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This study investigated the ability of lactic acid bacteria (LAB) isolated from oranges to use fish by-products (FB) and chicken by-products (CB) as nitrogen sources alternative to yeast extract for lactic acid (LA) production in a papaya by-product medium as a carbon source. Once the fermentation agents had been isolated, they were subjected to biochemical and molecular characterization. Inexpensive nitrogen sources, precisely CB and FB, were prepared, freeze-dried, and yield evaluated. Also, before to the fermentation experiments, the Total Kjehdahl Nitrogen (TKN) of these by-products and that of the yeast extract were determined. Then, three production media differing in terms of nitrogen source were formulated from these nitrogen sources. From the 22 LAB isolated from orange, two isolates of interest (NGO25 and NGO23) were obtained; all belonging to the Lactiplantibacillus plantarum species based on 16S rRNA gene sequencing. Furthermore, the production yield powder obtained after lyophilization of 1 L of CB and FB surpernatant were, respectively, 16.6 g and 12.933 g. The TKN of different nitrogen sources powder were 71.4 ± 0.000% DM (FB), 86.145 ± 0.001% DM (CB), and 87.5 ± 0.99% DM (yeast extract). The best kinetic parameters of LA production (LA (g/L): 31.945 ± 0.078; volumetric productivity (g/L.h): 1.331 ± 0.003; LA yield (mg/g) 63.89 ± 0.156; biomass (g/L) 7.925 ± 0.035; cell growth rate (g/L.h): 0.330 ± 0.001) were recorded by Lactiplantibacillus plantarum NGO25 after 24 h of fermentation. The latter data were obtained in the production medium containing CB as nitrogen sources. In addition, this production medium cost only $0.152 to formulate, compared to yeast extract which required $1.692 to formulate. Thus, freeze-dried CB can be used as an alternative to yeast extract in large-scale production of LA.

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Topology and bioactive molecules are crucial for stimulating cellular and tissue functions. To regulate the chronic wound microenvironment, mono-assembly technology is employed to fabricate a radial egg white hydrogel loaded with lyophilized adipose tissue-extracellular vesicles (radial EWH@L-EVs). The radial architecture not only significantly modified the gene expression of functional cells, but also achieved directional and controlled release kinetics of L-EVs. Through the synergy of topographical and inherent bioactive cues, radial EWH@L-EVs effectively reduced intracellular oxidative stress and promoted the polarization of macrophages toward an anti-inflammatory phenotype during the inflammatory phase. Afterward, radial EWH@L-EVs facilitated the centripetal migration and proliferation of fibroblasts and endothelial cells as the wound transitioned to the proliferative phase. During the latter remodeling phase, radial EWH@L-EVs accelerated the regeneration of granulation tissue, angiogenesis, and collagen deposition, thereby promoting the reorganization chronic wound. Compared with the gold standard collagen scaffold, radial EWH@L-EVs actively accommodated the microenvironment via various functions throughout all stages of diabetic wound healing. This can be attributed to the orientation of topological structures and bioactive molecules, which should be considered of utmost importance in tissue engineering.

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Lyophilization (aka freeze drying) has been shown to provide long-term stability for many crucial biotherapeutics, e.g., mRNA vaccines for COVID-19, allowing for higher storage temperature. The final stage of lyophilization, namely secondary drying, entails bound water removal via desorption, in which accurate prediction of bound water concentration is vital to ensuring the quality of the lyophilized product. This article proposes a novel technique for real-time estimation of the residual moisture during secondary drying in lyophilization. A state observer is employed, which combines temperature measurement and mechanistic understanding of heat transfer and desorption kinetics, without requiring any online concentration measurement. Results from both simulations and experimental data show that the observer can accurately estimate the concentration of bound water in real time for all possible concentration levels, operating conditions, and measurement noise. This framework can also be applied for monitoring and control of the residual moisture in other desorption-related processes.

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Combined drying, an energy-efficient method that includes osmotic pretreatment in molasses and shortened successive lyophilization, was used to obtain celery root powder and incorporate it in the formulation of cookies, with the aim of obtaining a new product. Wheat flour was substituted with combinedly dehydrated celery root powder at levels from 0 to 30%, and optimization of the amount of wheat flour substitution regarding technological, sensory and nutritive characteristics was performed. The optimal level of 20% substitution was determined using Z-score analysis, from the aspect of the best nutritive improvement and the mildest adverse impact on the technological and sensory quality. In the second research phase, comparison of the cookies with the 20% celery root powder substitution, dehydrated by different methods, indicated that combined dehydration showed upgraded results in terms of the overall quality of the final product, for 28.85 percentile points higher than cookies with lyophilized and for 65.24 percentile points higher than cookies with the addition of convectively dried celery root powder. The cookie containing celery powder previously osmodehydrated in molasses had higher contents of analyzed minerals (1.2-3.3 times), total phenols (10.8%) and antioxidant activities (14% for DPPH and 4% for ABTS) compared to the cookie with lyophilized powder.

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BACKGROUND: Facilitating the healing process of injured anterior cruciate ligament (ACL) tissue is crucial for patients to safely return to sports. Stem cell derived exosomes have shown positive effects on enhancing the regeneration of injured tendons/ligaments. However, clinical application of exosomes in terms of storage and pre-assembly is challenging. We hypothesized that lyophilized exosomes derived from human umbilical cord stem cells (hUSC-EX) could enhance the cell activity of chronically injured ACL cells. MATERIALS AND METHODS: We harvested the 8 weeks injured ACL cells from rabbit under IACUC (No. 110232) approval. The studied exosomes were purified from the culture medium of human umbilical cord stem cells (IRB approval No. A202205014), lyophilized to store, and hydrated for use. We compared exosome treated cells with non-exosome treated cells (control group) from the same rabbits. We examined the cell viability, proliferation, migration capability and gene expression of type I and III collagen, TGFß, VEGF, and tenogenesis in the 8 weeks injured ACL cells after hUSC-EX treatment. RESULTS: After hydration, the average size of hUSC-EX was 84.5 ± 70.6 nm, and the cells tested positive for the Alix, TSG101, CD9, CD63, and CD81 proteins but negative for the α-Tubulin protein. After 24 h of treatment, hUSC-EX significantly improved the cell viability, proliferation and migration capability of 8 weeks injured ACL cells compared to that of no exosome treatment group. In addition, the expression of collagen synthesis, TGFß, VEGF, and tenogenesis gene were all significantly increased in the 8 weeks injured ACL cells after 24 h hUSC-EX delivery. DISCUSSION: Lyophilized exosomes are easily stored and readily usable after hydration, thereby preserving their characteristic properties. Treatment with lyophilized hUSC-EX improved the activity and gene expression of 8 weeks injured ACL cells. CONCLUSION: Lyophilized hUSC-EX preserve the characteristics of exosomes and can improve chronically injured (8 weeks) ACL cells. Lyophilized hUSC-EX could serve as effective and safe biomaterials that are ready to use at room temperature to enhance cell activity in patients with partial ACL tears and after remnant preservation ACL reconstruction.

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In the majority of pharmaceutical applications, polymers are employed extensively in a diverse range of pharmaceutical products, serving as indispensable components of contemporary solid oral dosage forms. A comprehensive understanding of the properties of polymers and selection the appropriate methods of characterization is essential for the design and development of novel drug delivery systems and manufacturing processes. Orally disintegrating film (ODF) formulations are considered to be a potential substitute to traditional oral dosage forms and an alternative method of drug administration for children and uncooperative adult patients, including those with swallowing difficulties. A multitude of pharmaceutical formulations with varying mechanical and biopharmaceutical properties have emerged from the modification of the original polymeric bulk. Here we propose different formulation approaches, i.e. solvent casting (SC), 3D printing (3DP), electrospinning (ES), and lyophilization (LP) that enabled us to adjust the disintegration time and the release profile of poorly water soluble haloperidol (HAL, BCS class II) from PVA (polyvinyl alcohol) based polymer films while maintaining similar hydrogel composition. In this study, the solubility of haloperidol in aqueous solution was improved by the addition of lactic acid. The prepared films were evaluated for their morphology (SEM, micro-CT), physicochemical and biopharmaceutical properties. TMDSC, TGA and PXRD were employed for extensive thermal and structural analysis of fabricated materials and their stability. These results allowed us to establish correlations between preparation technology, structural characteristics and properties of PVA films and to adapt the suitable manufacturing technique of the ODFs to achieve appropriate HAL dissolution behaviour.

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The in vitro permeation testing (IVPT) of topical products is performed across the human cadaver skin, which is stored frozen for a prolonged duration. The cryo-preservation technique is not economical and is a cumbersome process. Moreover, prolonged skin preservation in a frozen state and frequent freeze-thawing are known to affect the integrity of the skin barrier. Therefore, lyophilization was explored as an alternative to protect the skin tissue from microbial contamination and degeneration. Notably, the project's objective was to investigate the impact of the freeze-drying process on the skin's barrier properties. The morphometrics of the lyophilized skin were measured. Histological studies did not reveal any notable changes in the organization and intactness of the layers due to the freeze-drying process. The biophysical attributes of the skin, such as transepidermal water evaporation rate and transepidermal electrical resistivity (TEER), were not significantly different between the control skin (not subjected to the freeze-drying process) and the freeze-dried skin (FDS). The permeability of caffeine, a hydrophilic model permeant, and nicotine, a lipophilic model permeant, were consistent across the control and the FDS. It is evident from the studies that the lyophilization process did not significantly impact the barrier properties and permeability of the skin.

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Current lead coronavirus vaccines require continuous cold or ultra-cold storage from the manufacturing site to the field to maintain protective efficacy. Since cold chain capacity is limited and complex, logistics planning is crucial to limit vaccine wastage.[1] The restrictive storage concerns also make it difficult to share vaccines between public health departments and neighboring states, leading to increased vaccine wastage.[2] A Newcastle Disease Virus (NDV) vector-based severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) vaccine candidate, NDV-HXP-S, offers a cost-effective alternative which aims to improve global access to SARS CoV-2 vaccines.[3] The NDV-HXP-S vaccine candidate can be mass-produced in chicken eggs and has demonstrated efficacy in preclinical studies, as well as acceptable safety and potent immunogenicity in clinical studies.[3,4-10] To further advance the NDV-HXP-S vaccine candidate, this manuscript describes work focused on the development of multidose thermotolerant vaccine formulations (i.e., those which would not require continuous extended refrigeration), making it convenient to use and store, and simplifying transport and distribution logistics, especially in outbreak settings. Liquid and lyophilized formulations for parenteral administration were rigorously screened for the vaccine formulation's ability to maintain S-antigen stability after exposure to temperature stress at 40 °C, 25 °C, and 2 °C to 8 °C storage for six months. Preservative efficacy was evaluated to enable a multidose liquid vaccine format as well as endotoxin testing in lyophilized formulations. Lead liquid vaccine formations were identified that were able to maintain S-antigen content at 2 °C to 8 °C and 25 °C storage for the entire six-month study. Lead lyophilized vaccine formulations were identified which were able to maintain S-antigen content for six months at 2 °C to 8 °C, 25 °C, and 40 °C. Both the liquid and lyophilized formulations identified are improved thermotolerant SARS-CoV-2 vaccine formulations.

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Near UV and visible light photodegradation can target therapeutic proteins during manufacturing and storage. While the underlying photodegradation pathways are frequently not well-understood, one important aspect of consideration is the formulation, specifically the formulation buffer. Citrate is a common buffer for biopharmaceutical formulations, which can complex with transition metals, such as Fe(III). In an aqueous solution, the exposure of such complexes to light leads to the formation of the carbon dioxide radical anion (•CO2-), a powerful reductant. However, few studies have characterized such processes in solid formulations. Here, we show that solid citrate formulations containing Fe(III) lead to the photochemical formation of •CO2-, identified through DMPO spin trapping and HPLC-MS/MS analysis. Factors such as buffers, the availability of oxygen, excipients, and manufacturing processes of solid formulations were evaluated for their effect on the formation of •CO2- and other radicals such as •OH.

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Over the past decade, continuous spin freeze-drying technology has emerged as a promising alternative to conventional batch freeze-drying, effectively addressing many of the latter's inherent disadvantages. Much of the focus during this period has been on controlling and optimizing the primary drying phase of this process. However, optimizing the secondary drying step is equally critical for the overall efficiency of the process. The primary aim of this study was to develop a comprehensive semi-mechanistic model for the secondary drying phase in continuous spin freeze-drying, accounting for the effects of process settings such as freezing rate and product temperature on desorption kinetics. Additionally, the study aimed to address discrepancies between conventional desorption models, typically applied in batch freeze-drying, and the observed data in this research. To achieve this, a residual moisture-dependent activation energy was introduced to improve the accuracy of the desorption model. Using NIR spectroscopy and IR-thermography, unknown model parameters could reliably be estimated using a simple and fast procedure. The calibrated model successfully predicted the final moisture content with an accuracy within 0.11% of the measured value under previously untested process conditions. Ultimately, the proposed semi-mechanistic model demonstrated its reliability in predicting the impact of new process conditions on both product temperature and residual moisture over time, enabling the development of a practical design space.

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The research context involves analyzing the potential benefits derived from integrating insect protein into everyday food items. Utilizing methods consistent with established food science protocols, wheat bread was prepared with variations of 0%, 5%, 10%, and 15% Tenebrio molitor larvae powder, derived from larvae cultivated on brewery spent grain. A substrate selected for its superior nutritional content and a substrate with agar-agar gels were used. The tests included basic bread tests; sugar, acrylamide, amino, and fatty acid (FA) tests; and sensory acceptability. The results have shown that the acrylamide levels in bread with larvae remained below harmful thresholds, suggesting that using T. molitor can be a safe alternative protein source. The incorporation of powdered T. molitor larvae (p-TMLs) into bread was observed to increase certain sugar levels, such as glucose, particularly at higher larval concentrations. The addition of T. molitor significantly raised the protein and fat levels in bread. The inclusion of larvae enriched the bread with essential amino acids, enhancing the nutritional value of the bread significantly. The FA profile of the bread was altered by the inclusion of p-TMLs, increasing the levels of monounsaturated FAs. Despite the nutritional benefits, higher concentrations of larvae decreased the sensory acceptability of the bread. This suggests that there is a balance to be found between enhancing the nutritional content and maintaining consumer appeal. These findings highlight the potential for using p-TMLs as a sustainable, nutritious ingredient in bread making, although the sensory qualities at higher concentrations might limit consumer acceptance.

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To ensure product stability, it is critical to maintain the monohydrate state of cyclophosphamide following lyophilization, as this is the most stable solid form of the Cyclophosphamide. On the other hand, because of their limited aqueous solubility and stability, non-aqueous solvents are preferred for determining the composition and stability of bulk solutions. Hence, the purpose of this study was to use non-aqueous solvents for determining the composition and stability of bulk solutions, and to shorten the lyophilization process by retaining the cyclophosphamide monohydrate. Furthermore, prior to selecting the solvent for the bulk solution consisting of 90:10 tertiary butyl alcohol (TBA) and acetonitrile (ACN), various factors were taken into account, including the freezing point, vapor pressure of solvents, solubility, and stability of cyclophosphamide monohydrate. The concentration of the bulk solution was adjusted to 200 mg/mL in order to optimize the fill volume, enhance sublimation rates at lower temperatures during primary drying, and eliminate the need for secondary drying. The differential scanning calorimetry (DSC) measurements of bulk solution were used to improve the lyophilization cycle. The lyophilization cycle opted was freezing at a temperature of -55 °C with annealing step at -22 °C by which the reconstitution time was significantly reduced. The drying was performed at below - 25 °C while maintaining a chamber pressure of 300 mTorr. The complete removal of non-aqueous solvents was achieved by retaining water within the system. The presence of cyclophosphamide monohydrate was confirmed using X-ray diffraction (XRD). The reduction of lyophilization process time was established by conducting mass transfer tests and evaluating the physicochemical properties of the pharmaceutical product. Using non-aqueous solvents for freeze-drying cyclophosphamide is a viable option, and this study provides significant knowledge for the advancement of future generic pharmaceuticals.

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The surge in RNA therapeutics has revolutionized treatments for infectious diseases like COVID-19 and shows the potential to expand into other therapeutic areas. However, the typical requirement for ultra-cold storage of mRNA-LNP formulations poses significant logistical challenges for global distribution. Lyophilization serves as a potential strategy to extend mRNA-LNP stability while eliminating the need for ultra-cold supply chain logistics. Although recent advancements have demonstrated the promise of lyophilization, the choice of lyoprotectant is predominately focused on sucrose, and there remains a gap in comprehensive evaluation and comparison of lyoprotectants and buffers. Here, we aim to systematically investigate the impact of a diverse range of excipients including oligosaccharides, polymers, amino acids, and various buffers, on the quality and performance of lyophilized mRNA-LNPs. From the screening of 45 mRNA-LNP formulations under various lyoprotectant and buffer conditions for lyophilization, we identified previously unexplored formulation compositions, e.g., polyvinylpyrrolidone (PVP) in Tris or acetate buffers, as promising alternatives to the commonly used oligosaccharides to maintain the physicochemical stability of lyophilized mRNA-LNPs. Further, we delved into how physicochemical and structural properties influence the functionality of lyophilized mRNA-LNPs. Leveraging high-throughput small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM), we showed that there is complex interplay between mRNA-LNP structural features and cellular translation efficacy. We also assessed innate immune responses of the screened mRNA-LNPs in human peripheral blood mononuclear cells (PBMCs), and showed minimal alterations of cytokine secretion profiles induced by lyophilized formulations. Our results provide valuable insights into the structure-activity relationship of lyophilized formulations of mRNA-LNP therapeutics, paving the way for rational design of these formulations. This work creates a foundation for a comprehensive understanding of mRNA-LNP properties and in vitro performance change resulting from lyophilization.

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Bacterial cellulose (BC) presents significant promise as a biomaterial, boasting unique qualities such as exceptional cellulose purity, robust mechanical strength, heightened crystalline structure, and biodegradability. Several studies have highlighted specific effects, such as the impact of dehydration/rehydration on BC tensile strength, the influence of polymer treatment methods on mechanical properties, the correlation between microorganism type, drying method, and Young's modulus value, and the relationship between culture medium composition, pH, and crystallinity. Drying methods are crucial to the structure, performance, and application of BC films. Research findings indicate that the method used for drying can influence the mechanical properties of BC films, including parameters such as tensile strength, Young's modulus, and water absorption capacity, as well as the micromorphology, crystallinity, and thermal characteristics of the material. Their versatility makes them potential biomaterials applicable in various fields, including thermal and acoustic insulation, owing to their distinct thermal and mechanical attributes. This review delves into the thermal and mechanical behavior of bacterial cellulose aerogels, which are profoundly impacted by their drying mechanism.

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With the emergence of diseases, the U.S. catfish industry is under challenge. Current trends prefer autochthonous bacteria as potential probiotic candidates owing to their adaptability and capacity to effectively colonize the host's intestine, which can enhance production performance and bolster disease resistance. The objective of this study was to isolate an autochthonous bacterium as probiotic for hybrid catfish. Initially, an analysis of the intestinal microbiota of hybrid catfish reared in earthen ponds was conducted for subsequent probiotic development. Twenty lactic acid bacteria were isolated from the digesta of overperforming catfish, and most of the candidates demonstrated probiotic traits, including proteolytic and lipolytic abilities; antagonistic inhibition of catfish enteric bacterial pathogens, negative haemolytic activity and antibiotic susceptibility. Subsequent to this screening process, an isolate of Lactococcus lactis (MA5) was deemed the most promising probiotic candidate. In silico analyses were conducted, and several potential probiotic functions were predicted, including essential amino acids and vitamin synthesis. Moreover, genes for three bacteriocins, lactococcin A, enterolysin A and sactipeptide BmbF, were identified. Lastly, various protectant media for lyophilization of MA5 were assessed. These findings suggest that Lactococcus lactis MA5 can be an autochthonous probiotic from hybrid catfish, holding promise to be further tested in feeding trials.

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Resveratrol (RES) has demonstrated advantages as anti-cancer, anti-inflammatory, blood sugar-lowering agent and as cardioprotective agent, among others. Despite RES therapeutic advantages its use in pharmaceutical applications is limited by its low oral bioavailability, mainly due to its poor water solubility. Formulation of poorly water-soluble compound as solid dispersion (SD) converts a crystalline into a more soluble in water amorphous drug. Lyophilization or freeze-drying is a process in which water, an organic solvent, or a co-solvent system is frozen, followed by its removal from the sample, initially by sublimation (primary drying) and then by desorption (secondary drying). This study aimed the development and optimization of a bulk freeze-drying cycle by critical process parameters assessment in each phase to prepare a RES third-generation SD, containing Eudragit E PO as hydrophilic polymer at 1:2 ratio, and Gelucire 44/14 as surfactant at 16 % (w/w) to RES, using a tert-butanol (TBA)/Acetate buffer pH 4.5 (75:25) co-solvent system. A RES third-generation SD with good appearance, not cracked, collapsed, or melted was prepared by an optimized and robust bulk lyophilization process. A physicochemical characterization confirmed the conversion of RES to the amorphous state in the SD and formulation stability after 1 month at 40 °C/75 % RH. Increased solubility and higher dissolution rate compared with pure RES were also obtained.

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The remarkable success of two FDA-approved mRNA-encapsulating vaccines (Comirnaty® and Spikevax®) indicated the importance of lipid nanoparticles (LNPs) delivery systems in clinical use. Currently, mRNA-encapsulating LNPs (mRNA-LNPs) vaccines are stored as frozen liquid at low or ultralow temperatures. We designed lyophilized LNPs utilizing FDA-approved lipids to expedite the clinical application of our developed lyophilized mRNA-LNPs in the future. The key parameters of sucrose concentration and the selection and molar ratio of the four lipids in these vaccines were optimized for long-term stability with high transfection efficiency after lyophilization. We demonstrated that 8.7% sucrose is the optimal cryoprotectant concentration to maintain the transfection efficiency of lyophilized mRNA-LNPs. Optimal lipid formulations with high transfection efficiency both before and after lyophilization were screened using an orthogonal experimental design. The ratios of distearoylphosphatidylcholine (DSPC)/cholesterol and the selection of the ionizable and PEGylated lipids are the main factors influencing the long-term stability of mRNA-LNPs. Comparative mouse transfection experiments showed that the optimal lyophilized mRNA-LNPs maintained high mRNA expression after lyophilization, predominantly in the spleen or liver, with no expression in the kidneys or eyes. Our studies demonstrated the importance of the sucrose concentration and of the selection and molar ratio of the four lipids composing LNPs for maintaining mRNA-LNP stability under lyophilization and for long-term storage under mild conditions.

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The encapsulation of ß-carotene was investigated using pullulan and whey protein isolate (WPI) as a composite matrix at a weight ratio of 20:80, employing both spray-drying and freeze-drying techniques. The influence of processing parameters such as the concentration of wall material, flow rate, and inlet temperature for SP encapsulants, as well as wall-material concentration for FZ encapsulants, was examined in terms of encapsulation efficiency (EE). The morphology, structural characterization, moisture sorption isotherms, and thermal properties of the resulting encapsulants at optimum conditions were determined. Their stability was investigated under various levels of water activity, temperature conditions, and exposure to UV-Vis irradiation. ß-carotene was efficiently encapsulated within SP and FZ structures, resulting in EE of approximately 85% and 70%, respectively. The degradation kinetics of ß-carotene in both structures followed a first-order reaction model, with the highest rate constants (0.0128 day-1 for SP and 0.165 day-1 for FZ) occurring at an intermediate water-activity level (aw = 0.53) across all storage temperatures. The photostability tests showed that SP encapsulants extended ß-carotene's half-life to 336.02 h, compared with 102.44 h for FZ encapsulants, under UV-Vis irradiation. These findings highlight the potential of SP encapsulants for applications in functional foods, pharmaceuticals, and carotenoid supplements.

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Protein biotherapeutics typically require expensive cold-chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold-chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., "cryoprotection" and "lyoprotection"), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake-forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1-16) were extracted via ethanol precipitation. PG size was generally consistent at ~70-100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1-16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, ß-galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate-buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein-PG13 mixtures had a cake-like appearance and all frozen protein-PG13 mixtures had a Tg' of ~ -26°C. The lyoprotection and cake-forming properties of PG13 were density-dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

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Probiotics offer health advantages when consumed in adequate quantities. As ongoing research identifies promising new strains, ensuring their viability and functionality through simple preservation methods is vital for success within the probiotic industry. This study employed a factorial design to investigate the combined effects of four cryoprotectants [C1: MRS broth + 14 % (w/v) glycerol, C2: Aqueous solution containing 4 % (w/v) trehalose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate, C3: Aqueous solution containing 10 % (w/v) skimmed milk and 4 % (w/v) sodium glutamate, C4: Aqueous solution containing 4 % (w/v) sucrose, 6 % (w/v) skimmed milk, and 4 % (w/v) sodium glutamate] and three methods of preservation (P1: -86 °C freezing, P2: -196 °C liquid nitrogen freezing, and P3: storing at 4 °C after lyophilization) on the cell viability of three potentially probiotic strains over 12 months. Pediococcus sp P15 and Weissella cibaria ml6 had the highest viability under treatments C3 and C2, after 12 months of storage, respectively. Meanwhile, Lactococcus lactis ml3 demonstrated the highest viability in both treatments C2 and C4 (P ≤ 0.05). According to the results freezing, either P1 or P2, is the most effective preservation method for P. sp P15 and W. cibaria ml6. Meanwhile, L. lactis ml3 showed the highest colony count under treatment (P1) after 12 months of storage (P ≤ 0.05). Among the tested conditions, P. sp P15 and L. lactis ml3 exhibited the highest viability and bile salt resistance when stored under P1C1. For W. cibaria ml6, the optimal storage condition was P2C2 (frozen in liquid nitrogen with cryoprotectant C2).

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