Incorporating transgender and nonbinary participants in phase 1 clinical drug trials: Current knowledge gaps and considerations.

Phase 1 clinical drug trials critically depend on the participation of healthy volunteers to evaluate the safety and pharmacokinetics of new medicinal products. Current selection criteria and health definitions often overlook the unique health profiles of transgender and nonbinary individuals, potentially excluding them from participating in these essential early-stage studies. This review aims to identify and discuss current knowledge gaps and considerations regarding the inclusion of transgender and nonbinary participants in phase 1 clinical drug trials. We highlight the need for research on how gender-affirming hormone therapy may affect drug pharmacokinetics and call for the development of inclusive biological reference ranges that account for the physiological effects of hormone therapies.

A Cell-Based Assessment of the Muscle Anabolic Potential of Blue Whiting (Micromesistius poutassou) Protein Hydrolysates.

Blue whiting (BW) represents an underutilised fish species containing a high-quality protein and amino acid (AA) profile with numerous potentially bioactive peptide sequences, making BW an economic and sustainable alternative source of protein. This study investigated the impact of three different BW protein hydrolysates (BWPH-X, Y and Z) on growth, proliferation and muscle protein synthesis (MPS) in skeletal muscle (C2C12) myotubes. BWPHs were hydrolysed using different enzymatic and heat exposures and underwent simulated gastrointestinal digestion (SGID), each resulting in a high degree of hydrolysis (33.41-37.29%) and high quantities of low molecular mass peptides (86.17-97.12% <1 kDa). C2C12 myotubes were treated with 1 mg protein equivalent/mL of SGID-BWPHs for 4 h. Muscle growth and myotube thickness were analysed using an xCelligence™ platform. Anabolic signalling (phosphorylation of mTOR, rpS6 and 4E-BP1) and MPS measured by puromycin incorporation were assessed using immunoblotting. BWPH-X significantly increased muscle growth (p < 0.01) and myotube thickness (p < 0.0001) compared to the negative control (amino acid and serum free media). Muscle protein synthesis (MPS), as measured by puromycin incorporation, was significantly higher after incubation with BWPH-X compared with the negative control, but did not significantly change in response to BWPH-Y and Z treatments. Taken together, these preliminary findings demonstrate the anabolic potential of some but not all BWPHs on muscle enhancement, thus providing justification for human dietary intervention studies to confirm and translate the results of such investigations to dietary recommendations and practices.

In Vitro Digestibility, Biological Activity, and Physicochemical Characterization of Proteins Extracted from Conventionally and Organically Cultivated Hempseed (Cannabis sativa L.).

The proteins from two conventionally (CC1 and CC2) and one organically cultivated (OC) hempseed samples were extracted (by alkaline solubilization followed by isoelectric precipitation) and compared in terms of their physicochemical, digestibility and in vitro bioactivity properties. The OC hempseed had higher total protein and lower nonprotein nitrogen content. Protein extracts showed bimodal particle size distributions, with OC showing the smallest and CC1 the largest mean particle diameter (d(0.5)), i.e., 89.0 and 120.0 µm, respectively. Chromatographic analysis showed similar protein profiles for all three protein extracts. The protein extracts were subjected to in vitro simulated gastrointestinal digestion (SGID). Degree of hydrolysis (DH) measurement showed that the highest extent of digestion upon SGID was associated with CC1 (11.0 ± 1.5%), which also had the lowest in vitro antioxidant activity. Only the OC and OC digested samples had lipase inhibitory activity. The results indicate that the cultivation method impacted the composition, physicochemical, digestibility, and biofunctional properties of hempseed proteins.

Pharmacokinetics of ß-d-N4-Hydroxycytidine, the Parent Nucleoside of Prodrug Molnupiravir, in Nonplasma Compartments of Patients With Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

ß-d-N4-hydroxycytidine (NHC), the parent nucleoside of molnupiravir, a COVID-19 antiviral, was quantified at SARS-CoV-2 transmission sites in 12 patients enrolled in AGILE Candidate-Specific Trial-2. Saliva, nasal, and tear NHC concentrations were 3%, 21%, and 22% that of plasma. Saliva and nasal NHC were significantly correlated with plasma (P < .0001). Clinical Trials Registration. NCT04746183.

Insolubility in milk protein concentrates: potential causes and strategies to minimize its occurrence.

Milk protein concentrates (MPCs), which are produced from skim milk following a series of manufacturing steps including pasteurization, membrane filtration, evaporation and spray drying, represent a relatively new category of dairy ingredients. MPC powders mainly comprise caseins and whey proteins in the same ratio of occurrence as in milk. While bovine MPCs have applications as an ingredient in several protein enriched food products, technofunctional concerns, e.g., reduced solubility and emulsification properties, especially after long-term storage, limit their widespread and consistent utilization in many food products. Changes in the surface and internal structure of MPC powder particles during manufacture and storage occur via casein-casein and casein-whey protein interactions and also via the formation of casein crosslinks in the presence of calcium ions which are associated with diminishment of MPCs functional properties. The aggregation of micellar caseins as a result of these interactions has been considered as the main cause of insolubility in MPCs. In addition, the occurrence of lactose-protein interactions as a result of the promotion of the Maillard reaction mainly during storage of MPC may lead to greater insolubility. This review focuses on the solubility of MPC with an emphasis on understanding the factors involved in its insolubility along with approaches which may be employed to overcome MPC insolubility. Several strategies have been developed based on manipulation of the manufacturing process, along with composition, physical, chemical and enzymatic modifications to overcome MPC insolubility. Despite many advances, dairy ingredient manufacturers are still investigating technical solutions to resolve the insolubility issues associated with the large-scale manufacture of MPC.

Blue Whiting (Micromesistius poutassou) Protein Hydrolysates Increase GLP-1 Secretion and Proglucagon Production in STC-1 Cells Whilst Maintaining Caco-2/HT29-MTX Co-Culture Integrity.

Inducing the feeling of fullness via the regulation of satiety hormones presents an effective method for reducing excess energy intake and, in turn, preventing the development of obesity. In this study, the ability of blue whiting soluble protein hydrolysates (BWSPHs) and simulated gastrointestinal digested (SGID) BWSPHs, to modulate the secretion and/or production of satiety hormones, such as glucagon-like peptide-1 (GLP-1), cholecystokinin (CCK) and peptide YY (PYY), was assessed in murine enteroendocrine STC-1 cells. All BWSPHs (BW-SPH-A to BW-SPH-F) (1.0% w/v dw) increased active GLP-1 secretion and proglucagon production in STC-1 cells compared to the basal control (Krebs-Ringer buffer) (p < 0.05). The signaling pathway activated for GLP-1 secretion was also assessed. A significant increase in intracellular calcium levels was observed after incubation with all BWSPHs (p < 0.05) compared with the control, although none of the BWSPHs altered intracellular cyclic adenosine monophosphate (cAMP) concentrations. The secretagogue effect of the leading hydrolysate was diminished after SGID. Neither pre- nor post-SGID hydrolysates affected epithelial barrier integrity or stimulated interleukin (IL)-6 secretion in differentiated Caco-2/HT-29MTX co-cultured cells. These results suggest a role for BWSPH-derived peptides in satiety activity; however, these peptides may need to be protected by some means to avoid loss of activity during gastrointestinal transit.

In Vivo and In Vitro Comparison of the DPP-IV Inhibitory Potential of Food Proteins from Different Origins after Gastrointestinal Digestion.

Dipeptidyl-peptidase IV (DPP-IV) plays an essential role in glucose metabolism by inactivating incretins. In this context, food-protein-derived DPP-IV inhibitors are promising glycemic regulators which may act by preventing the onset of type 2 diabetes in personalized nutrition. In this study, the DPP-IV-inhibitory potential of seven proteins from diverse origins was compared for the first time in vitro and in vivo in rat plasma after the intestinal barrier (IB) passage of the indigested proteins. The DPP-IV-inhibitory potentials of bovine hemoglobin, caseins, chicken ovalbumin, fish gelatin, and pea proteins were determined in rat plasma thirty minutes after oral administration. In parallel, these proteins, together with bovine whey and gluten proteins, were digested using the harmonized INFOGEST protocol adapted for proteins. The DPP-IV half-maximal inhibitory concentration (IC50) was determined in situ using Caco-2 cells. The DPP-IV-inhibitory activity was also measured after IB passage using a Caco2/HT29-MTX mixed-cell model. The peptide profiles were analyzed using reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC-MS/MS) with MS data bioinformatics management, and the IC50 of the identified peptides was predicted in silico. The in vitro and in vivo DPP-IV-inhibitory activity of the proteins differed according to their origin. Vegetable proteins and hemoglobin yielded the highest DPP-IV-inhibitory activity in vivo. However, no correlation was found between the in vivo and in vitro results. This may be partially explained by the differences between the peptidome analysis and the in silico predictions, as well as the study complexity.

Refocusing dental care: A risk-based preventative oral health programme for dentate older people in UK care homes.

BACKGROUND: There is strong evidence for managing the risk of dental caries, notably dose-dependent use of fluoride based on risk. Specific guidance is lacking on higher fluoride use in older people in care homes and prevention is often omitted from dental care plans. OBJECTIVES: To introduce a risk-based preventative approach to existing routine dental care for older people in care homes. METHODS: Three mixed residential and nursing care homes for the frail and elder (>65 years) were selected to participate. All residents were risk assessed based on dependency, dentition status and self-care abilities and consequently placed on the appropriate evidence-based intervention (2800 ppm high dose fluoride toothpaste and/or quarterly fluoride varnish placement). Full mouth ICDAS dental examinations were completed at baseline, 6 months and 12 months. RESULTS: At baseline, 127 risk assessments were completed in which most dentate residents (58.2%, n = 74) were assessed as Risk Level 2/3 (mod/high) whilst edentulous residents were all Risk Level 1 (low) (41.7%, n = 53). Only 13 (26.5%) of the 49 eligible residents completed the 12-month preventative programme. There was a significant difference in root caries (P < .0001), with 17 (51.5%) root lesions changing from active at baseline to arrested at 12 months. CONCLUSIONS: The findings provide early indication of fluoride efficacy, especially on root caries in this vulnerable group, and highlight the challenges of delivering programme's in these complex, changing environments.

Optimal dose and safety of molnupiravir in patients with early SARS-CoV-2: a Phase I, open-label, dose-escalating, randomized controlled study.

OBJECTIVES: AGILE is a Phase Ib/IIa platform for rapidly evaluating COVID-19 treatments. In this trial (NCT04746183) we evaluated the safety and optimal dose of molnupiravir in participants with early symptomatic infection. METHODS: We undertook a dose-escalating, open-label, randomized-controlled (standard-of-care) Bayesian adaptive Phase I trial at the Royal Liverpool and Broadgreen Clinical Research Facility. Participants (adult outpatients with PCR-confirmed SARS-CoV-2 infection within 5 days of symptom onset) were randomized 2:1 in groups of 6 participants to 300, 600 and 800 mg doses of molnupiravir orally, twice daily for 5 days or control. A dose was judged unsafe if the probability of 30% or greater dose-limiting toxicity (the primary outcome) over controls was 25% or greater. Secondary outcomes included safety, clinical progression, pharmacokinetics and virological responses. RESULTS: Of 103 participants screened, 18 participants were enrolled between 17 July and 30 October 2020. Molnupiravir was well tolerated at 300, 600 and 800 mg doses with no serious or severe adverse events. Overall, 4 of 4 (100%), 4 of 4 (100%) and 1 of 4 (25%) of the participants receiving 300, 600 and 800 mg molnupiravir, respectively, and 5 of 6 (83%) controls, had at least one adverse event, all of which were mild (≤grade 2). The probability of ≥30% excess toxicity over controls at 800 mg was estimated at 0.9%. CONCLUSIONS: Molnupiravir was safe and well tolerated; a dose of 800 mg twice daily for 5 days was recommended for Phase II evaluation.

A multi-stakeholder approach to the co-production of the research agenda for medicines optimisation.

BACKGROUND: Up to 50% of medicines are not used as intended, resulting in poor health and economic outcomes. Medicines optimisation is 'a person-centred approach to safe and effective medicines use, to ensure people obtain the best possible outcomes from their medicines'. The purpose of this exercise was to co-produce a prioritised research agenda for medicines optimisation using a multi-stakeholder (patient, researcher, public and health professionals) approach. METHODS: A three-stage, multiple method process was used including: generation of preliminary research questions (Stage 1) using a modified Nominal Group Technique; electronic consultation and ranking with a wider multi-stakeholder group (Stage 2); a face-to-face, one-day consensus meeting involving representatives from all stakeholder groups (Stage 3). RESULTS: In total, 92 research questions were identified during Stages 1 and 2 and ranked in order of priority during stage 3. Questions were categorised into four areas: 'Patient Concerns' [e.g. is there a shared decision (with patients) about using each medicine?], 'Polypharmacy' [e.g. how to design health services to cope with the challenge of multiple medicines use?], 'Non-Medical Prescribing' [e.g. how can the contribution of non-medical prescribers be optimised in primary care?], and 'Deprescribing' [e.g. what support is needed by prescribers to deprescribe?]. A significant number of the 92 questions were generated by Patient and Public Involvement representatives, which demonstrates the importance of including this stakeholder group when identifying research priorities. CONCLUSIONS: A wide range of research questions was generated reflecting concerns which affect patients, practitioners, the health service, as well the ethical and philosophical aspects of the prescribing and deprescribing of medicines. These questions should be used to set future research agendas and funding commissions.

Physicochemical, Nutritional and In Vitro Antidiabetic Characterisation of Blue Whiting (Micromesistiuspoutassou) Protein Hydrolysates.

Protein hydrolysates from low-value underutilised fish species are potential sources of high-quality dietary protein and health enhancing peptides. Six blue whiting soluble protein hydrolysates (BW-SPH-A_F), generated at industrial scale using different hydrolysis conditions, were assessed in terms of their protein equivalent content, amino acid profile and score and physicochemical properties in addition to their ability to inhibit dipeptidyl peptidase IV (DPP-IV) and stimulate the secretion of insulin from BRIN-BD11 cells. Furthermore, the effect of simulated gastrointestinal digestion (SGID) on the stability of the BW-SPHs and their associated in vitro antidiabetic activity was investigated. The BW-SPHs contained between 70-74% (w/w) protein and all essential and non-essential amino acids. All BW-SPHs mediated DPP-IV inhibitory (IC50: 2.12-2.90 mg protein/mL) and insulin secretory activity (2.5 mg/mL; 4.7 to 6.4-fold increase compared to the basal control (5.6 mM glucose alone)). All BW-SPHs were further hydrolysed during SGID. While the in vitro DPP-IV inhibitory and insulin secretory activity mediated by some BW-SPHs was reduced following SGID, the activity remained high. In general, the insulin secretory activity of the BW-SPHs were 4.5-5.4-fold higher than the basal control following SGID. The BW-SPHs generated herein provide potential for anti-diabetic related functional ingredients, whilst also enhancing environmental and commercial sustainability.

Intrapulmonary Pharmacokinetics of Cefepime and Enmetazobactam in Healthy Volunteers: Towards New Treatments for Nosocomial Pneumonia.

Cefepime-enmetazobactam is a novel ß-lactam-ß-lactamase inhibitor combination with broad-spectrum antimicrobial activity against a range of multidrug-resistant Enterobacteriaceae This agent is being developed for a range of serious hospital infections. An understanding of the extent of partitioning of ß-lactam-ß-lactamase inhibitor combinations into the human lung is required to better understand the potential role of cefepime-enmetazobactam for the treatment of nosocomial pneumonia. A total of 20 healthy volunteers were used to study the intrapulmonary pharmacokinetics of a regimen of 2 g cefepime-1 g enmetazobactam every 8 h intravenously (2 g/1 g q8h i.v.). Each volunteer contributed multiple plasma samples and a single epithelial lining fluid (ELF) sample, obtained by bronchoalveolar lavage. Concentrations of cefepime and enmetazobactam were quantified using liquid chromatography-tandem mass spectrometry. The pharmacokinetic data were modeled using a population methodology, and Monte Carlo simulations were performed to assess the attainment of pharmacodynamic targets defined in preclinical models. The concentration-time profiles of both agents in plasma and ELF were similar. The mean ± standard deviation percentage of partitioning of total drug concentrations of cefepime and enmetazobactam between plasma and ELF was 60.59% ± 28.62% and 53.03% ± 21.05%, respectively. Using pharmacodynamic targets for cefepime of greater than the MIC and free enmetazobactam concentrations of >2 mg/liter in ELF of 20% of the dosing interval, a regimen of cefepime-enmetazobactam of 2 g/0.5 g q8h i.v. infused over 2 h resulted in a probability of target attainment of ≥90% for Enterobacteriaceae with cefepime-enmetazobactam MICs of ≤8 mg/liter. This result provides a rationale to further consider cefepime-enmetazobactam for the treatment of nosocomial pneumonia caused by multidrug-resistant Enterobacteriaceae.

Current knowledge on the extraction, purification, identification, and validation of bioactive peptides from seaweed.

Seaweed (macroalgae) is considered as a sustainable bioresource rich in high-quality nutrients such as protein. Seaweed protein can be used as an alternative to other protein sources. Furthermore, these proteins are natural reservoirs of bioactive peptides (BAPs) associated with various health benefits such as antioxidant, antihypertensive, and antidiabetic activities. However, seaweed-derived BAPs remain underexploited due to challenges that arise during protein extraction from algal biomass. Coupled with this, limited proteomic information exists regarding certain seaweed species. This review highlights the current state of the art of seaweed protein extraction techniques, e.g., liquid, ultrasound, microwave, pulsed electric field, and high hydrostatic pressure assisted extraction. The review also focuses on the enzymatic hydrolysis of seaweed proteins and characterization of the resultant hydrolysates/peptides using electrophoretic and chromatographic techniques. This includes reference to methods employed for separation, fractionation, and purification of seaweed BAPs, as well as the methodologies used for identification, e.g., analysis by mass spectrometry. Furthermore, a bioinformatics or in silico approach to aid discovery of seaweed BAPs is discussed herein. Based on the information available to date, it is suggested that further research is required in this area for the development of seaweed BAPs for nutraceutical applications.

Nitisinone causes acquired tyrosinosis in alkaptonuria.

For over two decades, nitisinone (NTBC) has been successfully used to manipulate the tyrosine degradation pathway and save the lives of many children with hereditary tyrosinaemia type 1. More recently, NTBC has been used to halt homogentisic acid accumulation in alkaptonuria (AKU) with evidence suggesting its efficacy as a disease modifying agent. NTBC-induced hypertyrosinaemia has been associated with cognitive impairment and potentially sight-threatening keratopathy. In the context of a non-lethal condition (ie, AKU), these serious risks call for an evaluation of the wider impact of NTBC on the tyrosine pathway. We hypothesised that NTBC increases the tyrosine pool size and concentrations in tissues. In AKU mice tyrosine concentrations of tissue homogenates were measured before and after treatment with NTBC. In humans, pulse injection with l-[13 C9 ]tyrosine and l-[d8 ]phenylalanine was used along with compartmental modelling to estimate the size of tyrosine pools before and after treatment with NTBC. We found that NTBC increased tyrosine concentrations in murine tissues by five to nine folds. It also significantly increased the tyrosine pool size in humans (P < .001), suggesting that NTBC increases tyrosine not just in serum but also in tissues (ie, acquired tyrosinosis). This study provides, for the first time, the experimental proof for the magnitude of NTBC-related acquired tyrosinosis which should be overcome to ensure the safe use of NTBC in AKU.

Homogentisic acid is not only eliminated by glomerular filtration and tubular secretion but also produced in the kidney in alkaptonuria.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P < 0.001), and was significantly associated with decreased HGA clearance (CLHGA ) (P < 0.001) and FEHGA (P < 0.001). CLHGA and FEHGA were increased beyond the theoretical maximum renal plasma flow, confirming renal production and emphasising the greater contribution of net tubular secretion than glomerular filtration to renal elimination of HGA. The kidneys are crucial to elimination of HGA. Elimination of HGA is impaired with age resulting in worsening disease over time. The kidney is an important site for production of HGA. Tubular secretion of HGA contributes more to elimination of HGA in AKU than glomerular filtration.

Investigating the clinical factors and comedications associated with circulating levels of atorvastatin and its major metabolites in secondary prevention.

Aims The lipid-lowering drug, atorvastatin (ATV), is 1 of the most commonly prescribed medications worldwide. The aim of this study was to comprehensively investigate and characterise the clinical factors and comedications associated with circulating levels of ATV and its metabolites in secondary prevention clinical practice. Methods The plasma concentrations of ATV, 2-hydroxy (2-OH) ATV, ATV lactone (ATV L) and 2-OH ATV L were determined in patients 1 month after hospitalisation for a non-ST elevation acute coronary syndrome. Factors were identified using all subsets multivariable regression and model averaging with the Bayesian information criterion. Exploratory genotype-stratified analyses were conducted using ABCG2 rs2231142 (Q141K) and CYP2C19 metaboliser status to further investigate novel associations. Results A total of 571 patients were included; 534 and 37 were taking ATV 80 mg and 40 mg daily, respectively. Clinical factors associated with ATV and/or its metabolite levels included age, sex, body mass index and CYP3A inhibiting comedications. Smoking was newly associated with increased ATV lactonisation and reduced hydroxylation. Proton pump inhibitors (PPIs) and loop diuretics were newly associated with modestly increased levels of ATV (14% and 38%, respectively) and its metabolites. An interaction between PPIs and CYP2C19 metaboliser status on exposure to specific ATV analytes (e.g. interaction P = .0071 for 2-OH ATV L) was observed. Overall model R2 values were 0.14-0.24.ConclusionMultiple factors were associated with circulating ATV and metabolite levels, including novel associations with smoking and drug-drug(-gene) interactions involving PPIs and loop diuretics. Further investigations are needed to identify additional factors that influence ATV exposure.

Enzymatic Modification of Porphyra dioica-Derived Proteins to Improve their Antioxidant Potential.

Enzymatic hydrolysis has been employed to modify protein functional properties and discover new sources of antioxidants. In this study, the effect of different enzymatic treatments on antioxidant activity of Porphyra dioica (blades and protein isolate (PI)) was investigated. Protein nitrogen content of P. dioica blades and PI were 23 and 50% (dry weight), respectively. Blades and PI were hydrolyzed with Prolyve® and Prolyve® plus Flavourzyme®. Peptide profiles and molecular mass distribution of the hydrolysates were investigated. The hydrolysis promoted generation of peptides and low molecular mass components <1 kDa. Antioxidant activity was assessed using ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) scavenging, 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) inhibition, and reactive oxygen species scavenging ability, i.e., oxygen radical absorbance capacity (ORAC) and hypochlorous acid (HOCl) scavenging assays. In general, enzymatic hydrolysis of P. dioica blades and PI enhanced the in vitro antioxidant activity. Direct hydrolysis of blades improved ORAC values up to 5-fold (from 610 to 3054 µmol Trolox eq./g freeze dried sample (FDS). The simultaneous release of phenolic compounds suggested a potential synergistic activity (ORAC and ABTS·+ assays). Such hydrolysates may be of value as functional food ingredients.

Variable Glycemic Responses to Intact and Hydrolyzed Milk Proteins in Overweight and Obese Adults Reveal the Need for Precision Nutrition.

Background: Dietary modifications can contribute to improved pancreatic ß cell function and enhance glycemic control. Objectives: The objectives of this study were as follows: 1) to investigate the potential of milk protein hydrolysates to modulate postprandial glucose response; 2) to assess individual responses; and 3) to explore the inter- and intraindividual reproducibility of the response. Methods: A 14-d randomized crossover study investigated interstitial glucose levels of participants in response to 12% w/v milk protein drinks (intact caseinate and casein hydrolysate A and B) consumed in random order with a 2-d washout between treatments. Milk protein drinks were consumed immediately prior to study breakfast and evening meals. Twenty participants (11 men, 9 women) aged 50 ± 8 y with a body mass index (in kg/m2) of 30.2 ± 3.1 were recruited. Primary outcome was glucose levels assessed at 15-min intervals with the use of glucose monitors. Results: Repeated-measures ANOVA revealed that for breakfast there was a significant difference across the 3 treatment groups (P = 0.037). The ability to reduce postprandial glucose was specific to casein hydrolysate B in comparison with intact caseinate (P = 0.039). However, despite this significant difference, further examination revealed that only 3 out of 18 individuals were classified as responders (P < 0.05). High intraclass correlation coefficients were obtained for glucose response to study meals (intraclass correlation coefficient: 0.892 for breakfast with intact caseinate). The interindividual CVs were higher than the intraindividual CVs. Mean inter- and intraindividual CVs were 19.4% and 5.7%, respectively, for breakfast with intact caseinate. Conclusion: Ingestion of a specific casein hydrolysate successfully reduced the postprandial glucose response; however, at an individual level only 3 participants were classified as responders, highlighting the need for precision nutrition. Exploration of high interindividual responses to nutrition interventions is needed, in combination with the development of precision nutrition, potentially through an n-of-1 approach. This clinical trial was registered as ISRCTN61079365 (https://www.isrctn.com/).

Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

Technical note: Fourier transform infrared spectral analysis in tandem with 31P nuclear magnetic resonance spectroscopy elaborates detailed insights into phosphate partitioning during skimmed milk microfiltration and diafiltration.

Our previous study identified peaks in the 31P nuclear magnetic resonance (31P NMR) spectra of skim milk, denoting the interaction of different phosphate species such as inorganic and casein-associated phosphate during the separation of colloidal and serum phases of skim milk by microfiltration (MF) and diafiltration (DF). In the current study, we investigated the same samples generated by the aforementioned separation using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy analysis. The results confirmed that the technique was not only capable of differentiating between the mineral equilibrium of the casein phosphate nanocluster (CPN) and milk serum, but also complemented the application of 31P NMR. An ATR-FTIR broad band in the region of 1,055 to 1,036 cm-1 and a specific band at 1,076 cm-1 were identified as sensitive to the repartitioning of different phosphate species in milk in accordance with the 31P NMR signals representing casein-associated phosphate and inorganic phosphate in the serum. A third ATR-FTIR signal at 1,034 cm-1 in milk, representing precipitated inorganic calcium phosphate, had not previously been detected by 31P NMR. Thus, the results indicate that a combination of ATR-FTIR and 31P NMR spectroscopies may be optimally used to follow mineral and protein phase changes in milk during membrane processing.