Mycoprotein ingestion within or without its whole-food matrix results in equivalent stimulation of myofibrillar protein synthesis rates in resting and exercised muscle of young men

Abstract

Ingestion of mycoprotein stimulates skeletal muscle protein synthesis (MPS) rates to a greater extent than concentrated milk protein when matched for leucine content, potentially attributable to the whole-food nature of mycoprotein. We hypothesised that bolus ingestion of mycoprotein as part of its whole food matrix would stimulate MPS rates to a greater extent compared with a leucine matched bolus of protein concentrated from mycoprotein. Twenty-four healthy young (age; 21±2 y, BMI; 24±3 kg.m²) males received primed, continuous infusions of L-[ring-²H₅]phenylalanine and completed a bout of unilateral resistance leg exercise before ingesting either 70 g mycoprotein (MYC; 31.4 g protein, 2.5 g leucine; n=12) or 38.2 g of a protein concentrate obtained from mycoprotein (PCM; 28.0 g protein, 2.5 g leucine; n=12). Blood and muscle samples (vastus lateralis) were taken pre- and (4 h) post- exercise/protein ingestion to assess postabsorptive and postprandial myofibrillar protein fractional synthetic rates (FSRs) in resting and exercised muscle. Protein ingestion increased plasma essential amino acid and leucine concentrations (P<0.0001), but more rapidly (both 60 vs 90 min; P<0.0001) and to greater magnitudes (1367 vs 1346 μmol·L⁻¹ and 298 vs 283 μmol·L⁻¹, respectively; P<0.0001) in PCM compared with MYC. Protein ingestion increased myofibrillar FSRs (P<0.0001) in both rested (MYC, Δ0.031±0.007%·h⁻¹ and PCM, Δ0.020±0.008%·h⁻¹) and exercised (MYC, Δ0.057±0.011%·h⁻¹ and PCM, Δ0.058±0.012%·h⁻¹) muscle, with no differences between conditions (P>0.05). Mycoprotein ingestion results in equivalent postprandial stimulation of resting and post-exercise myofibrillar protein synthesis rates irrespective of whether it is consumed within or without its whole-food matrix.

Introduction

Dietary protein is essential to support the maintenance of skeletal muscle mass, and to facilitate muscle reconditioning and/or tissue accrual in response to exercise. This is accomplished by the ingestion of dietary protein stimulating muscle protein synthesis (MPS) rates resulting in postprandial muscle protein accretion ⁽¹⁾. The postprandial increase in MPS rates is achieved by a transient rise in circulating amino acids following protein ingestion, which act as both stimulus and substrate ⁽¹⁾. Although essential amino acids are the bulk drivers of postprandial MPS rates ⁽²⁾, the relative increase in MPS is often suggested to be primarily dictated by rapid and/or large postprandial rises in plasma leucine concentrations (coined ‘the leucine trigger’ or ‘threshold’ hypothesis; ^{(3, 4)}). This notion of the nutritional regulation of postprandial MPS has helped understanding of key parameters of protein nutrition, such as optimal amounts ^{(5, 6)}, timing ^{(7, 8)} and daily distribution ⁽⁷⁾, to maximise MPS responses to isolated proteins. However, recent data imply that when protein is consumed within a whole-food source, the role that leucine plays in the postprandial regulation of MPS is more nuanced ^{(9, 10)}.

Mycoprotein is a fungal derived protein rich (~45%) whole-food source, produced by continuous flow fermentation of the filamentous fungi Fusarium venenatum. We reported that mycoprotein ingestion provides a bioavailable source of amino acids but, due to being a fibre rich whole-food, a relatively slow (and low) plasma leucine response ⁽¹¹⁾. Nevertheless, we recently showed that bolus ingestion of mycoprotein increases MPS rates in rested and exercised muscle to a greater extent than a leucine matched bolus of concentrated milk protein ⁽¹²⁾. While the multitude of differences between two entirely different protein sources precluded our being able to determine the causative mechanism, the data ran contrary to the leucine trigger hypothesis and implied non-protein constituents may be involved.

In line with others’ interpretations ^{(9, 10)}, we have speculated such data may be attributable to a dietary protein’s anabolic potency being augmented when present within its specific whole-food matrix. In support, ingestion of whole eggs ⁽¹³⁾ or whole milk ⁽¹⁴⁾ result in greater anabolic responses compared with their isolated counterparts (egg whites and skimmed milk, respectively), despite the whole-food forms eliciting slower and lower plasma leucine responses ⁽¹³⁾. Contrary to these data, casein co-ingested with a serum milk matrix did not increase MPS rates compared with isolated casein ⁽¹⁵⁾, perhaps highlighting a subtle difference from ingesting protein within its natural whole-food as opposed to with added constituent parts (i.e. ‘co-ingestion’). What is presently lacking from the literature are any comparisons of the MPS response to non-animal derived protein sources within and without natural whole-food matrices.

In the present study we hypothesized that the MPS response to bolus mycoprotein ingestion is more contingent on its existence within a whole-food matrix than its ability to elicit rapid or high levels of postprandial leucinaemia. To test this hypothesis, we compared the myofibrillar protein synthesis (MyoPS) response to bolus ingestion of a novel dietary protein concentrate extracted from mycoprotein (PCM; 73 % protein) to that of a naturally produced mycoprotein (MYC; 45 % protein) in resting and exercised muscle of healthy young men.

Methods

Participants

Twenty-four resistance trained, young and healthy men volunteered to take part in the present study (age; 21±2 y, body mass; 78±10 kg, BMI; 24±3 kg.m², 22 Caucasian, 2 Mixed race). Participants’ characteristics are presented in Table 1. Participants were considered resistance trained if they were engaged in resistance training >3 times per week for >3 months prior to taking part in the study. This population was selected as training status impacts the anabolic response to exercise ⁽¹⁶⁾. Therefore, selecting resistance trained individuals ensured optimal exercise execution, (more) ecological validity (to exercise training) and an assumed greater homogeneity of responses to exercise. Subjects had not undergone any previous stable isotope tracer protocols in the previous 6 months ensuring negligible background stable isotope enrichments. Exclusion criteria included any metabolic impairment, cardiovascular complications or allergies to mycoprotein/Quorn/edible fungi or environmental moulds. Subjects were admitted onto the study after being deemed healthy based on blood pressure (<140/90 mmHg), BMI (18-30 kg/m²) and responses to a routine medical health questionnaire. Experimental procedures, potential risks, and the purpose of the study were explained to the participants prior to obtaining informed written consent. This study was approved by the Sport and Health Sciences ethics committee of the University of Exeter (190206/B/07) in accordance with the Declaration of Helsinki and is registered at ClinicalTrials.Gov (NCT04084652). Recruitment and data collection were carried out in the Nutritional Physiology Research Unit at the University of Exeter between April 2019 and December 2019.

Table 1.

Participants’ characteristics

	MYC (n=12)	PCM (n=12)
Age, y	21.3 ± 0.5	21.5 ± 0.9
Height, cm	179.5 ± 1.5	178.1 ± 1.5
Weight, kg	77.8 ± 3.4	77.2 ± 2.1
BMI, kg/m2	24.1 ± 0.9	24.3 ± 0.6
Systolic blood pressure (mmHg)	127 ± 1	126 ± 2
Diastolic blood pressure (mmHg)	70 ± 2	68 ± 2
Fat, % body mass	11 ± 2	10 ± 1.3
Lean mass, kg	69 ± 3	70 ± 2
Leg press 1RM (dominant), kg	157 ± 10	159 ± 9
Leg press 1RM (non-dominant), kg	152 ± 9	156 ± 8
Leg extension 1RM (dominant), kg	61 ± 3	66 ± 4
Leg extension 1RM (non-dominant), kg	61 ± 3	66 ± 4

MYC, mycoprotein; PCM, protein concentrated from mycoprotein; BMI, body mass index; 1RM, one repetition maximum.

Values are mean ± SEM. No significant differences between groups.

Pre-testing

Once accepted onto the study, all participants underwent a pre-testing protocol at least 5 days before a single experimental trial. Participants reported to the laboratory to assess body composition, unilateral leg strength and to become familiarised with the exercise protocol to be used during the experimental trial (described below). Body composition (body fat [%] and lean mass [kg]) was assessed using Air Displacement Plethysmography (BodPod, Life measurement, Inc. Concord, CA, USA). Participants were also provided with a 3-day food diary (two weekdays and one weekend day) to assess habitual dietary intake.

Experimental protocol

Participants were randomly assigned to one of two parallel groups, and completed a single experimental trial in a double-blind manner. An overview of the experimental protocol can be found in Figure 1. Participants were asked to avoid vigorous physical activity and alcohol in the 48 h preceding the trial. All participants were provided with a standardised meal to consume as their last food intake ~10 h before arriving at the laboratory (4.6 MJ [1110 kcal], 29% energy from fat, 46% energy from carbohydrate, 25% energy from protein). On the day of the trial, participants arrived at the laboratory at 8.00 am after a 10 h overnight fast. A Teflon^™ cannula was inserted into an antecubital vein of one arm for the infusion of the stable isotope tracer. Before the infusion was initiated, a baseline venous blood sample was taken from this site to measure background isotopic enrichments. Following the baseline blood sample, the infusion protocol began with a single intravenous priming dose of L-[ring-²H₅]phenylalanine (2.12 μmol/kg) (t= −210 min). After the priming dose, a continuous tracer infusion was initiated (t=−210 min) at a rate of 0.05 μmol·kg⁻¹·h⁻¹ for L-[ring-²H₅]phenylalanine for the duration of the protocol. Once this infusion was in progress, a second Teflon^™ cannula was inserted retrogradely into a dorsal hand vein of the contralateral arm and placed in a heated hand unit (55 °C) to allow for subsequent arterialised venous blood sampling ⁽¹⁷⁾. Following 90 min of continuous infusion (t= −120 min), arterialised venous blood samples were then taken throughout the remainder of the infusion at the following time (t) points: −120, −60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 210 and 240 min. A baseline muscle biopsy sample was collected at t= −120 min from the non-dominant (resting) leg. Muscle biopsies were collected from the m. vastus lateralis (~15 cm from the patella) with a modified Bergström suction needle under local anaesthetic (2% lidocaine). All biopsy samples were immediately freed from any visible blood, connective and adipose tissue before being frozen in liquid nitrogen (within 30-60 s) and stored at −80°C until analysis. At −60 min, participants were taken by wheelchair to the research gymnasium in order to execute a bout of unilateral leg resistance exercise, as described below. Participants were taken by wheelchair back to the laboratory such that no weight bearing was performed with the resting leg throughout the experiment. Following exercise, bilateral muscle biopsies were collected from the rested (at least 1-2 cm distal from the baseline incision) and exercised legs (120 min after the initial biopsy; t= 0). Immediately following the biopsies (t=0), participants consumed either a mycoprotein (MYC, 45% protein, 10% carbohydrate, 13% fat, 25% fibre) or protein concentrated from mycoprotein (PCM, 73% protein, 0.1% carbohydrate, 2% fat, 18% fibre) beverage (administered randomly in a double-blind fashion) within an allotted 5 min time period. Following beverage consumption, participants were asked to identify the protein beverage they consumed, with 67% (11 from 12 in MYC and 5 from 12 in PCM) guessing correctly, implying partial success of the blinding procedures. Participants then rested in a semi-supine position for 4 h, after which another set of bilateral muscle biopsies were collected at least 2 cm distal to the previous incisions, completion of which indicated the end of the experiment after which participants were provided with food and transport home. Average incorporation time between the collection of biopsies for calculation of postabsorptive MyoPS did not differ between groups in either rested (MYC, 128±2min; PCM, 126±1min; P>0.05) or exercised legs (MYC, 123±1min; PCM 122±1: P>0.05). Average incorporation times between the collection of biopsies used to calculate postprandial MyoPS rates did not differ between groups in either rested (MYC, 245±2 min; PCM 244±2 min; P>0.05) or exercised (MYC, 244±1 min; PCM, 244±1 min; P>0.05) legs.

FIGURE 1.

Protocol schematic of the experimental visit.

Resistance exercise protocol

During the pre-testing visit, unilateral 3 repetition max (RM) was assessed to estimate 1RM for leg press and leg extension exercises ⁽¹⁸⁾. The dominant leg (only) was used for the exercise throughout to maximise weight lifted, optimise movement execution and thereby create a maximal exercise stimulus. 3RM, rather than 1RM, was selected to predict 10RM to minimise safety risks. Strength testing began with a brief warm up/practice of each exercise. Thereafter, participants attempted a self-selected weight for 3RM. Weight was increased for each subsequent attempt with final 3RM being accepted as the last weight lifted correctly before a failed attempt (±5kg from failed attempt). 10RM was then calculated as 70% of estimated 1RM. Once 3RM testing had finished, participants rested for ~5 min and were then asked to complete one set (~10 repetitions) at the calculated 70% 1RM for familiarisation and verification purposes.

For the experimental trial, participants completed a brief warm-up of leg press exercise consisting of 10-repetitions at 50% 1RM followed by 8-repetitions at 60% 1RM performed unilaterally with the dominant leg as per during pre-testing. Thereafter, participants executed 4 unilateral sets of leg press followed by 4 sets of leg extension, each separated by 120 s rest. Sets were performed at 70% 1RM and participants were encouraged to work to volitional failure during each set, aiming to fail at ~10 repetitions. For subsequent sets, the weight was increased when participants were able to perform >12 repetitions and decreased when participants were unable to perform 8 repetitions. Verbal encouragement was provided throughout, and the rested, non-dominant leg was kept relaxed and unloaded throughout.

Protein beverage preparation

Freeze dried mycoprotein and protein isolated from mycoprotein were produced and provided by Marlow Foods Ltd, Quorn Foods, Stokesley, UK. Natural mycoprotein was produced as previously described ⁽¹⁹⁾. Briefly, Fusarium venenatum for mycoprotein production was grown via continuous aerobic flow fermentation, with the addition of carbohydrate and ammonia substrates, under tightly controlled conditions (temperature 28-30°C and pH 6.0). The mycelium of the fungus is heat-treated (72-74°C for 30-45 min) to reduce ribonucleic acid concentrations, then further heat treated at 90°C. The suspended hyphae are then centrifuged with the resultant solid mass further concentrated by vacuum chilling. The produced mycoprotein was then milled and freeze dried to produce a protein powder (45% protein). For the production of the novel protein concentrate, 250 kg of frozen mycoprotein pads (dry matter 23.9%; protein concentration 140 g/kg) was passed through a frozen meat grinder. This was then added to an agitator extraction tank with 7.4 L water + 0.167 L of 20% sodium hydroxide per kg of mycoprotein pad. This was then centrifuged and any non-soluble material removed. The remaining protein solution was then precipitated by the addition of sulphuric acid, reducing the pH to 3.5. The precipitated protein was then centrifuged and the supernatant removed. The resulting precipitate was suspended and washed in 1500 L of water. This wash step was repeated before being centrifuged and the water supernatant removed. Following removal of the water, the pH was then adjusted to 5.5 by adding sodium hydroxide and the sample was then dried. Finally, the protein went through an ethanol wash and evaporation step to maximise the removal of residual lipids, producing 8.1 kg of protein concentrate powder (73% protein). Both protein sources were independently analysed (Premier Analytical Services) for energy, macronutrient content and amino acid composition, the details of which are displayed in Table 2.

TABLE 2.

The nutritional composition of the experimental beverages (70 g and 38.2 g of mycoprotein and protein concentrated from mycoprotein, respectively).

	MYC	PCM
Macronutrients
Protein, g	31.5	28.0
Carbohydrate, g	7	0
Fat, g	9	0.6
Fibre, g	17.5	7.0
Energy, kcal	238	132
Energy, kJ	996	558
Amino acid content, g
Alanine	2.0	1.8
Arginine	2.2	2.0
Asparagine	3.3	3.0
Cysteine	0.2	0.2
Glutamine	3.9	3.7
Glycine	1.5	1.4
Histidine	0.8	0.7
Isoleucine	1.5	1.6
Leucine	2.5	2.5
Lysine	2.6	2.4
Methionine	0.4	0.5
Phenylalanine	1.5	1.4
Proline	1.6	1.3
Serine	1.6	1.4
Threonine	1.7	1.6
Tryptophan	0.4	0.4
Tyrosine	1.2	1.2
Valine	1.9	1.9

Protein content (g) is calculated from the sum of the amino acids measured after complete hydrolysis.

MYC, mycoprotein; PCM, protein concentrated from mycoprotein.

The evening before the experimental trial the powdered protein sources were assimilated with 400 mL water and 20 mL energy free flavouring (Clearwater, US.), blended for ~2 min and refrigerated overnight (420 mL final volume). Drinks were enriched (2%) with L-[ring-²H₅]phenylalanine to maintain systemic isotopic steady state following protein ingestion ⁽¹²⁾. During the experimental trial, once participants had consumed the drink, an additional 100 mL water were added to ‘rinse’ the bottle and ensure that all the protein had been consumed. All drinks were well tolerated and consumed within the allotted 5 min (average time to consume was 3.7±0.5 and 3.6±0.6 min for MYC and PCM, respectively; P>0.05) with no adverse effects reported during the test day or on follow-up calls on subsequent days. Once consumed, participants were asked to subjectively rate the drink from 1 (bad) to 10 (good) for tolerability (MYC, 4.2±0.5; PCM, 5.0±0.5; P>0.05). Double blinding of the drinks was achieved by having a separate researcher from those carrying out the experimental trial visits prepare the drinks in an opaque bottle. The drinks were matched for leucine content (2.5 g) requiring 70 and 38.2 g powder thereby providing 31.4 and 28.0 g protein in MYC and PCM, both respectively.

Blood sample collection and analyses

Ten mL of arterialised venous (with the exception of the baseline sample which was a venous collection) blood were collected into a syringe at each time point. Five mL of that sample were added to EDTA containing tubes (BD vacutainer LH; BD Diagnostics, Nu-Care) and centrifuged for 10 min at 4000 rpm at 4°C. The plasma supernatant was then removed, aliquoted and stored at −80°C for later analyses. The remaining 5 mL of blood were added to additional vacutainers (BD vacutainers SST II, BD Diagnostics, Nu-Care) and left upright to clot at room temperature for 30 min and then centrifuged for 10 min at 4000 rpm at 4°C. The serum supernatant was then removed, aliquoted, and stored at −80°C for future analyses.

Serum insulin concentrations were measured using a commercially available ELISA kit (DRG Insulin ELISA, EIA-2935, DRG International Inc, Springfield, USA). Plasma L-[ring-²H₅]phenylalanine enrichments (tracer/tracee ratio and MPEs) and concentrations of phenylalanine, leucine, valine, isoleucine, lysine, histidine, glutamic acid, methionine, proline, serine, threonine, tyrosine and alanine were determined in tert-butyldimethylsilyl derivatives by GC-MS with electron impact ionization (Agilent, Santa Clara, CA, USA) as described previously ⁽²⁰⁾. Briefly, to prepare samples for GC-MS, 10 μL of 2 mM nor-leucine was added as an internal standard to 450 μL plasma and deproteinised on ice with 450 μL of 15% 5-sulfosalcylic acid. Samples were then vortexed and centrifuged at 4000 rpm for 10 min at 4°C. The supernatant was then loaded onto cation exchange columns. Columns were then filled with ddH₂O, followed by 6 mL 0.5 M acetic acid and then washed once more with ddH₂O, with the columns allowed to drain between each step. The amino acids were then eluted with 2 mL of 6 M ammonia hydroxide (NH₄OH). The eluate was dried using a Speed-Vac for 8 h at 60°C.

Muscle tissue analyses

Myofibrillar protein extractions were performed as previously described ⁽²¹⁾. The process was carried out with ~50 mg of muscle tissue, which was homogenised using a mechanical glass pestle in a glass tube in homogenization buffer (in mM: 50 Tris·HCl pH 7.4, 1 EDTA, 1 EGTA, 10 b-glycerophosphate salt, 50 NaF, and 0.5 activated Na3VO4; [Sigma-Aldrich Company Ltd., Poole, UK] with a complete protease inhibitor cocktail tablet [1 tablet per 50 mL of buffer; Roche, Burgess Hill, UK]). The homogenate was transferred into a clean 2 mL eppendorf and centrifuged at 2200 g for 10 min at 4°C. The supernatant (sarcoplasmic fraction) was aliquoted and stored at −80°C for subsequent western blot analysis. The remaining pellet was then washed in 500 μL of homogenization buffer and centrifuged again at 700 g for 10 min at 4°C and the resultant supernatant discarded. The remaining protein portion (myofibrillar and collagen) ⁽²²⁾ was then solubilised in 750 μL of 0.3 M sodium hydroxide and heated at 50°C for 30 min and centrifuged at 10,000 g for 5 min at 4°C. The supernatant (myofibrillar fraction) was then aliquoted into a new 2 mL eppendorf and precipitated in 500 μL of 1 M perchloric acid. These samples were centrifuged at 700 g for 10 min at 4°C and the resultant supernatant discarded. The remaining myofibrillar pellet was washed in 1 mL 70% ethanol and centrifuged at 700 g for 5 min at 4°C before the ethanol was removed. This step was repeated once more before the amino acids were then hydrolysed by adding 2 mL of 6 M hydrochloric acid and heating at 110°C for 24 h. Once hydrolysed, the amino acids were then dried using a Speed-Vac for 4 h at 80°C. Samples were then reconstituted in 1.5 mL 25% acetic acid and pipetted into the cation exchange column. The Eppendorf was then rinsed with another 1.5 mL 25% acetic acid. The columns were then eluted with 2 mL of 6M NH₄OH into a 2 mL Eppendorf and the eluate dried in a Speed-Vac for 8 h at 60°C. Samples were cleaned by adding 1 mL ddH₂O and 1 mL 0.1% formic acid in acetonitrile and centrifuged at 10,000 g for 3 min at 4°C. The supernatant was aliquoted into a new Eppendorf and dried in the Speed-Vac for 5 h at 60°C. In order to derivatise the muscle sample, 50 μL MTBSTFA + 1% tertbutyl-dimethylchlorosilane and 50 μL acetonitrile were added to the dry samples, vortexed and heated at 95°C for 45 min ⁽²³⁾. The samples were analysed by GC-MS (7890 GC coupled with a 5975 MSD; Agilent Technologies) in triplicate using electron impact ionization and selected ion monitoring for measurement of isotope ratios ⁽²⁴⁾. One μL of the sample was injected in splitless mode (injector temperature: 280°C). Peaks were resolved using an HP5-MS 30 m × 0.25 mm ID × 0.25 μm capillary column (Agilent). Helium was used as the carrier gas at 1.2 mL/min constant flow rate. The temperature ramp was set from 80-245°C at 11°C/min, then to 280°C at 40°C/min ⁽²⁴⁾. Selected ion recording conditions were used to monitor fragments m/z 237 and 239 for the m + 3 and m + 5 fragments of phenylalanine-bound protein and m/z 336 and 341 for the m + 0 and m + 5 fragments of the phenylalanine-free fraction. A single linear standard curve from mixtures of known m+5/m+0 ratios for L-[ring-2H5]phenylalanine was used to determine the enrichments of the protein-bound samples using the m+5/m+3 ratio.

The sarcoplasmic fractions obtained from the myofibrillar amino acid extractions were analysed for total and phosphorylated mammalian target of rapamycin (mTOR and p-mTOR Ser²⁴⁴⁸, respectively) contents as an indirect measure of mTOR activity. Total protein content of the sarcoplasmic fraction was measured using a colorimetric assay (DC protein assay, Bio-Rad Laboratories, Inc.). Proteins were unfolded at 95°C in XT sample buffer (Bio-Rad Laboratories, Inc.) and 20 μg of protein was then loaded per lane in duplicate onto a 3-8% tris acetate polyacrylamide gel and separated by electrophoresis in XT tricine running buffer for 65 min at 150 V. Proteins were transferred to a 0.2 μM nitrocellulose membrane using Trans-blot turbo transfer system (Bio-Rad Laboratories, Inc.) at 2.5 A and 25 V for 10 min. Membranes were blocked in 5% bovine serum albumin in tris buffered saline, 0.1% Tween (TBST) for 1 h, before incubation in rabbit anti-phospho-mTOR Ser²⁴⁴⁸ monoclonal antibody (5536, Cell Signaling Technology, Inc.; 1:1000 in TBST) and rabbit anti-α-tubulin (11H10, Cell Signaling Technology, Inc.; 1:20,000 in TBST) loading control overnight at 4°C. Membranes were washed (3 × 10 min) in TBST before 1 h incubation in secondary horseradish peroxidase conjugated anti-rabbit IgG antibody (ab6721, Abcam PLC; 1:3000 in TBST) at room temperature. Following another wash (3 × 10 min) in TBST, membranes were sub-merged in Clarity Western chemiluminescent detector solution for 5 min (Bio-Rad Laboratories, Inc.). The membranes were then imaged using a Chemidoc scanner (Bio-Rad Laboratories, Inc.) and band intensities quantified using Image Lab software (Bio-Rad Laboratories, Inc.). The expected migration of p-mTOR (~289 kDa) and α-tubulin (~52 kDa) was confirmed using a kaleidoscope protein ladder (Bio-Rad Laboratories, Inc.). For total mTOR, the membranes were incubated in stripping buffer for 30 min at room temperature (Thermo Fisher Scientific), blocked for 1 h in 5% bovine serum albumin in TBST, and re-probed overnight with an anti-mTOR monoclonal primary antibody (2972, Cell Signaling Technology, Inc.; 1:1000 in TBST) plus anti-α-tubulin at 4°C, and the above steps were repeated to obtain corresponding bands for total mTOR. The band densities of both p-mTOR and total mTOR were calculated as a ratio against the α-tubulin band within each lane, p-mTOR calculated as a ratio of total mTOR (i.e., indicating ‘phosphorylation status’). Fold change in p-mTOR was calculated from basal to fed conditions in rested and exercised legs.

Calculations

The fractional synthetic rates (FSRs) of myofibrillar proteins were calculated using the standard precursor-product equation ⁽²⁰⁾:

$$\text{FSR}(\%\cdot \text{h}-1)=[\Delta {\text{E}}_{\text{p}}\times \text{t}/{\text{E}}_{\text{precursor}}\times \text{t}]\times 100$$

Where ΔEp is the increment in protein bound L-[ring-²H₅]phenylalanine in myofibrillar protein between two muscle biopsies, E_precursor is the average L-[ring-²H₅]phenylalanine enrichment in the plasma precursor pool over time and t indicates the time (h) between two muscle biopsies.

Statistical analysis

A 2-sided power analysis with expected effect sizes estimated from previous research ^{(12, 25)} revealed that n=10 in each group were sufficient to detect expected differences in postprandial MyoPS rates between protein conditions (MYC vs PCM) when using a repeated measures analysis of variance (ANOVA) (P< 0.05, Power 80%, f = 0.67; G*power version 3.1.9.2). Factoring in a 20% drop-out rate, 24 participants were therefore recruited for the study. Statistical significance was set at P<0.05. All calculations were performed on Graphpad 7.1. Participants’ characteristics, total work done and background L-[ring-²H₅]phenylalanine enrichments were analysed using independent samples t-tests. Differences in serum insulin concentrations, plasma amino acid concentrations, plasma tracer enrichments and myofibrillar L-[ring-²H₅]phenylalanine enrichments were compared using two-way (group [MYC vs PCM] x time) repeated measures ANOVA. Separate two-way ANOVA were performed on fasted and fed plasma L-[ring-²H₅]phenylalanine enrichments. Myofilbrillar FSRs were analysed using a three-way (group x time x exercise) ANOVA. Total postprandial amino acid availability was calculated as incremental area under the curve (iAUC) with baseline set as t = 0.

Results

Participants’ characteristics

No differences in body mass, height, BMI, body fat percentage, lean mass or leg strength (1RM) were found between groups (all P>0.05; see Table 1). Total work performed (repetition x load) during the experimental exercise bout did not differ between groups for leg press (MYC, 4401±227 kg; PCM, 4792±325 kg), leg extension (MYC, 1486±97 kg; PCM, 1573±86 kg) or across the full exercise protocol (MYC; 5887±296 kg; PCM; 6366±369 kg) (all P>0.05).

Serum insulin and plasma amino acid concentrations

All serum insulin and plasma amino acid concentration data are presented for n=24 (MYC, 12 and PCM, 12). Serum insulin concentrations over the time-course of the experiment are depicted in Figure 2. Fasting serum insulin concentrations were similar between groups (MYC, 12±5 mU·L⁻¹; PCM, 10±3 mU·L⁻¹). The ingestion of protein significantly increased fasting serum insulin concentrations (time effect; P<0.0001) and to a different extent between groups (time x group interaction; P<0.001). Postprandial serum insulin concentrations in the MYC group were elevated compared with baseline from 30-60 min (P<0.05), peaked at 45 min (20±6 mU·L⁻¹), and had returned to fasting levels by 90 min. In the PCM group, postprandial insulin concentrations rose more rapidly (greater than baseline from 15-60 min; P<0.05) peaked earlier (30 min; at 24±11 mU·L⁻¹), but also returned to fasting concentrations by 90 min). Despite these divergent temporal responses, serum insulin concentrations did not differ between groups at any specific time point. There was a trend for greater postprandial serum insulin iAUC in the PCM compared with MYC group (P=0.085).

FIGURE 2.

Time course (A) and incremental area under the curve (B) (iAUC; calculated as above postaborptive values) of serum insulin concentrations for a 3.5 h postabsorptive period (time course only) and a 4 h postprandial period in healthy resistance trained men. The dashed vertical line represents drink consumption (70 g of mycoprotein containing 31.5 g protein and 2.5 g leucine [MYC; n=12] or 38.2g of protein concentrated from mycoprotein containing 28.0 g protein and 2.5 g leucine [PCM; n=12]), and execution of a bout of unilateral resistance leg exercise. Time course data were analysed using a 2-way repeated measures ANOVA (group x time) with Sidak post-hoc tests used to detect differences at individual time points. iAUC data were analysed using an independent t-test. Time x group interaction; P<0.001. Values are mean ± SEM.

Plasma total (TAA), essential (EAA), branched-chain (BCAA), non-essential (NEAA) and individual amino acid concentrations over the time course of the experiment are presented in Figures 3 and 4. All plasma amino acid concentrations increased following protein ingestion (time effects; all P<0.0001). Plasma concentrations of TAA, NEAA, EAA and BCAA all displayed a more rapid increase following protein ingestion in the PCM compared with MYC group (time x group interactions; all P<0.0001). Following protein ingestion, plasma TAA concentrations remained elevated for longer in the MYC compared with PCM group with higher concentrations observed at 150, 180 and 210 min (all P<0.05). Similar responses were also observed for plasma EAA and BCAA concentrations, with significantly higher concentrations observed post protein ingestion between 150 and 240 min (P<0.05) and 180-240 min (P<0.05), respectively, in MYC compared with PCM. Plasma leucine and isoleucine concentrations increased more rapidly in the PCM group (time x group interaction; P<0.0001) with greater concentrations detected at 15 and 30 min compared with the MYC group (P<0.05). Plasma leucine and isoleucine concentrations remained elevated following protein ingestion for longer in the MYC compared with PCM group with greater concentrations observed between 150 and 240 min (P<0.05). Aside from glutamic acid and proline (time x group interaction, both P>0.05), all individual plasma amino acid and NEAA concentrations increased to differing degrees dependent on the protein source ingested (time x group interactions; all P<0.001).

FIGURE 3.

Time course and incremental area under the curve (iAUC; calculated as above postaborptive values) of amino acids (AA) (A, B), non-essential amino acids (NEAA) (C, D), essential amino acids (EAA) (E, F) and branch chain amino acids (BCAA) (G, H) over a 3.5 h postabsorptive period (time course only) and 4 h postprandial period in healthy resistance trained men. The dashed vertical line represents drink consumption (70 g of mycoprotein containing 31.5 g protein and 2.5 g leucine [MYC; n=12] or 38.2g of protein concentrated from mycoprotein containing 28.0 g protein and 2.5 g leucine [PCM; n=12]), and execution of a bout of unilateral resistance leg exercise. Time course data were analysed using a 2-way repeated measures ANOVA (group x time) with Sidak post-hoc tests used to detect differences at individual time points. iAUC data were analysed using an independent t-test. * denotes individual differences between conditions at that time point and a difference between conditions on the bar graphs (P<0.05). Time x group interaction; all P<0.001. Values are mean ± SEM.

FIGURE 4.

Time course and incremental area under the curve (iAUC; calculated as above postaborptive values) of valine (A, B), leucine (C, D), isoleucine (E, F), alanine (G, H), lysine (I, J), histidine (K, L), glutamic acid (M, N), methionine (O, P), proline (Q, R), serine (S, T), threonine (U, V) and tyrosine (W, X) over a 3.5 h postabsorptive period (time course only) and 4 h postprandial period in healthy resistance trained men. The dashed vertical line represents drink consumption (70 g of mycoprotein containing 31.5 g protein and 2.5 g leucine [MYC; n=12] or 38.2g of protein concentrated from mycoprotein containing 28.0 g protein and 2.5 g leucine [PCM; n=12]), and execution of a bout of unilateral resistance leg exercise. Time course data were analysed using a 2-way repeated measures ANOVA (group x time) with Sidak post-hoc tests used to detect differences at individual time points. iAUC data were analysed using an independent t-test. * denotes individual differences between conditions at that time point and a difference between conditions on the bar graphs (P<0.05). Time x group interaction (all P<0.001) except glutamic acid and proline (both P>0.05). Values are mean ± SEM.

Total postprandial plasma amino acid availabilities, as indicated by iAUC, are also displayed in Figures 3 and 4 for all amino acid parameters (inset graphs). Greater postprandial plasma availability was observed in the MYC compared with PCM group for EAA, serine and threonine (all P<0.05), and trends (all P<0.1) observed for TAA, BCAA, leucine and isoleucine. Postprandial plasma availability for all other amino acid parameters did not differ between groups (all P>0.05).

Plasma and skeletal muscle tracer analysis

The samples of two participants from the PCM group were excluded from tracer analyses due to insufficient muscle tissue. Therefore, all data for plasma and muscle L-[ring-²H₅]phenylalanine are for n=22 (12 MYC; 10 PCM).

The time course of plasma L-[ring-²H₅]phenylalanine enrichments are displayed in Figure 5. Plasma L-[ring-²H₅]phenylalanine enrichments changed over time (time effect; P<0.05) but to the same extent between groups (P>0.05) in the postabsorptive period (time x group interaction; P>0.05). Plasma L-[ring-²H₅]phenylalanine enrichments decreased transiently (between 60 and 120 min, and between 30 and 90 min in MYC and PCM, respectively) following protein ingestion in both groups (time effect; P<0.0001) and more rapidly so in PCM compared with the MYC group (time x group interaction; P<0.0001). By 120 min a steady state had been regained in both groups.

FIGURE 5.

Time course of plasma phenylalanine concentrations (A) and plasma L-[ring-²H₅]phenylalanine enrichments (B) during the experimental trial over a 3.5 h postabsorptive period and 4 h postprandial period in healthy resistance trained men. The dashed vertical line represents drink consumption (70 g of mycoprotein containing 31.5 g protein and 2.5 g leucine [MYC; n=12] or 38.2g of protein concentrated from mycoprotein containing 28.0 g protein and 2.5 g leucine [PCM; n=12]), and execution of a bout of unilateral resistance leg exercise. Time course data were analysed using a 2-way repeated measures ANOVA (group x time) with Sidak post-hoc tests used to detect differences at individual time points. iAUC data were analysed using an independent t-test. * denotes individual differences between conditions at that time point and a difference between conditions on the bar graphs (P<0.05). Time x group interaction; all P<0.001. Values are mean ± SEM. MPE, mole percent excess.

Myofibrillar protein-bound L-[ring-²H₅]phenylalanine enrichments were equivalent between groups at baseline (P>0.05). Myofibrillar protein-bound L-[ring-²H₅]phenylalanine enrichments increased and to the same extent in both groups (time effect; P<0.0001, time x group interaction; P>0.05) during the postabsorptive period in the resting (from 0.0028±0.0003 to 0.0062±0.0008 in MYC and from 0.0035±0.0007 to 0.0068±0.0014 in PCM) and exercised (from 0.0028±0.0003 to 0.0057±0.0006 in MYC and from 0.0035±0.0007 to 0.0059±0.0009 in PCM) legs, with no differences between legs. Protein ingestion increased myofibrillar protein-bound L-[ring-²H₅]phenylalanine enrichments to the same extent between groups (time effect; P<0.0001, time x group interaction; P>0.05) in the resting (from 0.0062±0.0008 to 0.0186±0.0017 in MYC and from 0.0068±0.0014 to 0.0172±0.0021 in PCM; P > 0.05) and exercised leg (from 0.0057±0.0006 to 0.0221±0.0015 in MYC and from 0.0059±0.0009 to 0.0216±0.0021 in PCM; time effect; P>0.05) but to a greater extent in the exercised leg compared with the rested leg (exercise x time interaction; P<0.01).

Myofibrillar FSRs were calculated using the average plasma L-[ring-²H₅]phenylalanine enrichments during the prandial period of interest as the precursor pool (Figure 6). Postabsorptive myofibrillar FSRs were similar between groups in both rested (MYC, 0.031±0.005%·h⁻¹ and PCM, 0.031±0.008%·h⁻¹; P>0.05) and exercised (MYC, 0.026±0.003%·h⁻¹ and PCM, 0.022±0.004%·h⁻¹; P > 0.05) muscle, with no difference between legs. Protein ingestion increased myofibrillar FSRs in rested and exercised muscle in both groups (time effect; P<0.0001) but to a greater extent in exercised compared with rested tissue (exercise x time interaction; P<0.01). The increase in myofibrillar FSRs following protein ingestion was equivalent in both groups in rested and exercised tissue (group x time interaction and group x time x exercise interaction; P>0.05). Myofibrillar FSRs increased from 0.031±0.008 to 0.062±0.007%·h⁻¹ and from 0.026±0.003 to 0.082±0.010%·h⁻¹ in rested and exercised tissue, respectively, in the MYC group, and from 0.031±0.009 to 0.052±0.006%·h⁻¹ and 0.022±0.005 to 0.080±0.010%·h⁻¹ in rested and exercised tissue, respectively, in the PCM group. This also meant that the delta increases from postabsorptive to postprandial myofibrillar FSRs were equivalent between groups in both rested (MYC, Δ0.031±0.007%·h⁻¹ and PCM, Δ0.020±0.008%·h⁻¹) and exercised (MYC, Δ0.057±0.011%·h⁻¹ and PCM, Δ0.058±0.012%·h⁻¹) muscle (group effect; P > 0.05), and values greater in the exercised compared with rested leg (exercise effect; P<0.01).

FIGURE 6.

Myofibrillar protein fractional synthetic rates (FSR) calculated using the plasma L-[ring-²H₅]phenylalanine precursor pool for a postabsorptive (fasted) and postprandial (fed) period (A) and delta FSR change from postabsorptive to postprandial state (B) for both MYC and PCM conditions in rested and exercised (unilateral leg press and leg extension) muscle in health young resistance trained males. Postprandial state represents a 4 h period following drink consumption (70 g of mycoprotein containing 31.5 g protein and 2.5 g leucine [MYC; n=12] or 38.2g of protein concentrated from mycoprotein containing 28.0 g protein and 2.5 g leucine [PCM; n=10]), and execution of a bout of unilateral resistance leg exercise. Data was analysed using a 3-way (protein ingestion x group x exercise) ANOVA. Delta FSR data was analysed using a 2-way (group x exercise) ANOVA. † denotes significant difference between fasting and fed conditions (main effect of protein ingestion; P<0.0001). # denotes a significant difference between rested and exercised tissue in fed conditions (exercise x feeding interaction; P<0.01). Values are mean ± SEM.

Myocellular signalling responses

Myocellular signalling responses (Figure 7) were determined in n=24. Basal muscle total mTOR content and mTOR phosphorylation status did not differ between conditions (P>0.05). Total mTOR content did not change in either group throughout the experiment nor differ between legs (time, exercise and group x time interaction; P>0.05) (Figure 7a). A three-way ANOVA of muscle mTOR phosphorylation status revealed a significant effect of time (P<0.05) and exercise (P<0.05) but did not detect a significant effect of group (P>0.05) (Figure 7b). However, the time effect observed here is likely driven by the high mTOR phosphorylation status in the exercised leg immediately after exercise and therefore does not represent an effect of protein ingestion. Given the effect this time point has on the statistical analysis, and that the primary interest of this experiment is to look at the change from basal to postprandial conditions, this time point (rested fasted and exercise fasted) has been excluded from further statistical analysis. Therefore, all muscle mTOR phosphorylation status data have subsequently been analysed from basal to fed (rested and exercise) conditions within a three-way ANOVA. Muscle mTOR phosphorylation status increased following protein ingestion (time effect; P<0.05); however, the postprandial increase in mTOR phosphorylation did not differ between groups (group x time interaction; P>0.05) or between rested and exercised muscle (exercise x time interaction; P>0.05). There was a trend for differences in mTOR phosphorylation status fold change (from basal to fed state) between groups (group effect; P=0.072) (Figure 7c). However, there was no difference in fold change between rested and exercised legs (exercise effect; P>0.05).

FIGURE 7.

Total skeletal muscle mTOR content (A) muscle mTOR phosphorylation status (as a ratio of phosphorylated to total protein) (B), mTOR phosphorylation status (as a ratio of phosphorylated to total protein) fold change from basal to postprandial conditions in rested and exercised muscle tissue (C) and representative blots (D) for both groups. Basal bars represent total and phosphorylation status of mTOR before exercise and protein ingestion. Fasted bars represent total and phosphorylation status of mTOR immediately following exercise in the rested (rested fasted) and exercised (exercise fasted) legs. Fasted data points were excluded from the statistical analysis as the primary interest in the study is investigating change from basal to fed conditions. Postprandial bars (rested fed and exercise fed) represent total and phosphorylation status of mTOR 4 h following drink consumption (70 g of mycoprotein containing 31.5 g protein and 2.5 g leucine [MYC; n=12] or 38.2g of protein concentrated from mycoprotein containing 28.0 g protein and 2.5 g leucine [PCM; n=12]), and execution of a bout of unilateral resistance leg exercise. Data were analysed using a 3-way (time x group x exercise) ANOVA. Fold change data were analysed using a 2-way ANOVA (group x exercise). All interactions and main effects for total skeletal muscle mTOR content (P>0.05). Significant effect of feeding on mTOR phosphorylation status (time effect P<0.05). All interactions and main effects for mTOR phosphorylation status fold change (P>0.05). † denotes a significant difference from basal condition (P<0.01). Values are mean ± SEM. mTOR, mammalian target of a rapamycin.

Discussion

In the present work we compared the myofibrillar protein synthetic (MyoPS) response to the ingestion of leucine matched boluses of mycoprotein and a protein concentrated from mycoprotein in resting and exercised muscle tissue of young men. We hypothesised that the whole food matrix of mycoprotein would confer anabolic properties resulting in a greater MyoPS response compared with the protein concentrate. In line with our previous work ^{(12, 26)}, mycoprotein ingestion robustly increased resting and post-exercise MyoPS rates; however, contrary to our hypothesis, this stimulation was of a comparable magnitude following ingestion of the protein concentrate, implying no additive anabolic properties of the wholefood matrix.

We developed a novel protein concentrate with the aim of removing the protein from the wholefood matrix, thereby providing a direct comparison between protein delivered both within and without its wholefood matrix. This was a successful model as the removal of the non-protein constituents produced a concentrated protein source (73%) resulting in divergent protein digestion and amino acid absorption kinetics (see Figures 3 and 4) compared with the whole-food version. Despite this, however, we observed equivalent postprandial MyoPS rates across groups in both rested and exercised muscle tissue (see Figures 6). In corroboration, we saw no differences in the muscle mTOR signalling response (Figures 7) between conditions. Though the lack of differences observed in mTOR phosphorylation may be partially attributed to the muscle sampling timing, given that mTOR phosphorylation peaks 90-120min following protein ingestion ^{(27, 28)}, and the lack of assessment of downstream targets (p70SK1/4E-BP1). Nonetheless, collectively these data indicate that the wholefood matrix of mycoprotein did not augment the postprandial muscle anabolic response.

As expected, our novel protein concentrate increased the speed and magnitude at which leucine appeared in the plasma post ingestion compared with its whole food counterpart (also observed in most amino acids measured and, resultantly, insulin; sees Figure 4 and 2). The rapid appearance of leucine and insulin in plasma in the PCM condition likely resulted in greater (and/or quicker) muscle leucine uptake, supported by the tendency for lower postprandial plasma leucine AUC. Therefore, despite leucine matched conditions, the PCM bolus clearly provided a greater (and earlier) plasma and intramuscular leucine stimulus which the leucine trigger hypothesis would imply would confer a greater anabolic response ⁽²⁹⁾. Though the timing of the biopsies in the present study prohibited us from being able to detect a potential earlier (e.g. 2 rather than 4 h) stimulation of MyoPS and mTOR phosphorylaion in the PCM condition, our data contribute towards a growing number of studies that imply the plasma leucinaemic response is more, but not consistently so ^{(30, 31)}, predictive of postprandial MPS rates when comparing consumption of isolated rather than whole-food protein sources ^{(12, 13, 26, 29, 32)}. However, taking the pivotal role of leucine at face-value, it is interesting to speculate as to the role that the wholefood matrix may still have played in the stimulation of MyoPS in the present study. First, an indirect effect is plausible whereby the whole-food/matrix sensitised the muscle to the amino acid/leucine stimulus, such that a lower peak was required to elicit the same MyoPS response compared with the isolated source ⁽¹⁰⁾. Second, the whole-food and/or its matrix may have acted directly upon myocellular anabolic signalling pathways, providing additional (and independent) stimulation of MyoPS from the protein (leucine) alone. Either of these possibilities could plausibly still have been evident in the present work, allowing for an inferior postprandial leucine response to facilitate the same MyoPS.

Our data do not discount the possibility that a whole-food effect concerning postprandial muscle anabolism exists, considering these data are specific only to mycoprotein. However, it is of relevance to consider why a wholefood effect may not have been observable within the constraints of the present design. Although we produced a novel protein concentrate significantly purer in protein than the control condition (i.e. 73 vs 45%) it was not a protein isolate (i.e. >80%) and, therefore, still contained non-protein constituents (Table 2). It is therefore plausible that the concentrate had retained enough of its whole-food nature to maximise any additional anabolic properties. Production of a purer mycoprotein isolate (i.e. >80%) would provide valuable insight into whether the concentrate produced in this study was pure enough to properly test the wholefood hypothesis. Alternatively, it is also possible that a maximal postprandial MyoPS response had been achieved simply by the amount of protein provided, rendering any additional effect of the wholefood delivery redundant. In support, postprandial rates of MPS following unilateral resistance exercise have been reported as maximal following the ingestion of ~20 g of high-quality animal derived proteins ^{(5, 33)} or, alternatively, ~2.5 g of leucine ⁽³⁴⁾. Participants in the present study consumed in excess of this protein amount in both conditions (31.5 g and 28.0 g in MYC and PCM, respectively) and also reached the suggested leucine threshold of 2.5 g. Given that previous evidence for the additional anabolic potential of delivering protein as part of a whole-food has been shown with 18 g of egg protein (~1.6 g leucine) ⁽¹³⁾ or ~8 g of milk protein (~0.8 g leucine) ⁽¹⁴⁾ our data further this debate to imply that a potentiating anabolic effect of wholefood may only be present in response to sub-optimal doses of protein. It therefore appears likely that any non-protein related anabolic effects of a whole food source would be alternative to (and not in addition to) the role of sufficient amino acids.

The present data, nor the preceding discussion, do not offer a satisfactory explanation for our previous observations concerning mycoprotein ingestion stimulating MPS rates to a greater extent than isolated milk protein despite optimal protein (>20 g) and leucine (2.5 g) being provided in both conditions, and greater leucinaemic responses in the milk condition ⁽¹²⁾; neither proposed whole food effects nor plasma leucine kinetics appear responsible. Rather, the present data point towards amino acid factors independent of leucine ⁽²⁶⁾ (e.g. total protein and/or specific amino acids) being of relevance. While human data are lacking, amino acids such as arginine, lysine and methionine are thought to be important from a myocellular signalling ^(35–37) and substrate limitation perspective ^(38–40). Intriguingly, there is also early in vitro evidence suggesting that ergothioneine, a naturally occurring amino acid predominantly found in fungal derived protein sources, can upregulate anabolic signalling pathways ⁽⁴¹⁾. It will be of relevance for future work to establish optimal protein amounts with differing sources, as well as optimal amino acid compositions with consideration beyond leucine.

Finally, some broader context of the present data is worth discussion. Firstly, taking into account some of the wider metabolic impacts of ingesting protein with within a wholefood matrix, we observed an attenuated insulin response following the ingestion of MYC compared with PCM (Figure 2.). While a rise in circulating insulin is necessary to support an increase in MPS ⁽⁴²⁾, regular peaks in insulin have been linked with the development of cardiovascular disease ⁽⁴³⁾. Therefore, in the case of mycoprotein, it might be beneficial to consume as part of the wholefood matrix, given that this will robustly stimulate MPS while resulting in a lower peak in insulin. Secondly, considering the term ‘wholefood’ more specifically, it should be acknowledged that this encompasses a vast variety of food sources that vary in macro- and micro- nutrient content. Therefore, it is plausible, or even likely, that some food matrices may confer divergent anabolic effects. For instance, studies to date demonstrating an anabolic effect of wholefoods have used animal derived food sources only ^{(13, 14)}, rich in certain vitamins (e.g. vitamin D) and fat moieties (e.g. palmitate, cholesterol, omega 3 fatty acids, phospholipids, phosphatidylcholine) that are suggested to modulate myocellular anabolic signalling pathways ^(44–47). Conversely, it is suggested that the wholefood matrix of non-animal derived sources (especially plant-based sources) is inhibitory to plasma amino acid appearance ^(48–50), which may attenuate the postprandial MPS response. However, the present study is the first to investigate a wholefood effect of a non-animal derived protein source. Our data clearly show that the wholefood matrix is not inhibitory to the MyoPS response following ingestion of mycoprotein, as could also reasonably be expected of a high fibre protein source ⁽⁴⁸⁾. This highlights the complexity surrounding the ‘wholefood hypothesis’, suggesting that not all wholefood matrices possess the potential to potentiate (or attenuate) the MPS response. Instead, it is possible that specific nutrients within certain food sources (and delivery of these nutrients within a protein dense food matrix) are key to a food source’s anabolic potency. Given the increasing drive to identify (or optimise) alternative (non-animal) dietary protein sources to support increasing global demands ⁽⁵¹⁾, the future study of a wide range of protein rich whole food sources is clearly of paramount importance.

In conclusion, ingestion of leucine matched boluses of mycoprotein or protein concentrated from mycoprotein result in equivalent stimulation of MyoPS rates in rested and exercised tissue, suggesting that one can consume protein within a wholefood or as an isolated source without compromising the anabolic response. These data demonstrate that the whole-food nature of mycoprotein does not confer additional anabolic capacity, at least when sufficient protein is provided.

Abbreviations:

MYC

mycoprotein

PCM

protein concentrated from mycoprotein

AA

amino acids

BCAA

branch chain amino acids

MPE

mole % excess

mTOR

mammalian target of rapamycin

TBST

tris-buffered saline 0.1% Tween

MPS

muscle protein synthesis

FSR

fractional synthetic rate