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.

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.

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).

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.

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.

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.

Pulsed electric fields accelerate release of mannoproteins from Saccharomyces cerevisiae during aging on the lees of Chardonnay wine.

The potential of PEF for triggering autolysis of Saccharomyces cerevisiae and accelerating the release of mannoproteins during aging on the lees of Chardonnay wine was evaluated. Release of mannoproteins in Chardonnay wine increased drastically in samples containing PEF-treated (5 and 10 kV/cm, 75 µs) yeasts. No mannoprotein release was observed in the first seven days of aging on the lees in wine containing untreated yeast; however, after the same time interval, the concentration of those compounds increased by 40 and 60% in wines containing yeast treated by PEF at 5 and 10 kV/cm, respectively. After 30 days of incubation, the mannoprotein concentration in wines containing yeast treated under the most intense PEF conditions reached the maximum value. Control cells, on the other hand, required six months to reach that maximum level. Chromatic characteristics, total polyphenol index, total volatile acidity, pH, ethanol, and CIELAB parameters of the wine were not affected during aging on the lees with untreated and PEF-treated yeast. On the other hand, the capability of the mannoproteins released from yeast treated by PEF for decreasing wine turbidity, foaming, and interacting with tannins was similar to that of those released from untreated yeast; the differences observed were a consequence of the varying concentration of mannoproteins. The result obtained demonstrates that PEF permits the acceleration of the aging-on-lees step while avoiding or reducing the problems associated with it. To achieve this effect, intense treatment is not required. Therefore, wineries could process lees by using the most economical PEF devices on the market.

Factors influencing autolysis of Saccharomyces cerevisiae cells induced by pulsed electric fields.

The influence of temperature (7-43 °C), pH (3.5-7.0) and ethanol concentration (6-25%) on PEFinduced autolysis and the release of mannose from Saccharomyces cerevisiae was investigated. Changes in the release of intracellular compounds absorbing at 260 nm and 280 nm depended on storage conditions and differed among untreated and PEF-treated cells. For untreated cells, the increase of the Abs260 and Abs280 values during 3 weeks of storage was very low when incubated in media of different pH, different ethanol concentrations, or at 7° and 25 °C. Conversely, Abs260 and Abs280 values progressively increased for PEF-treated cells stored under the same conditions. Although the PEF treatment intensity was the same in all cases, the amount of intracellular material released depended on incubation conditions. Except for cells stored at 43 °C, for which the concentration of mannose in the media after 21 days was around 90 mg L-1, the amount of mannose released from untreated cells after 21 days of storage was lower than 60 mg L-1 under all other conditions assayed. After the same incubation time, the amount of mannose released from PEF treated cells ranged from 80 mg L-1, when they were stored in media with 25% ethanol, to 190 mg L-1 when they were stored at 43 °C. Interaction among assayed factors affecting mannose release was investigated in a medium containing 10% ethanol (v/v) and pH 3.5 for 21 days. Although the interaction of both factors delayed mannose release, the medium containing PEF-treated yeasts had approximately twice the amount of mannoproteins as those containing untreated yeasts.