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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.

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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.

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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.

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In this study, a mixture design with process variables was used to optimize the extraction of total phenolic compounds (TPC) from yerba mate leaves through high hydrostatic pressure extraction. The studied variables were pressure (50, 100, and 150 MPa), extraction time (10, 20, and 30 min), and solvent (water, glycerin, and 50% v/v water/50% v/v glycerin). The multiple linear regression model presented an excellent fit (R2 adjusted of 0.9792) and demonstrated the major influence of glycerin content on the water/glycerin mixture solvent for TPC extraction. Optimal process conditions obtained were 69% v/v water, 31% v/v glycerin, 50 MPa pressure, and 10 min time. PRACTICAL APPLICATION: The paper describes a novel extraction method to obtain phenolic compounds from yerba mate (compounds that can replace synthesized antioxidants in the food industry) using high hydrostatic pressure and environmentally friendly solvents. The extraction process was studied to optimize its performance, obtaining more phenolic compounds from the same amount of yerba mate.

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The high hydrostatic pressure (HHP) process has been studied for several applications in food technology and has been commercially implemented in several countries, mainly for non-thermal pasteurization and shelf-life extension of food products. HHP processing has been demonstrated to accelerate proteolytic hydrolysis at a specific combination of pressure and pressure-holding time for a given protein source and enzyme. The enzymatic hydrolysis of proteins is a well-known alternative to producing biologically active peptides, with antioxidant and antihypertensive capacity, from different food protein sources. However, some of these protein sources contain allergenic epitopes which are often not degraded by traditional hydrolysis. Moreover, the peptide profile and related biological activity of a hydrolysate depend on the protein source, the enzymes used, the parameters of the proteolysis process (pH, temperature, time of hydrolysis), and the use of other technologies such as HHP. The present review aims to provide an update on the use of HHP for improving enzymatic hydrolysis, with a particular focus on studies which evaluated hydrolysate antihypertensive and antioxidant capacity, as well as residual allergenicity. Overall, HHP has been shown to improve the biological properties of hydrolysates. While protein allergenicity can be reduced with traditional hydrolysis, HHP can further reduce the allergenicity. Compared with traditional hydrolysis methods, HHP-assisted protein hydrolysis offers a greater opportunity to add value to protein-rich products through conversion into high-end hydrolysate products with enhanced nutritional and functional properties.

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Wheat flour is the main ingredient used in the preparation of bread. Factors such as low gluten content and the addition of nontraditional ingredients in baking affect the quality of wheat flour and may limit its use in baking. With the increasing trend of "clean label" products, it may be interesting to develop and use physical processes to improve the quality of wheat flour and avoid the use of chemical additives. High hydrostatic pressure, non-thermal plasma, ultrasound, ozonation, ultraviolet light, and pulsed light treatments are non-thermal emerging technologies (NTETs) that have been studied for this purpose. They were originally developed to inactivate microorganisms and enzymes in foods. Additionally, these technologies can be used at low temperatures to modify the most important component of wheat flour, i.e., gluten and its fractions, which are responsible for the rheological properties of wheat flour dough. Thus, this review focuses on the effects of these NTETs by considering the following factors: (1) the technological properties of gluten, (2) gluten-starch interactions, (3) possible effects of NTETs on minor components of flours, and (4) the quality of wheat flour and the resulting final products.

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BACKGROUND: Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. RESULTS: In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield). CONCLUSIONS: We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.

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The extraction of bioactive compounds from fruits, such as lemon, has gained relevance because these compounds have beneficial properties for health, such as antioxidant and anticancer properties; however, the extraction method can significantly affect these properties. High hydrostatic pressure and ultrasound, as emerging extraction methods, constitute an alternative to conventional extraction, improving extractability and obtaining extracts rich in bioactive compounds. Therefore, lemon extracts (LEs) were obtained by conventional (orbital shaking), ultrasound-assisted, and high-hydrostatic-pressure extraction. Extracts were then microencapsulated with maltodextrin at 10% (M10), 20% (M20), and 30% (M30). The impact of microencapsulation on LEs physicochemical properties, phenolics (TPC), flavonoids (TFC) and relative bio-accessibility (RB) was evaluated. M30 promoted a higher microencapsulation efficiency for TPC and TFC, and a longer time required for microcapsules to dissolve in water, as moisture content, water activity and hygroscopicity decreased. The RBs of TPC and TFC were higher in microcapsules with M30, and lower when conventional extraction was used. The data suggest that microencapsulated LE is promising as it protects the bioactivity of phenolic compounds. In addition, this freeze-dried product can be utilized as a functional ingredient for food or supplement formulations.

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This review focuses on describing and discussing recent findings regarding the effects of high hydrostatic pressure (HHP) on the supramolecular structure and technofunctional properties of starch, as well as on analyzing the hypothesis to explain these changes. The non-thermal modification of starch through HHP involves complex supramolecular structural changes that depend on the botanical source, amylose content, and treatment intensity. Overall, the granular morphology, lamellar and crystalline structures, and double helices undergo different degrees of modification/disorganization during HHP, but these changes are distinguished from thermal modification by an improvement at the same gelatinization degree. The HHP-induced supramolecular modifications determine the properties of starch, including water solubility, swelling power, pasting, water and oil holding capacity, thermal properties, and in vitro digestibility.

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Phenolic compounds from fruits and vegetables have shown antioxidant, anticancer, anti-inflammatory, among other beneficial properties for human health. All these benefits have motivated multiple studies about preserving, extracting, and even increasing the concentration of these compounds in foods. A diverse group of vegetable products treated with High Hydrostatic Pressure (HHP) at different pressure and time have shown higher phenolic content than their untreated counterparts. The increments have been associated with an improvement in their extraction from cellular tissues and even with the activation of the biosynthetic pathway for their production. The application of HHP from 500 to 600 MPa, has been shown to cause cell wall disruption facilitating the release of phenolic compounds from cell compartments. HPP treatments ranging from 15 to 100 MPa during 10-20 min at room temperature have produced changes in phenolic biosynthesis with increments up to 155%. This review analyzes the use of HHP as a method to increase the phenolic content in vegetable systems. Phenolic content changes are associated with either an immediate stress response, with a consequent improvement in their extraction from cellular tissues, or a late stress response that activates the biosynthetic pathways of phenolics in plants.

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This study evaluated the effect of different extraction technologies and conditions in order to obtain jaboticaba skin extracts. Firstly, the skins were extracted by conventional extraction, according to a rotatable central composite design, varying ethanol concentration, solid:liquid ratio, and temperature. Next, ultrasound-assisted extraction was performed using different power densities and times. Finally, high-pressure extractions were performed with varying pressures and times. For agitated bed extraction, the highest anthocyanin content was observed for ethanol concentrations varying between 60% and 80%. Thus, the independent variables which more influenced anthocyanin content were ethanol concentration and solid:liquid ratio. Folin-Ciocalteu reducing capacity was linearly affected by the increase in temperature. Ethanol concentration was the variable that most influenced ABTS+. On the other hand, the increase in ethanol concentration decreased the antioxidant capacity by ABTS+. Considering the ultrasound extraction, increasing its power did not affect total monomeric anthocyanins content, while the increase in process time had better yields. The highest antioxidant capacity and total monomeric anthocyanins were found for the highest extraction time. Similarly, with ultrasound, the increase in high hydrostatic-assisted extraction time positively influenced anthocyanin content and antioxidant capacity. As a result, the ultrasound-assisted method was found to be the best extraction technology for anthocyanins recovery.

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Current methods for vanilla bean curing are long and reduce the enzymatic activity necessary for flavor development. High hydrostatic pressure (HHP) at 50-600 MPa was used to improve phenolic compounds formation and ß-d-glucosidase activity in vanilla beans compared with scalded beans. Phenolics were analyzed by HPLC and ß-d-glucosidase activity by spectrophotometry. Vanillin was the main phenolic and it was formed by ß-d-glucovanillin hydrolysis and vanillyl alcohol oxidation. HHP improved vanillin content and influenced ß-d-glucosidase activity. At the beginning of the curing the highest increments of vanillin were produced at 400 MPa (up to 15%), while at the end, this was observed at 50 (138%) and 600 MPa (74%). Maximum increment of up to 400% in ß-d-glucosidase activity was observed from 100 to 300 MPa, which was attributed to tissue decompartmentalization, and conformational changes induced by pressure. HHP could be used during vanilla curing to improve vanillin content and ß-d-glucosidase activity.

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The growing incidence of non-communicable diseases makes the search for natural sources of bioactive compounds a priority for such disease prevention/control. Achyrocline satureioides ('marcela'), a plant rich in polyphenols and native to Brazil, Uruguay, Paraguay, and Argentina, could be used for this purpose. Data on its antidiabetic/antiobesity properties and cellular uptake of bioactive compounds are lacking. The potentiality of non-thermal technologies such as high-hydrostatic pressure (HP) to enhance polyphenol extraction retains attention. Thus, in the present study aqueous and ethanolic marcela extracts with/without assisted-HP processing were chemically characterized and assessed for their in vitro antioxidant capacity, antidiabetic and antiobesity activities, as well as cellular cytotoxicity and uptake on intestinal cell monolayers (TC7-cells, a clone of Caco-2 cells). Aqueous and ethanolic conventional extracts presented different polyphenolic profiles characterized mainly by phenolic acids or flavonoids, respectively, as stated by reverse phase-high-performance liquid chromatography (RP-HPLC) analyses. In general, ethanolic extracts presented the strongest bioactive properties and HP had none or a negative effect on in vitro bioactivities comparing to conventional extracts. TC7-cell viability and cellular uptake demonstrated in conventional and HP-assisted extracts, highlighted the biological effects of marcela bioactive compounds on TC7-cell monolayers. TC7-cell studies showed no HP-induced cytotoxicity. In sum, marcela extracts have great potential as functional ingredients for the prevention/treatment of chronic diseases such as diabetes.

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Since conventional thermal processing can have detrimental consequences on aroma compounds, non-thermal technologies such as high hydrostatic pressure (HHP) have been explored. HHP may alter the weak chemical bonds of enzymes. These changes can modify the secondary, tertiary, and quaternary structures of key enzymes in the production of aroma compounds. This can result in either an increase or decrease in their content, along with reactions or physical processes associated with a reduction of molecular volume. This article provides a comprehensive review of HHP treatment's effects on the content of lipid-derived aroma compounds, aldehydes, alcohols, ketones, esters, lactones, terpenes, and phenols, on various food matrices of vegetable and animal origin. The content of aldehydes and ketones in food samples increased when subjected to HHP, while the content of alcohols and phenols decreased, probably due to oxidative processes. Both ester and lactone concentrations appeared to decline due to hydrolysis reactions. There is no clear tendency regarding terpenes concentration when subjected to HHP treatments. Because of the various effects of HHP on aroma compounds, an area of opportunity arises to carry out future studies that allow optimizing and controlling the effect.

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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.

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ABSTRACT: The effect of high hydrostatic pressure (HHP) application on whey protein concentrate was evaluated both before (pre-treatment - PT) and during (hydrolysis assisted - HA) hydrolysis processes. A factorial design 22 with 3 central points was used with pressure (100, 250, 400 MPa) and time (5, 20 and 35 minutes) as independent variables. The hydrolysis was evaluated and monitored by soluble protein, aromatic amino acid contents and RP-HPLC. ABTS and ORAC tests were used to evaluate the in vitro antioxidant capacity. The reduction of soluble protein content was approximately 20% for conventional hydrolysis and for all PT treatments up to 4 h of reaction, while HHP assisted hydrolysis at 100 MPa showed a 35% protein reduction after 35 minutes of reaction. In addition, pressurization favored peptic hydrolysis of β-lactoglobulin by up to 98% and also improved the in vitro antioxidant capacity of the hydrolysates, which increased from 34.25 to 60.89 μmoles TE g-1 of protein in the best treatment. The results suggest that the use of HHP assisted hydrolysis favored the peptic hydrolysis, with a reduction in hydrolysis time and increased antioxidant activity.

RESUMO: Neste estudo, o efeito da aplicação de alta pressão hidrostática (HHP) sobre o concentrado proteico de soro de leite foi avaliado antes (pré-tratamento - PT) e durante os processos de hidrólise (assistida por hidrólise - HA). Utilizou-se o delineamento fatorial 22 com três pontos centrais, onde as variáveis independentes foram pressão (100, 250, 400 MPa) e tempo (5, 20 e 35 minutos). A hidrólise foi avaliada pelo conteúdo de proteínas solúveis e aminoácidos aromáticos, além do perfil peptídico por RP-HPLC. As análises de ABTS e ORAC foram utilizadas para avaliar a capacidade antioxidante in vitro. A redução do teor de proteína solúvel foi de aproximadamente 20% para a hidrólise convencional e para todos os pontos de PT até 4h de reação, enquanto a hidrólise assistida por HHP a 100 MPa mostrou uma redução de 35% de proteína em 35 minutos de reação. Além disso, a pressurização favoreceu a hidrólise péptica da β-lactoglobulina em até 98% e também melhorou a capacidade antioxidante in vitro dos hidrolisados, que aumentaram de 34,25 para 60,89 μmoles de TE g-1 de proteína no melhor tratamento. Os resultados sugerem que o uso da hidrólise assistida por HHP favoreceu a hidrólise péptica, com redução no tempo de hidrólise e aumento da atividade antioxidante.

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The effect of high hydrostatic pressure (HHP) application on whey protein concentrate was evaluated both before (pre-treatment - PT) and during (hydrolysis assisted - HA) hydrolysis processes. A factorial design 22 with 3 central points was used with pressure (100, 250, 400 MPa) and time (5, 20 and 35 minutes) as independent variables. The hydrolysis was evaluated and monitored by soluble protein, aromatic amino acid contents and RP-HPLC. ABTS and ORAC tests were used to evaluate the in vitro antioxidant capacity. The reduction of soluble protein content was approximately 20% for conventional hydrolysis and for all PT treatments up to 4 h of reaction, while HHP assisted hydrolysis at 100 MPa showed a 35% protein reduction after 35 minutes of reaction. In addition, pressurization favored peptic hydrolysis of -lactoglobulin by up to 98% and also improved the in vitro antioxidant capacity of the hydrolysates, which increased from 34.25 to 60.89 moles TE g-1 of protein in the best treatment. The results suggest that the use of HHP assisted hydrolysis favored the peptic hydrolysis, with a reduction in hydrolysis time and increased antioxidant activity.(AU)

Neste estudo, o efeito da aplicação de alta pressão hidrostática (HHP) sobre o concentrado proteico de soro de leite foi avaliado antes (pré-tratamento - PT) e durante os processos de hidrólise (assistida por hidrólise - HA). Utilizou-se o delineamento fatorial 22 com três pontos centrais, onde as variáveis independentes foram pressão (100, 250, 400 MPa) e tempo (5, 20 e 35 minutos). A hidrólise foi avaliada pelo conteúdo de proteínas solúveis e aminoácidos aromáticos, além do perfil peptídico por RP-HPLC. As análises de ABTS e ORAC foram utilizadas para avaliar a capacidade antioxidante in vitro. A redução do teor de proteína solúvel foi de aproximadamente 20% para a hidrólise convencional e para todos os pontos de PT até 4h de reação, enquanto a hidrólise assistida por HHP a 100 MPa mostrou uma redução de 35% de proteína em 35 minutos de reação. Além disso, a pressurização favoreceu a hidrólise péptica da -lactoglobulina em até 98% e também melhorou a capacidade antioxidante in vitro dos hidrolisados, que aumentaram de 34,25 para 60,89 moles de TE g-1 de proteína no melhor tratamento. Os resultados sugerem que o uso da hidrólise assistida por HHP favoreceu a hidrólise péptica, com redução no tempo de hidrólise e aumento da atividade antioxidante.(AU)

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RESEARCH BACKGROUND: Passion fruit and carrot have a good antioxidant capacity, however, their consumption is low. There is no information on their use in beverages or in processes such as high hydrostatic pressure, which provides the safety of the drink without affecting its quality. EXPERIMENTAL APPROACH: In this study the effect of high hydrostatic pressure (HHP; 500 MPa for 250 s at 25 °C) and thermal processing (at 65 °C for 10 min, 75 °C for 2 min and 95 °C for 1 min) were evaluated in the formulation of a cold-pressed beverage from purple passion fruit, green passion fruit and carrot juice, taking into account antioxidant capacity, vitamin C concentration, sensorial evaluation and microbiological growth at 8 °C. RESULTS AND CONCLUSIONS: The formulation containing 67% purple passion fruit, 17% green passion fruit and 17% carrot was the one that stood out with its antioxidant capacity, high vitamin C concentration and sensorial evaluation. The HHP treatment preserved the antioxidant capacity and vitamin C concentration, and resulted in the best scent. Juices stored at 8 °C did not show microbial growth. NOVELTY AND SCIENTIFIC CONTRIBUTION: In this study, we used tropical raw materials with good sensory acceptance and antioxidant capacity that could be used in the production of high value-added foods. Additionally, the research demonstrated that HHP is a conservation method that maintains the antioxidant capacity, vitamin C and aroma of the beverage to a greater extent compared to thermal treatments; the latter is of interest for its use in minimally processed products and functional food.

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High hydrostatic pressure (HHP) promotes the release of bioactive compounds from their intracellular compartments making them more bioaccessible. Our aim was to propose a schematic tissue model to explain the release mechanisms of betalains and phenolic compounds in vegetable cells submitted to HHP by analyzing cell microstructure, cell morphology, cell viability and the localization of bioactive compounds in prickly pear fruits. Prickly pear slices were pressurized at 100, 350 and 600 MPa at 20 °C. Chlorenchyma cells (in peels) and parenchyma cells (in pulps) were analyzed by transmission electron microscopy, confocal laser scanning microscopy and optical microscopy. After pressurization, the respiration and ethylene production of processed fruits were measured every 6 h (during storage at 16 °C and 75% RH for 24 h). In chlorenchyma cells, HHP ruptured betalain-storing vesicles in the cytoplasm and possibly increased the activity of endogenous enzymes. Contrarily, HHP released betalains from the vacuoles of parenchyma cells due to breaking of the tonoplast where they presented higher stability. In both tissues, phenolic compounds were released from cell walls with increasing pressure and enhanced by cell wall ultrastructural modifications (100 MPa), rupture (350 MPa) and the rearrangement of microfibrillated cellulose (600 MPa). Prickly pears submitted to HHP presented advanced senescence marked by considerable ethylene increase and the gradual loss of CO2 production after 6 h. Cells were viable at 100 MPa by conserving intact cell membranes and after 24 h their respiration rates presented no significant differences compared to controls therefore indicating the possibility of synthesis of bioactive compound as a response to abiotic stress. We have proposed a new approach for analyzing the effects of HHP and have identified the storing of betalains in vesicles located in the cytoplasm of chlorenchyma cells for the first time. This study is the first to fathom the dynamic morphological changes and release mechanisms of bioactive compounds in vegetable cells subjected to HHP.

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The objectives of this study were: to assess the efficiency of high hydrostatic pressure or ultra-high pressure homogenization against Mycobacterium smegmatis in milk and to discuss whether M. smegmatis can be considered a suitable surrogate for other Mycobacterium spp. in high pressure inactivation trials using milk. Three strains of this specie (CECT 3017, 3020 and 3032) were independently inoculated into both skimmed (0.2% fat) and whole milk (3.4% fat) at an approximate load of 6.5 Log CFU/ml and submitted to HHP treatments at 300, 400 or 500 MPa for 10 m at 6°C and 20°C. Evolution of the surviving cells of the inoculated strains was evaluated analysing milk immediately after the treatments and after 5 and 8 d of storage at 6°C. HHP treatments at 300 MPa were seldom efficient at inactivating M. smegmatis strains, but lethality increased with pressure applied in all cases. Generation of sub-lethal injured cells was observed only after 400 MPa treatments since inactivation at 500 MPa was shown to be complete. Significant differences were not observed due to either temperature of treatment or fat content of milk, except for strain CECT3032, which was shown to be the most sensitive to HHP treatments. Milk inoculated with strain CECT3017 was submitted to ultra-high pressure homogenization (UHPH) treatments at 200, 300 and 400 MPa. Maximum reductions were obtained after 300 and 400 MPa treatments, although less than 3.50 Log CFU/ml were inactivated. UHPH did not cause significant number of injured cells. The usefulness of this species as a marker for pressure-based processing seems limited since it showed greater sensitivity than some pathogenic species including other Mycobacteria reported in previous studies.

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