Progress 04/15/18 to 04/14/21
Outputs
Target Audience:The target audience includes academic and industry experts in the field of food engineering, food science, and colloid science. This audience of graduate students, postdoctoral scholars, professors, and microencapsulation industry experts was reached through presentation and participation in three conferences/workshops over the project duration. Changes/Problems:Project efforts were paused from January 2020 through March 2020 in order to accommodate PD's appointment as a lecturer for an upper division course (kinetics and bioreactor design). Due to COVID-19, lab research activities were curtailed during spring 2020. During this time, PD drafted a manuscript summarizing the project. Also during this time, PD was offered a scientist position with an agricultural biotechnology company. PD has resigned from the university effective May 12, 2020 in order to accept this scientist position in industrial research and development. This career advancement will allow PD to gain valuable experience regarding the commercial use of microencapsulation for agricultural applications. What opportunities for training and professional development has the project provided?D attended two academic conferences and an industry-focused workshop during the project duration. PD attended the Conference of Food Engineering in Minneapolis, MN in September 2018 to deliver a research talk. This experience provided an opportunity to increase academic exposure, strengthen oral presentation skills, and network with academic and industry leaders in the food engineering field. In June 2019, PD attended the 9th International Colloids Conference in Sitges, Spain. There, PD presented a poster of project results, took advantage of networking opportunities, and communicated his scientific research to other conference attendees. PD attended the 21st Industrial Microencapsulation and Applications Workshop: Chemistry, Technologies, Economics, Materials, Applications, and Plant Visit. This industry-oriented microencapsulation conference / short course took place in Minneapolis, MN from August 20-21, 2019. At this event, PD attended seminars regarding microencapsulation technologies, built contacts with microencapsulation experts in industry and academia, and toured industrial and academic microencapsulation facilities. PD paused project activities from January to March 2020 to take a lecturer position within the university. During this time, PD taught an upper division course on bioreaction kinetics and bioreactor design. This provided PD an opportunity to bolster skills in scientific communication, lecture delivery, and course management. How have the results been disseminated to communities of interest?Results of this work have been presented in poster format at an international academic conference (9th International Colloids Conference). A manuscript is in preparation for eventual publication in a peer-reviewed scientific journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The food industry needs innovative solutions that reduce losses of volatile bioactives during food processing, facilitate their incorporation into functional foods, and ensure their delivery and uptake in the human digestive system. This project developed and investigated a new microencapsulation system for incorporating volatile bioactive compounds into functional foods. Limonene, a model volatile bioactive, was enveloped in microscopic protective polymer shells formulated with calcium carbonate nanoparticles (CCNP) to achieve unique particle microstructures. To form the most robust shells, the volatile bioactive cargo droplets were first coated with CCNP, which helped to densely crosslink the surrounding polymer shell. These surfactant-free microcapsules contained more bioactives after production, in some cases released less bioactive cargo when incorporated into aqueous environments simulating foods, reduced the release of bioactives in the stomach environment, and promoted release in the intestinal environment, the site of absorption. This microencapsulation system could ultimately improve process economics for food industry and provide consumers with fortified food products that provide both nutrition and health benefits. The microencapsulation system developed here produces novel microstructures while utilizing very few and very scalable processing steps, thus improving process economics relative to the cumbersome alternative process. Aside from economics, the microencapsulation system offers advantages for the production of functional foods that provide both nutritional and health benefits. The microcapsules mitigate the unwanted release (and subsequent loss or degradation) of bioactive cargo in the food and in the stomach prior to intestinal absorption, potentially improving bioavailability. Objective 1: To understand how the cross-linking structure within microcapsules influences volatile retention during spray-drying. D-limonene was microencapsulated in spray-dried cross-linked alginate microcapsules (CLAMs), with formulation variations designed to exhibit three different microstructures. CCNP served as a reservoir of divalent cations for cross-linking the alginate polymer. Alginate cross-linking was accomplished by in situ acidification, which partially dissolves the acid-soluble calcium salt to rapidly form calcium alginate during spray drying. CLAMs with interface-targeted cross-linking (iCLAMs) were compared to two control microcapsule formulations, both with unstructured internal interfaces (and hence random cross-linking distribution). iCLAMs were formed by first generating a Pickering emulsion comprising limonene stabilized by CCNP, followed by combining with alginate and ammonium succinate solutions prior to spray-drying. The location of CCNP at the limonene droplet surfaces resulted in iCLAMs with enhanced crosslinking at the internal interfaces within the spray dried particles. Surfactant-free control CLAMs (Ctrl-CLAMs) were prepared by homogenizing all components simultaneously, allowing the internal interfaces to be stabilized by both alginate and CCNP. In another control (T80-CLAMs), limonene was emulsified with the nonionic surfactant Tween 80 before combining with the other components. SEMs of sectioned iCLAMs revealed a layer of residual CCNP at the internal interface between the limonene cargo pocket and crosslinked alginate shell. In Ctrl-CLAMs and T80-CLAMs, CCNP were distributed throughout the volume of the polymer matrix. In the bulk powders, alginate crosslinking extent was unchanged between formulations. However, the crosslinking microstructure in iCLAMs was likely more organized, with the densest crosslinking occurring near the calcium-rich internal interfaces. Unfortunately, visualizing the crosslinking density in iCLAMs by confocal microscopy, as originally proposed, was unsuccessful. In experiments utilizing calcium responsive dyes, regions of high calcium content (and thus high crosslinking density) could not be differentiated from the background signal. Internal particle microstructure affected volatile losses during microencapsulation. While all formulations were prepared with the same target limonene content (25%), spray dried iCLAMs contained significantly more limonene than Ctrl-CLAMs or T80-CLAMs. When isolating only the losses that occurred during spray drying, iCLAMs retained significantly more limonene than Ctrl-CLAMs, suggesting that the enhanced crosslinking and residual CCNP at the internal interfaces of iCLAMs provided additional resistance to limonene mass transfer during spray drying. Volatile retention during spray-drying of iCLAMs was elevated but not significantly greater than that of T80-CLAMs, although the final limonene content of iCLAMs was significantly greater than that of T80-CLAMs. Limonene losses during homogenization may account for this discrepancy. Objective 2: To investigate how the incorporation of inert nanoparticles into the cargo-polymer interfaces within microcapsules affects volatile retention and cargo release kinetics in aqueous environments simulating food systems and the gastrointestinal tract. Although prepared as a dry powder, CLAMs would be suspended in aqueous environments in many envisioned food applications. Limonene loss from aqueous CLAM suspensions and emulsions was measured over time in open vessels subjected to thermal treatments and gentle agitation. The limonene remaining in the aqueous system was extracted and quantified by gas chromatography. Surprisingly, the kinetics of loss were similar for iCLAMs, Ctrl-CLAMs, and T80-CLAMs, and this held true at 37, 60, and 80°C. However, compared to all three CLAMs, limonene was lost much more rapidly from CCNP Pickering emulsions, and even more rapidly from Tween 80 emulsions; these emulsions systems offered less of a mass transfer barrier to limonene compared to the alginate hydrogel of rehydrated CLAMs. However, the microstructure of the CLAMs did not appear to significantly affect the kinetics of limonene loss in this environment. When suspended in water at room temperature in a closed vessel, iCLAMs and Ctrl-CLAMs released significantly less limonene cargo after 2h (approximately 5%) than T80-CLAMs. The nonionic surfactant likely facilitated the release of emulsified limonene from the T80-CLAMs. The differences in limonene release between the three CLAMs formulations was driven more by the presence or absence of surfactant than by the differences between particle microstructures. CLAMs prepared without surfactant exhibited improved gastrointestinal release properties compared to T80-CLAMs. In simulated gastric fluid for 2h, iCLAMs and Ctrl-CLAMs released significantly less limonene than T80-CLAMs. When subsequently transferred into simulated intestinal fluid for 2h, the alginate matrix disintegrated and completely released the remaining limonene. The similar release properties of iCLAMs and Ctrl-CLAMs suggested that the spatial distribution of CCNP within the CLAMs did not exert a significant effect on gastric release. Overall, the calcium enriched internal interfaces of iCLAMs did not improve cargo release properties. However, the elimination of nonionic surfactant in forming iCLAMs did help mitigate unwanted cargo release in water and gastric fluid. Considering the shift toward simpler ingredient labels in the food industry, a microcapsule formulation comprising fewer components that also exhibits improved functional properties is highly desirable. The work under this objective led to a change in knowledge regarding the release of volatile bioactive lipophilic compounds from CLAMs. By preparing iCLAMs with nanostructured internal interfaces without surfactants, the unwanted release of volatile bioactive cargo may be reduced, both during incorporation into foods and during the gastric phase of digestion, potentially improving bioavailability.
Publications
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Progress 04/15/18 to 08/12/20
Outputs
Target Audience:The target audience includes academic and industry experts in the field of food engineering, food science, and colloid science. This audience of graduate students, postdoctoral scholars, professors, and microencapsulation industry experts was reached through presentation and participation in three conferences/workshops over the project duration. Changes/Problems:Project efforts were paused from January 2020 through March 2020 in order to accommodate PD's appointment as a lecturer for an upper division course (kinetics and bioreactor design). Due to COVID-19, lab research activities were curtailed during spring 2020. During this time, PD drafted a manuscript summarizing the project. Also during this time, PD was offered a scientist position with an agricultural biotechnology company. PD has resigned from the university effective May 12, 2020 in order to accept this scientist position in industrial research and development. This career advancement will allow PD to gain valuable experience regarding the commercial use of microencapsulation for agricultural applications. What opportunities for training and professional development has the project provided?D attended two academic conferences and an industry-focused workshop during the project duration. PD attended the Conference of Food Engineering in Minneapolis, MN in September 2018 to deliver a research talk. This experience provided an opportunity to increase academic exposure, strengthen oral presentation skills, and network with academic and industry leaders in the food engineering field. In June 2019, PD attended the 9th International Colloids Conference in Sitges, Spain. There, PD presented a poster of project results, took advantage of networking opportunities, and communicated his scientific research to other conference attendees. PD attended the 21st Industrial Microencapsulation and Applications Workshop: Chemistry, Technologies, Economics, Materials, Applications, and Plant Visit. This industry-oriented microencapsulation conference / short course took place in Minneapolis, MN from August 20-21, 2019. At this event, PD attended seminars regarding microencapsulation technologies, built contacts with microencapsulation experts in industry and academia, and toured industrial and academic microencapsulation facilities. PD paused project activities from January to March 2020 to take a lecturer position within the university. During this time, PD taught an upper division course on bioreaction kinetics and bioreactor design. This provided PD an opportunity to bolster skills in scientific communication, lecture delivery, and course management. How have the results been disseminated to communities of interest?Results of this work have been presented in poster format at an international academic conference (9th International Colloids Conference). A manuscript is in preparation for eventual publication in a peer-reviewed scientific journal. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The food industry needs innovative solutions that reduce losses of volatile bioactives during food processing, facilitate their incorporation into functional foods, and ensure their delivery and uptake in the human digestive system. This project developed and investigated a new microencapsulation system for incorporating volatile bioactive compounds into functional foods. Limonene, a model volatile bioactive, was enveloped in microscopic protective polymer shells formulated with calcium carbonate nanoparticles (CCNP) to achieve unique particle microstructures. To form the most robust shells, the volatile bioactive cargo droplets were first coated with CCNP, which helped to densely crosslink the surrounding polymer shell. These surfactant-free microcapsules contained more bioactives after production, in some cases released less bioactive cargo when incorporated into aqueous environments simulating foods, reduced the release of bioactives in the stomach environment, and promoted release in the intestinal environment, the site of absorption. This microencapsulation system could ultimately improve process economics for food industry and provide consumers with fortified food products that provide both nutrition and health benefits. The microencapsulation system developed here produces novel microstructures while utilizing very few and very scalable processing steps, thus improving process economics relative to the cumbersome alternative process. Aside from economics, the microencapsulation system offers advantages for the production of functional foods that provide both nutritional and health benefits. The microcapsules mitigate the unwanted release (and subsequent loss or degradation) of bioactive cargo in the food and in the stomach prior to intestinal absorption, potentially improving bioavailability. Objective 1: To understand how the cross-linking structure within microcapsules influences volatile retention during spray-drying. D-limonene was microencapsulated in spray-dried cross-linked alginate microcapsules (CLAMs), with formulation variations designed to exhibit three different microstructures. CCNP served as a reservoir of divalent cations for cross-linking the alginate polymer. Alginate cross-linking was accomplished by in situ acidification, which partially dissolves the acid-soluble calcium salt to rapidly form calcium alginate during spray drying. CLAMs with interface-targeted cross-linking (iCLAMs) were compared to two control microcapsule formulations, both with unstructured internal interfaces (and hence random cross-linking distribution). iCLAMs were formed by first generating a Pickering emulsion comprising limonene stabilized by CCNP, followed by combining with alginate and ammonium succinate solutions prior to spray-drying. The location of CCNP at the limonene droplet surfaces resulted in iCLAMs with enhanced crosslinking at the internal interfaces within the spray dried particles. Surfactant-free control CLAMs (Ctrl-CLAMs) were prepared by homogenizing all components simultaneously, allowing the internal interfaces to be stabilized by both alginate and CCNP. In another control (T80-CLAMs), limonene was emulsified with the nonionic surfactant Tween 80 before combining with the other components. SEMs of sectioned iCLAMs revealed a layer of residual CCNP at the internal interface between the limonene cargo pocket and crosslinked alginate shell. In Ctrl-CLAMs and T80-CLAMs, CCNP were distributed throughout the volume of the polymer matrix. In the bulk powders, alginate crosslinking extent was unchanged between formulations. However, the crosslinking microstructure in iCLAMs was likely more organized, with the densest crosslinking occurring near the calcium-rich internal interfaces. Unfortunately, visualizing the crosslinking density in iCLAMs by confocal microscopy, as originally proposed, was unsuccessful. In experiments utilizing calcium responsive dyes, regions of high calcium content (and thus high crosslinking density) could not be differentiated from the background signal. Internal particle microstructure affected volatile losses during microencapsulation. While all formulations were prepared with the same target limonene content (25%), spray dried iCLAMs contained significantly more limonene than Ctrl-CLAMs or T80-CLAMs. When isolating only the losses that occurred during spray drying, iCLAMs retained significantly more limonene than Ctrl-CLAMs, suggesting that the enhanced crosslinking and residual CCNP at the internal interfaces of iCLAMs provided additional resistance to limonene mass transfer during spray drying. Volatile retention during spray-drying of iCLAMs was elevated but not significantly greater than that of T80-CLAMs, although the final limonene content of iCLAMs was significantly greater than that of T80-CLAMs. Limonene losses during homogenization may account for this discrepancy. Objective 2: To investigate how the incorporation of inert nanoparticles into the cargo-polymer interfaces within microcapsules affects volatile retention and cargo release kinetics in aqueous environments simulating food systems and the gastrointestinal tract. Although prepared as a dry powder, CLAMs would be suspended in aqueous environments in many envisioned food applications. Limonene loss from aqueous CLAM suspensions and emulsions was measured over time in open vessels subjected to thermal treatments and gentle agitation. The limonene remaining in the aqueous system was extracted and quantified by gas chromatography. Surprisingly, the kinetics of loss were similar for iCLAMs, Ctrl-CLAMs, and T80-CLAMs, and this held true at 37, 60, and 80°C. However, compared to all three CLAMs, limonene was lost much more rapidly from CCNP Pickering emulsions, and even more rapidly from Tween 80 emulsions; these emulsions systems offered less of a mass transfer barrier to limonene compared to the alginate hydrogel of rehydrated CLAMs. However, the microstructure of the CLAMs did not appear to significantly affect the kinetics of limonene loss in this environment. When suspended in water at room temperature in a closed vessel, iCLAMs and Ctrl-CLAMs released significantly less limonene cargo after 2h (approximately 5%) than T80-CLAMs. The nonionic surfactant likely facilitated the release of emulsified limonene from the T80-CLAMs. The differences in limonene release between the three CLAMs formulations was driven more by the presence or absence of surfactant than by the differences between particle microstructures. CLAMs prepared without surfactant exhibited improved gastrointestinal release properties compared to T80-CLAMs. In simulated gastric fluid for 2h, iCLAMs and Ctrl-CLAMs released significantly less limonene than T80-CLAMs. When subsequently transferred into simulated intestinal fluid for 2h, the alginate matrix disintegrated and completely released the remaining limonene. The similar release properties of iCLAMs and Ctrl-CLAMs suggested that the spatial distribution of CCNP within the CLAMs did not exert a significant effect on gastric release. Overall, the calcium enriched internal interfaces of iCLAMs did not improve cargo release properties. However, the elimination of nonionic surfactant in forming iCLAMs did help mitigate unwanted cargo release in water and gastric fluid. Considering the shift toward simpler ingredient labels in the food industry, a microcapsule formulation comprising fewer components that also exhibits improved functional properties is highly desirable. The work under this objective led to a change in knowledge regarding the release of volatile bioactive lipophilic compounds from CLAMs. By preparing iCLAMs with nanostructured internal interfaces without surfactants, the unwanted release of volatile bioactive cargo may be reduced, both during incorporation into foods and during the gastric phase of digestion, potentially improving bioavailability.
Publications
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Progress 04/15/18 to 04/14/19
Outputs
Target Audience:The academic audience was reached through presentation and participation in two technical conferences: one in the field of food engineering and another in the field of colloid science. This audience consisted of graduate students, postdoctoral scholars, professors, and industry experts. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?PD attended the Conference of Food Engineering in Minneapolis, MN in September 2018 to give an oral presentation. The research talk afforded the PD the opportunity to gain increased academic exposure and strengthen oral presentation skills. The conference provided a chance to network with academic and industry leaders in the food engineering field. PI Strobel attended the 9th International Colloids Conference in Sitges, Spain in June 2019 to present results pertaining to Objective 1. Results were summarized in a poster format, and PD used the opportunity to communicate his scientific research to other conference attendees. How have the results been disseminated to communities of interest?These results have been shared at an international conference (9th International Colloids Conference) and will ultimately be published in an academic journal. What do you plan to do during the next reporting period to accomplish the goals?Upcoming work will address objective (2): "to investigate how the incorporation of inert nanoparticles into the cargo-polymer interfaces affects volatile retention and cargo release kinetics in aqueous environments simulating food systems and the GI tract." Inert nanoparticles will be used in conjunction with calcium carbonate nanoparticles to form iNP-CLAMs, in which the internal interfaces between cargo (limonene) and polymer matrix are reinforced by polymer cross-linking and an inert nanoparticle barrier. A variety of inert nanoparticles (e.g. clay, silica, latex) will be screened for their ability to produce suitable Pickering emulsions at different particle ratios, and subsequently these will be incorporated into iNP-CLAMs via spray-drying. Volatile retention and powder yields will be tracked for all formulations. The release of limonene from microcapsules will be studied in aqueous environments: (1) simulated beverages at pH 3 and 5 and (2) simulated gastrointestinal fluids.
Impacts
What was accomplished under these goals?
This project aims to develop and investigate a new microencapsulation system for incorporating volatile bioactive compounds into functional foods. This system could ultimately (a) improve the economics of microencapsulation for food industry and (b) provide consumers with fortified food products that provide both nutrition and health benefits. The economics of microencapsulation may be improved by developing a process that minimizes the number of manufacturing steps, utilizes highly scalable equipment, and mitigates the unwanted loss of volatile bioactives during processing. Bioavailability of bioactives and nutrients may be improved by developing a microencapsulation system that minimizes undesired cargo release, both in foods and in the stomach, and which targets delivery to the small intestine where these compounds can be absorbed. Work in the period beginning September 2018 through spring 2019 primarily supported Objective 1: To understand how the cross-linking structure within microcapsules influences volatile retention during spray-drying. D-limonene, a model volatile bioactive compound, was microencapsulated in cross-linked alginate microcapsules (CLAMs) prepared with formulation variations designed to exhibit three different intended microstructures. In all formulations, calcium carbonate nanoparticles served as a reservoir of divalent cations for cross-linking the alginate polymer in situ during an industrially-scalable spray-drying process. CLAMs with interface-targeted cross-linking (iCLAMs) were prepared and compared to two control microcapsule formulations, both with unstructured internal interfaces (and hence random cross-linking distribution). iCLAMs were characterized by a layer of calcium carbonate nanoparticles at the internal interface between limonene cargo and cross-linked alginate, and the presence of this interfacial calcium carbonate was confirmed by SEM. In control CLAM formulations (prepared with and without nonionic surfactant), calcium carbonate nanoparticles were distributed throughout the volume of the polymer matrix. In the bulk powders, the extent of alginate cross-linking was unchanged between formulations, as measured by the quantity of solubilizable alginate. However, the cross-linking microstructure in iCLAMs was likely more organized, with the densest cross-linking targeted to the calcium-rich internal interfaces. A portion of volatile D-limonene was lost during the microencapsulation process, which involved steps for homogenization and spray-drying. Retention of volatile cargo during the spray-drying of iCLAMs was significantly greater relative to a control CLAM formulation with identical composition and random calcium distribution. Volatile retention was not significantly greater for iCLAMs relative to control CLAMs prepared with nonionic surfactant. However, the total limonene content of iCLAMs was greater than both controls, with all microcapsules prepared with the same target loading; this implies that the calcium-enriched interfaces improve the retention of volatile cargo during both homogenization and spray-drying. In addition, investigations into the release properties of iCLAMs were initiated to begin advancing Objective 2: To investigate how the incorporation of inert nanoparticles into the cargo-polymer interfaces within microcapsules affects volatile retention and cargo release kinetics in aqueous environments simulating food systems and the gastrointestinal tract. Relative to the control formulation prepared with nonionic surfactant, iCLAMs exhibited improved cargo release properties. When suspended in water, iCLAMs released less limonene after a two hour interval. When subjected to simulated gastric fluid for two hours, iCLAMs retained significantly more limonene compared to the control. Complete matrix dissolution and cargo release was achieved upon transferring microcapsules to simulated intestinal fluid within two hours. Eliminating surfactant from the CLAMs formulation appeared to minimize the unwanted release of cargo during the gastric phase of digestion, which may improve the bioavailability of encapsulated cargo once released in the intestine.
Publications
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