The effect of hyperbaric storage on Bacillus subtilis endospores, as a new food preservation methodology with potential to replace the conventional refrigeration processes, was assessed and compared to refrigeration. To do so, three different matrices (McIlvaine buffer, carrot juice and brain-heart infusion broth, BHI-broth) were inoculated with B. subtilis endospores and stored at 25, 50 and 100 MPa at variable/uncontrolled room temperature (18-23 °C), under refrigeration (4 °C), and room temperature at atmospheric pressure (0.1 MPa), up to 60 days. Two different quantification procedures were performed to assay both vegetative and endospores (unheated samples) and endospores (heated samples), to assess germination under pressure. The results showed that hyperbaric storage yielded pronounced endospore loads reductions in carrot juice and BHI-broth at 50 and 100 MPa, while in McIlvaine buffer, lower endospore loads reductions were observed. At 25 MPa, the endospores germinated and outgrew in carrot juice. Under refrigeration conditions, both carrot juice and BHI-broth underwent endospore germination and outgrowth after 60 and 9 days of storage, respectively, while in McIlvaine buffer there were no endospore outgrowth. These results suggest that hyperbaric storage at room temperature might not only be a feasible preservation procedure regarding endospores, but also that the food product (matrix characteristics) seems to influence the microbial inactivation that occurs during hyperbaric storage.

Control of endospores of Alicyclobacillus acidoterrestris in pasteurized apple juice using hyperbaric storage at 18 to 23 °C was compared to storage at atmospheric pressure and 18 to 23 °C, as well as refrigeration at ~4 °C for up to 30 days. The juice samples were inoculated with approximately 1 × 105 CFU/mL spores. The juice spoiled quickly at atmospheric pressure and ambient temperature, while under refrigeration spore levels remained unchanged for 30 days. Hyperbaric storage of inoculated apple juice at 25, 50 and 100 MPa at 18 to 23 °C resulted in spore inactivation at more rapid rates as pressure magnitudes increased, reaching levels below the detection limit of 10 CFU/mL at 50 and 100 MPa. In highly acid foods such as apple juice, hyperbaric storage at pressures ≤100 MPa and ambient temperature was effective in inactivating spores of A. acidoterrestris for periods up to 30 days. These results indicate hyperbaric storage at ambient temperature as a clearly more efficient preservation procedure to control the development of A. acidoterrestris endospores, compared to ambient temperature and refrigerated storage, in highly acidic foods as apple juice.

Food contamination with heat-resistant fungi (HRF), and their spores, is a major issue among fruit processors, being frequently found in fruit juices and concentrates, among other products, leading to considerable economic losses and food safety issues. Several strategies were developed to minimize the contamination with HRF, with improvements from harvesting to the final product, including sanitizers and new processing techniques. Considering consumers' demands for minimally processed, fresh-like food products, nonthermal food-processing technologies, such as high-pressure processing (HPP), among others, are emerging as alternatives to the conventional thermal processing techniques. As no heat is applied to foods, vitamins, proteins, aromas, and taste are better kept when compared to thermal processes. Nevertheless, HPP is only able to destroy pathogenic and spoilage vegetative microorganisms to levels of pertinence for food safety, while bacterial spores remain. Regarding HRF spores (both ascospores and conidiospores), these seem to be more pressure-sensible than bacterial spores, despite a few cases, such as the ascospores of Byssochlamys spp., Neosartorya spp., and Talaromyces spp. that are resistant to high pressures and high temperatures, requiring the combination of both variables to be inactivated. This review aims to cover the literature available concerning the effects of HPP at room-like temperatures, and its combination with high temperatures, and high-pressure cycling, to inactivate fungi spores, including the main factors affecting spores' resistance to high-pressure, such as pH, water activity, nutritional composition of the food matrix and ascospore age, as well as the changes in the spore ultrastructure, and the parameters to consider regarding their inactivation by HPP.

BACKGROUND: Raw ewe's milk, as used to manufacture Serra da Estrela Protected Designation of Origin cheese, was pre-treated y high pressure processing (HPP), using previously optimized conditions (121 MPa for 30 min), aiming to evaluate its effect on ilk technological properties for subsequent cheese production, namely the impact on resulting curd, whey and cheese hroughout ripening. RESULTS: The cheese yield increased 10.4% as a result of milk pre-treated by HPP, which also yielded inactivation of beneficial icrobial groups. After 60 days of ripening, both treated and control cheeses showed no significant differences (P ≥ 0.05) with espect to quantified microbial load or basic physicochemical quality parameters. CONCLUSION: HPP appears to be a promising non-thermal treatment for ewes' milk to inactivate contaminant bacteria but ith no negative effect on lactic acid bacteria, which is very important for the unique characteristics of Serra da Estrela cheese.

Hyperbaric storage (HS) is a novel technology for storing foods under mild pressures that, when used at room temperature (RT), offers much lower energy costs and greenhouse gas emissions than conventional refrigeration (RF). Watermelon juice (WJ), with interesting associated health benefits, is highly perishable due to its pH (5.20–6.70) and water activity (aW, 0.97–0.99). This work investigated aW’s impact on WJ’s preservation by HS/RT, studying the behavior of Escherichia coli, Listeria monocytogenes, and Saccharomyces cerevisiae inoculated in WJ at aW 0.930–0.971 stored at 25–75 MPa for up to 28 days, along with RT and RF atmospheric pressure controls. The results showed that HS could control microbial growth, and, during storage, inactivation was also observed, and that HS’s impact depended on the aW level, microorganism, and storage pressure. Inactivation was often increased at 50–75 MPa and at aW 0.930–0.950, while growth mostly occurred at aW 0.971. The inactivation curves were mathematically described by the first-order and Weibull kinetic models, with the Weibull model frequently obtaining better fits. These findings support HS’s potential for food preservation, showing better overall WJ growth control and inactivation effects than RF, without temperature control, making HS environmentally friendlier.

The systematic effect of pH (4.00, 5.00 and 6.50) on hyperbaric storage (HS, 25-100 MPa) at uncontrolled room temperature (RT, 18-23 °C) for up to 28 days was studied for the first time, using watermelon juice (WJ) as a case-study, due to its high perishability, focusing on Escherichia coli and Saccharomyces cerevisiae’s behaviour, along with RT and refrigerated controls at atmospheric pressure for comparison. Results showed that HS at RT could control E. coli and S. cerevisiae’s growth and that, during storage, inactivation also occurred, often reaching reductions over 5 log CFU/mL. HS’ effect varied according to the microorganism, the juice’s pH and storage pressure, and inactivation was observed regardless the pH and pressure level tested for both microorganisms. Higher pressures increased the inactivation rates of both microorganisms, while pH’s impact depended on the microorganism. E. coli showed greater inactivation at pH 4.0 whereas S. cerevisiae exhibited comparable or slightly higher reductions at pH 6.50. Microbial inactivation was well described by both first-order and Weibull kinetic models, with the latter generally providing a better fit. At pH 4.00, S. cerevisiae obtained lower zp-values than E. coli. This work showed HS’ potential for food preservation regardless of the pH (despite being influenced by it), without needing temperature control, which may lower energy costs and greenhouse gas emissions, being potentiality environmentally friendlier.

Honey is a value-added product rich in several types of phenolic compounds, enzymes, and sugars recently explored in biomedical and food applications. Nevertheless, even though it has a low water activity (aW = 0.65) that hinders the development of pathogenic and spoilage microorganisms, it is still prone to contamination by pathogenic microorganisms (vegetative and spores) and may constitute harm to special groups, particularly by immunosuppressed people and pregnant women. Thus, an efficient processing methodology needs to be followed to ensure microbial safety while avoiding 5-hydroxymethylfurfural (HMF) formation and browning reactions, with a consequent loss of biological value. In this paper, both thermal (pressure-assisted thermal processing, PATP) and nonthermal high-pressure processing (HPP), and another pressure-based methodology (hyperbaric storage, HS) were used to ascertain their potential to inactivate Bacillus subtilis endospores in honey and to study the influence of aW on the inactivation on this endospore. The results showed that PATP at 600 MPa/15 min/75 C of diluted honey (52.9 Brix) with increased aW (0.85 compared to 0.55, the usual honey aW) allowed for inactivating of at least 4.0 log units of B. subtilis spores (to below detection limits), while HS and HPP caused neither the germination nor inactivated spores (i.e., there was neither a loss of endospore resistance after heat shock nor endospore inactivation as a consequence of the storage methodology). PATP of undiluted honey even at harsh processing conditions (600 MPa/15 min/85 C) did not impact the spore load. The results for diluted honey open the possibility of its decontamination by spores’ inactivation for medical and pharmaceutical applications.

BACKGROUND: Raw ewe’s milk, used to manufacture Serra da Estrela Protected Designation of Origin (PDO) cheese, was pre-treated by high pressure processing (HPP), using previously optimized conditions (121 MPa/30 min), in order to evaluate its effect on milk technological properties for subsequent cheese production, namely the impact on resulting curd, whey, and cheese throughout ripening. RESULTS: The cheese yield increase 10.4% as a result of milk pre-treated by HPP, which also yielded inactivation of beneficial microbial groups. After 60 days of ripening, both treated and control cheeses showed neither significant differences (P ≥ 0.05) in quantified microbial load nor in basic physicochemical quality parameters. CONCLUSION: HPP seems to be a promising non-thermal treatment for ewes’ milk to inactivate contaminant bacteria yet with no negative effect on lactic acid bacteria, which are very important for the unique characteristics of Serra da Estrela cheese.

The potentiality of high pressure processing (HPP) to possibly enhance diffusion of asparaginase into raw potato sticks, and consequently on reduction of acrylamide levels in fried potatoes was evaluated. Raw potato sticks were immersed in asparaginase (10,000 ASNU/L) and immediately subjected to 0.1, 100, 200 and 400 MPa for 5 min, with total enzymatic reaction times of 5, 10 and 20 min and room temperature. Pressurized raw potato sticks became softer, more flexible, and required lower energy for cutting (up to 47% less); the roughness of potato surface and moisture content were slightly reduced; and the concentration of soluble solids in the exterior solutions increased, indicative of a leaching effect. Due to changes induced by asparaginase and/or HPP on raw potatoes, fried potatoes exhibited higher weight loss after frying, and higher hardness (crispness). The combined treatment with asparaginase and HPP showed to reduce acrylamide levels by 26 -47%, while with asparaginase or HPP alone there was no significant reduction.

Bone resident cells are constantly subjected to a range of distinct mechanical loadings, which generates a complex microenvironment. In particular, hydrostatic pressure (HP) has a key impact on modulation of cell function and fate determination. Although HP is a constant mechanical stimulus, its role in regulating the osteogenesis process within a defined 3D microenvironment has not been comprehensively elucidated. Perceiving how environmental factors regulate the differentiation of stem cells is essential for expanding their regenerative potential. Inspired by the mechanical environment of bone, this study attempted to investigate the influence of different ranges of cyclic HP on human adipose-derived mesenchymal stem cells (MSCs) encapsulated within a compartmentalized liquefied microenvironment. Taking advantage of the liquefied environment of microcapsules, MSCs were exposed to cyclic HP of 5 or 50 MPa, 3 times/week at 37 °C. Biological tests using fluorescence staining of F-actin filaments showed a noticeable improvement in cell-cell interactions and cellular network formation of MSCs. These observations were more pronounced in osteogenic (OST) condition, as confirmed by fluorescent staining of vinculin. More interestingly, there was a significant increase in alkaline phosphatase activity of MSCs exposed to 50 MPa magnitude of HP, even in the absence of osteoinductive factors. In addition, a greater staining area of both osteopontin and hydroxyapatite was detected in the 50 MPa/OST group. These findings highlight the benefit of hydrostatic pressure to regulate osteogenesis of MSCs as well as the importance of employing simultaneous biochemical and mechanical stimulation to accelerate the osteogenic potential of MSCs for biomedical purposes.