To evaluate the thermal stability of encapsulated bio-actives, the cells were heat-treated at 90 ◦C for 1, 2, 5, 10, 20, 40, and 60 min in a temperature-controlled water bath. The heating conditions were selected based on prior studies. After the heat treatment, the cell-encapsulated polyphenolics were extracted using the methods described in Section 3.10 of the material and methods section. The total antioxidant concentration of the extract was then measured using the FRAP assay. The control group of cells with encapsulated compounds but without the heat treatment were also extracted using the same approach and used for calculating the retention ratio during the treatment. The results in Figure 4 illustrate the percentage of total antioxidant capacity retained at each time point during the heating process. As observed in Figure 4, the bacterial carrier effectively protected the encapsulated compounds during thermal treatment. Approximately 93% of the antioxidant capacity for the encapsulated MG juice was retained after 1 h of heat treatment , square pot whereas only 74% of the initial antioxidants were preserved without using encapsulation after the heat treatment of juice for 1 h.
These observations indicated that cell carriers can effectively protect encapsulated antioxidant compounds against thermal stress. In addition to the total antioxidant capacity, the retention of anthocyanins was also monitored during the heating process at 90 ◦C. The cells encapsulated with polyphenols from the juice matrix were sampled at 1, 2, 5, 10, 20, 40, and 60 min, and compared to the non-heated polyphenols encapsulated in cells from the juice. As described previously, the anthocyanin content retained in the cells was extracted using methanol and measured using a UV-Vis spectrometer. Figure 5 shows similar patterns of enhanced stability of anthocyanin compounds on cell carriers similar to the results in Figure 4. Despite the fact that the MG juice contains more colored pigments , these compounds seemed to be more susceptible to heat. Only 61% of the anthocyanin pigments in the MG juice were retained after 60 min of heat treatment. In contrast, 90% of the encapsulated anthocyanin pigments were preserved in the cell carriers. These results demonstrated that the bacterial cell carriers effectively protected encapsulated anthocyanins from degradation caused by the thermal treatment.Overall, the results demonstrated that, after 60 min of heat treatment at 90 °C, more than 87% of the total antioxidant capacity and 90% of the anthocyanin content were recovered from the encapsulated MG as compared to the respective juice without encapsulation. The degradation of juice phenolics content including anthocyanin from heating were comparable with previous studies. The thermal stability of the encapsulated active compounds was significantly higher than non-encapsulated MG juice.
This protective effect of microcarriers has been observed in a range of encapsulation systems such as spray-drying particles and emulsions. However, comparable or higher percentages of antioxidant capacity and pigment content retention were observed using the cell carriers compared to the synthetic encapsulation carriers. For instance, more than 20% losses were observed for anthocyanins encapsulated in polymer matrices such as maltodextrin, mixture of maltodextrin and gum arabicadvantage of the cell carriers might be attributed to both the physical cellular structure and its complex chemical composition. As shown in Figure 1, the cell structures persisted through the encapsulation process, and literature has shown that some of the Lactobacillus strains can maintain structural integrity at elevated temperatures around 100–120 ◦C, for 30 to 60 min. The robust structure is essential for protecting encapsulated bio-actives, whereas colloidal encapsulation systems tend to destabilize both physically and chemically during encapsulation or in adverse environmental conditions. Besides the physical structure, the antioxidant property of intracellular content of L. casei has also been reported. Aguilar-Toalá et al. suggested that glutathione and other intracellular lipid and protein components might be involved in the antioxidant activities, which might in turn help stabilize and protect bio-active compounds encapsulated within the cell carrier.
Therefore, cell carriers are an efficient encapsulation material for preserving the bio-active functions of the extracted polyphenolics during thermal processing. In addition, encapsulation using cell carriers exhibits certain advantages in terms of the manufacturing process. In this study, we used L. casei cells to encapsulate a composite profile of polyphenolics with a basic temperature-controlled incubation. Currently, spray drying and freeze drying are the most commonly applied industrial techniques for microencapsulation and stabilization of plant polyphenolics from natural sources. Spray drying is a unit operation where liquid is atomized in a hot gas current to obtain a powder. While spray drying is prevalent with low cost, its limitations have also been extensively discussed. We observed 4 to 5 times higher amounts of anthocyanin content encapsulated in the cell carrier in this study when compared to spray-dried powder. Despite variations in the raw material, loss of heat in sensitive compounds during spray drying might be due to the exposure to oxygen and the thermal treatment . In addition, the drying process may cause the loss of dried material due to wall deposition, low thermal efficiency, broad size distribution, and irregular microstructures. Encapsulation using the preformed cellular structure of probiotic bacteria and passive incubation, on the other hand, significantly simplified the process with more uniform cellular size and microcellular structure.Total antioxidant capacity of juice matrix before and after encapsulation was quantified using the Ferric Reducing Antioxidant Power assay. Antioxidant activity was selected as a representation of the total bio-active compound concentration in the juice. The changes in the antioxidant content of the juice after encapsulation was evaluated to assess the encapsulation efficiency of diverse class of bio-active compounds. The protocol for measuring FRAP activity was adapted from Benzie and Strain. The stock solutions included 300 mM acetate buffer , 10 mM TPTZ solution in 40 mM HCl, and 20 mM FeCl3·6H2O solution. The fresh working solution was prepared by mixing 25 mL acetate buffer, 2.5 mL TPTZ solution, and 2.5 mL FeCl3·6H2O solution and then warmed at 37 ◦C before using. Fruit juice before and after encapsulation was allowed to react with 2850 µL of the FRAP solution for 30 min in the dark condition. Change in color of the solution was quantified using a UV-Vis measurement at 593 nm using a spectrometer. The standard curve was generated using a range of Trolox solutions between 25 and 800 µM. Results were expressed in µM T.E./mL fresh juice. The samples were diluted in case the absorbance value measured for the samples was over the linear range of the standard curve.Anthocyanin content in juice before and after encapsulation was also measured using a UV-Vis spectrometry. Grape juice is a significant source of plant anthocyanins and changes in the level of anthocyanins in a juice matrix before and after encapsulation also represent a measure of encapsulation of water-soluble pigments in bacterial cells. The absorbance value of the clarified samples was scanned from 250 nm to 600 nm, and a peak intensity was recorded at 530 nm for all the samples. The samples were diluted accordingly to avoid saturation in the absorbance signal. Standard curves were constructed using different concentrations of keracyanin chloride and anthocyanin content was represented as keracyanin equivalent content.Confocal Laser Scanning Microscopy images of bacterial cells after encapsulation with and without incubation with muscadine juice sample were collected using a Zeiss LSM 510 upright microscope with 40×/1.1 water objective.
Each sample was excited at 405 nm using an argon diode laser. Emission scans were acquired using a 500–550 nm bandpass emission filter. Lambda scans of each sample were collected over a range of 470–670 nm with 20 nm step size. The average intensity of the images acquired at different wavelengths during the lambda scan was measured using ImageJ software and plotted using an Origin 8.0 .Phenolic compounds were extracted by mixing 2 mL of the reconstituted MG juice sample with 13 mL of acidified methanol . After mixing using a vortexer, drainage collection pot the mixture was sonicated using a bath sonicator for 10 min and the extract was separated from the remaining juice solids by centrifugation at 5500 rpm for 5 min. The samples were then diluted 10-fold with milliQ water for HPLC-DAD analysis. To assess encapsulation efficiency and yield in cell-based carriers, phenolic content in the aqueous phase before and after encapsulation process was quantified. Chromatography separation and detection of phenolic compounds were performed on an Agilent 1260 Infinity reverse phase HPLC -D.A.D. system equipped with a thermostatic autosampler, thermostatic column compartment, and a diode array detector according to a method adapted from Plaza et al.. An Agilent PLRP-S 100 column with an Agilent 3 × 5 mm guard column was used at a temperature of 35 ◦C for all the analysis. Mobile phase A: 1.5% phosphoric acid solution. Mobile phase B: acetonitrile solution containing 20% mobile phase A. The gradient protocol for HPLC separation and analysis was as follows: 0 min, 94% solvent A; 73 min, 69% A; 78 min, 38% A; and 90 min, 94% A. The flow rate was 1 mL/min and the injection volume for all samples was 10 µL. Samples were filtered through 0.45 µm type H.A. Millipore filters prior to injection. Absorbance spectra were recorded from 250 nm to 600 nm. The eluted compounds were monitored and identified based on spectral and retention time comparisons with standards at multiple wavelengths, including 280 nm for Flavanol [gallic acid, -catechin and -epicatechin] and polymeric phenols, 320 nm for hydroxycinnamates , and 360 nm for flavonol and derivatives , respectively, using the D.A.D. detector. External calibration curves were constructed for gallic acid, -catechin, -epicatechin, caffeic acid, coutaric acid, quercetin, and myricetin glycosides were used for quantification of the target compounds. Polymeric phenols were quantified as catechin equivalents. Chromatograms were integrated using the Agilent CDSChemStation Software .The thermal stabilities of the encapsulated bio-active compounds were evaluated using a thermostatic water bath at 90 ◦C for up to 60 min. A 1 mL suspension of the cells with encapsulated compounds and 1 mL of juice alone were added to the prewarmed 20 mL glass vials and incubated in the dark for 1, 2, 5, 10, 20, 40, and 60 min. The concentration of the total antioxidant contents in the juice sample and the cell encapsulated sample were maintained the same. After the treatment, 1 mL acidified methanol was added to each vial. Bead-beating at 6.0 m/s for 30 s for 3 times was then carried out to facilitate thorough extraction. Finally, the homogenized samples were sonicated using a bath sonication device for 10 min. The methanolic extract was then centrifuged to remove cell Ecosystems are suffering from the pressures of on-going global change, including climate change and habitat loss. One of the main consequences of ecosystem disturbance is the local extinction of species, yet we have little understanding of the consequences of these extirpations for ecological interactions, community dynamics and ecosystem functions. To predict how ecosystems, which are naturally dynamic, will react to these pressures, we first need to understand how communities react to the natural dynamics that lead to changes in their composition of species, with special emphasis on changes in species interactions and the ability of the community to re-arrange itself and maintain its functioning. Up to now, understanding community-level rearrangements following changes in species composition has proved elusive, given the great complexity of ecological systems, which feature high levels of species diversity, interactions across species and environmental variability. The use of network analyses to represent some of the biotic interactions has allowed us to address part of this complexity. However, many network studies have used temporally and spatially aggregated data of observed interactions representing a snapshot of a community. Aggregating data omits important information regarding the dynamic nature of ecological interactions, and in particular concerning species functional roles, which can change due to competition for resources, the presence of parasites and pathogens or changes in species composition. These changes in species composition have been primarily assessed through studies focusing on species extinctions or invasions. Some of them have used experimental set-ups to explore community level dynamics following species extinctions. For example, Brosi & Briggs temporarily removed the most abundant bumblebee species and analysed how the rest of the pollinator community responded. They found that in manipulated sites, floral fidelity decreased, with consequences for plant reproductive success, and also that the loss of a single pollinator species changed pollination network structure.