Effect of added μ-calpain and post-mortem storage on the mechanical properties of bovine single muscle fibres extended to fracture

The effects of μ-calpain and post-mortem storage on the strength of single muscle fibres were investigated. During the 10 min of incubation at pH 7.5, μ-calpain became evenly distributed throughout the fibre. μ-Calpain-incubation resulted in thinner (P <0.001) Z-lines and reduced (P <0.001) the strength of the fibres compared to controls. These results demonstrate that μ-calpain is capable of mechanically weakening the muscle fibres. Post-mortem storage of meat for 10 days at 2?°C weakened (P <0.001) the muscle fibres compared to 24-h fibres. The presence or absence of Ca(2+) affected fibre stiffness. Fibres incubated at pH 7.5 in 100 μM Ca(2+) were less stiff than fibres incubated in 200 μM EGTA. Breaking stress and strain were not affected by Ca(2+). We hypothesise that Ca(2+) causes conformational changes in some of the load-bearing proteins, which alters their initial resistance to extension, but does not affect the breaking strength of the fibres.     

Protein denaturing conditions in beef deep semimembranosus muscle results in limited μ-calpain activation and protein degradation

The objective was to determine the effect of muscle location on protein solubility and protein degradation in deep (DSM) and superficial (SSM) portion of beef semimembranosus. At 24 h postmortem, the semimembranosus was removed from beef carcasses (n = 10), packaged in high-oxygen modified atmosphere (80% O₂ + 20% CO₂), and displayed for 7 d at 1 °C. DSM had higher (P < 0.05) L*, a*, b*, and hue values than SSM throughout display. DSM had significantly higher protein denaturation and less protein concentration than SSM. Western blotting for μ-calpain autolysis revealed that DSM maintained more (P < 0.05) unautolyzed μ-calpain than SSM. This result coincided with less desmin and troponin-T degradation in samples from the DSM. These results confirm the hypothesis that increased protein denaturation in DSM results in minimal proteolysis by negatively affecting μ-calpain activation. This demonstrates a potential to alter progression of proteolysis and improvement in tenderness associated with postmortem storage.     

The effect of temperature on the activity of μ- and m-calpain and calpastatin during post-mortem storage of porcine longissimus muscle

The experiment was conducted to determine the effect of temperature during post-mortem muscle storage on the activity of the calpain system, the myofibril fragmentation and the free calcium concentration. Porcine longissimus muscle were incubated from 2h post-mortem at temperatures of 2, 15, 25 and 30 °C and sampling times were at 2, 6, 24, 48 and 120 h post-mortem. After 120 h at 30 °C the free calcium concentration increased to 530 μM from 440 μM at 2 °C. Incubation at temperatures higher than 2 °C resulted in the appearance of autolyzed m-calpain activity and a decrease of native m-calpain activity. Native m-calpain decreased more slowly than native μ-calpain, and the autolysis process started later. Myofibril fragmentation increased with storage time and incubation temperature, while calpastatin activity decreased. The study showed that high temperature incubation not only rapidly activated μ-calpain but at higher temperatures and later time points also m-calpain.     

Effects of nutritional level on pork quality and gene expression of μ-calpain and calpastatin in muscle of finishing pigs

The study was designed to investigate the effects of nutritional level (control diet (CD), 14.19% crude protein, 13.81 MJ of DE/kg; low nutritional level diet (LND), 11.08% crude protein, 12.55 MJ of DE/kg) on pork quality and gene expression of μ-calpain and calpastatin in muscle of finishing pigs. The LND treatment increased drip loss (P < 0.05), had a trend to increase intramuscular fat (IMF) content (P = 0.09), decreased Warner–Bratzler shear force (WBSF) of pork (P < 0.05), improved mRNA level of μ-calpain (P < 0.05) in skeletal muscle, but had no effect on gene expression of calpastatin, compared with the CD treatment. These data suggest that a moderately reduced energy and protein diet increased pork tenderness and intramuscular fat. The increase in tenderness by LND treatment may be partly due to increased gene expression of μ-calpain in muscle.     

Characterization of peptides released from rabbit skeletal muscle troponin-T by μ-calpain under conditions of low temperature and high ionic strength

Rabbit skeletal muscle troponin-T (200 μg ml(-1)) was incubated with μ-calpain (2 μl ml(-1)) under conditions of low temperature and high ionic strength for 180 min at 4°C and the peptides released analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Troponin-T was hydrolyzed rapidly with the simultaneous appearance of eight peptides with masses of less than 14 up to 26 kDa. Two peptides produced by 10 min of incubation were electroblotted and analysis of these peptides by N-terminal sequencing and mass spectrometry showed that the principal cleavage sites of μ-calpain on troponin-T were at Thr(45)-Ala(46), Leu(69)-Met(70), Glu(220)-Lys(221) and Asn(231)-Val(232). The peptides present in insufficient quantity for electroblotting were isolated by reverse-phase high performance liquid chromatography (RP-HPLC). Cleavage sites were also identified at Met(151)-Gly(152), Asn(188)-Ile(189), Lys(223)-Arg(224), Arg(233)-Ala(234) and Ala(240)-Lys(241). In general, μ-calpain cleaved bonds containing one hydrophobic amino acid residue and mainly towards the C-terminus of troponin-T.     

Recombinant bovine interferon-τ enhances in vitro development of bovine embryos by upregulating expression of connexin 43 and E-cadherin

Interferon-τ (IFNT), produced in ruminants by embryonic trophoblastic cells before implantation, is involved in the maternal recognition of pregnancy. It is a pleiotropic molecule that alters the synthesis of endometrial proteins and inhibits the proliferation of some cells. The present study investigated the effects of recombinant bovine IFNT on the development of early-stage bovine embryos and the molecular mechanism underlying this effect. This study demonstrated that expression of mRNA encoding type I IFN receptor subunits was detectable from d 4 to 8 in in vitro fertilized (IVF) bovine embryos. A considerable number of IVF (n = 1,941) and parthenogenetic activated (n = 1,552) bovine embryos demonstrated that supplementing the culture medium with IFNT (100 ng/mL) produced a greater percentage of blastocysts, and the total cell number within the resulting blastocysts was higher. In addition, IFNT upregulated the expression levels of both mRNA and protein for connexin 43 (GJA1) and E-cadherin (CDH1) and expression levels for granulocyte-macrophage colony-stimulating factor and insulin-like growth factor 2 mRNA but not for their proteins in d 8 embryos. However, IFNT inhibited mRNA expression for leukemia inhibitory factor (LIF), LIF receptor α, and the sodium/potassium-transporting ATPase subunit β-1. We concluded that IFNT promoted the development of bovine embryos by upregulating the expression of GJA1 and CDH1. Thus, supplementing embryo cultures or transfer medium with IFNT may stimulate embryo development and improve embryo transfer efficiency.     

Identification of calpastatin, μ-calpain and m-calpain in Atlantic salmon (Salmo salar L.) muscle

A soft fish muscle is generally considered as a poor quality trait among consumers and producers. This degradation and softening of post mortem muscle is thought to be partly caused by proteolytic enzymes such as the calpain system. Separation and identification of μ-calpain and m-calpain and their inhibitor – calpastatin, from Atlantic salmon (Salmo salar) muscle were for the first time assessed in this study. A two-step chromatography approach was used, starting with a hydrophobic interaction column and followed by an anion exchange column. Calpastatin was successfully separated from calpain by hydrophobic interaction chromatography, and following the anion exchange chromatography, two forms of calpastatin (I and II) and two forms of calpain (micro (μ) and milli (m)) were revealed. The proteolytic activity of μ-calpain was detectable with column chromatography, but not consistently detected with casein zymography, and m-calpain was detected with both chromatography and casein zymogram. The proteolytic activity of m-calpain per g muscle was 15 times higher than that of μ-calpain. μ-Calpain had a temperature optimum of 15 °C and a maximum calcium requirement at 0.2 mM, while m-calpain had temperature optimum at 25 °C and a maximum calcium requirement of 0.6 mM. The two forms of calpastatin differed in inhibitory activity with calpastatin II having the highest activity. Both calpastatins tolerated heat treatment, as previously seen for mammals, and they kept their activity when stored at −80 °C, but not at −20 °C. The calpain to calpastatin ratio was 1:4.5 as observed for beef muscle. This study provides evidence that two calpain isoforms, likely to be μ- and m-calpain, in addition to two forms of calpastatin exist in Atlantic salmon muscle.     

Contribution of postmortem changes of integrin, desmin and μ-calpain to variation in water holding capacity of pork

The purpose of this study was to examine the relationship between integrin, desmin, μ-calpain and water holding capacity in fresh pork. High levels of intact integrin at one day postmortem were negatively correlated with day 1 (P<0.05) and days 1-5 (cumulative) drip loss (P<0.05). High levels of intact integrin at five days postmortem were negatively correlated with days 1-7 (cumulative) purge loss (P<0.05). Intensity of intact desmin at one day postmortem was positively correlated with days 1-7 purge loss (P<0.01). There were positive correlations between intensity of intact desmin at day 7 and day 1 (P<0.01), days 1-5 drip loss (P<0.01) and days 1-7 purge loss (P<0.05). Autolysis of μ-calpain was associated with the degradation of desmin and drip or purge loss postmortem. Our results indicate that low levels of degradation of integrin and high levels of desmin degradation were associated with low drip loss values in fresh pork.     

Mutations in calpastatin and μ-calpain are associated with meat tenderness,flavor and juiciness in Hanwoo (Korean cattle): Molecular modeling of the effects of substitutions in the calpastatin/μ-calpain complex

The objective of this study was to evaluate the effects of seven single nucleotide polymorphisms (SNPs) in Calpain 1 and Calpastatin genes previously associated with meat tenderness attributes in other cattle breeds in Korean Hanwoo cattle. The Hanwoo resource population was used to study association of 7 SNPs with beef tenderness, flavor, juiciness, intramuscular fat and shear force. In this association study, CAST:c.182A > G (+ 0.14, P = 0.04) and CAST:c.1985G > C (− 0.12, P = 0.02) had significant effects on juiciness, but no effects on other traits. In contrast, CAPN1:c.1589G > A was associated with meat tenderness (P = 0.01) and juiciness (P = 0.04). The CAPN1:c.1589G > A (Val530Ile) SNP marker displayed significant effect on the meat tenderness score which is strongly supported by molecular modeling of the CAPN1:c.1589G > A (Val530Ile) variant that inhibits CAST protein from binding more strongly than the wild-type protein, which may explain its effect on meat tenderness.     

Interactions between tea polyphenol and two kinds of typical egg white proteins—ovalbumin and lysozyme: Effect on the gastrointestinal digestion of both proteins in vitro

The promotion or inhibition of gastrointestinal digestion of tea polyphenol (TP) towards the two typical proteins from egg white (ovalbumin (OVA) and lysozyme (LYZ)) was examined. The results showed that TP made OVA/LYZ easier for digestion in the pepsin solution at pH 1.2 and inhibited OVA/LYZ digestion in pancreatin solution at pH 7.5. Non-covalent interactions between OVA/LYZ and TP and the secondary structure of OVA/LYZ were studied by using Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FTIR), respectively. Results suggested that stronger conformational change occurred at pH 1.2 compared with that of pH 7.5 affected by TP in both proteins. Non-covalent interactions between OVA/LYZ and TP at pH 1.2 increased random and β-sheet structures in both proteins at the expense of α-helix, which resulted in the proteins with looser structures. At pH 7.5, an opposite second structural change of both proteins caused by the non-covalent interactions between OVA/LYZ and TP. The conformational and second structural change of proteins (substrate) might be a reason for promoting and inhibiting digestion of OVA/LYZ affected by TP.     

Impact of a food-grade cationic biopolymer (ε-polylysine) on the digestion of emulsified lipids: In vitro study

ε-Polylysine (ε-PL) is a cationic biopolymer that may be used as a food ingredient because of its strong antimicrobial activity and potential to inhibit pancreatic lipase. We examined the effect of polylysine on the digestion of corn oil-in-water emulsions stabilized by either a natural anionic surfactant (quillaja saponin) or a synthetic non-ionic surfactant (Tween 20). Emulsions were prepared using high pressure homogenization (microfluidization) and then subjected to in vitro digestion in the absence or presence of polylysine at the maximum level allowed in foods by the FDA. Samples were characterized before and after in vitro digestion using electrophoresis, confocal microscopy, and static light scattering. The presence of polylysine decreased the hydrolytic activity of pancreatic lipase by around 53% and 28% in the Tween 20- and saponin-stabilized emulsions, respectively. The lipase-inhibiting properties of cationic polylysine were attributed to its electrostatic interaction with anionic components, such as bile salts, free fatty acids, and digestive enzymes. These results have important implications for the incorporation of polylysine into food systems, particularly those containing lipophilic nutrients.     

In vitro exposure of Acer negundo pollen to atmospheric levels of SO₂ and NO₂: effects on allergenicity and germination

In the last years, a rising trend of pollen allergies in urban areas has been attributed to atmospheric pollution. In this work, we investigated the effects of SO(2) and NO(2) on the protein content, allergenicity, and germination rate of Acer negundo pollen. A novel environmental chamber was assembled to exposure pollen samples with SO(2) or NO(2) at two different levels: just below and two times the atmospheric hour-limit value acceptable for human health protection in Europe. Results showed that protein content was lower in SO(2)-exposed pollen samples and slightly higher in NO(2)-exposed pollen compared to the control sample. No different polypeptide profiles were revealed by SDS-PAGE between exposed and nonexposed pollen, but the immunodetection assays indicated higher IgE recognition by all sera of sensitized patients to Acer negundo pollen extracts in all exposed samples in comparison to the nonexposed samples. A decrease in the germination rate of exposed in contrast to nonexposed pollen was verified, which was more pronounced for NO(2)-exposed samples. Our results indicated that in urban areas, concentrations of SO(2) and NO(2) below the limits established for human protection can indirectly aggravate pollen allergy on predisposed individuals and affect plant reproduction.     

Development of an HPLC–UV method for the simultaneous determination of tetracyclines in muscle and liver of porcine,chicken and bovine with accelerated solvent extraction

A simple and especially rapid method-using accelerated solvent extraction (ASE) and HPLC has been developed for the quantitative determination of oxytetracycline, tetracycline, chlortetracycline, minocycline, methacycline, demeclocycline and doxycycline in muscle and liver of porcine, chicken and bovine. Samples of muscle and liver were extracted with trichloracetic acid/acetonitrile using ASE instrument, parameters such as extraction temperature (40–80 °C) and pressure (45–85 bar) were investigated and the selected extraction (60 °C, 65 bar) was most effective. The limits of detection were lower than 10 μg/kg and limits of quantification no more than 15 μg/kg for all compounds in muscle and liver. The recoveries of tetracyclines spiked at levels of muscle 50–150 μg/kg, liver 150–450 μg/kg, averaged from 75.0% to 104.9% with the relative standard deviation values less than 10%. The method was applied to determine 30 real porcine livers. It is demonstrated that the new method is robust for detection and quantification of seven tetracycline residues in muscle and liver of porcine, chicken and bovine.     

Denaturation of soy proteins in solution and at the oil–water interface: A fluorescence study

Structural changes ensuing from denaturation of soy proteins in solution or occurring at the oil–water interface were studied by fluorescence spectroscopy. Studies were carried out on solutions and emulsions stabilized with β-conglycinin or glycinin. Tryptophan fluorescence spectroscopy was used to evaluate tertiary structural changes. The binding of fluorescent dyes and the accessibility of reactive cysteine thiols were also used to better identify structural changes of these proteins in solution. Protein conformational changes after interaction with the hydrophobic oil surface were compared with those ensuing from physical (temperature) or chemical denaturation (chaotropes). Results from solution denaturation experiments indicate that structural changes of β-conglycinin by both temperature and chaotropes are reversible under appropriate conditions, and result in a rearrangement of the supramacromolecular assembly of the protein structure. On the other hand, glycinin treated under the same conditions undergoes irreversible denaturation in solution at temperatures well below 90 °C. Both proteins undergo partial denaturation after adsorption on the lipid surface, and no further denaturation occurs upon heating of the emulsions prepared with either protein.     

Prospective randomized study to evaluate the efficacy and tolerability of Ectoin® containing Eye Spray (EES09) and comparison to the liposomal Eye Spray Tears Again® (TA) in the treatment of dry eye disease

PurposeTo subjectively and objectively evaluate the efficacy and tolerability of preservative-free Ectoin ® Eye Spray – Colloidal (EES09) and Tears Again ® Eye Spray (TA) in subjects with mild-moderate dry eye disease (DED), and to compare efficacy of these two eye sprays with each other.MethodsThirty-six volunteers (average age 32.3 ± 16.1 years; 26 females) were successfully recruited for this prospective double-blind study with between-subject design and randomly divided into two groups (gender and age balanced): Group A received EES09 and Group B received TA during the treatment phase. Inclusioncriteria were a minimum age of 18 years, a score of 18.0 or higher on the OSDI questionnaire, and a non-invasive tear break up time (NIKBUT, Oculus Keratograph M5, Oculus, Germany) of no more than 10s in at least one eye. The objective variables NIKBUT, conjunctival redness, lipid layer and osmolarity (TearLab Cooperation, USA) were assessed at baseline, 10 min. after spray application and after a treatment phase of 10±3 days (3x applications daily). Symptoms, tolerance and handling were evaluated with the OSDI and VAS questionnaires.ResultsA statistically significant increase in NIKBUT and improvement in DED symptoms were obtained for the overall group (mean NIKBUT 7.7 ± 1.7s vs. 11.6 ± 4.6s, p<0.001; mean OSDI score: 36.1 ± 12.7 vs. 20. 7± 12.7, p<0.001) during course of treatment. No statistically significant effect was established for the variables lipid layer (p = 0.406), conjunctival redness (p = 0.766) and osmolarity (p = 0.378). No statistically significant differences were observed between the groups, for any variable. The noninferioritycriterion for EES09 towards TA could be shown for the scores of the dry eye symptoms via VAS questionnaire and the variable NIKBUT.ConclusionsA beneficial treatment effect was confirmed for both, symptoms of DED and the objective variable NIKBUT. Both eye sprays were rated favourably in view of perceived tolerability and handling of the spray bottle.     

In vitro effect of some fungicides on growth and aflatoxins production by Aspergillus flavus isolated from Capsicum powder

The aim of this study was to determine the effect of some pre-harvest fungicides on growth and aflatoxin (AF) production of three Aspergillus flavus strains found in Capsicum powder. Each isolate, previously isolated from paprika, chilli and smoked paprika, was inoculated on yeast extract sucrose agar and on a 3% paprika extract agar medium supplemented with different fungicides and incubated at 20 and 30°C during 7 days. Growth measurements were obtained on days 3, 5 and 7, and the AF production was determined on day 7. The significance of the effects of the factors (strain, medium, temperature, time and fungicides) and their interaction over colony diameter and AF production was determined. Temperature constrained the effectiveness of fungicides in reducing growth, the fungicides being most effective at 20°C. The efficacy of the fungicides over AF production depended on the medium used and temperature. The most effective fungicides in inhibiting growth and AF production, regardless of the strain tested or applied conditions, were tebuconazole 25% and mancozeb 80% applied at a concentration of 0.75 and 3.5?g?l^?1, respectively. Care should thus be taken in the choice of a suitable fungicide because their effectiveness may depend on intra-specific variation and temperature. Moreover, it is necessary to take into account that the most efficient fungicide in reducing growth is not always the best choice for pre-harvest treatments because it may promote AF production. Thus, the best fungicide is the one that can simultaneous prevent growth and AF production.     

Effect of ascorbic acid and protein concentration on the molecular weight profile of bovine serum albumin and β-lactoglobulin by γ-irradiation

The effects of ascorbic acid and protein concentration on the molecular weight size distribution of BSA and β-lactoglobulin were examined after irradiation of proteins at various doses. Gamma-irradiation of protein solutions caused disruption of the ordered structure of protein molecules resulting in degradation, cross-linking, and aggregation of the polypeptide chains. SDS–PAGE and gel permeation chromatography study showed that ascorbic acid protected the aggregation and degradation of proteins by scavenging oxygen radicals produced by irradiation and the effect of irradiation on protein conformation was more significant at lower concentrations of proteins.     

In vitro effect of some fungicides on growth and aflatoxins production by Aspergillus flavus isolated from Capsicum powder

The aim of this study was to determine the effect of some pre-harvest fungicides on growth and aflatoxin (AF) production of three Aspergillus flavus strains found in Capsicum powder. Each isolate, previously isolated from paprika, chilli and smoked paprika, was inoculated on yeast extract sucrose agar and on a 3% paprika extract agar medium supplemented with different fungicides and incubated at 20 and 30°C during 7 days. Growth measurements were obtained on days 3, 5 and 7, and the AF production was determined on day 7. The significance of the effects of the factors (strain, medium, temperature, time and fungicides) and their interaction over colony diameter and AF production was determined. Temperature constrained the effectiveness of fungicides in reducing growth, the fungicides being most effective at 20°C. The efficacy of the fungicides over AF production depended on the medium used and temperature. The most effective fungicides in inhibiting growth and AF production, regardless of the strain tested or applied conditions, were tebuconazole 25% and mancozeb 80% applied at a concentration of 0.75 and 3.5 g l(-1), respectively. Care should thus be taken in the choice of a suitable fungicide because their effectiveness may depend on intra-specific variation and temperature. Moreover, it is necessary to take into account that the most efficient fungicide in reducing growth is not always the best choice for pre-harvest treatments because it may promote AF production. Thus, the best fungicide is the one that can simultaneous prevent growth and AF production.