Reactive Extrusion of Nonmigratory Active and Intelligent Packaging.

The environmental and economic burden of food waste demands new preservation technologies to reduce the degradative actions of spoilage such as moisture, oxygen, and microorganisms. Direct food additives can help maintain product quality; however, the limited life span of these additives combined with consumer desire for "clean label" products has motivated research into new food manufacturing technologies like active and intelligent packaging that can prevent and detect food spoilage. In this work, curcumin was grafted to polypropylene (PP-g-Cur) via reactive extrusion to produce nonmigratory active and intelligent packaging through a solvent-free, efficient, and continuous method. Immobilization of curcumin was confirmed by a standard migration assay exhibiting a maximum of 0.011 mg/cm2 migration, significantly below the EU migratory limit for food contact materials (0.1 mg/cm2). Compared to native PP films, PP-g-Cur films blocked 93% of UV light while retaining 64% transparency in the visible region, allowing for desirable product visibility while inhibiting UV degradation of packaged goods. While the ability of PP-g-Cur to inhibit growth of E. coli and L. monocytogenes was insignificant compared to control PP, free curcumin exhibited poor bacterial inhibition as well, suggesting that without hydrophilic modification, native curcumin has limited antimicrobial efficacy. PP-g-Cur films displayed significant radical scavenging in both organic (11.71 ± 3.02 TroloxEq (nmol/cm2)) and aqueous (3.18 ± 1.04 TroloxEq (nmol/cm2)) matrices, exhibiting potential for antioxidant behavior in both lipophilic and hydrophilic applications. Finally, when PP-g-Cur films were exposed to ammonia, an indicator of microbial growth, the color visually and quantitatively changed from yellow to red, demonstrating potential to indicate spoilage. These findings demonstrate the potential of a scalable technology to produce active and intelligent packaging to limit food waste and advance the capabilities of functional materials in a variety of applications.

Synthesis and Characterization of Antimicrobial Hydrophobic Polyurethane.

Food borne illness remains a major threat to public health despite new governmental guidelines and industry standards. Cross-contamination of both pathogenic and spoilage bacteria from the manufacturing environment can promote consumer illness and food spoilage. While there is guidance in cleaning and sanitation procedures, manufacturing facilities can develop bacterial harborage sites in hard-to-reach areas. New technologies to eliminate these harborage sites include chemically modified coatings that can improve surface characteristics or incorporate embedded antibacterial compounds. In this article we synthesize a 16 carbon length quaternary ammonium bromide (C16QAB) modified polyurethane and perfluoropolyether (PFPE) copolymer coating with low surface energy and bactericidal properties. The introduction of PFPE to the polyurethane coatings lowered the critical surface tension from 18.07 mN m-1 in unmodified polyurethane to 13.14 mN m-1 in modified polyurethane. C16QAB + PFPE polyurethane was bactericidal against Listeria monocytogenes (>6 log reduction) and Salmonella enterica (>3 log reduction) after just eight hours of contact. The combination of low surface tension from the perfluoropolyether and antimicrobial from the quaternary ammonium bromide produced a multifunctional polyurethane coating suitable for coating on non-food contact food production surfaces to prevent survival and persistence of pathogenic and spoilage organisms.

A high-throughput expression and screening platform for applications-driven PETase engineering.

The environmental consequences of plastic waste have impacted all kingdoms of life in terrestrial and aquatic ecosystems. However, as the burden of plastic pollution has increased, microbes have evolved to utilize anthropogenic polymers as nutrient sources. Of depolymerase enzymes, the best characterized is PETase, which hydrolyzes aromatic polyesters. PETase engineering has made impressive progress in recent years; however, further optimization of engineered PETase toward industrial application has been limited by lower throughput techniques used in protein purification and activity detection. Here, we address these deficiencies through development of a higher-throughput PETase engineering platform. Secretory expression via YebF tagging eliminates lysis and purification steps, facilitating production of large mutant libraries. Fluorescent detection of degradation products permits rapid screening of depolymerase activity in microplates as opposed to serial chromatographic methods. This approach enabled development of more stable PETase, semi-rational (SR) PETase variant containing previously unpublished mutations. SR-PETase releases 1.9-fold more degradation products and has up to 7.4-fold higher activity than wild-type PETase over 10 days at 40°C. These methods can be adapted to a variety of chemical environments, enabling screening of PETase mutants in applications-relevant conditions. Overall, this work promises to facilitate advancements in PETase engineering toward industrial depolymerization of plastic waste.

Consumers Respond Positively to the Sensory, Health, and Sustainability Benefits of the Rare Sugar Allulose in Yogurt Formulations.

Increased added sugar consumption is associated with type II diabetes, metabolic syndrome, and cardiovascular disease. Low and no-calorie alternative sweeteners have long been used as an aid in the reduction of added sugar. Unfortunately, these alternative sweeteners often have notable sensory deficits when compared to sucrose. Furthermore, many alternative sweeteners have synthetic origins, while consumers are increasingly turning to foods from natural origins, and from more sustainable sources. Such sweeteners include the rare sugar allulose, which can be manufactured from common agricultural waste and dairy co-product streams, and is reported to have a sensory profile similar to sucrose. This study aimed to determine the influence of the rare sugar allulose on consumer perception of sweetened vanilla yogurt. Participants were recruited to evaluate 4 vanilla yogurts sweetened with either sucrose, allulose, stevia or sucralose, and to rate their liking of the samples overall, and for flavor, texture, and their purchase intent. Statistical analysis of hedonic data from 100 consumers suggested that allulose performed similarly to sucrose in liking and purchase intent, and superior to other sweeteners tested in this study, with fewer off-flavors. Moreover, when consumers were queried on their purchase intent after learning details on the sweetener for each formulation, allulose scored significantly higher than all other formulations in purchase intent. This study highlights the potential of the rare sugar allulose as a low calorie, zero glycemic index, natural and better tasting sugar replacement in sweetened yogurt.

Directed Immobilization of PETase on Mesoporous Silica Enables Sustained Depolymerase Activity in Synthetic Wastewater Conditions.

Microplastic accumulation in terrestrial and aquatic environments is a growing environmental challenge. Biodegradation has shown promise as an intervention strategy for reducing the spread of microplastics. The wastewater treatment system is a key intervention point in microplastic biodegradation due to its pivotal role in the water cycle at the interface between human activity and the environmental. However, the best characterized microplastic degradation enzyme, PETase, lacks the stability to perform at scale in wastewater treatment. In this work, we show that genetic fusion of PETase to a silica binding peptide enables directed immobilization of the enzyme onto silica nanoparticles. PETase activity in simulated wastewater conditions is quantified by linear regression from time zero to the time of maximum fluorescence of a fluorescent oxidized product of PETase degradation of PET microfibers. Mesoporous silica is shown to be a superior support material to nonporous silica. The resulting biocatalytic nanomaterial has up to 2.5-fold enhanced stability and 6.2-fold increased activity compared to free enzyme in unbuffered, 40 °C simulated influent (ionic strength ∼15 mM). In unbuffered, 40 °C simulated effluent (ionic strength ∼700 µM), reaction velocity and overall catalytic activity were increased by the biocatalytic material 2.1-fold relative to free PETase. All reactions were performed in 0.2 mL volumes, and enzyme concentrations were normalized across both free and immobilized samples to 9 µg/mL. Site-directed mutagenesis is shown to be a complementary technique to directed immobilization, which may aid in optimization of the biomaterial for wastewater applications. PETase stabilization in application-relevant environments as shown here enables progress toward application of PETase for microplastic biodegradation in wastewater treatment.

Trehalose and its applications in the food industry.

Trehalose is a nonreducing disaccharide composed of two glucose molecules linked by α, α-1,1-glycosidic bond. It is present in a wide variety of organisms, including bacteria, fungi, insects, plants, and invertebrate animals. Trehalose has distinct physical and chemical properties that have been investigated for their biological importance in a range of prokaryotic and eukaryotic species. Emerging research on trehalose has identified untapped opportunities for its application in the food, medical, pharmaceutical, and cosmetics industries. This review summarizes the chemical and biological properties of trehalose, its occurrence and metabolism in living organisms, its protective role in molecule stabilization, and natural and commercial production methods. Utilization of trehalose in the food industry, in particular how it stabilizes protein, fat, carbohydrate, and volatile compounds, is also discussed in depth. Challenges and opportunities of its application in specific applications (e.g., diagnostics, bioprocessing, ingredient technology) are described. We conclude with a discussion on the potential of leveraging the unique molecular properties of trehalose in molecular stabilization for improving the safety, quality, and sustainability of our food systems.

In Vivo Capsid Engineering of Bacteriophages for Oriented Surface Conjugation.

The current state-of-the-art in bacteriophage (phage) immobilization onto magnetic particles is limited to techniques that are less expensive and/or facile but nonspecific or those that are more expensive and/or complicated but ensure capsid-down orientation of the phages, as necessary to preserve infectivity and performance in subsequent applications (e.g., therapeutics, detection). These cost, complexity, and effectiveness limitations constitute the major hurdles that limit the scale-up of phage-based strategies and thus their accessibility in low-resource settings. Here, we report a plasmid-based technique that incorporates a silica-binding protein, L2, into the T7 phage capsid, during viral assembly, with and without inclusion of a flexible linker peptide, allowing for targeted binding of the phage capsid to silica without requiring the direct modification of the phage genome. L2-tagged phages were then immobilized onto silica-coated magnetic nanoparticles. Inclusion of the flexible linker between the phage capsid protein and the L2 protein improved immobilization density compared to both wild type T7 phages and L2-tagged phages without the flexible linker. Taken together, this work demonstrates phage capsid modification without engineering the phage genome, which provides an important step toward reducing the cost and increasing the specificity/directionality of phage immobilization methods and could be more broadly applied in the future for other phages for a range of other capsid tags and nanomaterials.

Lactulose production from lactose isomerization by chemo-catalysts and enzymes: Current status and future perspectives.

Lactulose, a semisynthetic nondigestive disaccharide with versatile applications in the food and pharmaceutical industries, has received increasing interest due to its significant health-promoting effects. Currently, industrial lactulose production is exclusively carried out by chemical isomerization of lactose via the Lobry de Bruyn-Alberda van Ekenstein (LA) rearrangement, and much work has been directed toward improving the conversion efficiency in terms of lactulose yield and purity by using new chemo-catalysts and integrated catalytic-purification systems. Lactulose can also be produced by an enzymatic route offering a potentially greener alternative to chemo-catalysis with fewer side products. Compared to the controlled trans-galactosylation by ß-galactosidase, directed isomerization of lactose with high isomerization efficiency catalyzed by the most efficient lactulose-producing enzyme, cellobiose 2-epimerase (CE), has gained much attention in recent decades. To further facilitate the industrial translation of CE-based lactulose biotransformation, numerous studies have been reported on improving biocatalytic performance through enzyme mediated molecular modification. This review summarizes recent developments in the chemical and enzymatic production of lactulose. Related catalytic mechanisms are also highlighted and described in detail. Emerging techniques that aimed at advancing lactulose production, such as the boronate affinity-based technique and molecular biological techniques, are reviewed. Finally, perspectives on challenges and opportunities in lactulose production and purification are also discussed.

Bacteriophage Capsid Modification by Genetic and Chemical Methods.

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g., fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation, but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

General method for emulsion polymerization to yield functional terpolymers.

Copolymerization methods are used to impart specific, desired functional properties (e.g. mechanical or bioactive) to a material for targeted applications in biomedicine, food and agriculture, consumer products, advanced manufacturing, and more. Many polymerization methods exist to achieve tailored copolymer architectures. Of them, emulsion polymerization offers unique and industrially convenient features that make for easily scalable processes because the synthesis occurs in water and the latexes usually do not need further purification. Because of the breadth of copolymer architectures and thus wide range of potential applications for latexes produced by emulsion polymerization, there is great value in defining general methods for emulsion polymerizations to yield copolymers, including routes for synthesis of functional monomer building blocks, to permit consistency and optimization of these processes. Herein we present a general emulsion polymerization method for synthesis of a copolymer consisting of three functional monomers, suitable for adaptation to alternate base chemistries, curing chemistries, and functional ligands. This protocol includes the synthesis of the functional monomers glycidyl methacrylate-iminodiacetic acid (GMA-IDA) and 4-benzolylphneyl methacrylate (BPM).•Our synthesized copolymer includes a glycidyl methacrylate (GMA) monomer functionalized with a metal-chelating iminodiacetic acid (IDA) ligand, a UV-curable monomer, 4-benzoylphenyl methacrylate (BPM), and an inert hydrophobic monomer, n­butyl acrylate (BA).•The presented synthesis route demonstrates a general polymerization method that can be modified to copolymerize alternative functional monomers to create multi-functional polymers.

Reactive Extrusion of Nonmigratory Antioxidant Poly(lactic acid) Packaging.

Reactive extrusion of bio-derived active packaging offers a new approach to address converging concerns over environmental contamination and food waste. Herein, metal-chelating nitrilotriacetic acid (NTA) ligands were grafted onto poly(lactic acid) (PLA) by reactive extrusion to produce metal-chelating PLA (PLA-g-NTA). Radical grafting was confirmed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy with the introduction of secondary alkyl stretches (2919 and 2860 cm-1) and by X-ray photoelectron spectroscopy (XPS) with an increase in the atomic percentage of nitrogen. Compared to films prepared from native, granular PLA (gPLA), PLA-g-NTA films had lower contact angles and hysteresis values (86.35° ± 2.49 and 31.89° ± 2.27 to 79.91° ± 1.58 and 21.79° ± 1.72, respectively), supporting the surface orientation of the NTA ligands. The PLA-g-NTA films exhibited a significant antioxidant character with a radical scavenging capacity of 0.675 ± 0.026 nmol Trolox(eq)/cm2 and an iron chelation capacity of 54.09 ± 9.36 nmol/cm2. PLA-g-NTA films delayed ascorbic acid degradation, retaining ∼45% ascorbic acid over the 9-day study compared to <20% for control PLA. This research makes significant advances in translating active packaging technologies to bio-derived materials using scalable, commercially translatable synthesis methods.

Engineering Biorthogonal Phage-Based Nanobots for Ultrasensitive, In Situ Bacteria Detection.

Advances in synthetic biology, nanotechnology, and genetic engineering are allowing parallel advances in areas such as drug delivery and rapid diagnostics. Although our current visions of nanobots may be far off, a generation of nanobots synthesized by engineering viruses is approaching. Such tools can be used to solve complex problems where current methods do not meet current demands. Assuring safe drinking water is crucial for minimizing the spread of waterborne illnesses. Although extremely low levels of fecal contamination in drinking water are sufficient to cause a public health risk, it remains challenging to rapidly detect Escherichia coli, the standard fecal indicator organism. Current methods sensitive enough to meet regulatory standards suffer from either prohibitively long incubation times or requirement of expensive, impractical equipment. Bacteriophages, tuned by billions of years of evolution to bind viable bacteria and readily engineered to produce custom proteins, are uniquely suited to bacterial detection. We have developed a biosensor platform based on magnetized phages encoding luminescent reporter enzymes. This system utilizes bio-orthogonally functionalized phages to enable site-specific conjugation to magnetic nanoparticles. The resulting phage-based nanobots, when combined with standard, portable field equipment, allow for detection of <10 cfu/100 mL of viable E. coli within 7 h, faster than any methods published to date.

Antimicrobial and antifouling polymeric coating mitigates persistence of Pseudomonas aeruginosa biofilm.

Food wasted due to food spoilage remains a global challenge to the environmental sustainability and security of food supply. In food manufacturing, post-processing contamination of food can occur due to persistent bacterial biofilms, which can be resistant to conventional cleaning and sanitization. The objective was to characterize the efficacy of a polymeric coating in reducing Pseudomonas aeruginosa biofilm establishment and facilitating its removal. Viable cell density of a 48 h biofilm was reduced by 2.10 log cfu cm-2 on the coated surface, compared to native polypropylene. Confocal laser scanning and electron microscopy indicated reductions in mature biofilm viability and thickness on the coated material. The antifouling coating improved cleanability, with ∼2.5 log cfu cm-2 of viable cells remaining after 105 min cleaning by water at 65 °C, compared to 4.5 log cfu cm-2 remaining on native polypropylene. Such coatings may reduce the persistence of biofilms in food processing environments, in support of reducing food spoilage and waste.

Performance of photo-curable metal-chelating active packaging coating in complex food matrices.

Many packaged goods undergo transition metal-catalyzed oxidative spoilage. Recently, a nonmigratory photocurable metal-chelating coating was developed as an innovative active packaging approach to control oxidation of foods. In the present study, we investigate the influence of competing ions and increasing viscosity on the iron-chelating capacity and antioxidant efficacy of this coating in a model complex food system. The addition of calcium and magnesium causes a decrease in iron chelating capacity; however, 61% chelating capacity of materials was retained when 0.8 M sodium was present. Materials retained iron-chelating capacity even in solutions of 2700 cP, similar to the viscosity of salad dressing. Additionally, metal-chelating films significantly delayed transition metal-catalyzed ascorbic acid degradation, even in the presence of competing ions and at increased viscosity. These results suggest that metal-chelating active packaging coatings may present a new technological approach to addressing consumer demands for reduced additive use while controlling food spoilage and waste.

The Role of Solid Support Bound Metal Chelators on System-Dependent Synergy and Antagonism with Nisin.

Active packaging can enhance the performance of natural antimicrobials in controlling food spoilage and waste, while addressing consumer demands for cleaner labels. Yet, synergies are system dependent, with some conditions counterintuitively promoting antagonistic effects. In particular, metal chelators can improve performance of certain natural antimicrobials and have been incorporated in nonmigratory metal chelating active packaging technologies. However, the influence of chelating ligand chemistry on antimicrobial efficacy has not been investigated in microbial spoilage models. The effect of three commercial chelating resins on the growth of Alicyclobacillus acidoterrestris ATCC 49025, a thermoduric acidophilic spore-former, in growth media and apple juice was investigated. Dowex MAC-3, Chelex 100, and Lewatit TP260 were used as models for metal chelating active packaging containing carboxylic acid (CA), iminodiacetic acid (IDA), and aminomethylphosphonic acid (AMPA) ligands. Diameters (CA = 472.4 ± 117.2 µm, IDA = 132.93 ± 26.71 µm, and AMPA = 498.3 ± 29.24 µm), dissociation kinetics (CA = 6.44 ± 0.109, IDA = -0.977 ± 9.94, AMPA = 7.43 ± 0.193), and metal chelating capacities (CA = 1.16 × 10-4 mol/g, IDA = 1.52 × 10-3  mol/g, and AMPA = 4.67 × 10-4 mol/g) were used to distinguish differences in antimicrobial efficacies. Growth of A. acidoterrestris in acidified Potato Dextrose Broth over 24 hr with chelating resins indicated early death phase for CA and IDA resins and bactericidal for AMPA resin. However, viability in commercial apple juice with the inclusion of nisin and chelating resins was variable, with IDA resin significantly (P < 0.05) increasing viability while the effect of CA and AMPA resins remained elusive. This work emphasizes the importance of biological repeatability and correct statistical modeling in identifying conditions under which the antimicrobial intervention of nisin in real food systems, such as acidic beverages and juices, are synergistic or antagonistic. PRACTICAL APPLICATION: New technologies to control microbial food spoilage and waste need to be explored to address consumers on-going demands for reducing additive use.  Solid support bound metal chelators can both promote and control microbial growth when used in conjunction with nisin, a natural antimicrobial.  This work explores how system conditions can render a given technology either synergistic or antagonistic, and highlights the importance of sufficient biological replicates in experimental design.

Synthesis and characterization of lactose fatty acid ester biosurfactants using free and immobilized lipases in organic solvents.

In this work, lactose fatty acid esters were enzymatically synthesized from fatty acids and lactose using Candida antarctica B lipase (CALB) in organic solvents. Products were purified using a solvent extraction method and analyzed using ATR-FTIR and surface-active properties measurements. Results showed that hexanes and acetonitrile provide the highest conversions for both free and immobilized lipases, up to 77% and 93% respectively. The conversion rate of esterification is solvent-dependent for free lipase; the conversion rate of immobilized lipase still shows solvent dependency, but to a lesser degree. Surface tension, interfacial tension, critical micelle concentration (CMC), and contact angles were also measured for all of the samples, showing the potentials of these sugar esters as naturally derived surfactants for the food industry.

Facile Preparation of Epoxide-Functionalized Surfaces via Photocurable Copolymer Coatings and Subsequent Immobilization of Iminodiacetic Acids.

Herein, we report a simple coat/cure preparation of epoxide-functionalized surfaces using a photocurable copolymer technology. The photocurable copolymer, poly(glycidyl methacrylate- co-butyl acrylate- co-4-benzoylphenyl methacrylate) (GBB), was synthesized by single electron transfer-living radical polymerization (SET-LRP). The epoxide content in the copolymer was tuned by controlling the content of glycidyl methacrylate. Three copolymers, GBB(1), GBB(2), and GBB(3), with epoxide contents of 22, 63, and 91 mol %, respectively, were cast onto polypropylene films and photocured by UV-light exposure. Subsequently, iminodiacetic acids (IDA) were immobilized onto the GBB-coated materials via a ring-opening reaction. The IDA-functionalized coatings GBB(1)-IDA, GBB(2)-IDA, and GBB(3)-IDA presented IDA contents of 1.47 ± 0.08, 18.67 ± 1.46, and 49.05 ± 2.88 nmol/cm2, respectively, which increased as the epoxide content increased. The IDA-functionalized GBB coatings exhibited metal chelating capability toward transition metal ions (e.g., iron and copper). The reported photocurable copolymer technology offers a facile and tunable preparation of epoxide-functionalized surfaces, with potential extended applications in biopatterning, active packaging, and nanotechnology.

Photocurable coatings prepared by emulsion polymerization present chelating properties.

Herein, we present a method to synthesize a photocurable metal chelating copolymer coating via emulsion polymerization to enable a facile coat/cure preparation of metal chelating materials. The copolymer coating was a poly(n-butyl acrylate) based polymer (79 mol %) synthesized by emulsion polymerization, with iminodiacetic acid (2 mol %) and benzophenone moieties (19 mol %) to impart metal chelating and photocrosslinking properties, respectively. The copolymer was applied onto polypropylene films and was photocured (365 nm, 225 mW/cm2, 180 s) to produce metal chelating film. The resulting metal chelating film had activity towards Fe3+ by chelating 10.9 ± 1.9 nmol/cm2, 47.9 ± 5.3 nmol/cm2, and 156.0 ± 13.8 nmol/cm2 of Fe3+ at pH 3.0, pH 4.0, and pH 5.0, respectively. The metal chelating film controlled transition metal induced ascorbic acid degradation by extending half-life of ascorbic acid degradation from 6 days to 20 days at pH 3.0, and from 3 days to 6 days at pH 5.0, demonstrating its potential as an antioxidant active packaging material. Despite the introduction of polar iminodiacetic acid chelating moieties, the poly(n-butyl acrylate) based coatings retained low surface energies (24.0 mN/m) necessary to mitigate fouling and enable product release in packaging applications. This work overcomes a major knowledge gap in the area of functional coatings, by demonstrating a method by which critical properties such as control of surface energy, retention of mechanical properties, and scalability are integrated into the structure of a functional coating. The photocurable polymer coatings as reported here enable scalable production of active materials with metal chelating functionality, with applications in water treatment, trace metal detection, protein purification, and active packaging.

Stabilization of Lipase in Polymerized High Internal Phase Emulsions.

Candida antarctica lipase B is stabilized in a porous, high internal phase emulsion (HIPE) of polydicyclopentadiene to enable biocatalytic waste stream upcycling. The immobilized lipase is subjected to thorough washing conditions and tested for stability in extreme environments and reusability. A porous internal microstructure is revealed through scanning electron microscopy. After preparation, lipase activity increased to 139 ± 9.7% of its original activity. After 10 cycles of reuse, immobilized lipase retains over 50% activity. Immobilized lipase retains activity after 24 h of exposure to temperatures ranging from 20 to 60 °C and pH values of 3, 7, and 10. In the most extreme environments tested, lipase retained 42.8 ± 21% relative activity after exposure to 60 °C and 49.4 ± 16% relative activity after exposure to pH 3. Polymerized HIPEs stabilize lipase and, thus, extend its working range. Further synthesis optimization has the potential to increase enzyme stability, immobilization efficiency, and uniformity. The reported hierarchical stabilization technique shows promise for use of immobilized lipase in non-ideal, industrially relevant conditions.