Proteolysis Index Research Articles

Metabolic Profiling of Potential Probiotic or Synbiotic Cheeses by Nuclear Magnetic Resonance (NMR) Spectroscopy

To assess ripening of potential probiotic cheeses (containing either Lactobacillus casei -01 or Bifidobacterium lactis B94) or synbiotic cheeses with fructooligosaccharides (FOS) or a 50:50 mix of FOS/inulin, metabolic profiles have been obtained via classical biochemical analyses and by NMR spectroscopy. The addition of prebiotics to the cheeses resulted in lower proteolysis indices, especially in those synbiotic cheeses inoculated with B. lactis B94. Among synbiotic cheeses the combination of FOS and inulin resulted in an increase in lipolytic activity. The metabolic profiles of the cheeses analyzed by NMR spectroscopy, combined with multivariate statistics, allowed profiles to be distinguished by maturation time, added probiotic bacteria, or, in the case of B. lactis B94 cheese, added prebiotic. The NMR results are in agreement with the biochemical analyses and demonstrate the potential of NMR for the study of metabolic processes in probiotic/synbiotic food matrices.

Accuracy of near infrared spectroscopy for prediction of chemical composition, salt content and free amino acids in dry-cured ham

The capability of near infrared (NIR) spectroscopy was examined for the purposes of quality control of the traditional Slovenian dry-cured ham “ Kraški pršut.” Predictive models were developed for moisture, salt, protein, non-protein nitrogen, intramuscular fat and free amino acids in biceps femoris muscle (n = 135). The models' quality was assessed using statistical parameters: coefficient of determination (R 2) and standard error (se) of cross-validation (CV) and external validation (EV). Residual predictive deviation (RPD) was also assessed. Best results were obtained for salt content and salt percentage in moisture/dry matter (R CV 2 > 0.90, RPD > 3.0), it was satisfactory for moisture, non-protein nitrogen, intramuscular fat and total free amino acids (R CV 2 = 0.75–0.90, RPD = 2.0–3.0), while not so for protein content and proteolysis index (R CV 2 = 0.65–0.75, RPD < 2.0). Calibrations for individual free amino acids yielded R CV 2 from 0.40 to 0.90 and RPD from 1.3 to 2.9. Additional external validation of models on independent samples yielded comparable results. Based on the results, NIR spectroscopy can replace chemical methods in quality control of dry-cured ham.

Gene Expression of Muscle-Specific Ubiquitin Ligase, Atrogin-1/MAFbx, Positively Correlates with Skeletal Muscle Proteolysis in Food-Deprived Broiler Chickens

A relationship between skeletal muscle proteolysis and mRNA expression of atrogin-1/MAFbx, a muscle-specific ubiquitin ligase, was examined in food-deprived broiler chickens. In response to food deprivation for 24 and 48h, body and muscle weights decreased in a time-dependent manner. Muscle free Nτ-methylhistidine content, an index of myofibrillar proteolysis, was 1.8-fold and 2.5-fold greater than that of fed controls following 24 and 48h of food deprivation, respectively. Gene expression of atrogin-1/MAFbx drastically increased in response to food deprivation in a time-dependent manner. At 48h of food deprivation, the induction of atrogin-1/MAFbx mRNA expression level elevated up to 20-fold greater than that of fed controls. By contrast, mRNA expression levels of 20S proteasome C1 and C2 subunits tended to decrease with time. No significant difference was seen in the change of ubiquitin mRNA expression. A highly significant linear negative relationship (r2=0.754, P<0.001) was found between atrogin-1/MAFbx expression and muscle mass, while a highly significant linear positive relationship (r2=0.784, P<0.001) was found between atrogin-1/MAFbx expression and muscle free Nτ-methylhistidine content. These results indicate that atrogin-1/MAFbx plays a critical role in the development of muscle proteolysis and its gene expression is a reliable index of muscle proteolysis in broiler chickens.

Effect of free and encapsulated recombinant aminopeptidase on proteolytic indices and sensory characteristics of Cheddar cheese

The recombinant aminopeptidase of Lactobacillus rhamnosus S93 in free or encapsulated form was used in production of Cheddar cheese. The effects of these enzymes on the proteolytic indices as well as sensory characteristics have been investigated during Cheddar cheese ripening. An extrusion method was used to encapsulate the enzyme in alginate beads coated with chitosan. The free or encapsulated aminopeptidase were added at the renneting or salting stage at three different concentrations (50, 500, 2000 units per 18 L of milk). Indices of secondary proteolysis were enhanced by increasing the enzyme concentration. Cheeses with the highest concentration of the encapsulated enzyme had significantly higher concentrations of soluble nitrogen in phosphotungstic acid and total free amino acids and received the highest mean scores for the sensory characteristics.

The potential effect of FOS and inulin upon probiotic bacterium performance in curdled milk matrices

Inulin and fructooligosaccharides were studied for their prebiotic effect upon growth/survival of probiotic bacteria and technological potential in probiotic food processing, via characterization of glycolysis, proteolysis and lipolysis in curdled milk matrices; the ultimate goal is the manufacture of synbiotic cheeses. Prebiotic compounds did not significantly affect growth/viability of all strains studied, except Lactobacillus acidophilus La-5. Proteolysis indices revealed considerable casein degradation in probiotic and synbiotic matrices inoculated with Bifidobacterium lactis B94 and Lactobacillus casei-01; lower values were achieved in those inoculated with L. acidophilus La-5, yet a synbiotic effect was apparent in NPN values. Lipolysis was not extensive over storage, irrespective of matrix type; however, interesting differences in terms of the qualitative free fatty acids profile were observed. CLA isomers, and α-linolenic and γ−linolenic acids were detected upon 15 d of ripening of all inoculated matrices. Principal component analysis was able to discriminate the various matrices according to degree of maturation, throughout the ripening period. Microbiological and biochemical parameters unfolded a very good technological potential, especially of B. lactis B94 and L. casei-01, to produce novel types of functional dairy matrices – although extrapolation to actual cheeses should still be done with care, because e.g. syneresis was not considered.

MAFbx/Atrogin-1 expression is a poor index of muscle proteolysis

MAFbx/Atrogin-1 expression is a poor index of muscle proteolysis

Proteolysis indices related to cheese ripening and typicalness in PDO Grana Padano cheese

Different chemical indices related to proteolysis and ripening time have been investigated in Grana Padano ( n = 155) and Parmigiano-Reggiano ( n = 27) cheese samples using ion-exchange chromatography and capillary electrophoresis. Levels of intact caseins, γ-casein and free amino acids in cheese were not strictly related to the maturation period, varying among samples of the same age. A peptide, identified by HPLC/electrospray ionization-mass spectrometry as pyroglutamyl-γ 3-casein, resulted from cyclisation of N-terminal glutamic acid into a pyroglutamic acid residue. Based on the peak area ratio of pyroglutamyl-γ 3-casein and γ 3-casein, an equation, suitable for determination of the cheese age, is proposed. The commercial classes of Grana Padano ripened over 16 and 20 months can be identified by adopting a minimum threshold value for this peak area ratio.

Middle age is not associated with altered fibrinogen concentration and production in males

Whether ageing is associated with increased fibrinogen concentration and production remains unclear. We measured fibrinogen fractional (FSR) and absolute synthesis (ASR) rates in male volunteers, of either young (mean age: 28 years, range: 22-34) or middle age (mean age: 57 years, range: 38-72), using a leucine-tracer isotope dilution technique. In the middle-age group, neither fibrinogen FSR (20.8 +/- 1.6%/day) nor ASR (1.8 +/- 0.1 g/day), or concentration (274 +/- 15 mg/dl), were different from those of the younger group (FSR: 20.2 +/- 1.4; ASR: 1.7 +/- 0.2; concentration: 265 +/- 8, respectively). Leucine Ra, an index of endogenous proteolysis, was approximately 20% lower in the older than in the younger group (P < 0.02). Thus, middle age in males is not associated with increased fibrinogen concentration and turnover, whereas endogenous protein breakdown in decreased. Factor(s) different from age per se are likely to be involved in the dysfibrinogenemia possibly occurring with ageing. Protein turnover is already reduced in middle-age males.

Processing of dry-cured ham in a reduced-oxygen atmosphere: Effects on physicochemical and microbiological parameters and mite growth

The effects of a reduced-oxygen atmosphere (ROA) ([O(2)]<4.5%) during part or the whole of dry-cured ham processing on microbiological and physico-chemical parameters and mite growth were investigated in two independent experiments. In Experiment 1, six hams were processed in ROA and six in air for 275 days; in Experiment 2, where lower RH was used, six hams were processed in ROA for 289 days, six for 214 days in air+75 days in ROA, and six in air for 289 days. Microbiological analyses during the process and physicochemical analyses in final products were carried out. The use of ROA during the whole process increased the L* colour parameter in the subcutaneous fat and proteolysis index and decreased b* in the external part of the subcutaneous fat and cholesterol oxide concentration. The use of ROA combined with low RH retarded microbial growth and prevented mite growth.

Volatile composition and proteolysis in traditionally produced mature Kashar cheese

SummaryTwelve samples of raw milk mature Kashar cheese at different stages of ripening were collected from retail outlets. The average pH, moisture, fat‐in‐dry matter, protein, salt‐in‐dry matter and titratable acidity contents of the samples were 5.33, 39.39%, 45.20%, 27.33%, 6.62% and 0.65% (as lactic acid), respectively. Indices of proteolysis varied from 10.72% to 23.75% and 7.09% to 12.26% for pH 4.6‐soluble and 12% trichloroacetic acid‐soluble nitrogen fractions, respectively, and total free amino acid concentrations ranged from 6.36 to 36.03 mg Leu g−1 of cheese. The cheeses were analysed for volatile compounds by Solid Phase Microextraction and Gas Chromatography‐Mass Spectrometry (GC‐MS). A total of 113 compounds were detected and identified belonging to the following chemical groups: acids (eleven), esters (sixteen), ketones (sixteen), aldehydes (six), alcohols (twenty‐seven), sulphur compounds (seven), terpenes (seven) and miscellaneous compounds (twenty‐three). The potential effect of each compound on the flavour profile of Kashar cheese is discussed. Acids, esters, ketones and alcohols were found at considerable levels in the samples. Kashar cheeses obtained from different retail outlets displayed some differences in terms of chemical composition, proteolysis and patterns of aroma compounds; and may be attributed to their production technologies and age‐related variations.

Reduction of NaCl content in restructured dry-cured hams: Post-resting temperature and drying level effects on physicochemical and sensory parameters

The effects of post-resting temperature (5 °C, 15 °C and 25 °C) and drying level (low and high, corresponding to final water contents (w/w) of 57% and 50%, respectively) were evaluated in restructured dry-cured hams. The reduction of NaCl content, with and without the addition of K-lactate as NaCl substitute was also evaluated. Physicochemical and instrumental colour and texture (Stress Relaxation test) parameters and sensory attributes were measured. The main effects of reducing the NaCl addition from 30 g/kg to 15 g/kg in restructured dry-cured hams were the reduction of saltiness and the increase of a w , proteolysis and softness. The addition of K-lactate (19.7 g/kg) contributed to reduce these effects. Hams from high drying level had a longer processing time and a higher proteolysis index, but lower water content and harder texture. The increase of post-resting temperature to above 5 °C reduced the processing time and the metallic flavour, but at 25 °C restructured hams were spoiled. Therefore, the problems due to the reduction of NaCl to 15 g/kg in restructured dry-cured hams can be reduced by adding K-lactate and drying at 15 °C (after 3.5 months of resting at 5 °C) until a final weight loss of around 45% is reached.

Reliability of results and interpretation of measures of 3-methylhistidine in muscle interstitium as marker of muscle proteolysis

TO THE EDITOR: We were interested in the contribution by Tesch and colleagues to the evidence base concerning the events subsequent to acute immobilization in human muscle. Tesch and colleagues (14) used a microdialysis technique applied to human muscle in an attempt to measure an index of myofibrillar proteolysis 72 h after immobilization using the unilateral leg suspension technique. The authors discuss their results as though they had good evidence that the technique they used produces results that are sufficiently firm to be the basis of conjecture about mechanisms of protein balance in muscle after immobilization. We demur and propose that they are on very shaky ground. First, we wonder about the size of the effect reported. The authors claim it to be 44%, but calculation of the change shows it to be actually 29% with the SEs sufficiently large that they almost span the difference; SDs must overlap. Significance at the 5% level can only barely have been reached. Are the authors convinced that they have not ignored a type I error? The authors (14) begin their explanation of the advantages of the dialysis technique using 3-methylhistidine (3MeHis) by describing the gold standard method, i.e., the stable isotopelabeled amino acid tracer dilution method, as cumbersome and possibly unjustified in study of otherwise healthy subjects. We find this notion strange since Ferrando and colleagues (6, 12) have used the technique in many studies of how immobilization affects muscle protein turnover subsequent to disuse. We have also used the techniques in a wide variety of studies of proteolysis in healthy and infirm subjects (1, 2, 10, 11, 15) with no problems and, we would propose, producing results with a greater degree of certainty and biological appropriateness than those obtained by the 3MeHis dialysis technique.

The Effects of Growth Hormone and/or Testosterone on Whole Body Protein Kinetics and Skeletal Muscle Gene Expression in Healthy Elderly Men: A Randomized Controlled Trial

Alterations of protein turnover may contribute to the progressive decline of muscle mass with aging. Our objective was to examine the effects of near-physiological recombinant human GH and/or testosterone (T) administration to older men on whole body protein kinetics and muscle gene expression. A 6-month randomized, double-blind, placebo-controlled trial in 21 healthy elderly men aged 65-75 yr, was performed. Participants were randomized to receive placebo GH and placebo T, rhGH and placebo T (GH), T and placebo GH (T), or rhGH and T (GHT). The leucine rate of appearance (index of proteolysis), nonoxidative leucine disposal rate (an index of protein synthesis), and leucine oxidation rate were measured with an infusion of l-[1-(13)C] leucine. Muscle biopsies for the measurement of gene expression were performed. Body composition and aerobic capacity (maximal oxygen capacity) were measured. Serum IGF-I levels increased significantly with GH and GHT (P < 0.001) compared with placebo. T increased significantly only in the T group (P = 0.028). Leucine rate of appearance and nonoxidative leucine disposal rate increased with GH (P = 0.015, P = 0.019) and GHT (P = 0.017, P = 0.02), but leucine oxidation did not change significantly in any treatment group. Midthigh muscle mass and maximal oxygen capacity increased (P < 0.04) with GHT only. Expression of muscle function genes did not change significantly, but the within-group comparisons revealed a significant increase of androgen receptor expression in the GHT group (P = 0.001). This study showed that 6-month treatment with low-dose GH alone or with T in healthy elderly men produces comparable increments in whole body protein turnover and protein synthesis.

Changes of plasma free amino acid concentrations and myofibrillar proteolysis index by starvation in non‐pregnant dry cows

ABSTRACTThe effects of 5 day starvation on urinary metabolite excretion, plasma metabolites and free amino acid concentrations in four non‐pregnant dry cows were investigated. Starvation significantly reduced (P &lt; 0.05) the bodyweight and feces weight. The plasma glucose concentration decreased (P &lt; 0.05) during days 2–5 of starvation. Starvation significantly increased (P &lt; 0.05) the plasma nonesterified fatty acid concentration. The plasma urea nitrogen concentration and urinary urea nitrogen excretion increased transiently (P &lt; 0.05) at days 1 and 2 of starvation. The plasma 3‐methlhistidine concentration was significantly higher (P &lt; 0.05) at days 2, 3 and 5 of starvation. However, the urinary 3‐methylhistidine excretion was significantly higher (P &lt; 0.05) at days 4 and 5 of starvation, not corresponding to the change in the plasma. Starvation did not change the plasma proline, methionine, lysine and total amino acid concentrations. On the other hand, starvation reduced (P &lt; 0.05) the plasma aspartic acid, serine, asparagine, glutamic acid, glutamine, alanine, tyrosine, tryptophan and histidine concentrations. Plasma glycine increased during starvation. Plasma threonine, isoleucine, leucine and branched‐chain amino acid increased (P &lt; 0.05) by prolonged starvation. The results of this study indicate that prolonged starvation accelerated myofibril protein degradation in non‐pregnant dry cows and apparently consumes most glucogenic amino acids in preference to other amino acids.

Development of an indirect α‐actinin‐based immunoassay for the evaluation of protein breakdown and quality loss in fish species subjected to different chilling methods

Summary α‐Actinin release from the myofibrillar protein fraction to the sarcoplasm can be considered as an accurate proteolysis index in seafood muscle. The main objective of the present study was to develop a specific enzyme‐linked immunosorbent assay (ELISA), based on the use of a monoclonal antibody against α‐actinin to evaluate the degree of proteolysis in two different chilled fish species – European hake (Merluccius merluccius) and turbot (Psetta maxima) – kept under two different storage systems: flake ice and slurry ice. Comparison with sensory assessment, K‐value and sarcoplasmic protein profiles was carried out. A different degree of proteolysis could be observed in both fish species; thus, the immunoassay was shown to be useful in monitoring the protein degradation events in hake muscle especially under flake ice storage. In the case of turbot, as very low proteolysis development could be obtained, the assay was not suitable for assessing quality changes. A different break point of immunoassay values for each fish species is suggested.

Texture changes in dry-cured ham pieces by mild thermal treatments at the end of the drying process

The present study evaluated the effects of mild thermal treatments at the end of the drying process on physicochemical characteristics and instrumental and sensory texture in dry-cured ham. Experiment 1: effect of thermal treatments (4–46°C) for 4h and 24h. Experiment 2: time effect (4–168h) of thermal treatments at 30°C and 36°C. Both experiments were done on small dry-cured ham dices. Experiment 3: time effect (4–168h) of thermal treatment at 30°C on both instrumental and sensory texture of 4-cm-thick sections of dry-cured ham. The thermal treatment at 30°C for 168h on both dry-cured ham muscle dices (20mm×20mm×15mm) and dry-cured ham sections (4cmthick) decreased softness, adhesiveness and pastiness in BF muscle, without increasing hardness in SM muscle or affecting moisture, aw and proteolysis index.

Insulin effect on serum potassium and auto‐inhibition of insulin secretion is intact in a patient with leprechaunism despite severe impairment of substrates metabolism

The effect of insulin on glucose, protein metabolism, circulating fatty acids (FFA), potassium (K(+)) and C-peptide concentrations were investigated in a 12-year-old girl with leprechaunism. The mutations do not affect the insulin-receptor binding affinity and insulin-stimulated auto-phosphorylation of the receptor. The subject was studied with a primed-continuous infusion of [6,6 - (2)H(2)] glucose and [1-(13)C] leucine during a basal period followed by two steps of insulin infusion (1 and 10 mU/kg/min) of 2 h each, during which plasma glucose level decreased from 131 to 115 and then to 95 mg/dL. Whole body glucose disposal was virtually unaffected by insulin, slightly decreasing from 21 micromol/kg/min in the basal period to 20 and to 19 micromol/kg/min during the two steps of insulin infusion, respectively. The endogenous leucine flux, an index of proteolysis, was completely insensitive to insulin, being 182, 189 and 180 micromol/kg/min, in the three periods, respectively. The FFA concentration (an indirect index of lipolysis) decreased from 1135 to 799 during step 1. During step 2 the FFA concentration rebounded to 917 micromol/L. The concentration of K(+) decreased from 4.2 to 3.2 mmol/L and an infusion of 20 mEq/h of KCl was necessary to prevent further hypokalemia (final value 3.3 mmol/l). The C-peptide concentration declined from 1.85 to 0.97 and then to 0.29 pmol/mL. The dissociation of control exerted by insulin on K+ uptake and on beta-cell secretion may rely on a differential expression and folding of the mutated receptors in the different insulin target tissues.

Characterization of the chemistry, biochemistry and volatile profile of Kuflu cheese, a mould-ripened variety

The chemistry, biochemistry and volatile compounds of Kuflu cheese, a Turkish mould-ripened variety were studied. A total of 29 samples were analysed and the titratable acidity, moisture, salt-in-moisture, fat-in-dry matter and total protein contents (as mean values) were 0.96%, 49.97%, 7.49%, 12.18% and 37.84%, respectively, and the pH of the cheeses was 6.29. Indices of proteolysis (i.e., the levels of pH 4.6- and trichloroacetic acid-soluble nitrogen) were high; however, these values were lower than those of other Blue cheeses probably due to proportionally higher levels of total nitrogen and salt-in-moisture in Kuflu cheese samples. Urea-polyacrylamide gel electrophoresis of the pH 4.6-insoluble fractions of the cheeses showed that both α s1- and β-caseins were extensively degraded, but β-casein was less degraded than α s1-casein. RP-HPLC peptide profiles of the pH 4.6-soluble fractions from Kuflu cheeses showed that some minor quantitative differences were found between the samples, while peptide profiles of the samples were qualitatively similar. One hundred and thirty-eight compounds were identified in the volatile fractions of Kuflu cheese by gas chromatography–mass spectrometry (GC–MS) using a solid -phase microextraction technique. Ketones and alcohols were the principal class of volatile components in Kuflu cheeses, and terpenes and sulphur compounds were found at substantial levels in the majority of the samples, but aldehydes and lactones were present at low levels. The RP-HPLC and GC–MS data were analysed by principal component analysis based on their peptide and volatile profiles, respectively. Kuflu cheeses obtained from different markets had some differences in terms of chemical composition, proteolysis and patterns of aroma compounds.

Effect of the inoculation of a starter culture and vacuum packaging (during resting stage) on the appearance and some microbiological and physicochemical parameters of dry-cured ham

The effect of the inoculation of a starter culture and vacuum packaging (during the resting stage) on dry-cured ham appearance, microbiological and physicochemical parameters was studied. Half of the 36 processed hams were inoculated, after salting, with a commercial starter culture containing lactic-acid bacteria, Gram-positive catalase-positive cocci and yeasts. 18 hams per group (inoculated and non-inoculated) remained vacuum packaged during resting. Microbiological analyses were carried out on the lean surface during processing, on subcutaneous fat tissue at the drying stage and on lean tissue in the finished product. Appearance was evaluated during processing. Physicochemical analyses (NaCl, H 2O, proteolysis index, a w) were done on Semimembranosus and Biceps femoris in the final product. Inoculation caused a reduction of mould growth and oil drip. Vacuum packaging induced increased proteolysis and increases in all microbial counts and a reduction of oil drip, mould growth and weight loss during processing.

Smoking impairs muscle protein synthesis and increases the expression of myostatin and MAFbx in muscle

Smoking causes multiple organ dysfunction. The effect of smoking on skeletal muscle protein metabolism is unknown. We hypothesized that the rate of skeletal muscle protein synthesis is depressed in smokers compared with non-smokers. We studied eight smokers (> or =20 cigarettes/day for > or =20 years) and eight non-smokers matched for sex (4 men and 4 women per group), age (65 +/- 3 and 63 +/- 3 yr, respectively; means +/- SEM) and body mass index (25.9 +/- 0.9 and 25.1 +/- 1.2 kg/m(2), respectively). Each subject underwent an intravenous infusion of stable isotope-labeled leucine in conjunction with blood and muscle tissue sampling to measure the mixed muscle protein fractional synthesis rate (FSR) and whole body leucine rate of appearance (Ra) in plasma (an index of whole body proteolysis), the expression of genes involved in the regulation of muscle mass (myostatin, a muscle growth inhibitor, and MAFBx and MuRF-1, which encode E3 ubiquitin ligases in the proteasome proteolytic pathway) and that for the inflammatory cytokine TNF-alpha in muscle, and the concentration of inflammatory markers in plasma (C-reactive protein, TNF-alpha, interleukin-6) which are associated with muscle wasting in other conditions. There were no differences between nonsmokers and smokers in plasma leucine concentration, leucine rate of appearance, and plasma concentrations of inflammatory markers, or TNF-alpha mRNA in muscle, but muscle protein FSR was much less (0.037 +/- 0.005 vs. 0.059 +/- 0.005%/h, respectively, P = 0.004), and myostatin and MAFBx (but not MuRF-1) expression were much greater (by approximately 33 and 45%, respectivley, P < 0.05) in the muscle of smokers than of nonsmokers. We conclude that smoking impairs the muscle protein synthesis process and increases the expression of genes associated with impaired muscle maintenance; smoking therefore likely increases the risk of sarcopenia.