Myofibrillar Protein Fractions Research Articles

Effect of different precooling methods on the physicochemical changes, water migration and myofibrillar protein characteristics of pufferfish (Takifugu obscurus) during cold storage

SummaryThe objective of this study was to evaluate the effect of slurry ice (SI) in physicochemical changes, water migration, myofibrillar protein characterisation, and extended shelf life during cold storage of pufferfish (Takifugu obscurus). SI provided faster cooling rate, better structural stability of muscle tissue, and a significant reduction in total viable count (TVC). In addition, the use of SI treatment resulted in a considerable reduction in total volatile basic nitrogen (TVB‐N) and pH. The results of the texture profile analysis (TPA) demonstrated that SI had a significant inhibitory effect on the reduction of hardness, springiness, resilience, and chewiness. In addition, SI treatment better maintained a well‐structured myofibrillar protein stabilising the tertiary structure. Low‐field nuclear magnetic resonance (LF‐NMR) and magnetic resonance imaging (MRI) revealed that SI treatment could retard the water migration. Furthermore, the results of confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) indicated that SI treatment of pufferfish effectively slowed down the degradation of their myofibrillar protein fraction throughout the cold storage.

Changes of Raw Texture, Intramuscular Connective Tissue Properties and Collagen Profiles in Broiler Wooden Breast during Early Storage.

A recently identified broiler myopathy known as wooden breast (WB) is predominantly found in the pectoralis major muscle of fast-growing broiler hybrids and is causing significant losses to the poultry industry. The aim of this study was to investigate the effects of WB syndrome on raw meat texture, purge loss and thermal properties of intramuscular connective tissue of pectoralis major muscle in the early postmortem period (1-3 days). Results showed that the presence of the WB muscles condition at 1 day postmortem was associated with significantly increased stiffness (27.0 N vs. 23.1 N) and significantly increased purge loss (1.8% vs. 1.0%) compared to normal breast (NB). However, on 3 days postmortem, these parameters did not differ between WB and NB groups. Insoluble and total collagen content was significantly higher in WB muscles compared to NB muscles, and the extractability of intramuscular connective tissue (IMCT) of WB was also higher (0.42% vs. 0.37%) compared to NB and remained stable in the early postmortem period. There was significantly lower protein content in the sarcoplasmic protein fraction and myofibrillar protein fraction of WB muscles compared to NB muscles (p < 0.05). The IMCT of these two groups showed different thermal properties, as the enthalpy of denaturation (ΔH) was significantly lower in WB muscles compared to NB muscles. The WB syndrome had a great effect on the texture and connective tissue properties of the meat compared to normal muscle, with a tendency for having a lower purge loss and higher raw meat hardness.

Cytokine Pattern of Peripheral Blood Mononuclear Cells Isolated from Children Affected by Generalized Epilepsy Treated with Different Protein Fractions of Meat Sources.

The objective of the present study was the evaluation of cytokine patterns in terms of TNF-α, IL-10, IL-6, and IL-1β secretion in peripheral blood mononuclear cell (PBMC) supernatants isolated from blood of children affected by generalized epilepsy and treated in vitro with myofibrillar, sarcoplasmic, and total protein fractions of meat and fish sources. Children with generalized epilepsy (EC group, n = 16) and children without any clinical signs of disease, representing a control group (CC group n = 16), were recruited at the Complex Structure of Neuropsychiatry Childhood-Adolescence of Policlinico Riuniti (Foggia, Italy). Myofibrillar (MYO), sarcoplasmic (SA), and total (TOT) protein fractions were obtained from longissimus thoracis muscle of beef (BF) and lamb (LA); from pectoralis muscle of chicken (CH); and from dorsal white muscle of sole (Solea solea, SO), European hake (Merluccius merluccius, EH), and sea bass fish (Dicentrarchus labrax, SB), respectively. PBMCs were isolated from peripheral blood of EC and CC groups, and an in vitro stimulation in the presence of 100 μg/mL for each protein fraction from different meat sources was performed. Data were classified according to three different levels of cytokines produced from the EC group relative to the CC group. TNF-α, IL-10, and IL-6 levels were not affected by different meat fractions and meat sources; on the contrary, IL-1β levels were found to be significantly affected by the tested proteins fractions, as well as different meat sources, in high-level cytokine group. On average, the protein fractions obtained from LB, BF, and CH meat sources showed a higher level of IL-1β than the protein fractions obtained from EH and SB fish samples. When all cytokine classes were analyzed, on average, a significant effect was observed for IL-10, IL-1β, and TNF-α. Data obtained in the present study evidence that the nutritional strategy based on protein from fish and meat sources may modulate the immunological cytokine pattern of infants with generalized epilepsy.

Proximate composition and changes in muscle proteins of dried salted abo (Otolithes ruber)

Dried salted abo is a popular fishery product in San Miguel Bay (SMB), Philippines. However, the development of this food product has not been fully explored. In this study, the proximate composition and muscle protein profile of dried salted abo was investigated after confirming the molecular identification of abo found in SMB. Sequence analysis of 16S ribosomal RNA and cytochrome c oxidase subunit I genes suggested that abo in SMB could be Otolithes ruber. Abo fillets were brined in 4%, 8%, and 12% salt concentrations (w/w) and sun-dried for 6 and 12 hrs. In both 6 hrs and 12 hrs drying time, moisture content decreased in samples brined with 12% salt, suggesting that the salt concentration could be lowered between 8–12% with 6 hrs drying time. Additionally, the proteolytic profiling by SDS-PAGE analysis detected 17 bands in the myofibrillar protein fraction. Subsequent mass spectrometry identified nine proteins, of which the myosin heavy chain, actin, and keratin were degraded while the other proteins remained stable. Moreover, at 12% salt concentration, changes in staining intensities in several proteins were observed at both 6 hrs and 12 hrs drying time. These findings revealed that the applied salting and drying conditions led to the degradation of myosin heavy chain and polymerization of actin, in addition to an overall alteration of protein profile, which could affect the texture of dried abo product. Overall, this study demonstrated molecular evidence for species identification of abo for the first time. The information on proximate composition and changes in the protein profile of dried salted abo obtained from this study could help the local dried fish industry develop high-quality dried abo products with low salt content.

Analysis of Se and Hg biomolecules distribution and Se speciation in poorly studied protein fractions of muscle tissues of highly consumed fishes by SEC-UV-ICP-MS and HPLC-ESI-MS/MS

Distribution of Se and Hg in sarcoplasmic, myofibrillar and alkali-soluble protein fractions extracted from muscle tissue of tuna, swordfish and salmon (wild vs. farmed) is investigated for the first time. SEC-UV-ICP-MS analyses revealed that Se and Hg are mostly bound to proteins of 2–12 kDa and up to 574 kDa, respectively. Moreover, Se and Hg appeared associated to proteins of same molecular weight, evidencing that Se–Hg interaction may occur at the level of the fish tissue evaluated. Important differences were found between farmed and wild salmon, suggesting the effect of the type of feed and growing conditions on Se and Hg content and their distribution through protein fractions. Finally, Se speciation studies performed by HPLC-ICP-MS and confirmed by HPLC-ESI-MS/MS showed SeMeSeCys as the only Se specie found in soluble, sarcoplasmic, myofibrillar and alkali-soluble proteins of all fishes analysed, except in soluble proteins extracted from tuna, where SeMet was also identified.

Early resistance training-mediated stimulation of daily muscle protein synthetic responses to higher habitual protein intake in middle-aged adults.

The ingestion of protein potentiates the stimulation of myofibrillar protein synthesis rates after an acute bout of resistance exercise. Protein supplementation (eating above the protein Recommended Dietary Allowance) during resistance training has been shown to maximize lean mass and strength gains in healthy young and older adults. Here, contractile, oxidative, and structural protein synthesis were assessed in skeletal muscle in response to a moderate or higher protein diet during the early adaptive phase of resistance training in middle-aged adults. The stimulation of myofibrillar, mitochondrial or collagen protein synthesis rates during 0-3weeks of resistance training is not further enhanced by a higher protein diet. These results show that moderate protein diets are sufficient to support the skeletal muscle adaptive response during the early phase of a resistance training programme. Protein ingestion augments muscle protein synthesis (MPS) rates acutely after resistance exercise and can offset age-related loss in muscle mass. Skeletal muscle contains a variety of protein pools, such as myofibrillar (contractile), mitochondrial (substrate oxidation), and collagen (structural support) proteins, and the sensitivity to nutrition and exercise seems to be dependent on the major protein fraction studied. However, it is unknown how free-living conditions with high dietary protein density and habitual resistance exercise mediates muscle protein subfraction synthesis. Therefore, we investigated the effect of moderate (MOD: 1.06±0.22gkg-1 day-1 ) or high (HIGH: 1.55±0.25gkg-1 day-1 ) protein intake on daily MPS rates within the myofibrillar (MyoPS), mitochondrial (MitoPS) and collagen (CPS) protein fractions in middle-aged men and women (n=20, 47±1years, BMI 28±1kgm-2 ) during the early phase (0-3weeks) of a dietary counselling-controlled resistance training programme. Participants were loaded with deuterated water, followed by daily maintenance doses throughout the intervention. Muscle biopsies were collected at baseline and after weeks 1, 2 and 3. MyoPS in the HIGH condition remained constant (P=1.000), but MOD decreased over time (P=0.023). MitoPS decreased after 0-3weeks when compared to 0-1week (P=0.010) with no effects of protein intake (P=0.827). A similar decline with no difference between groups (P=0.323) was also observed for CPS (P=0.007). Our results demonstrated that additional protein intake above moderate amounts does not potentiate the stimulation of longer-term MPS responses during the early stage of resistance training adaptations in middle-aged adults.

The effect of a chemotherapy drug cocktail on myotube morphology, myofibrillar protein abundance, and substrate availability.

Cachexia, a condition prevalent in many chronically ill patients, is characterized by weight loss, fatigue, and decreases in muscle mass and function. Cachexia is associated with tumor burden and disease‐related malnutrition, but other studies implicate chemotherapy as being causative. We investigated the effects of a chemotherapy drug cocktail on myofibrillar protein abundance and synthesis, anabolic signaling mechanisms, and substrate availability. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4 μl/ml DMSO) or a chemotherapy drug cocktail (a mixture of cisplatin [20 μg/ml], leucovorin [10 μg/ml], and 5‐fluorouracil [5‐FLU; 50 μg/ml]) for 24–72 h. Compared to myotubes treated with vehicle, those treated with the drug cocktail showed 50%–80% reductions in the abundance of myofibrillar proteins, including myosin heavy chain‐1, troponin, and tropomyosin (p < 0.05). Cells treated with only a mixture of cisplatin and 5‐FLU had identical reductions in myofibrillar protein abundance. Myotubes treated with the drug cocktail also showed >50% reductions in the phosphorylation of AKTSer473 and of mTORC1 substrates ribosomal protein S6Ser235/236, its kinase S6K1Thr389 and eukaryotic translation initiation factor 4E‐binding protein 1 (all p < 0.05). Drug treatment impaired peptide chain initiation in myofibrillar protein fractions and insulin‐stimulated glucose uptake (p = 0.06) but increased the expression of autophagy markers beclin‐1 and microtubule‐associated proteins 1A/1B light chain 3B (p < 0.05), and of apoptotic marker, cleaved caspase 3 (p < 0.05). Drug treatment reduced the expression of mitochondrial markers cytochrome oxidase and succinate dehydrogenase (p < 0.05). The observed profound negative effects of this chemotherapy drug cocktail on myotubes underlie a need for approaches that can reduce the negative effects of these drugs on muscle metabolism.

Higher Protein Intake Does Not Augment Muscle Protein Synthetic Responses During the Early Stages of Resistance Training in Middle-Aged Adults

Abstract Objectives Dietary protein intake augments muscle protein synthesis rates during acute recovery from resistance exercise training (RET) and can help offset age-related loss in muscle mass. Skeletal muscle tissue contains a variety of different protein pools such as myofibrillar (contractile), mitochondrial (substrate oxidation), and collagen (structural support) proteins that have been reported to have different contraction and nutrient sensitivities in controlled laboratory studies. However, no study has investigated the impact of dietary protein intake on the synthesis of these specific muscle proteins fractions during RET in a free-living setting. Therefore, we investigated the effect of dietary protein intake on daily muscle protein synthesis rates (PS) within the myofibrillar (MyoPS), mitochondrial (MitoPS), and collagen (CPS) protein fractions throughout the early phase (0 – 3 wks) of RET in middle-aged adults. Methods Twenty middle-aged adults (47 ± 1 y, BMI 28 ± 1 kg/m2, M = 12, F = 8) were stratified and randomized to consume either moderate (MOD; 1.06 ± 0.22 g/kg/d) or high (HI; 1.55 ± 0.25 g/kg/d) amounts of protein during a dietary counseling-controlled resistance training program. On day 0, participants ingested intermittent boluses of deuterated (2H) water followed by daily maintenance doses throughout the study period. Muscle biopsies were collected prior to and at weeks 1, 2, and 3 of the intervention to assess MyoPS, MitoPS, and CPS by LC/MS. Results MitoPS decreased at 0–3 wks when compared to 0–1 wks (P = 0.010, CI: −0.77 – −0.10%/d) regardless of group (HI: 0.64 ± 0.32%/d and MOD 0.86 ± 0.55%/d; P = 0.827). A similar decline was also observed for CPS (group P = 0.323; time P = 0.007). The MyoPS in the HI condition remained constant (0–1 wk: 0.81 ± 0.42%/d and 0–3 wks: 0.81 ± 0.72%/d, P = 1.0), but MOD decreased over time (0–1 wk: 1.35 ± 0.9%/d and 0–3 wks: 0.77 ± 0.43%/d, P = 0.023). Conclusions Our results demonstrated that additional protein intake above moderate amounts does not meaningfully potentiate the stimulation of longer-term muscle protein synthetic responses within the myofibrillar, mitochondrial, or collagen protein fractions during the early stages of a RET in middle-aged adults. Funding Sources Funded in part by the Beef Checkoff. AFS funded by CAPES Scholarship.

Effects of Whey Protein Isolate Ingestion on Muscle Protein Synthesis at Rest and After Resistance-exercise in Middle-aged Men: A Research Protocol

Introduction: Sarcopenia is a syndrome primarily found in elderly individuals and is defined by decreased skeletal muscle mass and strength. Sarcopenia often increases the risk or is comorbid with various diseases. Thus, the objective of this study is to determine the appropriate dosage of whey protein isolate (WPI) for middle-aged men at rest and after resistance-exercise for maximum muscle protein synthesis (MPS) and to reduce the risk of sarcopenia. Methods: 50 healthy men (age 45± 5yrs, BMI &lt; 30 kg/m2) will be randomly assigned an amount of ingested WPI (0g,10g,20g,30g,40g). 24 hours prior, a baseline saliva sample and ingestion of 5 doses of 150 mL bolus (70% D2O) every 2 hours will be completed at the participants’ own homes. On the day of the study, a saliva sample will be taken 1 hour before 10 repetitions of maximum unilateral leg extension. After the exercise protocol (t = 0 h), saliva samples will be obtained every hour until t = 6 h and 3 muscle biopsies at t = 0 h, t = 3 h, and t = 6 h. After isolating myofibrillar protein fractions from the biopsies, the samples will be analyzed using gas chromatography-pyrolysis-isotope ratio mass spectrometer (GC-P-IRMS). Analysing saliva samples will be conducted using temperature conversion elemental analyzer (TC/EA) followed by isotope ratio mass spectrometer (IRMS). Atom percent excess for both deuterium enrichment of protein-bound alanine and precursor enrichment will be conducted to allow the calculation of fractional synthetic rate (FSR, %/h). Discussion: We anticipate FSR (%/h) will be significantly greater in the exercised leg compared to the unexercised leg at each WPI ingestion dosage. Furthermore, we anticipate that ≥20 g of ingested WPI will be sufficient for maximal MPS at rest. Finally, we anticipate that at ≥30 g ingestion of WPI will be sufficient for maximal MPS after resistance training. It is beneficial for physicians to know the optimal dose for MPS in middle-aged adults, so they can suggest more accurate recommendations of proactive protein intake. This will help alleviate the high prevalence of sarcopenia and its detrimental effects on health, healthcare, and the economy.

Comparative Study on Pale, Soft and Exudative (PSE) and Red, Firm and Non-Exudative (RFN) Pork: Protein Changes during Aging and the Differential Protein Expression of the Myofibrillar Fraction at 1 h Postmortem.

In this paper, the protein changes during aging and the differences in the myofibrillar protein fraction at 1 h postmortem of pale, soft and exudative (PSE), and red, firm and non-exudative (RFN) pork longissimus thoracis (LT) were comparatively studied. The PSE and RFN groups were screened out based on the differences in their pH and lightness (L*) at 1 h, and their purge loss at 24 h postmortem. Based on the measured MFI, desmin degradation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, PSE meat presented more significant changes in the myofibrillar protein fraction compared to RFN meat during postmortem aging. Through liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS) analysis, a total of 172 differential proteins were identified, among which 151 were up-regulated and 21 were down-regulated in the PSE group. The differential proteins were muscle contraction, motor proteins, microfilaments, microtubules, glycolysis, glycogen metabolism, energy metabolism, molecular chaperones, transport, and enzyme proteins. The AMP activated protein kinase (AMPK) signaling pathway, HIF-1 signaling pathway, calcium signaling pathway, and PI3K-Akt signaling pathway were identified as the significant pathways related to meat quality. This study suggested that the different changes of the myofibrillar protein fraction were involved in the biochemical metabolism in postmortem muscle, which may contribute to the molecular understanding of PSE meat formation.

Effects of Farm Management Practices and Transport Time on Post-Mortem Changes of Longissimus lumborum Muscle Proteins in Suckling Goat Kids.

The combined effect of farm management practices, transport time, and ageing time on the electrophoretic changes of sarcoplasmic (SPP) and myofibrillar (MFP) protein fractions of goat kids was studied. A total of 64 suckling goat kids were withdrawn from two farms with “high” (GW) and “low” (DW) welfare-friendly management practices, and they were transported for 2 or 6 h immediately before slaughtering. Longissimus lumborum samples were obtained at 3, 8, and 21 days post-mortem, and muscle proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE. Both protein extracts displayed significant changes attributable to meat maturation. Managing conditions of kids in DW farms increased the post-mortem susceptibility of muscle proteins. Some MFP of Longissimus lumborum muscle, such as troponin T, as well as 26–30 and 35–37 kDa fractions were influenced significantly by deficient on-farm management, and therefore, these protein fragments might be considered as indicators of low-welfare on-farm management in goat kids.

Walking Or Body Weight Squats May Improve Muscle Dietary Amino Acid Sensitivity During Prolonged Sitting

Low physical activity (e.g. reduced daily steps) reduces the ability of dietary amino acids (AA) to support muscle protein synthesis, leading to eventual muscle loss. Interrupting prolonged sitting with short bouts of intermittent exercise can improve carbohydrate and lipid metabolism, however its ability to sensitize skeletal muscle to dietary AA has yet to be investigated. PURPOSE: To determine the ability of interrupting prolonged sitting with practical ‘activity snacks’ to enhance the postprandial incorporation of dietary AA into myofibrillar protein. METHODS: As a subset of a larger study, twelve participants (7 males and 5 females; ~23y; ~40.0mlO2/kg/min; ~25.1kg/m2; ~4676 steps/d) completed three 7.5 hr trials in a randomized order consisting of prolonged sitting (SIT), sitting with intermittent walking (WLK; 2 min at 3.1mph every 30 min) or sitting with intermittent squatting (SQT; 15 ‘chair stands with calf raise’ every 30 min). Mixed-macronutrient meals (~55:30:15% carbohydrate:fat:protein) were provided at 20% (breakfast) and 30% (lunch) of daily energy requirements to be consistent with Western feeding patterns. Meals were enriched to 15% with ring-[2H5]phenylalanine or ring-[13C6]phenylalanine to model the metabolic fate of dietary AA. Muscle biopsies taken at the end of each trial as well as at the beginning of trial 2 were used to determine change in AA enrichment (LC/MS/MS) in the myofibrillar protein fraction (ΔMyo). RESULTS: ΔMyo was 0.032±0.004MPE in SIT and tended to be greater with SQT (0.038±0.003; P=0.10) and WLK (0.047±0.006; P=0.06) according to a prior comparisons (paired one-tail T-test). Relative to SIT, effect sizes were large for WLK (ES=0.88; 95% CI -0.30 - 2.07) and moderate for SQT (ES=0.55; 95% CI -0.60 - 1.71). CONCLUSION: Interrupting prolonged periods of sitting with intermittent bouts of body weight-dependent activity has the potential to improve the utilization of dietary AA for de novo muscle protein synthesis in young healthy adults. Our results add to the evidence that reducing sedentary time through ‘activity snacks’ may help maintain muscle mass and quality. Future research should determine whether at risk populations (e.g. aging, obese) may obtain a greater benefit from this simple lifestyle modification. Supported in part by an ACSM Research Endowment Grant.

Effect of Feeding a Low Vitamin a Diet to Beef Steers on Calpain 1 Activation during Meat Aging

ObjectivesThe objective of the study was to determine if a vitamin A deficient diet during beef finishing influences calpain 1 activation during meat aging.Materials and MethodsSixty-four steers of approximately 7 mo of age were subjected to a 14-d acclimation period followed by a 95-d growing period on a low vitamin A diet (1017 IU vitamin A/kg DM) designed to deplete liver vitamin A stores. Steers were assigned to a randomized complete blocked design with a 2 × 2 arrangement of treatments (breed: commercial Angus, n = 32, and purebred Simmental, n = 32; and a Low Vitamin A diet or a control diet). The low Vitamin A (LVA) treatment was a finishing diet with no supplemental vitamin A (723 IU vitamin A/kg DM). The control (CON) treatment was the LVA diet plus supplementation with 2200 IU vitamin A/kg DM for a total of 2923 IU vitamin A/kg DM. Serum retinol concentrations were monitored at the beginning and end of treatment. Upon completion of finishing, steers were slaughtered in two groups at a commercial plant. After fabrication, boneless strip loins (IMPS 180) were collected and transported to NDSU. Samples (approximately 40 g) were collected from the anterior portion of the strip loin on d-2 and d-7 of aging and immediately frozen. Protein was extracted from meat samples in fractionation buffers to yield sarcoplasmic and myofibrillar portions, separated by SDS-PAGE, and transferred to PVDF membranes. Immunoblot analysis was done using anti-desmin (d-2 and d-7) and anti-calpain 1 (d-2) antibodies, and results were visualized and documented. A pooled control was run on all membranes and set to a value of one for normalizing results. All experimental data were analyzed using the Proc Mixed procedure of SAS with breed of steers, dietary treatments, their interaction and slaughter date used as a fixed effect.ResultsCalpain 1 autolysis in the sarcoplasmic protein fraction of the d-2 aged loin samples were not affected by treatment or breed. The myofibrillar protein fraction from Angus loins had greater (P = 0.02) accumulation of the 76 kDa calpain 1 autolysis product than that from the Simmental loins; the myofibrillar fraction of the loins from the LVA treatment tended (P = 0.07) to have more 76 kDa calpain 1 autolysis product than that from the CON. There were not any differences (P &gt; 0.19) in the 80 kDa calpain 1 band or the 78 kDa calpain 1 intermediate autolysis product in the myofibrillar fraction. There was a treatment by breed interaction (P = 0.01) for desmin in the d-7 aged loins where Angus loins from the CON treatment had less accumulation of the 46 kDa band than Angus loins on the LVA treatment and Simmental loins from either treatment.ConclusionVitamin A restriction increased protein proteolysis in Angus but not in Simmental steers. The increased calpain 1 autolysis in Angus vs. Simmental, regardless of Vitamin A treatment, indicates a genetic difference that may be the driver for the increased protein degradation in steers a restricted vitamin A diet.

Maximizing Post-exercise Anabolism: The Case for Relative Protein Intakes.

Maximizing the post-exercise increase in muscle protein synthesis, especially of the contractile myofibrillar protein fraction, is essential to facilitate effective muscle remodeling, and enhance hypertrophic gains with resistance training. MPS is the primary regulated variable influencing muscle net balance with dietary amino acid ingestion representing the single most important nutritional variable enhancing post-exercise rates of muscle protein synthesis. Dose-response studies in average (i.e., ~80 kg) males have reported an absolute 20 g dose of high quality, rapidly digested protein maximizes mixed, and myofibrillar protein synthetic rates. However, it is unclear if these absolute protein intakes can be viewed in a “one size fits all” solution. Re-analysis of published literature in young adults suggests a relative single meal intake of ~0.31 g/kg of rapidly digested, high quality protein (i.e., whey) should be considered as a nutritional guideline for individuals of average body composition aiming to maximize post-exercise myofibrillar protein synthesis while minimizing irreversible amino acid oxidative catabolism that occurs with excessive intakes of this macronutrient. This muscle-specific bolus intake is lower than that reported to maximize whole body anabolism (i.e., ≥0.5 g/kg). Review of the available literature suggests that potential confounders such as the co-ingestion of carbohydrate, sex, and amount of active muscle mass do not represent significant barriers to the translation of this objectively determined relative protein intake. Additional research is warranted to elucidate the effective dose for proteins with suboptimal amino acid compositions (e.g., plant-based), and/or slower digestion rates as well as whether recommendations are appreciably affected by other physiological conditions such endurance exercise, high habitual daily protein ingestion, aging, obesity, and/or periods of chronic negative energy balance.

Comparative efficacy of actinidin from green and gold kiwi fruit extract on in vitro simulated protein digestion of beef Semitendinosus and its myofibrillar protein fraction

SummaryThis study was conducted to evaluate the comparative efficacy of actinidin from green and gold kiwifruit extract on in vitro simulated gastrointestinal protein digestion of cooked beef Semitendinosus and its myofibrillar protein fraction. Samples collected during gastrointestinal digestion were analysed for protein profile, protein digestibility (%), soluble protein (%) and free amino acid analysis. The addition of kiwifruit extracts significantly (P &lt; 0.05) increased protein digestibility, soluble protein and free amino acids released during in vitro gastrointestinal digestion. The in vitro digestibility (%) of the meat samples digested along the green or the gold kiwifruit extract was 2.08% and 3.47% higher than control samples respectively. The soluble protein (%) of the meat samples digested along the green kiwifruit extract was significantly (P &lt; 0.05) higher (18.28%) than control samples (13.03%) after gastrointestinal digestion. Comparatively, actinidin from green kiwifruit extract was more effective than gold kiwifruit extract.

Physico‐chemical and proteolytic changes during cold storage of dry‐cured pork loins with probiotic strains of LAB

SummarySelected physico‐chemical and proteolysis‐related parameters were evaluated in dry‐cured loins inoculated with probiotic (Lactobacillus rhamnosus LOCK900, Bifidobacterium animalis subsp. lactis BB12) and potentially probiotic (Lactobacillus acidophilus Bauer Ł0938) lactic acid bacteria (LAB) strains and compared with a control sample at five different storage times (28, 90, 180, 270 and 360 days). Physico‐chemical parameters of dry‐cured loins were significantly (P &lt; 0.01) affected by treatment, storage time and the interaction between them. The count of lactic acid bacteria (LAB) in dry‐cured loins was in accordance with their pH. Higher intensity of proteolysis, as assessed by non‐protein nitrogen content (NPN) and proteolysis index (PI), occurred in inoculated samples. Based on the SDS‐PAGE analysis, the modifications produced during storage on the sarcoplasmic proteins were more pronounced than those exerted on the myofibrillar protein fraction. However, inoculation effect was absent from SDS‐PAGE protein patterns.

Effect of Feeding a Low Vitamin a Diet to Beef Steers on Calpain 1 Activation during Meat Aging

ObjectivesThe objective of the study was to determine if a vitamin A deficient diet during beef finishing influences calpain 1 activation during meat aging.Materials and MethodsSixty-four steers of approximately 7 mo of age were subjected to a 14-d acclimation period followed by a 95-d growing period on a low vitamin A diet (1017 IU vitamin A/kg DM) designed to deplete liver vitamin A stores. Steers were assigned to a randomized complete blocked design with a 2 × 2 arrangement of treatments (breed: commercial Angus, n = 32, and purebred Simmental, n = 32; and a Low Vitamin A diet or a control diet). The low Vitamin A (LVA) treatment was a finishing diet with no supplemental vitamin A (723 IU vitamin A/kg DM). The control (CON) treatment was the LVA diet plus supplementation with 2200 IU vitamin A/kg DM for a total of 2923 IU vitamin A/kg DM. Serum retinol concentrations were monitored at the beginning and end of treatment. Upon completion of finishing, steers were slaughtered in two groups at a commercial plant. After fabrication, boneless strip loins (IMPS 180) were collected and transported to NDSU. Samples (approximately 40 g) were collected from the anterior portion of the strip loin on d-2 and d-7 of aging and immediately frozen. Protein was extracted from meat samples in fractionation buffers to yield sarcoplasmic and myofibrillar portions, separated by SDS-PAGE, and transferred to PVDF membranes. Immunoblot analysis was done using anti-desmin (d-2 and d-7) and anti-calpain 1 (d-2) antibodies, and results were visualized and documented. A pooled control was run on all membranes and set to a value of one for normalizing results. All experimental data were analyzed using the Proc Mixed procedure of SAS with breed of steers, dietary treatments, their interaction and slaughter date used as a fixed effect.ResultsCalpain 1 autolysis in the sarcoplasmic protein fraction of the d-2 aged loin samples were not affected by treatment or breed. The myofibrillar protein fraction from Angus loins had greater (P = 0.02) accumulation of the 76 kDa calpain 1 autolysis product than that from the Simmental loins; the myofibrillar fraction of the loins from the LVA treatment tended (P = 0.07) to have more 76 kDa calpain 1 autolysis product than that from the CON. There were not any differences (P > 0.19) in the 80 kDa calpain 1 band or the 78 kDa calpain 1 intermediate autolysis product in the myofibrillar fraction. There was a treatment by breed interaction (P = 0.01) for desmin in the d-7 aged loins where Angus loins from the CON treatment had less accumulation of the 46 kDa band than Angus loins on the LVA treatment and Simmental loins from either treatment.ConclusionVitamin A restriction increased protein proteolysis in Angus but not in Simmental steers. The increased calpain 1 autolysis in Angus vs. Simmental, regardless of Vitamin A treatment, indicates a genetic difference that may be the driver for the increased protein degradation in steers a restricted vitamin A diet.

Proteolysis during fermentation of Suanyu as a traditional fermented fish product of China

ABSTRACTProteolytic changes in Suanyu, a traditional fermented fish product of China, were determined during 6 weeks of fermentation. The gradual decrease of pH value and aw was detected, accompanied with an increasing trend detected in trichloroacetic acid–soluble C-1: peptides concentration. Protein composition changed throughout the fermentation. A decrease in sarcoplasmic and myofibrillar protein fractions was accompanied by an increase in the insoluble fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed most bands of sarcoplasmic, myofibrillar, and insoluble proteins lightened or disappeared due to proteolysis or denaturation. New bands emerged especially in insoluble proteins. Fluctuation of molecular weight distribution of the water-soluble nitrogen in Suanyu was found during fermentation. The highest percentage in Suanyu end-products was observed in the fraction of <0.18 kDa, followed by 0.18–0.5 kDa, 66.02 ± 0.38% and 10.79 ± 0.27%, respectively. Free amino acids analysis showed that nearly all amino acids increased, especially for Asp, Glu, Met, Phe, Leu, Val, and Ile. Overall, intense proteolysis in Suanyu brought numerous of small peptides and free amino acids, which contribute to the taste and flavor of Suanyu.

European Carp (Cyprinus carpio L.) Protein-Derived Ex Vivo Digests and In Vitro Hydrolysates Differ in the ACE I Inhibitory Activity and Composition of Released ACE Inhibitory Peptides.

Carp muscle tissue is a valuable source of biologically active constituents known to positively influence human health. In this study, carp protein digests/hydrolysates generated by human/ porcine digestive enzymes were analysed for their angiotensin I-converting enzyme inhibitory (ACEi) activity. The ex vivo digests and in vitro hydrolysates were used in a screening for ACEi peptides as well. Carp proteins were hydrolysed more rapidly by human gastrointestinal juices than by porcine enzymes. Sarcoplasmic protein fractions were digested/hydrolysed more easily than the myofibrillar ones. The inhibitory concentrations at 50% (IC50) for ex vivo digested sarcoplasmic and myofibrillar protein fractions were established at 1.50 and 1.04 mg/mL, respectively. While, for in vitro hydrolysed sarcoplasmic and myofibrillar protein fractions, the IC50 values were calculated as 2.57 and 1.12 mg/mL, respectively. The digested/hydrolysed samples of carp sarcoplasmic and myofibrillar protein fractions were separated by RP-HPLC-MS. Amino acid sequences were identified with the use of the LC-MS/MS method coupled with in silico systematic screening for ACEi peptides. ACEi peptides with IVY, IY, VY, ALPHA and VKAGF sequences were found in the carp digests/hydrolysates. In the ex vivo carp digest, an ACEi peptide TVY was also detected, while the ACEi peptide IW was identified in in vitro hydrolysate. Our study showed that different ACEi effects of carp digests and hydrolysates were generated with the use of human gastrointestinal and porcine enzymes.

Lipid and Protein Changes Related to Quality Loss in Frozen Sardine (Sardina pilchardus) Previously Processed Under High-Pressure Conditions

This research focuses on biochemical changes related to quality loss in frozen (−18 °C for 9 months) sardine (Sardina pilchardus) previously subjected to high-pressure (HP) processing (125–200 MPa). The inhibition (p < 0.05) of lipid hydrolysis development (lower free fatty acid formation and lipase activity), observed in frozen sardine as a result of the previous HP treatment, increased with the pressure level applied. Several parameters including peroxide value, thiobarbituric acid index, fluorescent compounds, and polyenes showed that the applied HP conditions prior to sardine freezing did not increase lipid oxidation. Also, HP did not induce a substantial modification of acid phosphatase and cathepsins B and D activities, and the electrophoretic patterns of sarcoplasmic and myofibrillar protein fractions did not change. However, HP processing led to a decrease in myofibrillar protein content in frozen pressure-treated fish, an effect that was higher in 175- and 200-MPa treated samples. In conclusion, this research showed that pressure treatments in the 125–200-MPa range with holding time of 0 min cause only minor modifications in biochemical indicators of deterioration throughout the subsequent frozen storage of samples for up to 9 months. This study shows the need to optimize HP conditions, particularly in the case of applications combining HP treatments, frozen storage, and thawing to obtain products with high quality and commercial viability.