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    Plant-based meat analogues and hybrid meats produced by high-moisture extrusion and high-temperature shear processing : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand. EMBARGOED until 15 November 2027.
    (Massey University, 2024-11-12) Mao, Boning
    Plant-based meat analogues (PBMAs) are primarily made from plant proteins as the foundational ingredients, which undergo thermomechanical processing (TMP) techniques to mimic the fibrous texture and flavour of meat but face issues such as a lack of fibrous structure, low digestibility and deficiencies in essential amino acids. High-moisture extrusion (HME) is the primary processing technique for preparing fibrous meat analogues. This study developed a novel high-temperature shear processing (HTSP) method for producing uniformly consistent hybrid meat analogues and PBMAs. A systematic comparison was conducted between meat analogues prepared via HME and HTSP, focusing on the impact of these TMP on protein conformational changes at the molecular level. It was found that these two techniques distinctly affect the secondary and tertiary conformations of soy, pea, and rice proteins. This study also found that the hybrid meat analogues demonstrated improved protein digestibility and bioavailability compared to PBMAs.
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    Muscle strength and muscle mass in older adults : a focus on protein intake, distribution, and sources : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Albany, New Zealand
    (Massey University, 2023) Hiol, Anne Nadine
    Background: Ageing and obesity, which impair muscle protein synthesis (MPS), are associated with muscle mass and muscle strength loss in older adults. It is recognised that adequate protein intake, distribution and sources contribute to increased MPS and muscle mass in older adults. However, little is known about protein intake, distribution, and sources in New Zealand (NZ) older adults. Furthermore, it is unclear whether dietary protein influences muscle strength. Objectives: This thesis explored muscle strength, muscle mass and dietary protein intake, distribution and sources in community-dwelling older adults living in NZ. To meet this objective, the role of obesity in the relationship between muscle mass and muscle strength was examined. This was followed by an investigation of protein intake, distribution and sources, and their association with muscle strength. Methods: Data were obtained from the Researching Eating Activity and Cognitive Health (REACH) study, a cross-sectional study aimed at investigating dietary patterns and associations with cognitive function and metabolic syndrome in older adults aged 65 to 74 years. Isometric grip strength was measured using a hand grip strength dynamometer (JAMAR HAND). Body fat percentage and appendicular skeletal muscle mass (ASM) (sum of lean mass in the arms and legs) were assessed using dual-energy X-ray absorptiometry (Hologic, QDR Discovery A). The ASM index was calculated by ASM (kilograms, kg) divided by height (meters, m) squared. Dietary intake was collected using a 4-day food record, and the data was entered into FoodWorks 10. Data on absolute daily protein intake (grams, g) were generated. According to the peaks of protein consumption throughout the day, days were divided into three meals: breakfast, mid-day, and the evening meals. Protein sources were classified as meat and fish; plant; or dairy and egg protein sources based on the primary type of protein found in food. The relative protein intakes (g/kg) per day, meal, and source were calculated by dividing the absolute protein intake (g) by each participant's body weight (kg). Statistical analyses: A linear regression analysis was performed to determine the association between muscle mass and muscle strength. This analysis was conducted on males and females based on obesity classifications using body fat percentage (obesity ≥ 30% males, ≥ 40% females). The relative protein intake was compared against a cut-off value of 1.2 g of protein per kg body weight (g/kg BW) per day. The distribution of protein across the three meals was expressed as the coefficient of variance (CV), the average of total protein intake per main meal and the number of meals exceeding 0.4 g/kg BW of protein across the day. Sources of protein intake were assessed at breakfast, mid-day and the evening meals. Results are presented as a percentage of the total protein intake for each meal. Finally, linear regression analyses were conducted separately in males and females to investigate the relationships between BMI- muscle strength and protein intake, distribution and sources, accounting for relevant confounders. Results: Muscle mass was a significant predictor of muscle strength in non-obese participants. However, in participants with obesity, muscle mass was no longer a significant predictor of muscle strength. More than half of the participants had a protein intake of < 1.2 g/kg BW per day (62% females, 57% males). Protein intake was unevenly distributed throughout the day (CV = 0.48 for males and females) and was inadequate for reaching 0.4 g/kg BW at breakfast (for both males and females) and at the mid-day meal for males. The main sources of protein at breakfast were milk (28%), breakfast cereals (22%), and bread (12%); at the mid-day meal, bread (18%), cheese (10%) and milk (9%); and at the evening meal, meat provided over half the protein (56%). In females, relative protein intake was positively associated with muscle strength adjusted BMI (BMI-muscle strength) (r2 = 0.15, ρ < 0.01). Protein derived from either dairy and egg (ρ = 0.03); and plant sources (ρ < 0.01) was related to BMI-muscle strength but not protein from meat and fish (ρ = 0.55). Greater frequency of protein consumption of at least 0.4 g/kg BW per meal was associated with BMI-muscle strength (ρ = 0.01), but the coefficient of variance for protein intake distribution was not related to BMI-muscle strength (ρ = 0.47). There was no relationship between BMI-muscle strength and total daily protein intake, protein from meat and fish; dairy and egg; and plant-based sources, or distribution defined as frequency of protein consumption of at least 0.4 g/kg BW per meal or CV in male older adults. Conclusions: Obesity should be considered when measuring associations between muscle mass and muscle strength in older adults. A higher BMI-adjusted muscle strength was associated with consuming more protein each day and a higher frequency of consumption of a meal containing at least 0.4 g/kg BW; and from dairy and egg; and plant food sources in female older adults. There was no correlation between protein intake, distribution and sources and muscle strength in males. Protein intake was less than 1.2 g/kg BW per day and 0.4 g/kg BW per meal for a large proportion of older adults. At breakfast and the mid-day meals the main sources of protein were from cereals and dairy products, and from meat sources at the evening meal. Further research is needed to investigate how best to optimise protein intake to increase and maintain muscle mass and muscle strength in older adults from the general population.
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    Effect of processing on muscle structure and protein digestibility in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
    (Massey University, 2021) Chian, Feng Ming
    The objective of this thesis was to investigate the effect of processing on meat protein properties, muscle structure and in vitro protein digestibility of beef. Meat processing techniques including pulsed electric field (PEF), shockwave (SW) processing, exogenous enzyme (actinidin) treatment, and sous vide (SV) cooking were explored, either alone or in combination, in this project. This thesis also aimed to study the diffusion of enzymes (actinidin from kiwifruit and pepsin in the gastric juice) into the meat. The first experiment investigated the effect of PEF processing alone on the ultrastructure and in vitro protein digestibility of bovine Longissimus thoracis, a tender meat cut (Chapter 3). It was observed that the moisture content of the PEF-treated samples (specific energy of 48 ± 5 kJ/kg and 178 ± 11 kJ/kg) was significantly lower (p < 0.05) by 1.3 to 4.6 %, compared to the untreated samples. The pH, colour, and protein thermal profile of the PEF-treated muscles remained unchanged. Pulsed electric field treatment caused the weakening of the Z-disk and I-band junctions and sarcomere elongation (25 to 38 % longer) of the muscles. The treatment improved in vitro meat protein digestibility by at least 18 %. In this thesis, the protein digestibility was determined in terms of the ninhydrin-reactive amino nitrogen released during simulated oral-gastro-small intestinal digestion. An enhanced proteolysis of the PEF-treated meat proteins (such as α-actinin and β-actinin subunit) during simulated digestion was also observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The improvement in protein digestibility of the PEF-treated meat was supported by more severe disruption of Z-disks and I-bands observed in PEF-treated samples, at the end of simulated digestion. In the second experiment, PEF treatment (specific energy of 99 ± 5 kJ/kg) was applied to bovine Deep and Superficial pectoral muscles in conjunction with SV cooking (60 ℃ for 24 h) (Chapter 4). This muscle cut was tested as it is a tough cut and requires slow cooking. There was no significant difference detected in the specific activities of the sarcoplasmic cathepsins present in the cytosol between the control and PEF-treated samples, both before and after cooking. In addition, similar micro- and ultrastructures were observed between the control SV-cooked and PEF-treated SV-cooked pectoral muscles. The combined PEF-SV treatment increased the in vitro protein digestibility of the pectoral muscles by approximately 29 %. An improvement in proteolysis of the treated meat proteins (e.g. myosin heavy chains and C-protein) during simulated digestion was also observed using SDS-PAGE. More damaged muscle micro- and ultrastructures were detected in PEF-treated SV-cooked muscles at the end of in vitro oral-gastro-small intestinal digestion, showing its enhanced proteolysis compared to the control cooked meat. Next, the effect of SW processing and subsequent SV cooking on meat protein properties, muscle structure and in vitro protein digestibility of bovine Deep and Superficial pectoral muscles were investigated (Chapter 5 and 6). Shockwave processing (11 kJ/pulse) alone decreased the enthalpy and thermal denaturation temperature of the collagen (p < 0.05) when compared to the raw control, studied using a differential scanning calorimeter. The purge loss, pH, colour, and the protein gel electrophoresis profile of the SW-treated raw muscles remained unaffected. Shockwave processing led to the disorganisation of the sarcomere structure and also modified the protein secondary structure of the myofibres. After subsequent SV cooking (60 ℃ for 12 h), more severe muscle fibre coagulation and denaturation were observed in the SW-treated cooked meat compared to the cooked control. An increase in cook loss and a decrease in the Warner-Bratzler shear force were detected in the SW-treated SV-cooked meat compared to the control cooked meat (p < 0.05). The in vitro protein digestibility of the SW-treated SV-cooked meat was improved by approximately 22 %, with an enhanced proteolysis observed via SDS-PAGE, compared to the control SV-cooked meat. These results were supported by the observation of more destruction of the micro- and ultrastructures of SW-treated cooked muscles, observed at the end of the simulated digestion. The effect of the kiwifruit enzyme actinidin on muscle microstructure was studied using Picro-Sirius Red staining (Chapter 7). Meat samples were subjected to two different conditions, simulating meat marination (pH 5.6) and gastric digestion in humans (pH 3). Actinidin was found to have a greater proteolytic effect on the myofibrillar proteins than the connective tissue under both conditions. When compared with pepsin under simulated gastric conditions, actinidin had a weaker proteolytic effect on the connective tissue of cooked meats. Nevertheless, incubating the cooked meat in a solution containing both actinidin and pepsin resulted in more severe muscle structure degradation, when compared to muscles incubated in a single enzyme system. Thus, the co-ingestion of kiwifruit and meat could promote protein digestion of meat in the stomach. In addition, both actinidin and pepsin were successfully located at the edges of the muscle cells and in the endomysium using immunohistofluorescence imaging. The observations suggest that the incubation solutions penetrate into the muscle through the extracellular matrix to the intracellular matrix, enabling the proteases to access their substrates. Overall, the present work demonstrated that there were strong interactions between processing, muscle protein properties and structure, and in vitro protein digestibility of the meat. Processing induces changes in meat protein properties and muscle structure, which in turn affects the digestion characteristics of muscle-based foods.
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    Development of a new meat analogue from soy protein-meat blends : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Riddet Institute, Palmerston North, New Zealand. EMBARGOED until 1 March 2027.
    (Massey University, 2019) Mao, Boning
    Meat analogues are plant protein-based products with similar sensory attributes to meat products. These products are increasingly prevalent, because they not only have high protein and less fat content, but they also have a fibrous nature that contributes to meat-like sensory attributes. Currently, meat analogues are mainly produced by high moisture extrusion using vegetable proteins, such as soy proteins and wheat gluten, because of their ability to form meatlike texturised fibre under thermal and mechanical stress conditions. This study focused on the development of a novel meat analogue with a high shear mixer by using soy protein isolate (SPI)–beef trimmings (BT) blends. It was assumed that the nutritional value, texture and flavour of traditional plant-based meat analogues will be improved by the addition of BT. This high shear mixer is a lab-scale mixer equipped with a shear and high pressure-temperature system, and it has potential to be a novel device for the production of food with a meat-like fibrous structure. Compared with high moisture extrusion, this high shear mixer requires less energy for rotation and heating. In addition, there are several factors (e.g., such as particle size, meat type, mixing time and fat content) that affect the texture of products during extrusion cooking.--Shortened abstract
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    High protein Chinese steamed bread : physico-chemical, microstructural characteristics and gastro-small intestinal starch digestion in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand
    (Massey University, 2019) Mao, Shiyuan
    In Asia, high protein low carbohydrate foods are in high demand because their consumption can provide improved nutritional benefits and help maintaining blood glucose levels close to normal. High protein versions of popular, highly consumed food products (staple foods) such as Chinese steamed bread (CSB) can be very useful to improve the health status of our populations. Thus, the objectives of this study were: to develop high protein Chinese steamed bread (HPCSB) using plant protein, dairy protein combinations. The high protein versions of the steamed breads were then compared with control 100% wheat flour based Chinese steamed bread for physico-chemical, microstructural, textural and in vitro starch digestion characteristics. In order to develop HPCSB, plant proteins (soy protein isolate) and dairy proteins (rennet casein and milk protein concentrate) were blended into wheat flour at two different levels. The addition of proteins has led to a change in colour characteristics (L*, a*, b*) and also resulted in a decreased specific volume of the breads. The textural characteristics measured through textural profile analysis of HPCSB showed an increased hardness and gumminess than control. The microstructure of HPCSB was observed to be more compact and had fewer air cells when observed through Scanning Electronic Microscopy. Furthermore, in vitro starch digestion of HPCSB depicted that the addition of proteins was capable of lowering the starch hydrolysis (%) and estimated glycaemic index (eGI), especially for RC I and RC II at significant levels. Addition of both proteins influenced the microstructure of HPCSBs, which in turn affected the textural and starch digestion properties. High protein Chinese steamed bread with low glycaemic properties can be prepared by critically selecting the protein sources with minimum changes in their physical and textural characteristics.
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    Insulin resistence in adult type-2 diabetic skeletal muscle : the effects of exercise and dietary-protein induced skeletal mucscle plasticity controlling microvascular blood flow and glucose transport : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Sport and Exercise Science), Massey University, Wellington, New Zealand
    (Massey University, 2019) Peeters, Wouter
    Introduction: Insulin-stimulated skeletal muscle glucose uptake is impaired in Type-2 Diabetes Mellitus (T2DM). Insulin resistance leads to reduced skeletal muscle microvascular function and insulin signalling. The purpose of the thesis was to evaluate and compare the effect of chronic intake of a novel keratin-derived protein (WDP) and whey protein, in conjunction with exercise training, on glucose homeostasis and skeletal muscle glucose uptake in T2DM. Methods: In a randomized, double-blinded clinical trial, thirty-five men and women with T2DM completed a 14-week exercise intervention but were randomly assigned to ingest either post-exercise and evening supplements of 20 g WDP-whey protein blend (WDP, n = 11), whey protein (WHEY, n = 12) or isocaloric maltodextrin (CON, n = 12). Before and after the intervention, fasting HbA1c and glucose clearance rate (GCR) during a hyperinsulinaemic isoglycaemic clamp were measured. Insulin-stimulated skeletal muscle blood flow and volume were measured during the clamps via near -infrared spectroscopy. Muscle from the m. vastus lateralis was harvested prior to and at 1-h into the clamps to determine skeletal muscle insulin signalling proteins. Results: Substantially bigger improvements in WDP compared to WHEY or CON were found for GCR, insulin-stimulated GLUT4 translocation and insulin-stimulated blood flow. Fasting eNOSser1177/eNOS possibly increased in WDP and WHEY compared to CON. Capillarization improved in all groups with unclear differences between groups. Conclusion: WDP-whey blend ingestion during 14 weeks of exercise training improved skeletal muscle plasticity and some processes involved in insulin-stimulated glucose uptake to a greater magnitude compared to whey protein or an exercise-only group in T2DM. WDP protein holds the potential to be an additional therapy to exercise as a treatment in T2DM.
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    Gastrointestinal endogenous proteins as a source of bioactive peptides : Doctor of Philosophy in Nutritional Sciences at Riddet Institute, Massey University, Palmerston North, New Zealand
    (Massey University, 2016) Acharya, Prasannalakshmi
    Gastrointestinal endogenous proteins (GEP) were investigated as a source of bioactive peptides. In silico and in vitro methods were used singly or in combination to study GEP-derived peptides after simulated digestion. The presence of bioactive peptides after in vivo digestion was determined using a porcine model. Bioactivity of the peptides was assessed using selected in vitro bioactivity assays, and peptides were characterised using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. In the in silico study, twenty six different GEP and seven dietary proteins were subjected to simulated in silico gastrointestinal (SIGIT) digestion. The predicted resultant peptides possessing amino acid sequences identical to those of known bioactive peptides were identified by screening them against an online database of bioactive peptides (BIOPEP). The predicted number of bioactive peptides released after the SIGIT digestion of GEP ranged from 1 (secretin) to 39 (mucin-5AC), while those for dietary proteins ranged from 1 (gliadin) to 55 (myosin). Angiotensin-I-converting enzyme (ACE-I) inhibitory peptide sequences were found in abundance in both GEP and dietary proteins. The GEP mucin-5AC and the dietary protein myosin were predicted to release the highest number of ACE-I inhibitory peptides (38 and 49 peptides respectively), and were found to be comparable in their potential to release ACE-I inhibitory peptides. Following SIGIT digestion of eleven representative GEP, nineteen novel GEP-derived peptide sequences were selected by applying quantitative structure-activity relationship rules, and were chemically synthesised. Two novel peptides with the amino acid sequences RPCF and MIM, showing dipeptidyl peptidase IV (DPP-IV) inhibitory activity and five novel antioxidant (2,2-diphenyl-1-picrylhydrazyl (DPPH)- inhibitory and, or ferric reducing antioxidant power (FRAP) activity) peptides with amino acid sequences CCK, RPCF, CRPK, QQCP and DCR were identified. These results indicate that GEP may contain novel bioactive peptide sequences. The potential release of bioactive peptides, from four GEP (trypsin, lysozyme, mucin, and serum albumin) and a dietary protein (chicken albumin), in the gastrointestinal tract (GIT) was investigated using an in vitro digestion model. The in vitro digests were screened for ACE-I-, renin-, platelet-activating factor acetylhydrolase (PAF-AH)-, and DPP-IV-inhibition, and antioxidant activity. All four in vitro GEP digests showed ACE-I inhibition comparable to that of the positive control captopril. In comparison to the unfractionated digests, the enriched fractions (<3 and <10 kDa) of lysozyme and serum albumin showed greater renin-, PAF-AH-, and DPP-IV-inhibition, and antioxidant potential. Over 190 peptide sequences were identified from these fractions using mass spectrometry. Stomach chyme (SC) and jejunal digesta (JD) were collected from growing pigs that were fed a protein-free diet for a period of 3 days. The peptides extracted from SC and JD samples were characterized by SDS-PAGE, and their ACE-I-, DPPH-, and microsomal lipid peroxidation (MLP)- inhibition, FRAP activity determined. Potential bioactive peptides responsible for bioactivity were identified using mass spectrometry. SDS-PAGE analysis showed that all of the samples contained a heterogeneous mixture of peptides. Porcine JD samples inhibited ACE-I and DPPH, while SC samples inhibited MLP. Characterization studies identified over 180 peptide sequences from the enriched fractions of SC and JD samples that showed the highest activity. Further, a porcine serum albumin peptide sequence (FAKTCVADESAENCDKS) was found to be a sub-sequence of a larger sequence identified in the in vitro digest of human serum albumin. There was considerable inter-animal variation for the bioactivities. This may be attributed to sampling effects and, or natural variations in the gut contents, thus underlining the complexity involved in in vivo release of bioactive peptides. Together, the results indicate: 1) GEP contain abundant encrypted bioactive peptide sequences; 2) GEP-derived bioactive peptides display a range of bioactivities; 3) GEP-derived bioactive peptides are released during gastrointestinal digestion in pigs; 4) GEP may contain numerous novel bioactive peptide sequences encoded within their primary sequence. In conclusion, the evidence reported here suggests that, like the dietary proteins, GEP are also a potentially rich source of exogenously-derived bioactive peptides in the gastrointestinal tract. Beyond their primary functions, GEP may act as an important cryptomic source of bioactive peptides, given that the amount of GEP secreted into the gut is equal to or greater than the dietary protein ingested per day, and that up to 80% of GEP are known to be digested.
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    Investigation of protein intakes of Māori in advanced age : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Nutrition and Dietetics, Massey University
    (Massey University, 2013) Bennett, Briar
    Aim: Current knowledge of protein intakes of Maori in advanced age is extremely limited. Methods: Dietary intakes of 216 Maori men and women aged 80-90 years were assessed using two 24 hour multiple pass recall dietary recalls. Energy, protein and nutrient intakes were analysed using FOODfiles 2010. Animal vs. plant protein intake, protein intake distribution and protein intake from Kai Maori and contemporary Maori foods protein intakes were examined. Results: Protein intake and percentage of energy as protein consumed met the nutrient reference values for both genders. The intake of animal protein (men = 52.?g, women= 36.6g) was higher than for plant protein (men= 19.Sg, women= 18.5g), and the animal: plant ratios for men and women were 2.63 and 1.94, respectively (p=0.009). Poultry, fish and seafood were the highest contributors of protein intake and percentage energy of protein in men and women. Protein intake at breakfast (men= 11.?g, women= 9.?g) and lunch (men= 16.Sg, women= 14.Sg) were inadequate (<30g protein per meal) and similar between the genders (p>0.05). Men consumed a larger median amount of protein at dinner than women (34.4g versus 23.3g, p<0.001). For men and women respectively there was a low contribution of protein from Kai Maori (median 1.31g and 1.08g) and contemporary Maori foods (median 3.28g and 2.65). Conclusion: Advanced age Maori met the total and percentage of energy protein requirements but protein distribution was inadequate in light of recent evidence. They had a higher intake of animal compared to plant protein foods. Traditional Maori foods contributed only a small proportion of protein to the diets of these advanced age Maori. Key words: protein intake, Maori, nutrition, animal protein, plant protein, protein distribution, traditional food, kai, advanced age, older adults.
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    The impact of post-exercise protein-leucine ingestion on subsequent performance and the systematic, metabolic and skeletal muscle molecular responses associated with recovery and regeneration : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Health), Massey University
    (Massey University, 2012) Nelson, Andre Richard
    The objective was to determine the effect of post-exercise protein-leucine coingestion with carbohydrate and fat on subsequent endurance performance and investigate whole-body and skeletal-muscle responses hypothesised to guide adaptive-regeneration. Methods. Study-JA Twelve trained-men ingested protein/leucine/carbohydrate/fat (20/7.5/89/22 g· h- 1 ) or carbohydrate/fat (control, 119/22 g· h- 1 ) supplements after intense cycling over six days. Glucose and leucine turnover, metabolomics, nitrogen balance and performance were examined. Study-] B Immune-function responses to supplementation were investigated via neutrophil 0 2- production, differential immune-cell count, hormones and cytokines. Study-2A Twelve trained-men ingested low-dose protein/leucine/carbohydrate/fat (23 .3/51180/30 g), high-dose (70115/180/30 g) or carbohydrate/fat control (274/30 g) beverages following 100- min of intense cycling. Vastus lateralis biopsies were taken during recovery (30-min/4-h) to determine the effect of dose on myofibrillar protein synthesis (FSR), and mTOR-pathway activity infened by western blot. Study-2B The transcriptome was intenogated to determine acute-phase biology differentially affected by protein-leucine dose. Results. Protein-leucine increased day-1 recovery leucine oxidation and synthesis, plasma and urinary branch-chain amino acids (BCAAs), products of their metabolism, and neutrophil-priming plasma metabolites versus control. Protein-leucine lowered serum creatine kinase 21-25% (±90% confidence limits 14%) and day 2-5 nitrogen balance was positive for both conditions, yet the impact on sprint power was trivial. Protein-leucine reduced day-1 neutrophil 02- production (15-17 ±20 mmol·02-·celr1 ) but on day-6 increased post-exercise production (33 ±20 mmol·02-·celr1 ) having lowered pre-exercise cortisol (21% ±15%). The increase in FSR with high-dose (0.103%· h-1 ± 0.027%· h-1 ) versus low-dose (0.092%· h-1 ± 0.017%· h-1 ) was likely equivalent. High-dose increased serum insulin (1.44-fold x/+90% confidence limits 1.18), 30- min phosphorylation ofmTOR (2.21-fold x/+1.59) and p70S6K (3.51-fold x/+1.93), and ii 240-min phosphorylation of rpS6 ( 4.85-fold x/-d .37) and 4E-BP1-a (1.99-fold x/-d .63) versus low-dose. Bioinformatics revealed a biphasic dose-responsive inflammatory transcriptome centred on interleukin (IL)-1~ at 30-min (high-dose) and IL6 at 240-min (highdose, low-dose) consistent with regulation of early-phase myeloid-cell associated muscle regeneration. Conclusions. Protein-leucine effects on performance during intense training may be inconsequential when in positive nitrogen balance, despite saturating BCAA metabolism, protein synthesis, and attenuating cell-membrane damage. 24 g of protein and 5 g leucine near saturated post-exercise myofibrillar FSR and simulated an early inflammatory promyogenic transcriptome common to skeletal muscle regeneration that was accentuated with 3-fold higher protein-leucine dose.