Monitoring of Staphylococcus xylosus DSM 20266 added as starter during fermentation and ripening of soppressata molisana, a typical Italian sausage

AIMS: "Soppressata molisana", a fermented sausage produced in southern Italy, is commonly obtained without starter addition. However, the use of starter cultures is more and more recommended in meat fermentation processes in order to guarantee stable production performance. In this study, the survival of the Staphylococcus xylosus DSM 20266 was evaluated during the ripening of "soppressata molisana" fermented sausage. METHODS AND RESULTS: The fastest method of RAPD-PCR was employed for discrimination of the added strain from those naturally present during the ripening of the "soppressata molisana". The results obtained were confirmed by analysis of the DNA macrorestriction profile by PFGE. The electrophoretic pattern of bacterial total proteins was also studied, but clear differences between the different strains could not be detected. CONCLUSIONS: The RAPD technique was a valid tool for monitoring Staph. xylosus DSM 20266 in "sopressata molisana". SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the possibility of monitoring the presence of Staph. xylosus strains during the ripening of fermented sausages by a reliable and repeatable technique such as RAPD.     

On the difference in stability between horse and sperm whale myoglobins

The work in the literature on apomyoglobin is almost equally divided between horse and sperm whale myoglobins. The two proteins share high homology, show similar folding behavior, and it is often assumed that all folding phenomena found with one protein will also be found with the other. We report data at equilibrium showing that horse myoglobin was 2.1 kcal/mol less stable than sperm whale myoglobin at pH 5.0, and aggregated at high concentrations as measured by gel filtration and analytical ultracentrifugation experiments. The higher stability of sperm whale myoglobin was identified for both apo and holo forms, and was independent of pH from 5 to 8 and of the presence of sodium chloride. We also show that the substitution of sperm whale myoglobin residues Ala15 and Ala74 to Gly, the residues found at positions 15 and 74 in horse myoglobin, decreased the stability by 1.0 kcal/mol, indicating that helix propensity is an important component of the explanation for the difference in stability between the two proteins.     

Kinetics and mechanism of the reduction of CrVI and CrV by D-lactobionic acid

The oxidation of D-lactobionic acid by Cr(VI) yields the 2-ketoaldobionic acid and Cr(3+) as final products when a 20-times or higher excess of the aldobionic acid over Cr(VI) is used. The redox reaction takes place through a complex multistep mechanism, which involves the formation of intermediate Cr(IV) and Cr(V) species. Cr(IV) reacts with lactobionic acid much faster than Cr(V) and Cr(VI) do, and cannot be directly detected. However, the formation of CrO(2)(2+), observed by the first time for an acid saccharide/Cr(VI) system, provides indirect evidence for the intermediacy of Cr(IV) in the reaction path. Cr(VI) and the intermediate Cr(V) react with lactobionic acid at comparable rates, being the complete rate laws for the Cr(VI) and Cr(V) consumption expressed by: -d[Cr(VI)]/dt=[k(I)+k(II)[H(+)]][lactobionicacid][Cr(VI)], where k(I)=(4.1+/-0.1) x 10(-3) M(-1) s(-1) and k(II)=(2.1+/-0.1) x 10(-2) M(-2) s(-1); and -d[Cr(V)]/dt=[k(III)[H(+)]+(k(IV)+k(V)[H(+)])[lactobionicacid]] [Cr(V)], where k(III)=(1.8+/-0.1) x 10(-3) M(-1) s(-1), k(IV)=(1.1+/-0.1) x 10(-2) M(-1) s(-1) and k(V)=(1.0+/-0.1) x 10(-2) M(-2) s(-1), at 33 degrees C. The Electron Paramagnetic Resonance (EPR) spectra show that five-co-ordinate oxo-Cr(V) bischelates are formed at pH 1-5 with the aldobionic acid bound to Cr(V) through the alpha-hydroxyacid group.     

NMR structure of the noncytotoxic alpha-sarcin mutant Delta(7-22): the importance of the native conformation of peripheral loops for activity

The deletion mutant Delta(7-22) of alpha-sarcin, unlike its wild-type protein counterpart, lacks the specific ability to degrade rRNA in intact ribosomes and exhibits an increased unspecific ribonuclease activity and decreased interaction with lipid vesicles. In trying to shed light on these differences, we report here on the three-dimensional structure of the Delta(7-22) alpha-sarcin mutant using NMR methods. We also evaluated its dynamic properties on the basis of theoretical models and measured its correlation time (6.2 nsec) by time-resolved fluorescence anisotropy. The global fold characteristic of ribotoxins is preserved in the mutant. The most significant differences with respect to the alpha-sarcin structure are concentrated in (1) loop 2, (2) loop 3, which adopts a new orientation, and (3) loop 5, which shows multiple conformations and an altered dynamics. The interactions between loop 5 and the N-terminal hairpin are lost in the mutant, producing increased solvent accessibility of the active-site residues. The degree of solvent exposure of the catalytic His 137 is similar to that shown by His 92 in RNase T1. Additionally, the calculated order parameters of residues belonging to loop 5 in the mutant correspond to an internal dynamic behavior more similar to RNase T1 than alpha-sarcin. On the other hand, changes in the relative orientation of loop 3 move the lysine-rich region 111-114, crucial for substrate recognition, away from the active site. All of the structural and dynamic data presented here reveal that the mutant is a hybrid of ribotoxins and noncytotoxic ribonucleases, consistent with its biological properties.     

Structural and kinetic characterization of native laccases from Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii

A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXA1b) whose sequences are known.     

Effects of transforming growth factor-beta and mechanical strain on osteoblast cell counts: an in vitro model for distraction osteogenesis

Factors known to regulate bone production during distraction osteogenesis include mechanical strain on bone forming cells and up-regulation of transforming growth factor-beta (TGF-beta) during the distraction, or strain phase of distraction osteogenesis. In the present study, an in vitro model was used to evaluate the functional effect of exogenous TGF-beta1 on mitogenesis in murine-derived MC3T3 osteoblasts during the period of active mechanical strain. The first hypothesis to be tested was that mitogenic suppression of MC3T3 osteoblasts by TGF-beta1 is further enhanced when these cells are also subjected to mechanical strain. To test this hypothesis, MC3T3 osteoblasts were seeded on flexible and rigid membranes. These were subjected to cyclic, vacuum-induced strain, simulating physiologic stress loads. After 24 hours, all cells were transferred to media containing TGF-beta1, and strain was continued for an additional 48 hours. The study was repeated by using two doses of TGF-beta1. This study demonstrated that final cell counts were significantly decreased in the presence of TGF-beta1 in both the nonstrained and strained groups (p < 0.0001). The final cell count in the strained group was significantly less than that in the nonstrained group (p < 0.0001) for both concentrations of TGF-beta1 tested, confirming the initial hypothesis. The second hypothesis to be tested was that alteration in the mitogenic response of MC3T3 osteoblasts after strain is not directly due to autocrine factors produced by the strained osteoblasts. To test this hypothesis, a proliferation assay was performed on nonconfluent MC3T3 osteoblasts by using conditioned media collected from strained and nonstrained osteoblasts. This study demonstrated no significant differences in cell counts after addition of conditioned media collected from strained versus nonstrained cells, confirming the latter hypothesis. The present study demonstrates the functional significance of mechanical strain on osteoblast cell counts. Furthermore, this may help to explain the temporal relationship observed during the early distraction (strain) phase of distraction osteogenesis in rodent models in which peak up-regulation of TGF-beta1 gene expression correlates with peak suppression of osteoblast function as measured by gene expression of extracellular matrix proteins.     

Blood-brain barrier dysfunctions following systemic injection of kainic acid in the rat.

Changes in blood-brain barrier (BBB) permeability and cerebral metabolic activity following intravenous injection of kainic acid (KA; 6, 12 mg/Kg) in rats were assessed by calculating respectively a blood-to-brain transfer constant (Ki) for [14C]alpha-aminoisobutyric acid and local cerebral glucose utilization (LCGU) values, at different times (1 h, or acute seizures phase, and 48 h, or chronic pathology phase) after the induction of seizures. A significant increase in the local permeability of the BBB was observed 1 h after the injection of KA 6 mg/Kg (eliciting no significant changes in cerebral metabolic activity, except within the frontal cortex and the hippocampus) and 12 mg/Kg (which induced a marked and widespread enhancement of LCGU). On the contrary, during the pathology phase, persistent regional increases in Ki values were evidenced in rats treated with the lowest dose of the convulsant, but not in rats injected with KA 12 mg/Kg (a dose able to cause extensive neuronal damage). Thus one can speculate that: 1) KA-induced regional changes in the permeability of the BBB are not correlated with changes in neuronal activity; 2) opening of the BBB is not reliably associated with neuronal injury.     

Different age-independent effects of nutraceutical combinations on endothelium-mediated coronary flow reserve

Background

Some components of Nutraceuticals (NUT) such as red yeast rice and Morus alba have demonstrated positive effects on the endothelial function in hypercholesterolemic subjects. Our aim was to compare the effects of two different NUT combinations on cold pressure test (CPT) derived coronary flow reserve (CFR) assessed by transthoracic echo-Doppler.

Results

In a randomized, single-blind study, 28 consecutive patients with a variety of cardiovascular risk factors received NUT A (LopiGLIK®: berberine, red yeast rice powder, and leaf extract of Morus alba) or B (Armolipid Plus®: policosanol, red yeast rice, berberine, astaxantine, folic acidandcoenzyme Q10). An echo-Doppler exam with evaluation of CFR was performed at baseline, 2?h (acute test) and 30?days after daily NUT assumption. Blood sampling for metabolic profile and platelet aggregometry was performed at baseline and after 30?days of daily NUT assumption. CFR was not significantly modified at the acute test. After 30?days, CFR improved with NUT A (p <?0.0001), because of the increase of hyperemic flow velocity (p =?0.007), but not with NUT B. CFR was comparable between the two groups at baseline but became significantly higher after 30?days in NUT A (p <?0.02), with a higher CFR percent variation versus baseline (p =?0.008). Total cholesterol and LDL-cholesterol were reduced with both NUT A (p <?0.001 and p <?0.002, respectively) and B (both p <?0.02), whereas platelet aggregation did not significantly change. In the pooled group of patients, after adjusting for age and percent changes of systolic blood pressure, heart rate, LDL-cholesterol and glycemia, NUT A – but not NUT B - was independently associated with CFR changes (β?=?0.599, p =?0.003).

Conclusions

LopiGLIK® improved endothelial-derived CFR, independently of the beneficial effects exerted on the lipid profile. These findings can have clinical reflections on the prevention of age-related inflammatory diseases including coronary artery disease.

Trial registration

(NUTRENDO)″(ClinicalTrials.gov, NCT02969070).