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Representative images of yeast S. cerevisiae during osmoporation. Microphotographs were acquired by SEM in different stages of osmoporation. Cells in iso-osmotic water-glycerol solution at 1.4 MPa (a–c); dehydrated at 30.0 MPa for 60 min leading to reduction of cellular volume (white arrow) (d–f) and rehydrated with the iso-osmotic solution (g–i). Stretch marks on cell surface (black arrow). BS budding scars. Bar scales: (a, d, and g) 10 μm, (b, e, and h) 5 μm, and (c, f, and i) 1 μm
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Osmoporation is an innovative method that can be used with food-grade yeast cells of Saccharomyces cerevisiae as natural encapsulating matrices. This technique overcomes barriers that difficult encapsulation and enables the internalization of fragile bioactive molecules such as fisetin into yeasts. In the present study, we assessed the effects of c...
Citations
... [64,66,67] However, those information does not seem to be a consensus in the literature. [68,69] ...
Lycopene (LYC), a carotenoid extracted mainly from tomatoes, has several biological properties, making its use desirable as a nutraceutical and pharmaceutical active ingredient. Among its various applications vulvovaginal candidiasis stands out. However, the use of LYC in therapy has limitations related to its solubil-ity and stability. In this study, mesoporous silica nanoparticles (MSNs) are used to load and protect LYC from degradation. The exact amount of drug incorporated was determined by analytical techniques, such as high-performance liquid chromatography (HPLC) and thermal analysis. For this, we developed and validated an HPLC method for LYC quantification and evaluated LYC impregnation in MSNs, followed by thermogravimetry analysis (TGA). Differential scanning calorimetry (DSC) was also used in order to confirm drug incorporation. Additionally , an in vitro release study in simulated vaginal fluid was also carried out. The HPLC method was duly validated for the range of 26-125 µg mL −1 and proved to be suitable for LYC quantification. DSC measurements suggest an improvement in the stability of the impregnated drug, which was reinforced by the release assay. Overall, the developed method is suitable to quantify LYC-loaded porous materials enabling its use in vaginal applications. K E Y W O R D S high-performance liquid chromatography, lycopene, mesoporous silica nanoparticles, thermo-gravimetry, validation This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
... The morphologies of andrographolide-treated yeast cells were observed by scanning electron microscopy (SEM) [28,29]. Briefly, samples were fixed with 2.5% glutaraldehyde (Electron Microscopy Science, Hatfield, PA, USA) for 24 h and washed twice with PBS. ...
Andrographolide, a bioactive compound found in Andrographis paniculata, has gained significant attention for its potential therapeutic properties. Despite its promising benefits, the understanding of its side effects and underlying mechanisms remains limited. Here, we investigated the impact of andrographolide in Saccharomyces cerevisiae and observed that andrographolide induced cytotoxicity, particularly when oxidative phosphorylation was active. Furthermore, andrographolide affected various cellular processes, including vacuole fragmentation, endoplasmic reticulum stress, lipid droplet accumulation, reactive oxygen species levels, and compromised cell integrity. Moreover, we unexpectedly observed that andrographolide induced the precipitation of biomolecules secreted from yeast cells, adding an additional source of stress. Overall, this study provides insights into the cellular effects and potential mechanisms of andrographolide in yeast, shedding light on its side effects and underlying cytotoxicity pathways.
... Being the indirect method considered more reliable by some authors, mainly due to the smallest standard deviation and reproducibility (between different indirect methods) (Amini et al., 2017;Bakonyi et al., 2017;Khoshneviszadeh et al., 2016). However, those information does not seem to be a consensus in the literature (Daneshmand et al., 2018;de Câmara et al., 2016). ...
Lycopene (LYC), a carotenoid extracted mainly from tomatoes, has several biological properties, making its use desirable as a nutraceutical and pharmaceutical active ingredient. However, the use of LYC in therapy has limitations related to its solubility and stability. In this study, mesoporous silica nanoparticles (MSNs) are used to load and protect LYC from degradation. The exact amount of drug incorporated was determined by analytical techniques, such as high performance liquid chromatography (HPLC) and thermal analysis. For this we developed and validated an HPLC method for LYC quantification and evaluated the LYC impregnated in MSNs, followed by thermogravimetry analysis (TGA) technique analysis. Differential scanning calorimetry (DSC) was also used in order to confirm drug incorporation. Additionally, an in vitro release study was also carried out. The HPLC method was duly validated for the range of 26–125 µg/mL and proved to be suitable for LYC quantification. DSC measurements suggest an improvement in the stability of the impregnated drug, which was reinforced by the release assay. Overall, the developed method is suitable to test LYC-loaded porous materials to enable the use in therapeutic applications.
... Being the indirect method considered more reliable by some authors, mainly due to the smallest standard deviation and reproducibility (between different indirect methods) [64,66,67]. However, those information does not seem to be a consensus in the literature [68,69]. Legend: p-value; **< 0.005; ***< 0.001; ****<0.0001. ...
Lycopene (LYC), a carotenoid extracted mainly from tomatoes, has several biological properties, making its use desirable as a nutraceutical and pharmaceutical active ingredient. Among its various applications vulvovaginal candidiasis stands out. However, the use of LYC in therapy has limitations related to its solubility and stability. In this study, mesoporous silica nanoparticles (MSNs) are used to load and protect LYC from degradation. The exact amount of drug incorporated was determined by analytical techniques, such as high performance liquid chromatography (HPLC) and thermal analysis. For this we developed and validated an HPLC method for LYC quantification and evaluated LYC impregnation in MSNs, followed by thermogravimetry analysis (TGA). Differential scanning calorimetry (DSC) was also used in order to confirm drug incorporation. Additionally, an in vitro release study in simulated vaginal fluid was also carried out. The HPLC method was duly validated for the range of 26–125 µg/mL and proved to be suitable for LYC quantification. DSC measurements suggest an improvement in the stability of the impregnated drug, which was reinforced by the release assay. Overall, the developed method is suitable to quantify LYC-loaded porous materials enabling its use in vaginal applications.
... Our research group has previously investigated the osmoporation bioprocess (da Silva Pedrini et al. 2014) for the incorporation of lipophilic compounds into yeasts (Câmara Jr. et al. 2016;Medeiros et al. 2018;de Medeiros et al. 2019) and probiotic cells (de Andrade et al. 2022b(de Andrade et al. , 2022c to improve their physicochemical stability and bioaccessibility. Now, we want to assess the performance of sonoporation coupled to drying to deliver targeted hydrophobes into food-grade cells and develop a straightforward and eco-friendly bioencapsulation strategy for food applications. ...
... The influence of process parameters on the encapsulation effectiveness has been demonstrated by various reports (Paramera et al. 2011;Young et al. 2017;Rubio et al. 2018Rubio et al. , 2021Karaman 2021;de Andrade et al. 2022c). In our previous work, it was shown that yeast encapsulation via osmoporation is regulated by temperature, cell density, osmotic pressure, ethanol, and bioactive concentration (Câmara Jr. et al. 2016;de Medeiros et al. 2019). In addition, it seems that the internalization of active molecules into yeast cells can be modulated through different drying protocols and cell permeabilization processes Dadkhodazade et al. 2018;Dimopoulos et al. 2021). ...
... strong influence on results. Although the use of highly concentrated fisetin solutions improved the EYs and AAs, lower EEs were found (Tables 1 and 2). Once again, this shows that cellular encapsulation systems do not support excessive bioactive concentration, which may be also associated with the total available intracellular space for entrapment . Câmara Jr. et al. (2016) andde Andrade et al. (2022c) observed that higher initial fisetin contents favored a more complete filling of both osmoporated S. cerevisiae and Lactobacillus acidophilus cells. Since their loading capacities reached a maximum level, these envelopes were not able to further internalize fisetin, resulting in poor EEs as a function of high no ...
The encapsulation of fisetin into S. cerevisiae cells through sonoporation coupled with drying is reported for the first time in the literature. To establish the best conditions to maximize the amount of internalized fisetin, the cell density (5–10% w/v), fisetin concentration (1–3 mg/mL), acoustic energy density (0–333.3 W/L), and drying method (freeze-drying and spray drying) were analyzed through a Box-Behnken experimental design (BBD) coupled with response surface methodology (RSM). Higher encapsulation efficiency (EE) was achieved with a cell density of 10% w/v, while fisetin concentration of 3 mg/mL favored the encapsulation yield (EY) and antioxidant activity (AA). Higher EE (67.7%), EY (25.7 mg/g), and AA (90%) were registered when an acoustic density of 333.3 W/L was used. Furthermore, both drying protocols promoted fisetin encapsulation, but through spray drying, the EE, EY, and AA were 11.5%, 11.1%, and 26.6% higher than via freeze-drying, respectively. This work proved that fully filled biocapsules were produced through sonoprocessing, and their morphology was influenced by the acoustic energy and drying process. Overall, these results open new perspectives for the application of sonoprocessing-assisted encapsulation, paving the way for developing innovative yeast-based delivery systems for lipophilic compounds such as fisetin.
Key points
• Sonoprocessing improves the encapsulation of fisetin into S. cerevisiae cells
• Spray drying promotes fisetin loading into yeasts’ intracellular space and cavities
• Fisetin binding with yeast extracellular agents are favored by freeze-drying
... For example, Young et al. (2017) demonstrated enhanced EEs of curcumin and fisetin, from 35Á9 and 34Á4% by mere impregnation to 64Á0 and 66Á6% via vacuum infusion, respectively. Osmoporation-assisted encapsulation of curcumin (de Medeiros et al. 2019) and fisetin (Câmara Jr. et al. 2016) led to EE levels of 34Á2 and 33Á3%, respectively. Previously, 85Á8% of fisetin were entrapped by poly-lactic acid nanoparticles (Feng et al. 2019), while a formulation of inulin, maltodextrin and tamarind gum encapsulated 82Á5 and 89Á4% of curcumin via spray drying and freeze drying, respectively (Guo et al. 2020). ...
... A similar trend was observed via osmoporation bioprocess. Improved EYs of curcumin (50Á5%; de Medeiros et al. 2019) and fisetin (0Á729 mg ;Câmara Jr. et al. 2016) were reached when higher bioactive concentrations in a range of 1-3 and 0Á32-3Á68 mg ml −1 were used, respectively. Moreover, a comparison between our current results and those obtained by the multistage osmoporation technique of Medeiros et al. (2018) demonstrates the benefits of adding non-loaded yeasts between each stage. ...
... Following the EY profiles in Fig. 2, it seems that the increment of ESs will reduce the total loaded content of the biocapsules, then, a reasonable balance between EE and EY is essential to find an adequate amount of ESs to maximize the efficiency of the encapsulation process. Previous reports have shown that encapsulation processes with increasing yeast densities usually led to poorly enriched final encapsulated products (Paramera et al. 2011;Câmara Jr. et al. 2016;de Medeiros et al. 2019;Rubio et al. 2021). Therefore, greater EYs may be achieved by batching the process into successive stages using less concentrated nonloaded yeast suspensions in each one of these ESs. ...
Some of the challenges of yeast encapsulation protocols are low phytochemical internalization rates and limited intracellular compartment of yeasts. This study uses an ultrasound‐assisted batch encapsulation (UABE) protocol to optimize the encapsulation of curcumin and fisetin by recovering non‐encapsulated biomaterial and further incorporating it into non‐loaded yeasts in three encapsulation stages (1ES, 2ES, and 3ES). The effect of selected acoustic energies (166.7 and 333.3 W L‐1) on the encapsulation efficiency (EE), yield (EY), and antioxidant activity retention were evaluated, and then, compared with a control process (without ultrasound treatment). Compared to the control, enhanced EEs were achieved for both curcumin (10.9% control to 58.5% UABE) and fisetin (18.6% control to 76.6% UABE) after 3ES and the use of 333.3 W L‐1. Similarly, the yeast maximum loading capacity was improved from 6.6 to 13.4 mg g‐1 for curcumin; and from 11.1 to 26.4 mg g‐1 for fisetin after UABE protocol. The antioxidant activity of produced biocapsules was positively correlated with the bioactive loaded content of yeasts when ultrasound treatment was applied. Overall, results from this study provide valuable information regarding UABE processes, and moreover, bring new and creative perspectives for the ultrasound technology in the food industry.
... Encapsulation via osmoporation has important advantages such as versatility, short processing time, viable scaling-up and use of safe reagents [13]. Our group has previously studied, adapted, and developed different osmoporation techniques [12][13][14][15][16]. For example, through osmoporation bioprocess, curcumin was efficiently encapsulated into yeasts [14], and further, Saccharomyces cerevisiae [15] and Lactobacillus acidophilus cells [16] were able to entrap fisetin at similar encapsulation efficiency levels. ...
... Our group has previously studied, adapted, and developed different osmoporation techniques [12][13][14][15][16]. For example, through osmoporation bioprocess, curcumin was efficiently encapsulated into yeasts [14], and further, Saccharomyces cerevisiae [15] and Lactobacillus acidophilus cells [16] were able to entrap fisetin at similar encapsulation efficiency levels. ...
... After Y-FIS fermentation, non-encapsulated fisetin was added at 0.05% (w/v). The encapsulated fisetin content was estimated by direct extraction as described by Câmara Jr. et al. [15] to guarantee similar contents of fisetin in Y-FIS and Y-LACF yoghurts. ...
Osmoporation is a novel encapsulation approach for bioactive compounds based on the osmoresistance mechanisms of microbial cells. To the best of our knowledge, this is the first study investigating the production of fisetin-enriched yoghurt using Lactobacillus acidophilus-based bio-capsules via osmoporation as the starter culture. Results showed that the milk acidification with fisetin-loaded L. acidophilus progressed at a slower pace due to complex mechanisms induced by osmoporation and internalized fisetin. Milk fermentation using fisetin bio-capsules reached a maximum acidification rate of 0.18 pH units/h after 23 h and pH 4.6 was achieved after 32 h. Besides, the antioxidant activity of yoghurts produced with fisetin bio-capsules did not change during cold storage, while the antioxidant activity of yoghurt produced with non-encapsulated fisetin was reduced by 2.5-fold after 28 days. Overall, this study shows that fisetin osmoporation using L. acidophilus is a versatile encapsulation bioprocess that enables the delivery of preserved phytoactives into fermented foods like yoghurt. This strategy has the potential to be extended to other applications in the dairy industry using lactic acid bacteria as both the encapsulation matrix and fermentation agent.
... Previously, our group has already demonstrated the effectiveness of osmoporation to encapsulate selected compounds. Both hydrophilic (FITC-Dextran, 20 kDa;da S Pedrini et al. 2014) and hydrophobic (fisetin, 286 Da; Câmara Junior et al. 2016) molecules have been successfully delivered to S. cerevisiae cell's cytoplasm using osmoporation. Moreover, it has been shown that the process efficiency can still be improved (Medeiros et al. 2018), which highlights the need for further investigations on how to take full advantage of this promising technique. ...
... CUR direct extraction and EE quantification were performed as described by Câmara Junior et al. (2016). Cell suspensions subjected to osmoporation process were divided into two groups. ...
... The use of statistical and mathematical methods has contributed for process development, improvement and optimisation over the years. RSM is a robust and valuable tool for evaluating and modelling experimental data in order to identify independent and combined influences of variables on process output (Câmara Junior et al. 2016). EE and EY data were analysed and converted into two independent representative regression models (Eqs. ...
This study proposes the investigation of curcumin encapsulation into Saccharomyces cerevisiae cells through osmoporation as an efficient way of increasing curcumin stability. The influence of three process parameters (cell, ethanol and curcumin concentrations) on the encapsulation process was evaluated, and the obtained biocapsules were characterised for physical and photochem-ical stabilisation. Results showed that encapsulation efficiency was favoured by the increase of cells/curcumin ratio and ethanol concentration up to 60%. Differential scanning calorimetry (DSC) curves revealed that yeast encapsulation delayed the curcumin melting point up to 207 °C. Encapsulated curcumin retained over 80% of antioxidant activity after thermal treatment (150 °C) and over 70% after a 50-day exposure to artificial light. Photochemical stability of yeast-encapsulated curcumin was increased by 5.7-fold, and half-life time reached 181 days under illumination conditions. Overall, osmoporation-produced yeast biocapsules confirmed the versatility of osmoporation as an encapsulation technique and successfully improved curcumin stability.
... The stabilization processes of yeasts in dried form are mainly applied to the yeast Saccharomyces cerevisiae, which is undoubtedly the most studied eukaryotic microorganism. Numerous studies have elucidated the mechanisms of passive, genetic or adaptive response of S. cerevisiae during nutritional, osmotic, dehydration and oxidation stresses (Câmara et al., 2016(Câmara et al., , 2018Da Silva Pedrini, Dupont, Câmara, Beney, & Gervais, 2014;Eleutherio et al., 2018;Garre, Raginel, Palacios, Julien, & Matallana, 2010;Nguyen et al., 2017). Nevertheless, precise reports about these mechanisms associated to other yeast species are currently scarce. ...
The development of new fermented foods and beverages requires more and more the use of new dehydrated yeasts species. In this context, the non-Saccharomyces (NS) yeasts Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans are developed especially in winemaking as co-culture in the fermentation of the must or for the must bioprotection. However, during formulation-dehydration the yeast cells are exposed to several stresses that reduce cellular activity. Among these, the oxidative stress induced by the stabilization processes has been described as one of the main causes of cell death. In this study, we analyzed the effects of growth medium associated with two dehydration kinetics on the accumulation of reactive oxygen species (ROS) and lipid peroxidation levels. The cultivability of tested yeast strains was dependent on growth and dehydration conditions. The L. thermotolerans strain was the most sensible to dehydration when growing in nutrient-poor media, which was characterized by high levels of ROS, lipid peroxidation and reduced cultivability. In contrast, this yeast was able to restore its cultivability when growing in nutrient-rich medium before dehydration. The other NS yeast strains acquired resistance to oxidative stress similarly but in a growth-dehydration condition less dependent manner. These results showed that modulation of growing medium composition is a simple way to improve resistance to oxidative attack imposed by dehydration in NS yeasts. This was the first time that multiple quantitative and qualitative indicators of oxidative stress response in these three NS yeast strains were explored.
... The plasma membrane has been described as the main target of injury induced by drying (Guyot et al., 2006;Lemetais et al., 2012;Ragoonanan et al., 2008;Simonin et al., 2007). In fact, changes in yeast cell membranes permeability caused by oxidative damages (de Souza Pereira and Geibel, 1999), osmotic perturbations (Da Silva Pedrini et al., 2014) and chemicals (Câmara Jr. et al., 2016) have been observed. ...
The use of yeasts in industry is inseparable from their ability to be produced and dehydrated. This dehydration process causes various dysfunctions in yeast cells that affect their functionality and viability. In order to protect yeasts from dehydration, food additives are often used as emulsifiers and antioxidants. However, yeasts are able to produce naturally protective substances, such as glutathione (GSH) and trehalose (TRE). In this context, three non-Saccharomyces (NS) strains, belonging to the different genera and species Torulaspora delbrueckii, Metschnikowia pulcherrima and Lachancea thermotolerans, were studied in this thesis. Despite the great interest aroused by their multiple agro-food applications, their dehydration resistance mechanisms associated with the synthesis of GSH and TRE, are currently unknown. This study is ultimately aimed at the formulation of new NS yeast dried strains without any food additives. In a first chapter, the impact of the “reference yeast” Saccharomyces cerevisiae dehydration in a pre-pilot fluidized bed has been correlated with the synthesis of GSH and TRE. It was possible to modulate the culture medium composition in order to optimize cell preservation before, during and after dehydration. In a second chapter, the previously defined conditions were applied to NS yeasts strains in order to understand the effects of dehydration on their microbial functionality. This study demonstrated that GSH plays an important role in NS yeasts protection, depending on the culture and dehydration conditions. In a third chapter, some oxidation resistance phenomena of the three NS strains were studied. It was clearly demonstrated that the susceptibility of cells to oxidative attack was dependent on culture-dehydration conditions and was yeast strain-dependent. Finally, in a fourth chapter, an in-depth biochemical study of the most dehydration-sensitive yeast strain, L. thermotolerans, was performed by synchrotron FTIR micro-spectroscopy. Cells grown in GSM (medium favoring the production of GSH), besides showing a better viability, showed a greater intensity in the spectral bands of lipids CH2 and CH3, associated with the plasma membrane fluidity. In addition, TSM grown cells (TSM is a medium favoring the production of TRE) exhibited a higher protein denaturation, suggested by the intensity of β-sheet peaks, and C=O (lipid oxides) bands correlated with lipid peroxidation. These data can explain the decreased of viability of this strain during production-dehydration process. The fundamental knowledge acquired in this study will be useful to obtain new dehydrated yeast strains without additives and with high performance. It will be useful also to improve the formulation and dehydration methods currently used in industry.
