Progress 01/26/15 to 09/30/17
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training for a postdoc. After working on this project part-time for three years, he successfully found a job at a startup company Profusa. How have the results been disseminated to communities of interest?We have presented a poster "Encapsulation of Lactic Acid Bacteria for Probiotic and Food Preservation Applications" for our annual departmental research symposium. We will also present the data at the ASABE annual conferenceJul 29 - Aug 1 2018. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Project summary: Lactic acid bacteria (LAB) have been shown to have both probiotic and food preservative properties. As such, their inclusion in food products, such as yogurt, is highly desirable. However, the low pH environment of certain food products, as well as harsh gastric conditions significantly reduces the number of surviving LAB, thus reducing their effectiveness. We have developed a novel set of capsules for protecting LAB from harsh environmental conditions based on our previously developed core-shell electrospray technology. We have shown that our capsules are capable of retaining nearly 100% viability of encapsulated LAB after a 2 hr exposure to simulated gastric fluid, yet are still capable of producing and releasing bacteriocins that can kill food pathogens. This first application of our core-shell encapsulation technology to LAB paves the way for the creation of an entirely new generation of bacterial capsules that could include characteristics such as multiple material types and complex geometries. Accomplishments year 1: In the first year of this project we optimized the parameters for creating core-shell capsules containing LAB. Specifically, we encapsulated two different species of LAB, Lactococcus Lactis, and Enterococcus Mundtii. The goal was to create uniformly spherical capsules of a consistent diameter and ensure that the LAB remained confined to the center of the capsules. Our capsules consisted of an alginate shell with a solid alginate core containing the bacteria and were between 300 and 400 µm in diameter. Having created the desired capsules, we showed that the bacteria could divide within the confines of the capsule over a culture period of a few hours and yet not burst open the capsule shell. Accomplishments year 2: Although the pure alginate capsules retained their integrity sufficiently to confine the bacteria over a period of a few hours in culture, as a polysaccharide, alginate has the potential to be digested over a long period of time (for example, during storage in a dairy product). To prevent capsule degradation, we exchanged the biodegradable alginate core for a mineral core made of silica gel. Although the formation of silica gel through the aggregation of silica colloids had been previously reported, we are not aware of any previous reports of employing silica gel in an electrospray process. The challenge is to ensure that the silica gel precursor solution remains liquid for a sufficiently long time that it can be forced through the spray nozzle, yet still form a solid gel sufficiently rapidly to retain a spherical shape. We achieved this result by careful optimization of the chemical conditions for gelation such that the solidification process took between 15 and 30 minutes. Once we created silica-core/ alginate-shell capsules, we demonstrated that LAB could be effectively confined in the capsule cores yet remain viable. Accomplishments year 3: In the third year of the project we applied the technology developed in years 1 and 2 to protect the LAB, specifically, L. Lactis, from simulated gastric conditions. We tested three types of capsules: the alginate core/alginate shell capsules developed in year 1, the silica core/alginate shell capsules developed in year 2, and clay hydrogel-core/ alginate shell capsules. The clay used was laponite XLG, which our lab has previously shown will form a hydrogel in the presence of calcium ions. We exposed the encapsulated bacteria to simulated gastric fluid (0.9% NaCl, pH 2.0, 3 g/L Pepsin) for up to 2 hrs and then measured the viability of the bacteria compared with free (unencapsulated) bacteria that had received the same treatment. In order to perform colony counts, the standard method of measuring bacterial viability, that bacteria needed to first be removed form the capsules. However, although sufficiently high concentrations of EDTA or other chelating agents will dissolve alginate and clay hydrogels, they also have the potential to harm encapsulated bacteria that depend on divalent in such as calcium for survival. In addition, silica gel cannot be easily degraded under biocompatible conditions. Therefore, we evaluated bacterial survival via a live/dead fluorescent stain that, combined with confocal microscopy, allowed us to measure bacterial viability in situ. We found that even after 2 hours of exposure to simulated gastric fluid, encapsulated bacteria retained nearly 100% viability (not significantly different from control capsules exposed only to PBS), while free bacteria experienced a significant reduction in viability after even a one-hour exposure. Having shown that the capsules were capable of protecting the LAB from gastric conditions, we next investigated if the encapsulated LAB could produce food-preserving bacteriocins, and if those bacteriocins could escape the capsules in sufficiently high concentrations to kill pathogens. We cultured the capsules containing the LAB overnight, collected the media, and removed any remaining capsules or bacteria through centrifugation. We exposed two different strains of the model food pathogen Bacillus Cereus to varying concentrations of this conditioned media to determine if it contained lethal concentrations of bacteriocins. We found that even 5% concentration of media conditioned by any of the three capsule types significantly inhibited the growth of both strains of B. Cereus. Conclusion: In conclusion, we have successfully created core-shell capsules capable of protecting LAB against harsh gastric conditions while retaining their ability to produce food -preserving bacteriocins. In the future, we hope to extend our encapsulation methodology to other food science applications to extend the usefulness of LAB in food production technology. As demonstrated in this study, the core-shell methodology not only allows encapsulated bacteria to be confined in the center of the capsules (rather than dispersed at the edges), but also provides flexibility to include multiple layers of different materials in a single capsule. Although here we only demonstrated dual material capsules, the same methodology can be applied to create triple layer capsules or even capsules with more complex architectures such as a janus structure. Thus, the technology presented here is only one illustration of a platform that could enable the development of a wide range of new capsule types for bacterial encapsulation or other food science applications.
Publications
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Pathogen growth inhibition We will test the pathogen growth inhibition at a variety ofLAB density and differentstages of LAB growth. Given the results of our control experiments with free LAB, we believethat increasing bacterial density per capsule, increasing LAB culture time prior to introducing the pathogen, and/or increasing capsule permeability (to more easily release bacteriocins) will have more profound effects in the pathogen growth inhibition. Bacterial viability Simultaneous with our pathogen growth inhibition experiments, we will continue to test and validate the viability of bacteria upon exposure to gastric fluid in both silica/alginate and alginate only core-shell capsules.
Impacts
What was accomplished under these goals?
Silica-core capsules Our previous experiments have shown that alginate is a promising material for forming protective capsules for lactic acid bacteria (LAB). However, alginate has a few inherent limitations. First, alginate is a readily digestible polysaccharide. While this property can be seen as a benefit in that it makes alginate capsules edible for humans, it also allows encapsulated bacteria to eventually digest and escape the capsules. Second, alginate hydrogel is a relatively porous material with high water content (typically > 98%) and therefore may not provide sufficient protection for the encapsulated bacteria. To preempt these issues, we developed novel core-shell capsules consisting of a silica gel core and an alginate shell. As silica is a mineral material, bacteria cannot easily digest it. Furthermore, recent studies have indicated that silica may provide additional protection for bacteria under gastric conditions compared with alginate alone. We optimized a silica gelation process such that silica gels could be formed slowly (over several hours) and under biocompatible conditions. The gelation process needs to be biocompatible to prevent damage to the bacteria during capsule formation. The slow rate of reaction allows the gelation process to be initiated and then core-shell capsules to be formed without the silica solidifying in the core-shell nozzle used for electrospray. We demonstrated that the size of the silica core can be easily tuned relative to the shell size and that encapsulated bacteria remain viable throughout the gelation process. Pathogen growth inhibition LAB have two principle benefits for the consumer. As probiotics they provide a direct health benefit upon reaching the intestines. In addition, they produce bacteriocins that inhibit pathogen growth, preventing food spoilage. We tested the efficacy of the Lactococcus Lactis at preventing the growth of Bacillus Cereus, a common food pathogen. We monitored the optical density at 600 nm (OD600) of cultures inoculated with B. Cereus that had either been exposed to MRS media conditioned by L. Lactis or fresh MRS media. While the B. Cereus in fresh media showed a typical sigmoidal growth curve over 15 hours, the culture exposed to conditioned media showed no significant increase in OD600 over the same time period. Furthermore, we showed that dropping live L. Lactis culture or conditioned media onto an agar plate inoculated with B. Cereus resulted in a distinct "kill-zone" around the drop, while fresh MRS media had no effect. We have begun experiments testing the efficacy of encapsulated LAB against B. Cereus. So far we have tested the efficacy of two species of LAB (L. Lactis and E. Mundtii) at preventing B. Cereus capsules in both alginate only and silica/alginate core-shell capsules. Our experiments have not yet shown any significant pathogen growth inhibition by encapsulated bacteria. However, we have only tested a single LAB density and only at the early stage of LAB growth. Given the results of our control experiments with free LAB we are confident that increasing bacterial density per capsule, increasing LAB culture time prior to introducing the pathogen, and/or increasing capsule permeability (to more easily release bacteriocins) will solve this issue. Bacterial viability Simultaneous with our pathogen growth inhibition experiments, we have begun testing the viability of bacteria upon exposure to gastric fluid in both silica/alginate and alginate only core-shell capsules. In our previous experiments, we found that colony count assays were not very effective at assessing viability of bacteria in capsules, as many bacteria tend to be lost during the capsule dissolution process. Instead, we used a fluorescent bacteria viability kit, which allows us to assess viability while bacteria are still contained in the capsules. We performed pilot experiments with this kit, measuring total fluorescence in the green (live) and red (dead) channels of confocal microscope images to quantify viability. Our initial results suggest that both alginate only and silica/ alginate capsules are capable of maintaining high viability of encapsulated bacteria after a 30 min exposure to simulated gastric fluid.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Y-C. Lu, W. Song, D. An, B. J. Kim, R. Schwartz, M. Wu and M. Ma "Designing Compartmentalized Hydrogel Microparticles for Cell Encapsulation and Scalable 3D Cell Culture"J. Mater. Chem. B 2015, 3, 353.
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Progress 01/26/15 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Capsule Optimization: We first performed a set of designed experiments to optimize the shape and size of the alginate capsules. The size of the capsules is influenced primarily by the concentration of alginate and the electrical voltage applied to the nozzle. Capsule shape is most affected by the relative flow rates of the inner and outer solutions that make up the core and shell of the capsules, respectively. We varied the concentration of alginate from 0.5% to 2%, the voltage from 5 kV to 9 kV and the inner flow rate from 5µL/ min to 25µL/min. The outer flow rate was held constant at 450µL/min. Each batch of capsules was examined under a microscope to assess quality of the shape, with a perfectly spherical capsule considered the ideal shape. We also measured capsule diameter to confirm that the capsules were sufficiently small (300 -400 µm). After completing a central composite design experiment, we concluded that the optimal parameters for the capsule formation were as follows: Alginate concentration (shell): 1.5% Voltage: 9 kV Outer solution (shell) flow rate: 450 µL/min Inner solution (core) flow rate: 5µL/min Bacterial Encapsulation: We applied our optimized protocol to encapsulate two different species of lactic acid bacteria: Lactococcus Lactis and Enterococcus Mundtii. Each species was grown in suspension culture overnight and then directly used as the core of alginate core-shell capsules. In addition, we created single layer capsules by mixing bacterial suspension culture with alginate directly. We found that Lactococcus Lactis grew slightly more rapidly than Enterococcus Mundtii and so for the purposes of optimization studies we focused on Lactococcus Lactis exclusively. In the future, we will apply the developed encapsulation technology to Enterococcus Mundtii as well. Figures 1 and 2 show images of Lactococcus Lactis encapsulated in both core-shell (Fig. 1) and single layer capsules (Fig. 2). As hypothesized, the core-shell method was effective at retaining the majority of the bacteria in the center of the capsules while the single layer method resulted in some bacteria at the capsule edge. Because of the low flow rate necessary to maintain a spherical capsule shape, the number of bacteria per capsule was lower than desired. We found that by suspending the capsules in growth media and culturing them for three hours increased the number of bacteria within the capsules without bursting the shell. This methodology allows for an exponentially larger number of bacteria to be encapsulated. Bacterial Survival under gastric conditions: We next assessed the relative survival rates of encapsulated bacteria in gastric conditions compared with free, non-encapsulated bacteria. We exposed bacteria to acidified saline solution (pH 2) at 37ºC to simulate the gastric environment. In our initial experiment we found that non-encapsulated bacteria experienced a 5-log reduction in number of colony forming units, while encapsulated bacteria experienced only a 3.5-log reduction. This suggests that even simple alginate capsules have the potential to improve bacterial survival in the stomach. We are currently performing more experiments to confirm this observation
Publications
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