How alginate properties influence in situ internal gelation in crosslinked alginate microcapsules (CLAMs) formed by spray drying.

Alginates gel rapidly under ambient conditions and have widely documented potential to form protective matrices for sensitive bioactive cargo. Most commonly, alginate gelation occurs via calcium mediated electrostatic crosslinks between the linear polyuronic acid polymers. A recent breakthrough to form crosslinked alginate microcapsules (CLAMs) by in situ gelation during spray drying ("CLAMs process") has demonstrated applications in protection and controlled delivery of bioactives in food, cosmetics, and agriculture. The extent of crosslinking of alginates in CLAMs impacts the effectiveness of its barrier properties. For example, higher crosslinking extents can improve oxidative stability and limit diffusion of the encapsulated cargo. Crosslinking in CLAMs can be controlled by varying the calcium to alginate ratio; however, the choice of alginates used in the process also influences the ultimate extent of crosslinking. To understand how to select alginates to target crosslinking in CLAMs, we examined the roles of alginate molecular properties. A surprise finding was the formation of alginic acid gelling in the CLAMs that is a consequence of simultaneous and rapid pH reduction and moisture removal that occurs during spray drying. Thus, spray dried CLAMs gelation is due to calcium crosslinking and alginic acid formation, and unlike external gelation methods, is insensitive to the molecular composition of the alginates. The 'extent of gelation' of spray dried CLAMs is influenced by the molecular weights of the alginates at saturating calcium concentrations. Alginate viscosity correlates with molecular weight; thus, viscosity is a convenient criterion for selecting commercial alginates to target gelation extent in CLAMs.

Chelator Regulation of In Situ Calcium Availability to Enable Spray-Dry Microencapsulation in Cross-Linked Alginates.

A recently patented one-step in situ cross-linked alginate microencapsulation (CLAM) by spray-drying (i.e., the UC Davis CLAMs technology) can overcome the high cost of scale-up that limits commercial applications. While increasing calcium loading in the CLAMs process can increase the extent of cross-linking and improve retention and protection of the encapsulated cargo, the potential for residual undissolved calcium salt crystals in the final product can be a concern for some applications. Here, we demonstrate an alternate one-step spray-dry CLAMs process using pH-responsive chelation of calcium. The "Chelate CLAMs" process is an improvement over the patented process that controls ion availability based on pH-responsive solubility of the calcium salt. Hyaluronic acid was encapsulated in CLAMs to minimize swelling and release in aqueous formulations. CLAMs with 61% (d.b.) hyaluronic acid (HA-CLAMs) demonstrated restricted plumping, limited water absorption capacity, and reduced leaching, retaining up to 49% hyaluronic acid after 2 h in water. Alternatively, "Chelate HA-CLAMs" formed by the improved process exhibited nearly full retention of hyaluronic acid over 2 h in water and remained visibly insoluble after 1 year of storage in water at 4 °C. Successful hyaluronic acid retention in CLAMs is likely due in part to its ability to cross-link with calcium.

Immobilization of chymotrypsin on hierarchical nylon 6,6 nanofiber improves enzyme performance.

Immobilized enzymes enable advances in bioprocessing efficiency and bioactive packaging. Enzyme immobilization onto macroscale solid supports is often limited by low protein loading, inadequate access to substrate, and non-ideal orientation to the solid support; immobilization on nanomaterials has improved activity retention, protein loading, and enabled improved performance in extreme environments, yet has practical limitations including handling, recovery. This work describes the immobilization of chymotrypsin to nylon 6,6 in two formats: electrospun nanofibers and planar films. Protein loading, enzyme activity, and kinetics were compared to that of commercially available systems (free chymotrypsin and chymotrypsin immobilized on agarose beads). Electrospun nylon 6,6 nanofibers had an average fiber diameter of 161±73nm, improving protein loading compared to its planar macroscale counterpart. Chymotrypsin immobilized onto nylon nanofibers exhibited shifts in both working optimum pH and temperature with an increase from pH 7.8 to pH 9, and increased optimum temperature by 10°C compared to free enzyme. The nanofibers also enhanced thermostability compared to native enzyme, enzyme on planar films, and the commercial standard agarose beads with 35% activity retained after 12h at 50°C. This work demonstrates the potential of hierarchical nanomaterials in improving enzyme performance, leveraging benefits of both nano and macroscale supports.

Inactivation of Salmonella on Sprouting Seeds Using a Spontaneous Carvacrol Nanoemulsion Acidified with Organic Acids.

Over the past decade, demand has increased for natural, minimally processed produce, including sprout-based products. Sanitization with 20,000 ppm of calcium hypochlorite is currently recommended for all sprouting seeds before germination to limit sprout-related foodborne outbreaks. A potentially promising disinfectant as an alternative to calcium hypochlorite is acidified spontaneous essential oil nanoemulsions. In this study, the efficacy of an acidified carvacrol nanoemulsion was tested against mung beans and broccoli seeds artificially contaminated with a Salmonella enterica Enteritidis cocktail (ATCC BAA-709, ATCC BAA-711, and ATCC BAA-1045). Treatments were performed by soaking inoculated seeds in acidified (50 mM acetic or levulinic acid) carvacrol nanoemulsions (4,000 or 8,000 ppm) for 30 or 60 min. After treatment, the number of surviving cells was determined via plate counts and/or the most probable number (MPN) approach. Treatment for 30 min successfully reduced Salmonella Enteritidis by 4 log CFU/g on mung beans (from an initial contamination level of 4.2 to 4.6 log CFU/g) and by 2 log CFU/g on broccoli seeds (from an initial contamination level of 2.4 to 2.6 log CFU/g) to below our detection limit (≤3 MPN/g). Treated seeds were sprouted and tested for the presence of pathogens and sprout yield. The final sprout product had no detectable pathogens, and total sprout yield was not influenced by any treatment.

Short communication: Effect of active food packaging materials on fluid milk quality and shelf life.

Active packaging, in which active agents are embedded into or on the surface of food packaging materials, can enhance the nutritive value, economics, and stability of food, as well as enable in-package processing. In one embodiment of active food packaging, lactase was covalently immobilized onto packaging films for in-package lactose hydrolysis. In prior work, lactase was covalently bound to low-density polyethylene using polyethyleneimine and glutaraldehyde cross-linkers to form the packaging film. Because of the potential contaminants of proteases, lipases, and spoilage organisms in typical enzyme preparations, the goal of the current work was to determine the effect of immobilized-lactase active packaging technology on unanticipated side effects, such as shortened shelf-life and reduced product quality. Results suggested no evidence of lipase or protease activity on the active packaging films, indicating that such active packaging films could enable in-package lactose hydrolysis without adversely affecting product quality in terms of dairy protein or lipid stability. Storage stability studies indicated that lactase did not migrate from the film over a 49-d period, and that dry storage resulted in 13.41% retained activity, whereas wet storage conditions enabled retention of 62.52% activity. Results of a standard plate count indicated that the film modification reagents introduced minor microbial contamination; however, the microbial population remained under the 20,000 cfu/mL limit through the manufacturer's suggested 14-d storage period for all film samples. This suggests that commercially produced immobilized lactase active packaging should use purified cross-linkers and enzymes. Characterization of unanticipated effects of active packaging on food quality reported here is important in demonstrating the commercial potential of such technologies.

Layer by layer assembly of a biocatalytic packaging film: lactase covalently bound to low-density polyethylene.

Active packaging is utilized to overcome limitations of traditional processing to enhance the health, safety, economics, and shelf life of foods. Active packaging employs active components to interact with food constituents to give a desired effect. Herein we describe the development of an active package in which lactase is covalently attached to low-density polyethylene (LDPE) for in-package production of lactose-free dairy products. The specific goal of this work is to increase the total protein content loading onto LDPE using layer by layer (LbL) deposition, alternating polyethylenimine, glutaraldehyde (GL), and lactase, to enhance the overall activity of covalently attached lactase. The films were successfully oxidized via ultraviolet light, functionalized with polyethylenimine and glutaraldehyde, and layered with immobilized purified lactase. The total protein content increased with each additional layer of conjugated lactase, the 5-layer sample reaching up to 1.3 µg/cm2 . However, the increase in total protein did not lend to an increase in overall lactase activity. Calculated apparent Km indicated the affinity of immobilized lactase to substrate remains unchanged when compared to free lactase. Calculated apparent turnover numbers (kcat ) showed with each layer of attached lactase, a decrease in substrate turnover was experienced when compared to free lactase; with a decrease from 128.43 to 4.76 s(-1) for a 5-layer conjugation. Our results indicate that while LbL attachment of lactase to LDPE successfully increases total protein mass of the bulk material, the adverse impact in enzyme efficiency may limit the application of LbL immobilization chemistry for bioactive packaging use.