Sequential extraction of compounds of interest from yeast biomass assisted by pulsed electric fields.

One strategy to reduce cost and improve feasibility of waste-yeast biomass valorization is to obtain a spectrum of marketable products rather than just a single one. This study explores the potential of Pulsed Electric Fields (PEF) for the development of a cascade process designed to obtain several valuable products from Saccharomyces cerevisiae yeast biomass. Yeast biomass was treated by PEF, which affected the viability of 50%, 90%, and over 99% of S. cerevisiae cells, depending on treatment intensity. Electroporation caused by PEF allowed access to the cytoplasm of the yeast cell without causing total breakdown of the cell structure. This outcome was an essential prerequisite to be able to perform a sequential extraction of several value-added biomolecules from yeast cells located in the cytosol and in the cell wall. After incubating yeast biomass previously subjected to a PEF treatment that affected the viability of 90% of cells for 24 h, an extract with 114.91 ± 2.86, 7.08 ± 0.64, and 187.82 ± 3.75 mg/g dry weight of amino acids, glutathione, and protein, respectively, was obtained. In a second step, the extract rich in cytosol components was removed after 24 h of incubation and the remaining cell biomass was re-suspended with the aim of inducing cell wall autolysis processes triggered by the PEF treatment. After 11 days of incubation, a soluble extract containing mannoproteins and pellets rich in ß-glucans were obtained. In conclusion, this study proved that electroporation triggered by PEF permitted the development of a cascade procedure designed to obtain a spectrum of valuable biomolecules from S. cerevisiae yeast biomass while reducing the generation of waste.

Pulsed electric field processing as an alternative to sulfites (SO2) for controlling saccharomyces cerevisiae involved in the fermentation of Chardonnay white wine.

The use of sulfites (SO2) for microbial control in the winemaking process is currently being questioned due to its potential toxicity. Pulsed Electric Fields (PEF) are capable of inactivating microorganisms at low temperatures, thus avoiding the negative effects of heat on food properties. In this study, the capacity of PEF technology for the decontamination of yeasts involved in the fermentation process of Chardonnay wine from a winery was evaluated. PEF treatments at 15 kV/cm of low (65 µs, 35 kJ/kg) and higher intensity (177 µs 97 kJ/kg) were selected for evaluating the microbial stability, physicochemical and volatile composition of wine. Even with the least intense PEF-treatment, Chardonnay wine remained yeast-free during 4 months of storage without sulfites. PEF-treatments did not affect the wine's oenological parameters or its aroma during storage. This study, therefore, reveals the potential of PEF technology as an alternative to sulfites for the microbiological stabilization of wine.

Evaluation of Pulsed Electric Fields (PEF) Parameters in the Inactivation of Anisakis Larvae in Saline Solution and Hake Meat.

Larvae of the nematode family Anisakidae are capable of causing parasitic infections in humans associated with the consumption of fishery products, leading to intestinal syndromes and allergic reactions. Anisakidae larvae are widely distributed geographically, with rates of parasitism close to 100% in certain fish species. Methods need to be established for their inactivation and elimination, especially in fishery products that are to be consumed raw, pickled, or salted, or which have been insufficiently treated to kill the parasite. Many strategies are currently available (such as freezing and heat treatment), but further ones, such as pulsed electric fields (PEF), have hardly been investigated until now. This study focuses on the experimental evaluation of the efficacy of PEF in the inactivation of Anisakis spp. larvae in terms of electric field strength, specific energy, and pulse width, as well as on the evaluation of the quality of fish samples after PEF treatment. Results show that viability of Anisakis was highly dependent on field strength and specific energy. Pulse width exerted a considerable influence at the lowest field strengths tested (1 kV/cm). Central composite design helped to define a PEF treatment of 3 kV/cm and 50 kJ/kg as the one capable of inactivating almost 100% of Anisakis present in pieces of hake, while affecting the investigated quality parameters (moisture, water holding capacity, and cooking loss) to a lesser extent than freezing and thawing. These results show that PEF could serve as an alternative to traditional freezing processes for the inactivation of Anisakis in fish.

Microbial Decontamination of Red Wine by Pulsed Electric Fields (PEF) after Alcoholic and Malolactic Fermentation: Effect on Saccharomyces cerevisiae, Oenococcus oeni, and Oenological Parameters during Storage.

New techniques are required to replace the use of sulfur dioxide (SO2) or of sterilizing filtration in wineries, due to those methods' drawbacks. Pulsed electric fields (PEF) is a technology capable of inactivating microorganisms at low temperatures in a continuous flow with no detrimental effect on food properties. In the present study, PEF technology was evaluated for purposes of microbial decontamination of red wines after alcoholic and malolactic fermentation, respectively. PEF combined with SO2 was evaluated in terms of microbial stability and physicochemical parameters over a period of four months. Furthermore, the effect of PEF on the sensory properties of red wine was compared with the sterilizing filtration method. Results showed that up to 4.0 Log10 cycles of S. cerevisiae and O. oeni could be eradicated by PEF and sublethal damages and a synergetic effect with SO2 were also observed, respectively. After 4 months, wine treated by PEF after alcoholic fermentation was free of viable yeasts; and less than 100 CFU/mL of O. oeni cells were viable in PEF-treated wine added with 20 ppm of SO2 after malolactic fermentation. No detrimental qualities were found, neither in terms of oenological parameters, nor in the sensory parameters of wines subjected to PEF after storage time.

Pulsed electric field increases the extraction yield of extra virgin olive oil without loss of its biological properties.

Introduction: Pulsed electric field (PEF) has been used for improving extraction of extra virgin olive oil (EVOO). However, the biological changes induced by the consumption of pulsed electric field-obtained extra virgin olive oil (PEFEVOO) have not been studied yet. Materials and methods: EVOO oils from Empeltre variety were prepared by standard (STD) cold pressure method involving crushing of the olives, malaxation and decanting and by this procedure including an additional step of PEF treatment. Chemical analyses of EVOO oils were done. Male and female Apoe-deficient mice received diets differing in both EVOOs for 12 weeks, and their plasma, aortas and livers were analyzed. Results: PEF application resulted in a 17% increase in the oil yield and minimal changes in chemical composition regarding phytosterols, phenolic compounds and microRNA. Only in females mice consuming PEF EVOO, a decreased plasma total cholesterol was observed, without significant changes in atherosclerosis and liver steatosis. Conclusion: PEF technology applied to EVOO extraction maintains the EVOO quality and improves the oil yield. The equivalent biological effects in atherosclerosis and fatty liver disease of PEF-obtained EVOO further support its safe use as a food.

Preclinical Studies of Granulysin-Based Anti-MUC1-Tn Immunotoxins as a New Antitumoral Treatment.

Two granulysin (GRNLY) based immunotoxins were generated, one containing the scFv of the SM3 mAb (SM3GRNLY) and the other the scFv of the AR20.5 mAb (AR20.5GRNLY). These mAb recognize different amino acid sequences of aberrantly O-glycosylated MUC1, also known as the Tn antigen, expressed in a variety of tumor cell types. We first demonstrated the affinity of these immunotoxins for their antigen using surface plasmon resonance for the purified antigen and flow cytometry for the antigen expressed on the surface of living tumor cells. The induction of cell death of tumor cell lines of different origin positive for Tn antigen expression was stronger in the cases of the immunotoxins than that induced by GRNLY alone. The mechanism of cell death induced by the immunotoxins was studied, showing that the apoptotic component demonstrated previously for GRNLY was also present, but that cell death induced by the immunotoxins included also necroptotic and necrotic components. Finally, we demonstrated the in vivo tumor targeting by the immunotoxins after systemic injection using a xenograft model of the human pancreatic adenocarcinoma CAPAN-2 in athymic mice. While GRNLY alone did not have a therapeutic effect, SM3GRNLY and AR20.5GRNLY reduced tumor volume by 42 and 60%, respectively, compared with untreated tumor-bearing mice, although the results were not statistically significant in the case of AR20.5GRNLY. Histological studies of tumors obtained from treated mice demonstrated reduced cellularity, nuclear morphology compatible with apoptosis induction and active caspase-3 detection by immunohistochemistry. Overall, our results exemplify that these immunotoxins are potential drugs to treat Tn-expressing cancers.

Two-Step PEF Processing for Enhancing the Polyphenol Concentration and Decontaminating a Red Grape Juice.

This study's aim is to evaluate Pulsed Electric Fields (PEF) technology as an alternative method for the processing of red grape juice. For this purpose, two PEF treatments were applied: first to grapes for polyphenol enrichment of the juice, and subsequently for microbial decontamination of the obtained juice. Juice obtained from PEF-treated grapes (5 kV/cm, 63.4 kJ/kg) had the polyphenol content 1.5-fold higher and colour intensity two times higher of control juices by spectrophotometric measurement (p ≤ 0.05). A subsequent decontamination treatment by PEF (17.5 kV/cm and 173.6 kJ/kg) achieved inactivation of the present microbiota (yeasts, moulds, and vegetative mesophilic bacteria) below detection level (<30 CFU/mL). Furthermore, PEF-treated juices were microbiologically stable up to 45 days, even at abusive refrigeration storage temperatures (10 °C). PEF juice quality and sensory characteristics were similar to a fresh juice; they were neither affected by the PEF decontamination treatment, nor by storage time and temperature. Results obtained in this study demonstrate the considerable potential of PEF for the production of a polyphenol-enriched and microbially stabilized red grape juice as a unique and sustainable alternative for the juice industry, while avoiding enzymatic and heat treatments.

Pulsed Electric Fields to Improve the Use of Non-Saccharomyces Starters in Red Wines.

New nonthermal technologies, including pulsed electric fields (PEF), open a new way to generate more natural foods while respecting their organoleptic qualities. PEF can reduce wild yeasts to improve the implantation of other yeasts and generate more desired metabolites. Two PEF treatments were applied; one with an intensity of 5 kV/cm was applied continuously to the must for further colour extraction, and a second treatment only to the must (without skins) after a 24-hour maceration of 17.5 kV/cm intensity, reducing its wild yeast load by up to 2 log CFU/mL, thus comparing the implantation and fermentation of inoculated non-Saccharomyces yeasts. In general, those treated with PEF preserved more total esters and formed more anthocyanins, including vitisin A, due to better implantation of the inoculated yeasts. It should be noted that the yeast Lachancea thermotolerans that had received PEF treatment produced four-fold more lactic acid (3.62 ± 0.84 g/L) than the control of the same yeast, and Hanseniaspora vineae with PEF produced almost three-fold more 2-phenylethyl acetate than the rest. On the other hand, 3-ethoxy-1-propanol was not observed at the end of the fermentation with a Torulaspora delbrueckii (Td) control but in the Td PEF, it was observed (3.17 ± 0.58 mg/L).

Direct Contact Ultrasound in Food Processing: Impact on Food Quality.

Consumers' demand for "minimally processed" products that maintain the "fresh-like" characteristics has increased in recent years. Ultrasound (US) is a non-thermal technology that enhances mass and energy transfer processes resulting in improved food quality. A new method of applying US to food without using a liquid or gaseous medium for the propagation of acoustic waves has recently been under research. It is known as direct contact US, since the food is directly placed on a plate where the transducers are located. In this type of systems, the main effect is not cavitation but acoustic vibration, which encourages mass and energy transfer processes due to the "sponge effect." Furthermore, as the product is not immersed in a liquid medium, the loss of hydrophilic nutritional compounds is reduced; systems such as these can thus be more easily implemented on an industrial level. Nevertheless, the very few studies that have been published about these systems mainly focus on dehydration and freezing. This article summarizes published research on the impact of direct contact US in nutritional and organoleptic quality of food in order to assess their potential to meet new market trends.

Pulsed electric field-assisted extraction of valuable compounds from microorganisms.

Microorganisms (bacteria, yeast, and microalgae) are a promising resource for products of high value such as nutrients, pigments, and enzymes. The majority of these compounds of interest remain inside the cell, thus making it necessary to extract and purify them before use. This review presents the challenges and opportunities in the production of these compounds, the microbial structure and the location of target compounds in the cells, the different procedures proposed for improving extraction of these compounds, and pulsed electric field (PEF)-assisted extraction as alternative to these procedures. PEF is a nonthermal technology that produces a precise action on the cytoplasmic membrane improving the selective release of intracellular compounds while avoiding undesirable consequences of heating on the characteristics and purity of the extracts. PEF pretreatment with low energetic requirements allows for high extraction yields. However, PEF parameters should be tailored to each microbial cell, according to their structure, size, and other factors affecting efficiency. Furthermore, the recent discovery of the triggering effect of enzymatic activity during cell incubation after electroporation opens up the possibility of new implementations of PEF for the recovery of compounds that are bounded or assembled in structures. Similarly, PEF parameters and suspension storage conditions need to be optimized to reach the desired effect. PEF can be applied in continuous flow and is adaptable to industrial equipment, making it feasible for scale-up to large processing capacities.

Enzymatic Processes Triggered by PEF for Astaxanthin Extraction From Xanthophyllomyces dendrorhous.

The aim of this study was to evaluate the potential of pulsed electric fields (PEF) to improve the extraction of the lipid-soluble astaxanthin from fresh biomass of a wild-type (CECT 11028) and mutant (ATCC 74219) Xanthophyllomyces dendrorhous strain using ethanol as solvent. Inactivation and propidium uptake studies revealed that inactivation is a good index for estimated the proportion of irreversible permeabilized cells when inactivation is higher than 70% in the two strains. Ethanol was ineffective for extracting carotenoids from the PEF-treated cells (20 kV/cm, 135 µs) of the two strains. However, after aqueous incubation of PEF-treated X. dendrorhous ATCC 74219 cells for 12 h, up to 2.4 ± 0.05 mg/g dried weight (d.w.) of carotenoids were extracted in ethanol. From total carotenoid extracted, around 84% corresponded to all-trans astaxanthin. The detection and quantification of esterase activity in the supernatant and the relationship between the percentage of esterase activity quantified and the amount of carotenoids extracted indicate that the extraction of astaxanthin was mediated by enzymatic esterase activity triggered by PEF during incubation. On the other hand, the formation of a large lipid globule into the cytoplasm of PEF-treated X. dendrorhous CECT 11028 cells during aqueous incubation prevented carotenoid extraction. The process developed in this investigation represents a more sustainable and greener method that those previously used for extracting astaxanthin from yeast.

Production of a Granulysin-Based, Tn-Targeted Cytolytic Immunotoxin Using Pulsed Electric Field Technology.

Granulysin is a protein present in the granules of human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells, with cytolytic activity against microbes and tumors. Previous work demonstrated the therapeutic effect of the intratumoral injection of recombinant granulysin and of the systemic injection of an immunotoxin between granulysin and the anti-carcinoembryonic antigen single-chain Fv antibody fragment MFE23, which were produced in the yeast Pichia pastoris. In the present work, we developed a second immunotoxin combining granulysin and the anti-Tn antigen single-chain Fv antibody fragment SM3, that could have a broader application in tumor treatment than our previous immunotoxin. In addition, we optimized a method based on electroporation by pulsed electric field (PEF) to extract the remaining intracellular protein from yeast, augmenting the production and purificiation yield. The immunotoxin specifically recognized the Tn antigen on the cell surface. We also compared the thermal stability and the cytotoxic potential of the extracellular and intracellular immunotoxins on Tn-expressing human cell lines, showing that they were similar. Moreover, the bioactivity of both immunotoxins against several Tn+ cell lines was higher than that of granulysin alone.

Understanding the occurrence of tailing in survival curves of Salmonella Typhimurium treated by pulsed electric fields.

This study aimed to gain more in-depth knowledge of the mechanisms involved in microbial inactivation by pulsed electric fields (PEF) to understand the tailing observed in survival curves of Salmonella Typhimurium (STCC 878). The comparison of the inactivation achieved by the application of one train of pulses with those obtained with pulses applied in two trains shows that the tail of the survival curves was a consequence of a transient increment of the microbial resistance to the effect of the electric field in a proportion of the cells. After some time following the application of the first pulse train, cells became again sensitive to the second train, and tailing tended to disappear. The required time was highly dependent on the characteristics of the incubation medium. Similar effects were observed when the treatments were validated on whole milk and orange juice. This study has demonstrated by the first time on microbial cells the benefits of splitting the delivered PEF treatment in two trains with a period of delay between them. Therefore, this insight opens up the possibility of developing new strategies to achieve the required inactivation levels to guarantee food safety by moderate PEF treatments.

Evolution of Polyphenolic Compounds and Sensory Properties of Wines Obtained from Grenache Grapes Treated by Pulsed Electric Fields during Aging in Bottles and in Oak Barrels.

The evolution of polyphenolic compounds and sensory properties of wines obtained from Grenache grapes, either untreated or treated with pulsed electric fields (PEF), in the course of bottle aging, as well as during oak aging followed by bottle aging, were compared. Immediately prior to aging in bottles or in barrels, enological parameters that depend on phenolic extraction during skin maceration were higher when grapes had been treated with PEF. In terms of color intensity, phenolic families, and individual phenols, the wine obtained with grapes treated by PEF followed an evolution similar to untreated control wine in the course of aging. Sensory analysis revealed that the application of a PEF treatment resulted in wines that are sensorially different: panelists preferred wines obtained from grapes treated with PEF. Physicochemical and sensory analyses showed that grapes treated with PEF are suitable for obtaining wines that require aging in bottles or in oak barrels.

Organic-solvent-free extraction of carotenoids from yeast Rhodotorula glutinis by application of ultrasound under pressure.

The extraction of Rhodotorula glutinis carotenoids by ultrasound under pressure (manosonication) in an aqueous medium has been demonstrated. The influence of treatment time, pressure, and ultrasound amplitude on R. glutinis inactivation and on the extraction of carotenoids was evaluated, and the obtained data were described mathematically. The extraction yields were lineal functions of those three parameters, whereas inactivation responded to a more complex equation. Under optimum treatment conditions, 82% of carotenoid content was recovered. Extraction of carotenoids in an aqueous medium was attributed to the capacity of ultrasound for cell disruption and emulsification. Cavitation caused the rupture of cell envelopes and the subsequent formation of small droplets of carotenoids surrounded by the phospholipids of the cytoplasmic membrane that would stabilize the emulsion. Analysis of the dispersed particle size of the extracts demonstrated that a fine, homogeneous emulsion was formed after treatment (average size: 230 nm; polydispersity <0.22). This research describes an innovative green process for extracting carotenoids from fresh biomass of R. glutinis in which only two unit operations are required: ultrasonic treatment, followed by a centrifugation step to discard cell debris. The extract obtained thanks to this procedure is rich in carotenoids (25 mg/L) and could be directly incorporated as a pigment in foods, beverages, and diet supplements; it can also be utilized as an ingredient in drugs or cosmetics.

Potential of Pulsed Electric Fields for the preparation of Spanish dry-cured sausages.

The aim of this investigation was to lay the groundwork of the potential application of Pulsed Electric Fields (PEF) technology for accelerating the drying process of meat and meat products, and specifically in this work of Spanish dry-cured sausages "longaniza". PEF treatments were applied to pork loin samples, and the influence of different PEF parameters on the process were evaluated. An optimal PEF treatment of 1 kV/cm, 200 µs of pulse width and 28 kJ/kg was determined as the most suitable to electroporate meat cells and to improve water transfer by achieving a water content reduction of 60.4% in treated-meat samples dried at 4 °C. The influence of PEF on meat drying rate was also studied on minced pork and the results showed that with a particle size of 4.0 mm higher drying rates were achieved. To validate the results, Spanish cured sausages were prepared from treated and untreated minced pork and stuffed into gauzes and natural pork casings at pilot plant scale. After the curing process, the application of PEF to sausages stuffed into gauze reduced the drying time from 17 to 9-10 days, a reduction of 41-47%, confirming the effects described at lab scale and the potential of PEF for accelerating the sausage-drying process.

Dataset on the use of the Ratkowsky model for describing the influence of storage temperature on microbial growth in hake fillets (Merluccius merluccius) stored under MAP.

This article presents the results obtained after applying the Ratkowsky model for developing secondary models describing the influence of storage temperature on microbial growth in hake fillets packaged under a modified atmosphere (MAP) rich in CO2 (50% CO2/50% N2). For this purpose the growth parameters (λ, µmax) already calculated in the related article "Modelling microbial growth in Modified-Atmosphere-Packed hake (Merluccius merluccius) fillets stored at different temperatures" [1] were used. The data include the fit and goodness of the fit parameters calculated as well as the comparison between fitted and observed data.

Characterization of the Spoilage Microbiota of Hake Fillets Packaged Under a Modified Atmosphere (MAP) Rich in CO2 (50% CO2/50% N2) and Stored at Different Temperatures.

The aim of this study was to characterize the spoilage microbiota of hake fillets stored under modified atmospheres (MAP) (50% CO2/50% N2) at different temperatures using high-throughput 16S rRNA gene sequencing and to compare the results with those obtained using traditional microbiology techniques. The results obtained indicate that, as expected, higher storage temperatures lead to shorter shelf-lives (the time of sensory rejection by panelists). Thus, the shelf-life decreased from six days to two days for Batch A when the storage temperature increased from 1 to 7 °C, and from five to two days-when the same increase in storage temperature was compared-for Batch B. In all cases, the trimethylamine (TMA) levels measured at the time of sensory rejection of hake fillets exceeded the recommended threshold of 5 mg/100 g. Photobacterium and Psychrobacter were the most abundant genera at the time of spoilage in all but one of the samples analyzed: Thus, Photobacterium represented between 19% and 46%, and Psychrobacter between 27% and 38% of the total microbiota. They were followed by Moritella, Carnobacterium, Shewanella, and Vibrio, whose relative order varied depending on the sample/batch analyzed. These results highlight the relevance of Photobacterium as a spoiler of hake stored in atmospheres rich in CO2. Further research will be required to elucidate if other microorganisms, such as Psychrobacter, Moritella, or Carnobacterium, also contribute to spoilage of hake when stored under MAP.

Modelling microbial growth in modified-atmosphere-packed hake (Merluccius merluccius) fillets stored at different temperatures.

Market globalization and changes in purchasing habits pose a challenge to the fishery industry because of the short shelf life of fish products. In view of this scenario, it would be very helpful if tools capable of predicting the shelf-life of fish could be developed. Thus, the objective of this study was to employ a modelling approach capable of predicting the evolution of the microbiota of hake fillets packaged under a modified atmosphere (MAP) rich in CO2 (50% CO2 / 50% N2) when stored at temperatures ranging between 1 and 10 °C. Growth curves of ten microbial groups were obtained at four different temperatures and fitted with the Baranyi model. Photobacterium showed high growth rates in hake fillets (0.99 days-1 at 4 °C), similar to those of Shewanella, lactic acid bacteria, and non-specific microbial groups investigated, and significantly higher than those of Pseudomonas. Furthermore, no lag phase was observed for Photobacterium regardless of the temperature investigated. On the other hand, Enterobacteriaceae and moulds and yeasts displayed low growth fitness, and their counts increased by <1.5-2 Log10 cycles along the incubation period regardless of storage temperature. The influence of storage temperature on growth parameters (λ, µmax and Yend) was subsequently studied, and secondary models were developed for the eight most relevant microbial groups. All of the final equations developed in this study showed R2 values ≥0.90, and RMSE values ≤0.50. In addition, results obtained in this investigation strongly suggest that Photobacterium would be the main responsible microorganism for the spoilage of hake fillets stored under MAP conditions (50% CO2/50% N2) along the entire range of temperatures investigated (1-10 °C).

Use of pulsed electric field permeabilization to extract astaxanthin from the Nordic microalga Haematococcus pluvialis.

The Nordic microalgal strain Haematococcus pluvialis was exposed to various stress conditions to induce astaxanthin accumulation. Highest carotenoid content (19.1 mg·g-1dw) was achieved in nitrogen-free culture medium at a high light intensity. The efficiency of Pulsed Electric Field (PEF) pre-treatment of stressed fresh biomass of H. pluvialis followed by incubation in the growth medium was compared to classical disruption methods (bead-beating, freezing-thawing, thermal treatment or ultrasound) for the subsequent extraction of astaxanthin in ethanol. N-starved cells treated with PEF followed by aqueous incubation for 6 h resulted in extraction of 96% (18.3 mgcar·gdw-1) of the total carotenoid content compared to 80% (15.3 mgcar·gdw-1) using other physical methods. The proportion of free forms of astaxanthin was higher in PEF-treated samples compared to mechanical disruption, suggesting PEF triggering an esterase activity. PEF pre-treatment of the cells followed by incubation in growth medium improved astaxanthin extraction in the eco-friendly solvent ethanol.