The growing global population and climate crisis demand expanding non-animal protein options. Single-cell protein biomass, referred to as "Solein", is produced by the hydrogen-oxidising bacterium Xanthobacter sp. SoF1 and is a promising, sustainable source of protein and dietary fibre, especially when created using renewable energy. This study investigates Solein protein powder (SPP) for its composition and techno-functional properties, comparing it to pea protein isolate (PPI). SPP had a lower fat content and higher dietary fibre, while matching the protein content of PPI. SPP met all indispensable amino acid requirements for adults over the age of three, as outlined by the FAO in 2013. A milk alternative resembling semi-skimmed cow's milk was produced from SPP and PPI. These emulsions were fermented with a commercial starter culture containing Streptococcus thermophilus. The fermentation process was monitored by tracking pH, total titratable acidity, and microbial growth. The resulting yoghurt alternative (YA) underwent textural and rheological analysis. Solein protein powder yoghurt alternative (SPP-YA) exhibited faster acidification, greater microbial growth, improved water retention, and a texture similar to dairy yoghurt. Static in vitro digestion revealed moderate protein digestibility of the non-fermented SPP emulsion (63.8-67.5%), based on total amino acids, free amino groups, and total nitrogen, with an in vitro Digestible Indispensable Amino Acid Score (DIAAS) of (51.0 ± 6.1%). Fermentation slightly reduced digestibility (57.8-59.6%) and DIAAS (48.3 ± 1.4%), with isoleucine as the limiting amino acid. This work provides the first insight into the structural and nutritional performance of hydrogen-oxidising bacterial protein in non-dairy YA.

This protocol describes a standardized in vitro method to determine the digestibility and digestible indispensable amino acid score (DIAAS) of dietary proteins. This ‘INFOGEST Quant’ method is an extension of our previous INFOGEST static digestion model (INFOGEST 2.0) and adds a workflow for the quantification of total protein digestibility, individual amino acid digestibility and DIAAS. The protocol was validated using in vivo data obtained by digesting the same food samples, and the results showed a high degree of agreement, confirming its relevance for nutritional assessments. To establish the DIAAS of a protein source, nonabsorbable peptides and proteins are precipitated after in vitro digestion and the resulting absorbable fraction is analyzed using ultrahigh-performance liquid chromatography with ultraviolet detection (to assess total and individual amino acids). Two alternative quantification strategies are also described: protein titration using the Kjeldahl method (to assess total nitrogen) and spectrophotometric analysis with o-phthalaldehyde (to assess total amino groups). Both alternative methods are valid only for the calculation of total digestibility and the proxy-digestible indispensable amino acid ratio, which gives an approximation of the DIAAS of the tested protein sources. Compared to existing approaches, this protocol is suitable for routine application in nutrition and food science laboratories. The preparatory steps take ~6 d, whereas the full workflow can be completed in triplicate in ~8 d. Analysis of the digesta takes an additional 3–5 d, depending on the method. The procedure requires only standard laboratory equipment and reagents and can be performed by anyone with basic training in biochemistry or a related discipline. This Protocol Extension is an adapted version of the authors’ previous INFOGEST static digestion model (INFOGEST 2.0), with an added workflow for the quantification of total protein digestibility, individual amino acid digestibility and digestible indispensable amino acid score.

Background Human milk (HM) is a unique nutritional source, tailored to meet infants’ needs during early life. However, declining breastfeeding rates, influenced by lactation problems, health-related factors, psychological state, and work-related constraints, have increased reliance on infant milk formula (IMF). Most IMF is derived from bovine milk, which differs substantially from HM in protein composition, fat structure, carbohydrate profile, and bioactive components. These compositional gaps limit IMF’s ability to replicate HM functionality with respect to digestibility, nutrient absorption, immune support, and microbiome maturation. Consequently, formula-fed infants may experience accelerated growth, increased susceptibility to allergic reactions, and gastrointestinal issues. Scope and approach This review examines the compositional and functional limitations of IMF relative to HM, focusing on proteins and human milk oligosaccharides (HMOs), and explores how precision fermentation (pFerm), an emerging biotechnology for large-scale production of bioidentical human milk proteins and oligosaccharides, can narrow the HM-IMF gap. We review the pFerm protein and HMO sectors, analysing case studies of both compounds and highlighting challenges and opportunities. Key findings and conclusions Market analysis shows that pFerm-derived bovine proteins and HMOs are well established, whereas human-derived proteins remain underdeveloped. Case studies of recombinant human lactoferrin and pFerm-derived HMOs demonstrate the ability of these innovations to narrow the HM-IMF gap. Key challenges to adoption include regulatory hurdles, economic scalability, and consumer apprehension regarding the perceived ‘unnatural’ nature of such ingredients. Nevertheless, the rapidly expanding pFerm sector - driven by biotechnological investment and growing consumer acceptance through effective communication - offers considerable opportunities to enhance IMF development through application of pFerm-derived HM compounds.

The study aimed to determine the amount of (3-casomorphin-7 released during semi-dynamic in vitro gastrointestinal digestion of A1/A1 and A2/A2 milk, yogurt, and Cheddar cheese. Samples were collected at 10 and 120 min during the intestinal phase for each of the four gastric emptying points, following the INFOGEST digestion model. (3-Casomorphin-7 was quantified using liquid chromatography coupled to triple quadrupole mass spectrometry. The levels of (3-casomorphin-7 were similar in A1/A1 (8.65-11.72 mu g/100 g protein) and A2/A2 (9.64-15.29 mu g/100 g protein) milk digests, higher in A1/A1 yogurt, while A1/A1 cheese digest exhibited the highest levels among all samples (36.19-62.73 mu g/100 g protein), nearly ten times higher than A2/A2 cheese digest. After in vitro gastrointestinal digestion of a serving size of the studied dairy products the released amounts may not align with levels required for in vivo opioid activity. However, further human clinical trials are warranted.

Microencapsulation, widely used across industries, enhances the delivery of bioactive compounds and probiotics in the gastrointestinal tract. The growing demand for functional foods has highlighted pectin as a promising encapsulation material, due to its favorable physicochemical properties and fermentability by gut microbiota, which supports intestinal health. In this study, we focused on encapsulating the pH sensitive probiotic Lactobacillus plantarum WCFS1 using various alginate-pectin compositions to evaluate their variability and functionality under simulated gastrointestinal conditions. Pectins with different degrees of methyl-esterification (DM) and blockiness distribution (DB) were enzymatically modified, characterized and tested. Results indicated that capsules with a 3:1 alginate to pectin ratio provided optimal stability. According to a three-way ANOVA analysis, the source, the DB, the pH, and the interaction between the resource and DB impact the viability of encapsulated L. plantarum WCFS1. Pectin with a lower DB enhanced the protection of L. plantarum WCFS1 under pH 3, 5, and 7. Under simulated digestive fluid conditions, the source, the DB, and the simulated fluids independently affect the viability of encapsulated L. plantarum WCFS1. Notably, orange pectin with a high DB (98 %) demonstrated the greatest efficacy in maintaining bacterial viability under simulated gastrointestinal conditions. The findings suggest that the pectin source and structural properties significantly influence the viability and survival of encapsulated probiotics under gastrointestinal conditions, which may lead to the tailored design of pectin-based capsules for controlled delivery of bacteria to specific parts of the gastrointestinal tract.

The development of plant protein-based delivery systems is often limited by poor stability and low retention efficiency under gastrointestinal conditions. This study investigated how pH (4 and 7) during external gelation influences the physicochemical properties, entrapment efficiency (EE), and in vitro gastrointestinal behaviour of alginate beads loaded with hemp protein concentrate (HPC), pea protein concentrate (PPC), or soy protein isolate (SPI). Zeta potential and Fourier transform infrared (FTIR) analyses suggested that at pH 4, the charge profiles of plant proteins favoured electrostatic association with anionic alginate, which was associated with higher EE values, with HPC achieving the maximum of 89.5% at pH 4. Conversely, at pH 7, electrostatic repulsion between biopolymers was associated with reduced EE. During in vitro digestion, beads formulated at pH 4 exhibited greater protein release than those prepared at pH 7, consistent with the expansion of the polymeric network under intestinal conditions. Size exclusion chromatography showed that released proteins underwent extensive proteolysis, generating low-molecular-weight fractions smaller than 300 Da. These results indicate that gelation pH is a relevant processing parameter for modulating protein retention and the extent of protein release of simulated digestion in alginate-based systems, with potential applications in protein-enriched food formulations.

BACKGROUND:Accurate probiotic enumeration ensures the dose delivered to the mammalian host. Plate count (PC) detects only culturable cells and can underestimate the total number of viable cells, particularly when some cells are injured or dormant and do not form colonies. OBJECTIVE:To qualify a flow cytometry assay reporting active fluorescent units (AFU) and to compare counts with the PC method for micro-encapsulated Lacticaseibacillus rhamnosus GG in a snack product. METHODS:Following AOAC INTERNATIONAL, International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), and United States Pharmacopeia (USP) principles, precision, accuracy, ruggedness, specificity, and robustness of plate count and flow cytometry methods were evaluated for the first time in a commercial yogurt-bite snack product. RESULTS:Both methods fulfilled the pre-specified performance targets (RSD ≤15%; AFU recovery 100-104%; live/dead R2 ≥0.95). Analyst-to-analyst differences were not significant. Flow cytometry spike recoveries were 100-104% across targets. Live/dead mixtures tracked linearly (R2 ≥0.95). Across matched samples, AFU and CFU values were equivalent within a pre-specified ±0.5 log band; batch means typically differed by ≤0.2 log with no systematic bias. CONCLUSION:The qualified flow cytometry method provides same-day counts and enumerates all membrane-intact cells, complementing plate counts while meeting analytical performance criteria. Findings endorse the use of AFU in tandem to CFU assessment for probiotic quantification, label accuracy and estimation of microbiome-relevant dose in snack and finished-product matrixes. HIGHLIGHTS:Flow cytometry provided same-day results and enumerated all membrane-intact cells for label verification in snack matrixes.

Bovine diet and stage of lactation can influence milk fat composition, flavour, and texture. The impact of these on sensory perception and consumer acceptance of butter remain poorly understood. In Ireland, commercial production typically uses a 95 % grass-fed (GRS) system. Previous research has focused on comparisons between 100 % GRS butter and total mixed ration. This study evaluated the sensory attributes and consumer perceptions of butters derived from 95 % GRS, partial mixed ration (PMR), and total mixed ration (TMR) feeding systems. 300 consumers evaluated GRS, TMR and PMR butter samples, providing hedonic and intensity ratings for taste, texture, colour, and flavour. Principal Component Analysis (PCA) revealed three independent perceptual dimensions accounting for 60.3 % of variance: hedonic (liking of appearance, smell, flavour, and overall impression), textural/visual (spreadability, creaminess, colour), and taste/aftertaste (flavour intensity, saltiness, aftertaste). No clear separation between consumer preference segments was observed in the PCA. Across feeding systems, GRS butter received significantly higher ratings than TMR for overall liking, appearance, smell, taste/ flavour, spreadability, colour intensity, flavour intensity, saltiness, and creaminess (p < 0.05). Multiple regression indicated that flavour and creaminess intensity were the strongest positive predictors of overall liking. Overall, while production system influenced measurable sensory properties, consumer preferences were heterogeneous. These findings suggest that butter liking is primarily driven by flavour and texture attributes, but the relative importance of these factors varies across consumer groups.

This study evaluated meat analogues using high-moisture extrusion (HME) using faba protein concentrate (FPC) alone (Control) and blends with single-cell proteins (SCPs): microalgae Chlorella vulgaris (SCP1) and bacteria Xanthobacter spp. (SCP2). Three blends were formulated via linear programming based on the beneficial nutrients content in meat (beef, pork and chicken): Blend1 (60 % FPC + 40 % SCP1), Blend2 (22.5 % FPC + 77.5 % SCP2), and Blend3 (13.5 % FPC + 11 % SCP1 + 75.5 % SCP2). Composition, texture, phytic acid and in vitro digestibility analyses assessed protein quality and mineral bioaccessibility. Samples were oven cooked before assays to simulate typical consumption. Cooking caused minor structural changes, without significantly affecting protein denaturation or phytic acid levels, as extrusion was the dominant thermal process. Protein digestibility was high (close to 100 %) across all samples and generally unaffected by cooking. SCP inclusion significantly improved amino acid profiles, with Blend1 and Blend2 classified as excellent sources and Control and Blend3 as good sources of essential amino acids. Minerals such as manganese and potassium showed enhanced bioaccessibility linked to reduced phytic acid levels due to SCP incorporation and extrusion. Compared to average meat and dietary reference values, extruded blends demonstrated promising nutritional equivalency, supporting their potential as sustainable, nutrient-dense meat analogues. This study highlights the benefit of combining alternative protein blends with high-impact extrusion to enhance meat substitute nutritional quality.

Despite growing interest in faba bean (Vicia faba L.) as a sustainable plant protein source, limited studies address process-induced structural mechanisms linking functionality with gastro-intestinal (GI) digestion. This study elucidates the effects of pre-extraction (soaking, dehulling) and post-extraction thermal treatment on the structure-digestibility relationship of faba bean protein concentrates. Simulated GI digestion was performed using the INFOGEST static model, assessing in vitro protein digestibility (%) and in vitro digestible indispensable amino acid score (DIAAS). Pre-processing had a significant impact on total phenolic content (TPC); soaking yielded the highest TPC (7.34 +/- 0.19 mg GAE/g DM), whereas dehulling (6.44 +/- 0.15 mg GAE/g DM) and coarse milling (5.63 +/- 0.09 mg GAE/g DM) resulted in comparatively lower values. Heating treatment (100 degrees C, 30 min) induced protein aggregation (500-2000 kDa), and enhanced peptide release, confirmed by SDS-PAGE, SEC, and LC-MS/MS. Heated faba concentrates showed a reduced abundance (%) of alpha-helix and an increase in beta-sheet structures (including intramolecular and aggregated forms), demonstrating changes in their secondary structure. Thermal treatment significantly improved digestibility (TN, R-NH2, TAA) (88.5-93.6 %) and highest in vitro DIAAR (140.9 %) values, particularly in DPI-H. Although several amino acids showed DIAAR values greater than 100 %, sulfur amino acids remained limited, keeping DIAAS values (based on 8 quantified indispensable amino acids) below 100 % across all samples. These findings provide valuable insights into chemical and structural changes induced by both pre-and post-extraction processing steps, which collectively drive enhanced digestibility and nutritional quality of faba bean isolates.

The development of plant protein-based delivery systems is often limited by poor stability and low retention efficiency under gastrointestinal conditions. This study investigated how pH (4 and 7) during external gelation modulates electrostatic interactions between anionic alginate and plant proteins as hemp protein isolate (HPI), pea protein isolate (PPI), and soy protein isolate (SPI), and how these interactions determine the structure of alginate beads and their gastrointestinal stability in vitro. Physicochemical properties, entrapment efficiency (EE), and protein release kinetics were evaluated using static INFOGEST in vitro digestion protocol. Zeta potential and FTIR analyses confirmed that at pH 4, positive surface charges on the plant proteins promoted electrostatic attraction and hydrogen bonding with the anionic alginate. This interaction significantly enhanced protein retention, leading to higher EE values where HPI achieved the maximum of 89.5% at pH 4. Conversely, electrostatic repulsion dominated at pH 7, restricting biopolymer association and reducing overall EE. During in vitro digestion, beads formulated at pH 4 exhibited greater protein release than those prepared at pH 7, a behaviour driven by the expansion of the polymeric network under intestinal conditions. High-performance size exclusion chromatography verified that both systems underwent extensive proteolysis, successfully generating highly bioaccessible peptides smaller than 300 Da. In conclusion, the formulation pH during external gelation serves as a critical mechanism to modulate the structural arrangement, entrapment efficiency, and gastrointestinal release profiles of plant proteins in mixed alginate systems, offering potential as targeted delivery vehicles for bioactive food applications.

Plant-based meat analogues (PBMAs) have gained attention as sustainable alternatives to meat, but consumer acceptance remains a key limitation due to challenges of replicating meat properties. This work showed the effects of replacing textured pea protein with fermented chickpea (FC) obtained through solid-state fermentation with Pleurotus ostreatus, at three replacement levels: 50% (FC-50), 75% (FC-75), and 100% (FC-100), in a vegan meatball analogue. The effects on physicochemical, nutritional, and sensory properties were assessed. The inclusion of FC reduced cooking loss, with a 36% reduction in FC-75 compared to the control. Colour was slightly affected, with an increase in lightness and a reduction in redness and yellowness. The nutritional results revealed a gradual increase in dietary fibre by 27% as the FC content increased, along with an increase in low-molecular-weight dietary fibre. Protein was reduced by 64% but remained within the values required to use the high-protein claim. Protein digestibility was >90%, with improved in vitro DIAAR (Digestible Indispensable Amino Acid Ratio) in all formulations. FC-75 obtained the best DIAAS (Digestible Indispensable Amino Acid Score) with 121%. The sensory evaluation showed that the inclusion of FC resulted in reduced beany off-flavour and increased mushroom perception, while decreasing elasticity and crumbliness and increasing pastiness texture. Among all formulations, FC-75 provided the greatest improvement in cooking loss and protein quality, while maintaining a sensory profile similar to the control. Overall, these results highlight the use of FC as a promising strategy to obtain PBMAs with more balanced physicochemical, nutritional, and sensory properties.

Background: Cheese serves as a dietary source of vitamin K; however, its impact on vitamin K status biomarkers in humans and the role of dietary vitamin K in modulating lipid profiles have yet to be elucidated. Objective: To explore the effect of six weeks of daily consumption of pasture-derived and total mixed ration (TMR)-derived Cheddar cheese on vitamin K status biomarkers and lipid profiles. Design: Biobanked samples (n = 60), including pasture-derived (n = 33) and TMR-derived (n = 27) Cheddar cheese groups from a previous human intervention study, were analysed. The original study examined the effects of six weeks of daily intake of 120 g of Cheddar cheese on metabolic health biomarkers in adults over 50 years with BMI ≥ 25 kg m-2. Vitamin K-dependent proteins, including dephosphorylated-uncarboxylated matrix Gla protein (dp-ucMGP), undercarboxylated osteocalcin (ucOC) and carboxylated osteocalcin (cOC), were measured using ELISA kits. The dp-ucMGP level and the ucOC : cOC ratio were used as vitamin K status biomarkers. Lipid profiles, including triglycerides, total cholesterol, HDL, LDL, and VLDL cholesterol, and apolipoprotein B, were measured by NMR spectroscopy. Results: Overall, Cheddar cheese intake (n = 60) led to decreases in dp-ucMGP (-34.73 pmol L-1; 95% CI: -47.14, -22.33) and ucOC : cOC (-0.047; 95% CI: -0.07, -0.02) after 6 weeks of consumption, with no differences between the groups. There were no differences in the changes in anthropometric markers or lipid profiles between groups. The sex-by-treatment interaction showed a significant impact on total cholesterol (P < 0.001), HDL cholesterol (P < 0.001) and LDL cholesterol (P = 0.002) levels. Among males, the TMR-derived cheese group exhibited a significantly greater increase in HDL cholesterol (P < 0.05). Among females, TMR-derived cheese consumption was associated with significantly greater decreases in total, HDL and LDL cholesterol levels (P < 0.05) compared with the pasture-derived group. Conclusion: Cheddar cheese intake may improve vitamin K status, and vitamin K intake from cheese may induce sex-specific effects on blood lipid profiles in overweight middle-aged adults.

The study aimed to determine the amount of β-casomorphin-7 released during semi-dynamic in vitro gastrointestinal digestion of A1/A1 and A2/A2 milk, yogurt, and Cheddar cheese. Samples were collected at 10 and 120 min during the intestinal phase for each of the four gastric emptying points, following the INFOGEST digestion model. β-Casomorphin-7 was quantified using liquid chromatography coupled to triple quadrupole mass spectrometry. The levels of β-casomorphin-7 were similar in A1/A1 (8.65-11.72 μg/100 g protein) and A2/A2 (9.64-15.29 μg/100 g protein) milk digests, higher in A1/A1 yogurt, while A1/A1 cheese digest exhibited the highest levels among all samples (36.19-62.73 μg/100 g protein), nearly ten times higher than A2/A2 cheese digest. After in vitro gastrointestinal digestion of a serving size of the studied dairy products the released amounts may not align with levels required for in vivo opioid activity. However, further human clinical trials are warranted.

Concerns about current food systems have prompted increased exploration of sustainable alternative protein sources, such as microalgae. This study investigated honey Chlorella vulgaris, a chlorophyll-deficient mutant, distinguished by its consumer-friendly honey colour, milder flavour and improved texture. To facilitate the nutritional transition towards this source, a standardised in vitro semi-dynamic INFOGEST digestion model was employed to analyse the digestive behaviour of C. vulgaris, focusing on the biochemical and structural changes during in vitro digestion. Gastric digestion was conducted over 67.5 min with dynamic fluid addition and gastric emptying. Results indicated slow gastric digestion of C. vulgaris due to the initially low pepsin activity and low protein solubility. Significant protein breakdown commenced when the pH dropped to 3.5. By the end of the gastric phase, 11.8 % of the protein and 3.0 % of free amine groups were released, generating new peptides of 0.3-1 kDa. Followed by 2 h static intestinal digestion, some cell structures remained intact, indicating a barrier to nutrient release. Pancreatic enzymes caused substantial protein hydrolysis, generating a higher fraction of 0.1-0.3 kDa peptides, with a notable release of essential amino acids as well as phenolic compounds. This study highlighted that protein insolubility and the cell wall structure of C. vulgaris may impede enzyme effectiveness, leading to a reduced protein breakdown. Furthermore, introduction of processing steps may enhance bioaccessibility in microalgae-derived foods, thereby contributing to the development of nutritional and sustainable food productions.

Whole milk powder (WMP) produced from pasture-fed dairy herds has been shown to have increased concentrations of unsaturated fatty acids (FA), including, but not limited to, ALA (C18:3 n-3) and oleic acid (C18:1 cis-9), compared to WMP derived from indoor herds consuming total mixed ration (TMR). Dairy products have been shown to have neutral or beneficial effects on cardiometabolic health, however, evidence on WMP in this area is lacking. Given the global market for WMP as a food ingredient, an investigation into the effect of TMR-fed vs pasture-fed WMP on human health is warranted. Therefore, the aim of this study was to test the effect of WMP derived from TMR-fed and pasture-fed herds on circulating FA concentrations and other markers of cardiometabolic health in adults. Healthy subjects were randomized to receive up to 200 g WMP/day as part of a 6-week crossover trial, with a 4-week washout period. WMP was produced from milks of TMR-fed and pasture-fed cows. WMPs were isocaloric and macro-nutrient matched. The primary outcome was difference in circulating FA between weeks 0 and 6. Changes in cholesterol concentrations, glycemic control, blood pressure and anthropometry were secondary outcomes. Dietary intake was also analyzed. A total of n 29 participants, 58.6% male, with a mean±SE age of 39.8±2.3 years and BMI of 26.12±1.39 kg/m2 completed the study protocol. No time × treatment interactions were observed for cholesterol concentrations, markers of glycemic control, blood pressure or anthropometry. There was no time × treatment interaction for classes of FA, however, the very long-chain saturated FA, lignoceric acid (C24:0), decreased in response to pasture-fed WMP consumption (-0.05±0.04 %TFA) with no change observed in response to the TMR WMP consumption (0.00±0.04 %TFA, P=.041), although this was not significant after Bonferroni correction for Type 2 error. Chronic, high-dose consumption of TMR and pasture-fed WMP had no effect on cardiometabolic health in healthy adults, despite different fatty acid composition. Comparison of foods with a proven dairy matrix effect, e.g., cheese made from TMR-fed and pasture-fed milks, may have more promising effects on human health. This trial was registered as ISRCTN10490434 (https://doi.org/10.1186/ISRCTN10490434).

The degree of digestion of gluten proteins is thought to be directly linked to their capacity to elicit immune-mediated responses in predisposed individuals. Levels and timing of detection of gluten epitopes in stool samples exhibit high interindividual variability. However, the reasons behind this variability remain unclear and existing data do not allow distinctions between the effects of the upper gastrointestinal tract and colonic fermentation. This ileostomy study aimed to analyse the structural and biochemical degradation of a gluten-containing meal in the terminal ileum and to investigate interindividual variability. Eleven participants consumed the test meal (oat porridge and a wheat breakfast cereal) and ileal effluent was collected once every hour over eight hours for quantification of soluble protein, free amines, potentially immunogenic gliadin fragments, and insoluble nitrogen. The molecular weight distribution of soluble proteins was assessed by SEC-HPLC. Microstructural features were studied using scanning electron and confocal laser scanning microscopy. Total ileal effluent output exhibited a relatively high interindividual coefficient of variation (CV) (40.2%, range: 169.4 to 553.1 g), similar to protein and free amines (46.0% and 40.2%, respectively). The ratios of protein to ileal effluent and of free amines to protein exhibited lower CVs (26.9% and 18.6%, respectively). In contrast, potentially immunogenic gliadin fractions exhibited markedly higher variability (CV = 69.4%, range: 3.1 to 65.7 mg of gliadin equivalents), even after normalization by the gliadin-to-soluble-protein ratio (CV = 63.1%, range: 0.8 to 15.1 mg g-1 of protein). Amino acid sequences recognized by the R5 antibody were detected in both 60% ethanol and aqueous extracts, with water-soluble components dominating gliadin output for almost all participants. The disproportionately higher variability in gliadin excretion compared to total nitrogen, protein, and free amines, suggests that interindividual differences in gluten hydrolysis originate primarily in the upper gastrointestinal tract, indicating that individual characteristics likely modulate the specific capacity to degrade gluten proteins.

The dairy matrix is thought to be responsible for the cholesterol-lowering effects of cheese compared to butter, despite having a high saturated fatty acid (SFA) content. Cheese derived from pasture-fed cows has been shown to have increased concentrations of unsaturated fatty acids, compared to cheese derived total mixed ration (TMR)-fed cows. In n 58 middle-aged, overweight adults, circulating total SFA were significantly lower following consumption of pasture-fed vs TMR-fed cheese (-3.87 +/- 1.09 vs-2.69 +/- 0.79 % total fatty acids, P = 0.038) after 6-weeks when consumed at a dose of 120 g/day. Behenic acid (C22:0, P = 0.015) and alpha-linolenic acid (C18:3 n-3, P = 0.004) decreased to a greater extent following pasture-fed vs TMR-fed cheese. However, these were no longer significant after Bonferroni correction. No other differences were observed between the intervention groups, including blood cholesterol concentrations. Further work with a longer duration and different doses is warranted.