Microstructural Changes in Vanilla planifolia Beans after Using High-Hydrostatic-Pressure Treatment in the Curing Process.

During vanilla bean curing, the cell arrangement derived from the killing technique applied to start bean ripening is essential to obtain the characteristic aroma and flavor of vanilla. Hence, killing is an important step to release the enzymes and compounds required for vanillin production. In this work, high hydrostatic pressure (HHP) at 100-400 MPa for 5 min, using water at 7 °C as the pressure-transmitting medium, was applied as the killing method, and its effect on the microstructural changes in vanilla beans during different curing cycles (C0-C20) was evaluated and compared with that observed after scalding by using water at 100 °C for 8 s. Microstructural changes in the cross-sectioned beans were analyzed using a stereomicroscope (SM), confocal laser scanning microscopy (CLSM), and environmental scanning electron microscopy (ESEM). The vanilla beans were cross-sectioned and three main sectors were analyzed: the total, annular, and core. The morphometric descriptors, namely, area, Feret's diameter, and circularity, were quantified via digital image analysis (DIA), from which a shrinkage ratio was calculated. The results show that the total area in the beans presented a maximum decrease in the C16 of curing. The core area was most affected by the HHP treatment, mainly at 400 MPa, rather than scalding. CSLM observations revealed the autofluorescence of the compounds inside the beans. In conclusion, the use of microscopy techniques and DIA allowed us to determine the microstructural changes in the HHP-treated pods, which were found to be more numerous than those found in the scalded beans.

Enzymatic Activity and Its Relationships with the Total Phenolic Content and Color Change in the High Hydrostatic Pressure-Assisted Curing of Vanilla Bean (Vanilla planifolia).

Diverse enzymatic reactions taking place after the killing of green vanilla beans are involved in the flavor and color development of the cured beans. The effects of high hydrostatic pressure (HHP) at 50-400 MPa/5 min and blanching as vanilla killing methods were evaluated on the total phenolic content (TPC), polyphenoloxidase (PPO), and peroxidase (POD) activity and the color change at different curing cycles of sweating-drying (C0-C20) of vanilla beans. The rate constants describing the above parameters during the curing cycles were also obtained. The TPC increased from C1 to C6 compared with the untreated green beans after which it started to decrease. The 400 MPa samples showed the highest rate of phenolic increase. Immediately after the killing (C0), the highest increase in PPO activity was observed at 50 MPa (46%), whereas for POD it was at 400 MPa (25%). Both enzymes showed the maximum activity at C1, after which the activity started to decrease. As expected, the L* color parameter decreased during the entire curing for all treatments. An inverse relationship between the rate of TPC decrease and enzymatic activity loss was found, but the relationship with L* was unclear. HHP appears to be an alternative vanilla killing method; nevertheless, more studies are needed to establish its clear advantages over blanching.

Assessment of the Efficiency of Technological Processes to Modify Whey Protein Antigenicity.

Whey is a by-product that represents a cheap source of protein with a high nutritional value, often used to improve food quality. When used as a raw material to produce hypoallergenic infant formulas (HIF), a processing step able to decrease the allergenic potential is required to guarantee their safe use for this purpose. In the present paper, thermal treatments, high hydrostatic pressure (HHP), and enzymatic hydrolysis (EH) were assessed to decrease the antigenicity of whey protein solutions (WPC). For monitoring purposes, a competitive ELISA method, able to detect the major and most allergenic whey protein ß-lactoglobulin (BLG), was developed as a first step to evaluate the efficiency of the processes. Results showed that EH together with HHP was the most effective combination to reduce WPC antigenicity. The evaluation method proved useful to monitor the processes and to be employed in the quality control of the final product, to guarantee the efficiency, and in protein antigenicity reduction.

Effect of High Hydrostatic Pressure and Pulsed Electric Fields Processes on Microbial Safety and Quality of Black/Red Raspberry Juice.

Black and red raspberries are fruits with a high phenolic and vitamin C content but are highly susceptible to deterioration. The effect of high hydrostatic pressure (HHP 400−600 MPa/CUT-10 min) and pulsed electric fields (PEF, frequency 100−500 Hz, pulse number 100, electric field strength from 11.3 to 23.3 kV/cm, and specific energy from 19.7 to 168.4 kJ/L) processes on black/red raspberry juice was studied. The effect on the inactivation of microorganisms and pectin methylesterase (PME) activity, physicochemical parameters (pH, acidity, total soluble solids (°Brix), and water activity (aw)), vitamin C and phenolic compounds content were also determined. Results reveal that all HHP-treatments produced the highest (p < 0.05) log-reduction of molds (log 1.85 to 3.72), and yeast (log 3.19), in comparison with PEF-treatments. Increments in pH, acidity, and TSS values attributed to compounds' decompartmentalization were found. PME activity was partially inactivated by HHP-treatment at 600 MPa/10 min (22% of inactivation) and PEF-treatment at 200 Hz/168.4 kJ/L (19% of inactivation). Increment in vitamin C and TPC was also observed. The highest increment in TPC (79% of increment) and vitamin C (77% of increment) was observed with PEF at 200 Hz/168.4 kJ/L. The putative effect of HHP and PEF on microbial safety, enzyme inactivation, and phytochemical retention is also discussed in detail. In conclusion, HHP and PEF improve phytochemical compounds' content, microbial safety, and quality of black/red raspberry juice.

Effect of High Hydrostatic Pressure Processing on the Anthocyanins Content, Antioxidant Activity, Sensorial Acceptance and Stability of Jussara (Euterpe edulis) Juice.

Jussara (Euterpe edulis) fruit is a strong candidate for exportation due to its high content of anthocyanins. However, its rapid perishability impairs its potential for further economic exploration, highlighting the relevance of producing ready-to-drink juices by applying innovative processing, such as high hydrostatic pressure (HHP). The effect of HHP (200, 350, and 500 MPa for 5, 7.5, and 10 min) on anthocyanins content and antioxidant activity (AA) by FRAP and TEAC assays, and the most effective HHP condition on overall sensory acceptance and stability of jussara juice, were investigated. While mild pressurization (200 MPa for 5 min) retained anthocyanins and AA, 82% of anthocyanins content and 46% of TEAC values were lost at the most extreme pressurization condition (500 MPa for 10 min). The addition of 12.5% sucrose was the ideal for jussara juice consumer acceptance. No significant difference was observed for overall sensory acceptance scores of unprocessed (6.7) and HHP-processed juices (6.8), both juices being well-accepted. However, pressurization was ineffective in promoting the retention of anthocyanins and AA in jussara juice stored at refrigeration temperature for 60 days, probably due to enzymatic browning.

High Hydrostatic Pressure Processing of Whole Carrots: Effect of Static and Multi-Pulsed Mild Intensity Hydrostatic Pressure Treatments on Bioactive Compounds.

In this study, the effects of static and multi-pulsed mild-intensity high hydrostatic pressure (HHP) treatments (60 or 100 MPa, ~23 °C) on the extractability and accumulation of phenolics and carotenoids in whole carrots were evaluated. HHP treatments were applied for the time needed to reach the desired pressure (come-up-time, CUT) either as a single pulse or multi-pulse (2P, 3P, and 4P). Likewise, a single sustained treatment (5 min) applied at 60 or 100 MPa was evaluated. Individual carotenoids, free and bound phenolics were quantified after HHP treatment and subsequent storage (48 h, 15 °C). As an immediate HHP response, phenolic extractability increased by 66.65% and 80.77% in carrots treated with 3P 100 MPa and 4P 60 MPa, respectively. After storage, CUT 60 MPa treatment accumulated free (163.05%) and bound (36.95%) phenolics. Regarding carotenoids, total xanthophylls increased by 27.16% after CUT 60 MPa treatment, whereas no changes were observed after storage. Results indicate that HHP processing of whole carrots at mild conditions is a feasible innovative tool to enhance the nutraceutical properties of whole carrots by increasing their free and bound phenolic content while maintaining carotenoid levels. HHP treated carrots can be used as a new functional food or as raw material for the production of food and beverages with enhanced levels of nutraceuticals.

Combined Treatments of High Hydrostatic Pressure and CO2 in Coho Salmon (Oncorhynchus kisutch): Effects on Enzyme Inactivation, Physicochemical Properties, and Microbial Shelf Life.

This study focused on applying different high hydrostatic pressure + carbon dioxide (HHP + CO2) processing conditions on refrigerated (4 °C, 25 days) farmed coho salmon (Oncorhynchus kisutch) to inactivate endogenous enzymes (protease, lipase, collagenase), physicochemical properties (texture, color, lipid oxidation), and microbial shelf life. Salmon fillets were subjected to combined HHP (150 MPa/5 min) and CO2 (50%, 70%, 100%). Protease and lipase inactivation was achieved with combined HHP + CO2 treatments in which lipase activity remained low as opposed to protease activity during storage. Collagenase activity decreased approximately 90% during storage when applying HHP + CO2. Combined treatments limited the increase in spoilage indicators, such as total volatile amines and trimethylamine. The 150 MPa + 100% CO2 treatment was the most effective at maintaining hardness after 10 days of storage. Combined treatments limited HHP-induced color change and reduced the extent of changes caused by storage compared with the untreated sample. Microbial shelf life was extended by the CO2 content and not by the HHP treatments; this result was related to an increased lag phase and decreased growth rate. It can be concluded that combining HHP and CO2 could be an effective method of inactivating endogenous enzymes and extend salmon shelf life.

Synergistic effect of ultraviolet radiation and high hydrostatic pressure on texture, color, and oxidative stability of refrigerated tilapia fillets.

BACKGROUND: The isolated application of high hydrostatic pressure (HHP) and ultraviolet-C (UV-C) radiation may induce physicochemical changes, thus jeopardizing fish quality attributes during refrigerated storage, which could be minimized by the use of synergistic treatments. Therefore, this study investigated the combined effect of UV-C at 0.103 ± 0.002 J cm−2 and HHP at 220 MPa for 10 min on quality parameters of tilapia fillets stored at 4 °C for 15 days. RESULTS: HHP and UV-C+HHP showed higher myoglobin concentration, higher metmyoglobin-reducing activity, and lower a* than control and the UV-C treatment (P < 0.05), reaching 2.49, 2.58, 1.62, 1.54 mg myoglobin g−1 , 13.30, 13.81, 2.29, 2.29%, and 2.32, 2.58, 4.29, 3.98 respectively on the last day of storage. UV-C, HHP, and UV-C+HHP increased water-holding capacity and decreased the pH levels, hardness, and chewiness (P < 0.05). HHP retarded lipid oxidation and UV-C increased protein oxidation compared with control (P < 0.05), achieving 0.81 and 2.80 mg malondialdehyde per kilogram of fish tissue, and 5.50 and 4.15 nmol carbonyl per milligram of protein respectively on 15th day of the storage. CONCLUSION: Both technologies (alone or together) enhanced texture parameters and water-holding capacity. UV-C did not induce color changes and lipid oxidation, whereas HHP showed high potential to prevent meat discoloration, lipid oxidation, and protein oxidation. In addition, this combination of methods could be an alternative to prevent protein oxidation induced by UV-C.

Hydrostatic High-Pressure Post-Processing of Specimens Fabricated by DLP, SLA, and FDM: An Alternative for the Sterilization of Polymer-Based Biomedical Devices.

In this work, we assess the effects of sterilization in materials manufactured using additive manufacturing by employing a sterilization technique used in the food industry. To estimate the feasibility of the hydrostatic high-pressure (HHP) sterilization of biomedical devices, we have evaluated the mechanical properties of specimens produced by commercial 3D printers. Evaluations of the potential advantages and drawbacks of Fused Deposition Modeling (FDM), Digital Light Processing (DLP) technology, and Stereolithography (SLA) were considered for this study due to their widespread availability. Changes in mechanical properties due to the proposed sterilization technique were compared to values derived from the standardized autoclaving methodology. Enhancement of the mechanical properties of samples treated with Hydrostatic high-pressure processing enhanced mechanical properties, with a 30.30% increase in the tensile modulus and a 26.36% increase in the ultimate tensile strength. While traditional autoclaving was shown to systematically reduce the mechanical properties of the materials employed and damages and deformation on the surfaces were observed, HHP offered an alternative for sterilization without employing heat. These results suggest that while forgoing high-temperature for sanitization, HHP processing can be employed to take advantage of the flexibility of additive manufacturing technologies for manufacturing implants, instruments, and other devices.

Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3.

Enzymes from the thermolysin family are crucial factors in the pathogenesis of several diseases caused by bacteria and are potential targets for therapeutic interventions. Thermolysin encoded by the gene LIC13322 of the causative agent of leptospirosis, Leptospira interrogans, was shown to cleave proteins from the Complement System. However, the production of this recombinant protein using traditional refolding processes with high levels of denaturing reagents for thermolysin inclusion bodies (TL-IBs) solubilization results in poor recovery and low proteolytic activity probably due to improper refolding of the protein. Based on the assumption that leptospiral proteases play a crucial role during infection, the aim of this work was to obtain a functional recombinant thermolysin for future studies on the role of these metalloproteases on leptospiral infection. The association of high hydrostatic pressure (HHP) and alkaline pH was utilized for thermolysin refolding. Incubation of a suspension of TL-IBs at HHP and a pH of 11.0 is non-denaturing but effective for thermolysin solubilization. Soluble protein does not reaggregate by dialysis to pH 8.0. A volumetric yield of 46 mg thermolysin/L of bacterial culture and a yield of near 100% in relation to the total thermolysin present in TL-IBs were obtained. SEC-purified thermolysin suffers fragmentation, likely due to autoproteolysis and presents proteolytic activity against complement C3 α-chain, possibly by a generation of a C3b-like molecule. The proteolytic activity of thermolysin against C3 was time and dose-dependent. The experience gained in this study shall help to establish efficient HHP-based processes for refolding of bioactive proteins from IBs.

Refolding of the recombinant protein Sm29, a step toward the production of the vaccine candidate against schistosomiasis.

Schistosomiasis is an important parasitic disease, with about 240 million people infected worldwide. Humans and animals can be infected, imposing an enormous social and economic burden. The only drug available for chemotherapy, praziquantel, does not control reinfections, and an efficient vaccine for prophylaxis is still missing. However, the tegumental protein Sm29 of Schistosoma mansoni was shown to be a promising antigen to compose an anti-schistosomiasis vaccine. Though, recombinant Sm29 is expressed in Escherichia coli as insoluble inclusion bodies requiring an efficient process of refolding, thus, hampering its production in large scale. We present in this work studies to refold the recombinant Sm29 using high hydrostatic pressure, a mild condition to dissociate aggregated proteins, leading to refolding on a soluble conformation. Our studies resulted in high yield of rSm29 (73%) as a stably soluble and structured protein. The refolded antigen presented protective effect against S. mansoni development in immunized mice. We concluded that the refolding process by application of high hydrostatic pressure succeeded, and the procedure can be scaled-up, allowing industrial production of Sm29.

Pressure-temperature folding landscape in proteins involved in neurodegenerative diseases and cancer.

High hydrostatic pressure (HHP) is a valuable tool to study processes such as protein folding, protein hydration and protein-protein interactions. HHP is a nondestructive technique because it reversibly affects internal cavities excluded from the solvent present in the hydrophobic core of proteins. HHP allows the solvation of buried amino acid side chains, thus shifting the equilibrium towards states of the studied molecule or molecular ensemble that occupy smaller volumes. HHP has long been used to dissociate multimeric proteins and protein aggregates and allows investigation of intermediate folding states, some of which are formed by proteins involved in human degenerative diseases, such as spongiform encephalopathies and Parkinson's disease, as well as cancer. When coupled with nuclear magnetic resonance and spectroscopic methods such as infrared and fluorescence spectroscopy, HHP treatment facilitates the understanding of protein folding and misfolding processes; the latter is related to protein aggregation into amyloid or amorphous species. In this review, we will address how HHP provides information about intermediate folding states and the aggregation processes of p53, which is related to cancer, and prion proteins, transthyretin and α-synuclein, which are related to human degenerative diseases.