during the establishment of the enteropathy model. Changes in metabolism, hematological and biochemical blood alterations, intestinal inflammation and oxidative damage were analyzed. Integrity barrier markers, macroscopic intestinal damage and survival rate were also evaluated. GMP treatment prevented anorexia and weight loss in animals. Furthermore, prophylaxis with GMP ameliorated the decline in hemoglobin, hematocrit, albumin and total protein levels. The treatment had no therapeutic efficacy on the decrease of occludin and mucin (MUC)-2 expression in intestinal tissue. However, GMP markedly decreased neutrophil infiltration, and CXCL1, interleukin-1ß and inducible nitric oxide synthase expression. Nitric oxide production and lipid hydroperoxide level in the small intestine were also diminished. These beneficial effects were mirrored by preventing ulcer development and increasing animal survival. These results suggest that GMP may protect against NSAID enteropathy through anti-inflammatory and antioxidant properties.

Postprandial metabolic responses to ingestion of bovine glycomacropeptide compared to a whey protein isolate in prediabetic volunteers.

PURPOSE: Whey protein was shown to reduce blood glucose responses in humans and various other positive effects have been attributed to this protein. In contrast, studies using glycomacropeptide (GMP) as part of the whey fraction of bovine milk are rare. We, therefore, studied the postprandial responses to GMP administration in humans with impaired glucose tolerance compared to the effects of pure whey protein in a random design. METHODS: Fifteen prediabetic volunteers received on different occasions one of three test drinks containing 50 g of maltodextrin19 (MD19) alone or in combination with either 50 g GMP or 50 g whey protein isolate (WPI). Blood was collected over 4 h with analysis of blood glucose and hormones, gastric emptying rate as well as plasma amino- and fatty acids, ß-hydroxybutyrate and acylcarnitines. RESULTS: The WPI drink reduced the AUC of venous blood glucose compared to the MD19 drink in the prediabetic group by 11% (p = 0.0018) whereas GMP reduced the AUC by 18% (p < 0.0001), significantly different to the WPI drink (p = 0.0384). The reduction in blood glucose after the GMP drink was accompanied by a significantly lower AUC of insulin (- 34%) than for the WPI drink. Levels of C-peptide and of glucose insulinotropic polypeptide (GIP) were highly increased after the WPI drink over the MD19 control drink but remained in essence unaffected by the GMP. CONCLUSION: GMP reduced the glycemic response more potently than whey protein, whereas insulin output was less affected making GMP an interesting protein to control postprandial glucose responses.

Optimising amino acid absorption: essential to improve nitrogen balance and metabolic control in phenylketonuria.

It has been nearly 70 years since the discovery that strict adherence to a diet low in phenylalanine prevents severe neurological sequelae in patients with phenylalanine hydroxylase deficiency (phenylketonuria; PKU). Today, dietary treatment with restricted phenylalanine intake supplemented with non-phenylalanine amino acids to support growth and maintain a healthy body composition remains the mainstay of therapy. However, a better understanding is needed of the factors that influence N balance in the context of amino acid supplementation. The aim of the present paper is to summarise considerations for improving N balance in patients with PKU, with a focus on gaining greater understanding of amino acid absorption, disposition and utilisation. In addition, the impact of phenylalanine-free amino acids on 24 h blood phenylalanine/tyrosine circadian rhythm is evaluated. We compare the effects of administering intact protein v. free amino acid on protein metabolism and discuss the possibility of improving outcomes by administering amino acid mixtures so that their absorption profile mimics that of intact protein. Protein substitutes with the ability to delay absorption of phenylalanine and tyrosine, mimicking physiological absorption kinetics, are expected to improve the rate of assimilation into protein and minimise fluctuations in quantitative plasma amino acid levels. They may also help maintain normal glycaemia and satiety sensation. This is likely to play an important role in improving the management of patients with PKU.

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

Repeated dose 90-day oral toxicity test of G-7% NANA in rats: An application of new criterion for toxicity determination to test article-induced changes.

G-7% NANA is N-acetylneuraminic acid(NANA) containing 7% sialic acid isolated from glycomacropeptide (GMP), a compound of milk. Since NANA is likely to have immunotoxicity, the need to ensure safety for long-term administration has been raised. In this study, a 90-day repeated oral dose toxicity test was performed in rats using G-7% NANA in the dosages of 0, 1250, 2500 and 5000 mg/kg/day.A toxicity determination criterion based on the significant change caused by the administration of the substancewas developed for estimating NOEL, NOAEL and LOAELapplied to this study. When analyzing the immunological markers, no significant changes were observed, even if other significant changes were observed in the high dose group. In accordance with the toxicity determination criterion developed, the NOEL in male and female has been determined as 2500 mg/kg/day, and the NOAEL in females has been determined as 5000 mg/kg/day. The toxicity determination criterion, applied for the first time in the repeated dose toxicity tests, could provide a basis for distinguishing NOEL and NOAEL more clearly; nevertheless, the toxicity determination criterion needs to be supplemented by adding differentiating adverse effects and non-adverse effects based on more experiences of the repeated dose toxicity tests.

Bovine glycomacropeptide promotes the growth of Bifidobacterium longum ssp. infantis and modulates its gene expression.

Bovine milk glycomacropeptide (GMP) is derived from κ-casein, with exclusively o-linked glycosylation. Glycomacropeptide promoted the growth of Bifidobacterium longum ssp. infantis in a concentration-dependent manner, and this activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. longum ssp. infantis following exposure to GMP revealed a substantial response to GMP relative to bacteria treated with GMP-P, with a greater number of differentially expressed transcripts and larger fold changes versus the control. Therefore, stimulation of B. longum ssp. infantis growth by GMP is intrinsically linked to the peptide's O-linked glycosylation. The pool of differentially expressed transcripts included 2 glycoside hydrolase (family 25) genes, which were substantially upregulated following exposure to GMP, but not GMP-P. These GH25 genes were present in duplicated genomic islands that also contained genes encoding fibronectin type III binding domain proteins and numerous phage-related proteins, all of which were also upregulated. Homologs of this genomic arrangement were present in other Bifidobacterium species, which suggest it may be a conserved domain for the utilization of glycosylated peptides. This study provides insights into the molecular basis for the prebiotic effect of bovine milk GMP on B. longum ssp. infantis.

Milk adulteration with acidified rennet whey: a limitation for caseinomacropeptide detection by high-performance liquid chromatography.

BACKGROUND: High-performance liquid chromatography (HPLC) is widely employed to determine the caseinomacropeptide (CMP) index and to detect milk tampering with rennet whey. Prior to HPLC analysis, CMP is subject to a trichloracetic acid isolation, causing further soluble proteins in the sample to precipitate. On this basis, we aimed to determine whether rennet whey acidification could adversely affect the HPLC sensitivity with respect to detecting this peptide. RESULTS: As hypothesized, the CMP index from milk with added acidified rennet whey was, on average, half that quantified from milk with added rennet whey. Moreover, the quantum satis of acidified whey added to milk sufficient to demonstrate a HPLC CMP > 30 mg L-1 was 94% greater than that required for this threshold to be reached with rennet whey. CONCLUSION: Milk tampering with acidified rennet whey may limit the analytical sensitivity of the reversed-phase HPLC employed for the screening of CMP and, ultimately, disguise the fraudulent addition of whey to milk. © 2017 Society of Chemical Industry.