Exploring structural and computational contrasts in Myoglobins: Implications for thermal treatment-induced Sulfmyoglobin formation.

Greening of tuna metmyoglobin (MetMb) by thermal treatment (TT) and free cysteine is associated with sulfmyoglobin (SulfMb) production. This greening reaction (GR) was once thought to occur only in tuna species. However, recent research has revealed that not all tuna species exhibit this behavior, and it can also occur in horse MetMb. Thus, the present study aimed to compare the GR-reactive (Katsuwonus pelamis and Equus caballus) and GR-unreactive (Sarda chiliensis and Euthynnus lineatus) MetMb using UV-vis spectrometry during TT (60 °C/30 min and free cysteine) to monitor the GR. We used molecular dynamics (MD) simulation to assess the stability of the heme group during TT. We discovered that using GR-unreactive MetMb resulted in an incomplete GR without producing SulfMb. Additionally, our MD simulations indicated that Met85 presence in the heme cavity from GR-unreactive is responsible for the heme group instability and displacement of distal His during TT.

Lippia graveolens Essential Oil to Enhance the Effect of Imipenem against Axenic and Co-Cultures of Pseudomonas aeruginosa and Acinetobacter baumannii.

This research focuses on assessing the synergistic effects of Mexican oregano (Lippia graveolens) essential oil or carvacrol when combined with the antibiotic imipenem, aiming to reduce the pathogenic viability and virulence of Acinetobacter baumannii and Pseudomonas aeruginosa. The study highlighted the synergistic effect of combining L. graveolens essential oil or carvacrol with imipenem, significantly reducing the required doses for inhibiting bacterial growth. The combination treatments drastically lowered the necessary imipenem doses, highlighting a potent enhancement in efficacy against A. baumannii and P. aeruginosa. For example, the minimum inhibitory concentrations (MIC) for the essential oil/imipenem combinations were notably low, at 0.03/0.000023 mg/mL for A. baumannii and 0.0073/0.000023 mg/mL for P. aeruginosa. Similarly, the combinations significantly inhibited biofilm formation at lower concentrations than when the components were used individually, demonstrating the strategic advantage of this approach in combating antibiotic resistance. For OXA-51, imipenem showed a relatively stable interaction during 30 ns of dynamic simulation of their interaction, indicating changes (<2 nm) in ligand positioning during this period. Carvacrol exhibited similar fluctuations to imipenem, suggesting its potential inhibition efficacy, while thymol showed significant variability, particularly at >10 ns, suggesting potential instability. With IMP-1, imipenem also displayed very stable interactions during 38 ns and demonstrated notable movement and positioning changes within the active site, indicating a more dynamic interaction. In contrast, carvacrol and thymol maintained their position within the active site only ~20 and ~15 ns, respectively. These results highlight the effectiveness of combining L. graveolens essential oil and carvacrol with imipenem in tackling the difficult-to-treat pathogens A. baumannii and P. aeruginosa.

Review of the Greening Reaction by Thermal Treatment: New Insights Exploring the Structural Implications of Myoglobin.

Myoglobin is the main factor responsible for muscle pigmentation in tuna; muscle color depends upon changes in the oxidative state of myoglobin. The tuna industry has reported muscle greening after thermal treatment involving metmyoglobin (MetMb), trimethylamine oxide (TMAO), and free cysteine (Cys). It has been proposed that this pigmentation change is due to a disulfide bond between a unique cysteine residue (Cys10) found in tuna MetMb and free Cys. However, no evidence has been given to confirm that this reaction occurs. In this review, new findings about the mechanism of this greening reaction are discussed, showing evidence of how free radicals produced from Cys oxidation under thermal treatment participate in the greening of tuna and horse muscle during thermal treatment. In addition, the reaction conditions are compared to other green myoglobins, such as sulfmyoglobin, verdomyoglobin, and cholemyoglobin.

Sulfmyoglobin production by free cysteine during thermal treatment: Involvement of heme iron in the production of free radicals.

The meat greening is an abnormal pigmentation related to microbiological contamination and lipid oxidation during storage. This color change results from sulfmyoglobin (SulfMb) production promoted by the reaction between metmyoglobin (MetMb), H2O2, and thiol compounds. Spectral studies on cooked meat suggested the production of SulfMb, probably due to the increment of free radicals during thermal treatment. Thus, we evaluated the involvement of free radicals and heme iron in the SulfMb production from horse MetMb and free cysteine (Cys) during thermal treatment. The results confirm that the reaction of SulfMb production at meat muscle pH (5.7-7.2) during heat treatment is a product of free radicals formed from Cys oxidation (SH) and reactive oxygen species (O2-, H2O2). This is catalyzed by the release of heme iron, thus promoting a consecutive reaction having MbFe(IV)O as a reaction intermediate.

The greening reaction of skipjack tuna (Katsuwonus pelamis) metmyoglobin promoted by free cysteine during thermal treatment.

Background: Tuna muscle greening is a problem that occurs after heating. A hypothesis has been postulated to address this problem, involving a conserved Cys residue at position 10 (Cys-10) present on tuna myoglobin (Mb) that is exposed during the thermic treatment, forming a disulfide bond with free cysteine (Cys) in the presence of trimethylamine oxide (TMAO), resulting in the greening of the tuna Mb. Methods: We present a study using skipjack tuna (Katsuwonus pelamis) metmyoglobin (MbFe(III)-H2O) where the effect of free Cys (1-6 mM), TMAO (1.33 mM), and catalase on the greening reaction (GR) was monitored by UV-vis spectrometry during thermal treatment at 60 °C for 30 min. Moreover, the participation of Cys-10 on the GR was evaluated after its blocking with N-ethymaleimide. Results: The GR occurred in tuna MbFe(III)-H2O after heat treatment with free Cys, forming sulfmyoglobin (MbFe(II)-S) as the responsible pigment for the tuna greening. However, the rate constants of MbFe(II)-S production depended on Cys concentration (up to 4 mM) and occurred regardless of the TMAO presence. We postulate that two consecutive reactions involve an intermediate ferrylmyoglobin (promoted by H2O2) species with a subsequent MbFe(II)-S formation since the presence of catalase fosters the reduction of the rate reaction. Moreover, GR occurred even with blocked Cys-10 residues in tuna Mb and horse Mb (without Cys in its sequence). Discussion: We found that GR is not exclusive to tuna Mb´s, and it can be promoted in other muscle systems. Moreover, Cys and thermal treatment are indispensable for promoting this pigmentation anomaly.

Partial Characterization of a Low-Molecular-Mass Fraction with Cryoprotectant Activity from Jumbo Squid (Dosidicus gigas) Mantle Muscle.

Freezing conditions affect fish muscle protein functionality due to its denaturation/aggregation. However, jumbo squid (Dosidicus gigas) muscle protein functionality remains stable even after freezing, probably due to the presence of low-molecular-mass compounds (LMMC) as cryoprotectants. Thus, water-soluble LMMC (<1 kDa) fraction obtained from jumbo squid muscle was evaluated by Fourier transform infrared spectrometry. From its spectra, total carbohydrates, free monosaccharides, free amino acids and ammonium chloride were determined. Cryoprotectant capacity and protein cryostability conferred by LMMC were investigated by differential scanning calorimetry. Fraction partial characterization showed that the main components are free amino acids (18.84 mg/g), carbohydrates (67.1 µg/mg) such as monosaccharides (51.1 µg/mg of glucose, fucose and arabinose in total) and ammonium chloride (220.4 µg/mg). Arginine, sarcosine and taurine were the main amino acids in the fraction. LMMC, at the mass fraction present in jumbo squid muscle, lowered the water freezing point to -1.2 °C, inhibiting recrystallization at 0.66 °C. Significant myofibrillar protein stabilization by LMMC was observed after a freeze-thaw cycle compared to control (muscle after extraction of LMMC), proving the effectiveness on jumbo squid protein muscle cryo- stability. Osmolytes in LMMC fraction inhibited protein denaturation/aggregation and ice recrystallization, maintaining the muscle structure stable under freezing conditions. LMMC conferred protein cryostability even at the very low mass fraction in the muscle.

Effect of Amidated Low-Methoxyl Pectin on Physicochemical Characteristics of Jumbo Squid (Dosidicus gigas) Mantle Muscle Gels.

Jumbo squid (Dosidicus gigas) muscle proteins show low functionality with limited use in gel products. This work aims to assess the influence of adding the natural and commercially available fibre, amidated low-methoxyl pectin (at 0.5, 1.0, 1.5, 2.0 and 3.0%), on the physicochemical and functional characteristics of jumbo squid (Dosidicus gigas) mantle muscle gels. The addition of 0.5% fibre showed an immediate effect on the gel texture profile analysis, improving hardness (p<0.05) from (3.4±0.7) N of the control (no added fibre) to (5.2±0.9) N, and increasing elasticity (p≥0.05). Shear force was significant only at 3.0% fibre addition. Water holding capacity also improved (p<0.05) with fibre addition (from 75% in the control to 90-95% after the treatments). Whiteness was affected (p<0.05) when 3.0% fibre was added. Differential scanning calorimetry showed two endothermic transition peaks in the gels. The second peak (actin) increased (p<0.05) by 1-2 °C with fibre addition. Therefore, the present study demonstrates that amidated low-methoxyl pectin (0.5-3.0%) is an excellent ingredient to improve jumbo squid mantle muscle protein functionality, increasing the gel texture and water retention characteristics.