The effectiveness of the selected bacterial carrier to protect encapsulated phytochemical compounds was assessed based on the thermal treatment of both the encapsulated phytochemicals and the phytochemicals in a control juice matrix. In summary, this study demonstrates the potential of using inactivated probiotic bacterial cell carrier for the binding and encapsulation of phytochemicals from a complex juice matrix and characterizes the binding and encapsulation efficiency of diverse phytochemicals and stability of encapsulated compounds in bacterial carriers using a combination of chemical analysis, spectral imaging, and antioxidant properties.Fruit juice contain a variety of phytochemicals, which in general can be classified into alkaloids, carotenoids, nitrogen-containing compounds, organosulfur compounds, and phenolics. Many in vitro and in vivo studies support that the antioxidant property of the phytochemicals plays a major role in their essential health benefits such as anti-inflammation and anti-carcinogen. To characterize the overall efficiency of encapsulating complex profiles of phytochemicals using the bacteria cell carriers, the relative encapsulation efficiency was measured based on the difference in antioxidant concentration of the juice sample before and after incubation with cells. To quantify this ratio, drainage pot antioxidant concentration in juice sample and juice residue after the encapsulation process was measured using the FRAP assay.
The method for FRAP assay is described in detail in Section 3.6. These differences in the FRAP values before and after the encapsulation process reflect the relative amount of antioxidant compounds, including phenolics that are infused or bound to a selected cell-based micro-carrier. Table 1 shows the total antioxidant capacity of MG juice sample measured using the FRAP assay. The encapsulation efficiency in the selected bacterial carrier was 72.67% for MG. This percentage indicates the total fraction of antioxidant compounds bound and encapsulated in a bacterial cell carrier compared to the total antioxidant content in the juice sample. This result suggests that a simple incubation method allows phytochemicals to passively diffuse from a juice matrix to inactivated L. casei cells and results in an efficient binding and encapsulation of the antioxidant compounds in the cell carrier.In addition to characterizing the encapsulated antioxidant content, the encapsulation efficiency of the anthocyanin pigments from the juice to the cells was also evaluated. Anthocyanin, being water soluble, is one of the major polyphenolic fractions in fruit juice and has a significant contribution to its antioxidant properties. To assess the anthocyanin content in the juice before and after encapsulation, the juice matrix was ex-tracted using methanol as described in the materials and methods section and the total anthocyanin content in the extract before and after incubation with cells was measured using a UV-Vis spectrophotometry.
The measured absorbance at 530 nm was converted to an equivalent keracyanin chloride concentration using a standard curve. Results show that MG juice had approximately 8.21 µM/mL of the equivalent keracyanin content. After incubation with inactivated bacterial cells, 66.97% of the total anthocyanin from the MG juice was encapsulated or bound to the cell carriers. In summary, these results highlight a significant potential of the selected bacterial strain for encapsulating antioxidants and anthocyanin family of compounds from a complex juice sample.To help visualize the encapsulated compounds and their intracellular distribution in the cell carriers, confocal multispectral fluorescence images were acquired based on the endogenous fluorescence signals of phytochemicals. The images were collected with a 405 nm excitation and an emission in the FITC channel from 500 to 550 nm. The fluorescence intensity of the cells in each image was quantified by randomly selecting 20 cells and measuring their mean pixel intensity using the ImageJ software. The mean background intensity was subtracted from the cell signals to remove the background signal. As shown in Figure 1, the signal intensity of L. casei carriers increased approximately 24-fold upon incubation of cells with an MG juice as compared to the auto- fluorescence signal from the control cells . Differences in the fluorescence signal intensity between the controls and the modified cells with juice phenolics was statistically significant with a p-value ≤ 0.05. The zoomed-in views in Figure 1b indicated that the cell carriers retained the cellular structure after the encapsulation process, and the encapsulated material was localized relatively uniformly across the intracellular compartment.
The broad emission range is usually associated with the presence of a diverse class of polyphenolic compounds. Based on the previous literature related to fluorescence properties of polyphenolics, the emission band between 533 nm and 595 nm mostly corresponds to anthocyanin content. MG spectra with the secondary emission around 590 nm indicates the presence of anthocyanin compounds in the cell carriers from juice matrix. In addition, the major peak in the MG spectra around 515 nm suggests a possible encapsulation of other phenolic compounds. Plant phenolics such as ferulic acid are known to have fluorescence emissions centered around 520 nm–530 nm. The broadening of the peaks observed in Figure 2 could be attributed to other photo active compounds present in the complex juice matrix. The shift in the emission range compared to the peaks observed from prior literature could also be caused by multiple factors. Anthocyanin polymerization during the juice processing and storage process could cause the emission to shift towards shorter wavelengths. In addition, fluorescence emission spectrums are known to be sensitive to environmental factors, including the excitation wavelength, medium pH and polarity, present macromolecules, etc.. Thus, it may contribute to the shifts observed in Figure 2. In this measurement, phenolic compounds that emit blue fluorescence were not captured in Figure 2 due to the limitation of the available wavelength range in this imaging system. To address these gaps in the compositional analysis of encapsulated compounds, analytical measurements using a HPLC method with known standards were conducted.Among the diverse groups of bio-active compounds present in the fruit and fruit juices, phenolic compounds constitute one of the largest and most diverse groups of phytochemicals. To characterize the phenolic compounds profile of the juice and the encapsulation efficiency, the MG juice before and after incubation with cells was analyzed using HPLC and, based on these measurements, the encapsulation efficiency of the selected polyphenolics was quantified. The protocols for the evaluation of phenolics in a grape juice matrix were already developed by Oberholster et al.. Target compound classes included in this study were flavanols , phenolic acid , flavonols , and polymeric polyphenols . Catechin, epicatechin, gallic acid, and polymeric phenols were quantified using chromatograms at signal 280 nm, caffeic acid at 320 nm, and quercitin and glycosylated myricetin at 360 nm. These polyphenolic compounds have been determined to be among the leading polyphenolic compounds in a grape juice. The chromatograms of 20% MG juice matrix at signals 280, 320, and 360 nmbefore and after encapsulation are shown in Figure 3. Peaks corresponding to each analyzed phenolic compounds were assigned in chromatograms collected at each signal.As observed from Table 2 and Figure 3, most of the investigated compounds were present in MG juice at different concentrations and had different levels of encapsulation efficiency. For flavanols, catechin and epicatechin were both present in the MG juice matrix. Despite these differences in absolute concentration levels, 17.40% of catechin and 18.77% of epicatechin were encapsulated upon incubation of cells with MG juice. For the phenolic acids, large pot with drainage the concentration of gallic acid in MG juice was 1.69 mg/mL and its encapsulation efficiency was 18.43% in L. casei cells. The content and encapsulation efficiency for gallic acid was significantly higher than the amount of coutaric acid and its encapsulation efficiency in L. casei cells. The amount of caffeic acid was below the detection limit in MG juice. Among flavonols, 20% MG juice contains 21.70 mg/mL of quercetin. The encapsulation efficiency of quercetin in L. casei was limited as compared to the quantified flavanols and phenolic acids. Glucoside derivatives are commonly found in grapes and wines, particularly delphinidin-3-glucoside, petunidin-3-glucoside, and malvidin-3-glucoside . MG juice contains 3.53 mg/mL myricetin 3-glycoside and its encapsulation efficiency was 69.85% upon incubation of cells with MG juice. Another common abundant polyphenolic compound in MG juice was polymeric phenols. The 20% juice contains 26.09 mg/mL of polymeric phenols.
The polymeric phenols identified using this protocol represent a mixture of polymeric pigments, which are formed based on reactions between grape anthocyanins and other components in the juice such as tannin, catechins, and proanthocyanidins. A total of 97.97% of the polymeric phenol was infused into the cell carriers upon incubation with MG juice. Taken together, the imaging and HPLC measurement results illustrate that cell carriers can simultaneously encapsulate diversity of bio-active compounds from a complex juice matrix. Compared to previous studies that have predominantly focused on yeast cells for the encapsulation of purified hydrophobic polyphenolic compounds, the results of this study suggest a potential of diverse cell carriers, including bacterial cell carriers, to simultaneously encapsulate multiple compounds from mixtures. Furthermore, since the encapsulation process was conducted using water soluble compounds in fruit juice, this study demonstrates that bacterial cell carriers can bind and encapsulate compounds from water extracts and juices. Together with prior studies, the results of this study illustrate the potential of cell carriers to encapsulate both hydrophobic and hydrophilic bio-actives. The encapsulation process of these compounds from cell carriers can be attributed to both composition and structure of cell carriers. Besides the structural integrity that withstood the encapsulation process as shown in Figure 1, bacterial and yeast cell carriers have a relatively high fraction of protein content on a dry basis. In the case of L. casei cells, the protein content can be as high as 80% or higher on a dry basis. Similarly, the protein content in yeast cells can range from 25 to 60% on a dry basis. In addition, cell carriers also express both soluble and structural proteins including membrane associated proteins. In previous studies, protein–polyphenolic interactions have been explored and the binding between protein isolates and polyphenolic compounds from juice or other plant extracts has been demonstrated. Thus, it is likely that a relatively high concentration and diversity of proteins in micro-scale cells carriers significantly promote the binding of diverse polyphenols from a juice matrix. In addition to proteins, bacteria and yeast cells also contain a diversity of carbohydrate bio-polymers mostly concentrated in cell walls and lipids that are integral parts of the cell membranes. Prior studies have shown interactions between polyphenols and cellular polysaccharides, and the binding mechanism could be attributed to a range of physical and chemical interactions. The complex and porous structures and surface properties of the cell wall has also been proposed to be important for the binding process. These compositions and cellular structures can provide a rich environment for the partitioning and compartmentalization of diverse compounds in cell-based carriers.The results illustrate that the encapsulated content and efficiencies varied by the chemical class, compounds, and juice matrix. Quercetin as a monomeric flavonol showed low incorporation rates from MG juice, while the glycosylated myricetin has a significantly higher encapsulation efficiency . In contrast, polymeric polyphenols yielded the highest encapsulation efficiency among all compounds tested from MG juice . This trend of differences in encapsulation efficiency of compounds of the same class was also observed in the case of flavonols and phenolic acids. Furthermore, based on these measurements, no clear correlation between encapsulation efficiency and relative hydrophilicity of the compounds was observed. These observations suggest that the partitioning of compounds in cells from a juice matrix significantly depends on the interactions among the polyphenolic compounds and the composition of cells. The characterization of these interactions is beyond the scope of this study, but these results suggest that it may be possible to select cellular compositions among the diverse class of microbes that may promote the binding of selective polyphenols from a given plant extract and juice.One of the important functionalities of encapsulation carriers is to protect the bio-active compounds from adverse environmental factors and food processing conditions. These adverse conditions may cause damage by oxidation, less favorable pH, and thermal induced reactions in bio-active compounds in food matrices. In order to produce a shelf-stable and microbially safe food, thermal processing methods such as pasteurization or sterilization are commonly used. Thus, in this study the effectiveness of the selected bacterial carrier in protecting and stabilizing the encapsulated juice polyphenols was evaluated.