Optimization of Ultrasound-Assisted Pretreatment for Accelerating Rehydration of Adzuki Bean (Vigna angularis).

Adzuki bean (Vigna angularis), which provides plant-based proteins and functional substances, requires a long soaking time during processing, which limits its usefulness to industries and consumers. To improve this, ultrasonic treatment using high pressure and shear force was judged to be an appropriate pretreatment method. This study aimed to determine the optimal conditions of ultrasound treatment for the improved hydration of adzuki beans using the response surface methodology (RSM). Independent variables chosen to regulate the hydration process of the adzuki beans were the soaking time (2-14 h, X1), treatment intensity (150-750 W, X2), and treatment time (1-10 min, X3). Dependent variables chosen to assess the differences in the beans post-immersion were moisture content, water activity, and hardness. The optimal conditions for treatment deduced through RSM were a soaking time of 12.9 h, treatment intensity of 600 W, and treatment time of 8.65 min. In this optimal condition, the values predicted for the dependent variables were a moisture content of 58.32%, water activity of 0.9979 aw, and hardness of 14.63 N. Upon experimentation, the results obtained were a moisture content of 58.28 ± 0.56%, water activity of 0.9885 ± 0.0040 aw, and hardness of 13.01 ± 2.82 g, confirming results similar to the predicted values. Proper ultrasound treatment caused cracks in the hilum, which greatly affects the water absorption of adzuki beans, accelerating the rate of hydration. These results are expected to help determine economically efficient processing conditions for specific purposes, in addition to solving industrial problems associated with the low hydration rate of adzuki beans.

Retrograded octenylsuccinylated maize starch-based emulgels for a promising oral delivery system of curcumin.

Emulgels are a type of soft solid delivery system that exploit the merits of both emulsions and gels, namely, bioactive encapsulability and structural stability, respectively. We utilized retrograded/octenylsuccinylated maize starch (ROMS) to fabricate the curcumin-loaded emulgel. Emulgels (oil volume fraction, 0.20) prepared with 1-4 % w/w ROMS exhibited fluid-like behaviors while emulgels with 5-8 % w/w ROMS exhibited a gel-like consistency. Compared to a fluidic emulsion stabilized with 3 % w/w octenylsuccinylated maize starch, the emulgels showed more sustained lipolysis and controlled curcumin release patterns. These results were attributed to rigid ROMS structures at the outer layer of oil droplets, hindering the lipase approach onto the oil/water interface and curcumin diffusion from the interface. Additionally, the bioaccessibility of curcumin in ROMS-stabilized emulgels was enhanced >9.6-fold compared to that of a curcumin solution. Furthermore, emulgels prepared with 8 % w/w ROMS exhibited a high yield stress (376.4 Pa) and maintained appearance and droplet size for 60 days of storage at 4 °C. Consequently, this emulgel has potential as a lipophilic bioactive-containing soft gel with sustained digestion and controlled release properties. Our findings may provide insights into rational delivery system designs.

Comparison of the pretreatment methods for enhancing hydration of water-soaked adzuki beans (Vigna angularis).

Five pretreatments methods, cold plasma, pressure drop, heating, and bath-type and probe-type sonications were compared to shorten the rehydration process of adzuki bean (Vigna angularis) soaked before the cooking in terms of the hydration and softening efficacies. Moisture content and water activity of the probe-type sonicated beans were most dramatically increased as 11-45% and 0.59-0.97 after soaking for only 2 h, respectively (non-treated: 11-12% and 0.59-0.66). Accordingly, the probe-type sonicated beans were most rapidly softened as 27-5 N in the 2 h-soaking and exhibited the lowest hardness after soaking/cooking as ~ 0.97 N (non-treated: 27-21 N and ~ 5.5 N, respectively). According to scanning electron micrographs, these results can be attributed to formation of prominent fissures or scars in the hilum of the probe-type sonicated beans. Consequently, this study will be provide valuable information for developing a rational process in food industry to shorten the rehydration of the adzuki beans.

Enhancing stability and bioavailability of sulforaphene in radish seed extracts using nanoemulsion made with high oleic sunflower oil.

The effect of nanoemulsions on the stability and bioavailability of sulforaphene (SFEN) in radish seed extract (RSE) was investigated. Four types of oil were used as lipid ingredients of the nanoemulsions: soybean, high oleic acid sunflower, coconut, and hydrogenated palm oils. SFEN in RSE nanoemulsions showed greater stability to temperature, acid, and alkaline conditions than SFEN in RSE suspended in water (RSE-S). Particularly under alkaline conditions, the half-life of SFEN in the nanoemulsion with high oleic sunflower oil (RSE-HOSO) was 8 times longer than that of RSE-S. Furthermore, in the pharmacokinetics study, it was observed that AUC0-8 increased and oral clearance (CL/F) decreased significantly in rats orally administered RSE-HOSO compared with RSE-S (p < 0.05). This study indicates that the type of oil used in nanoemulsions affects the stability and bioavailability of SFEN in RSE. These results may provide a guideline for the development of functional foods containing RSE. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01304-2.

Synthesis and physicochemical characterization of acyl myricetins as potential anti-neuroexocytotic agents.

Acyl myricetins (monopropionyl-, dipropionyl-, and monooctanoyl-myricetin, termed as MP1, MP2, and MO1, respectively) were synthesized through enzymatic or non-enzymatic esterification reaction of myricetin aglycone. Structure study indicated the hydroxyl group at C4' in B-ring was highly susceptible to acylation. Over its parental myricetin, acylated compounds showed enhanced lipophilicity (from 7.4- to 26.3-fold) and oxidative stability (from 1.9- to 3.1-fold) on the basis of logP and decay rate, respectively. MO1, presenting the physicochemical superiority compared to the others, provided lowest EC50 value of 2.51 µM on inhibition of neutrotransmitter release and CC50 value of 59.0 µM, leading to widest therapeutic window. All myricetin esters did not show any irritation toxicity when assessed with a chicken embryo assay. This study describes information on acylation of myricetin that has not yet been explored, and suggests that MO1 has membrane fusion-arresting and anti-neuroexocytotic potential for industrial application due to its enhanced biological properties.

Applications and perspectives of polyphenol-loaded solid lipid nanoparticles and nanostructured lipid carriers for foods.

Imbalanced nutrition in modern society is one of the reasons for disorders, such as cancer, cardiovascular disease, and diabetes, which have attracted the interest in bioactives (particularly polyphenols) to assist in the balanced diet of modern people. Although stability can be maintained during preparation and storage, the ingested polyphenols undergo harsh gastrointestinal digestion processes, resulting in limited bioaccessibility and low gut-epithelial permeation and bioavailability. Several lipid-based formulations have been proposed to overcome these issues. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) have also been highlighted as carrier systems for the oral delivery of lipophilic bioactives, including polyphenols. This paper summarizes the research on the ingredients, production methods, post-processing procedures, general characteristics, and advantages and disadvantages of SLNs and NLCs. Overall, this paper reviews the applications and perspectives of polyphenol-loaded SLNs and NLCs in foods, as well as their regulation, production, storage, and economic feasibility.

Delayed Quality Deterioration of Low-Moisture Cereal-Based Snack by Storing in an Active Filler-Embedded LDPE Zipper Bag.

This study focused on controlling the vapor permeability of an active zipper bag and preserving the quality of cereal-based snacks during the storage period at home. The active zipper bag was prepared by extruding low-density polyethylene with active fillers obtained from natural mineral materials. The active zipper bag showed the same transparent appearance as the existing one but showed 21% lower water vapor capability. As a result, during a 20-day storage period, three types of grain-based snacks (biscuits, shortbread cookies, and puffed snacks) showed delayed increases in weight, moisture content, and moisture activity when stored in an active zipper bag. In addition, this also affected the texture of the biscuits and shortbread cookies, in which the area under the curve was reduced significantly after appearing at a peak during the hardness measurement. On the other hand, the decrease in the number of air cell fracture events in puffed snacks was remarkable. This result suggests that the inner microstructure is preserved better when stored in an active zipper bag. In conclusion, the active zipper bag showed poor water vapor permeability, suggesting that the prepared zipper bag can be developed as snack packaging.

Neuron-recognizable characteristics of peptides recombined using a neuronal binding domain of botulinum neurotoxin.

Recombinant peptides were designed using the C-terminal domain (receptor binding domain, RBD) and its subdomain (peptide A2) of a heavy chain of botulinum neurotoxin A-type 1 (BoNT/A1), which can bind to the luminal domain of synaptic vesicle glycoprotein 2C (SV2C-LD). Peptide A2- or RBD-containing recombinant peptides linked to an enhanced green fluorescence protein (EGFP) were prepared by expression in Escherichia coli. A pull-down assay using SV2C-LD-covered resins showed that the recombinant peptides for CDC297 BoNT/A1, referred to EGFP-A2' and EGFP-RBD', exhibited ≥ 2.0-times stronger binding affinity to SV2C-LD than those for the wild-type BoNT/A1. Using bio-layer interferometry, an equilibrium dissociation rate constant (KD) of EGFP-RBD' to SV2C-LD was determined to be 5.45 µM, which is 33.87- and 15.67-times smaller than the KD values for EGFP and EGFP-A2', respectively. Based on confocal laser fluorescence micrometric analysis, the adsorption/absorption of EGFP-RBD' to/in differentiated PC-12 cells was 2.49- and 1.29-times faster than those of EGFP and EGFP-A2', respectively. Consequently, the recombinant peptides acquired reasonable neuron-specific binding/internalizing ability through the recruitment of RBD'. In conclusion, RBDs of BoNTs are versatile protein domains that can be used to mark neural systems and treat a range of disorders in neural systems.

Objective Quantitation of Focal Sweating Areas Using a Mouse Sweat-assay Model.

In vivo sweat quantitation assays are required for the development of drugs for the management of focal hyperhidrosis before clinical trials; however, in vivo assays, particularly mouse models, are rare. Even in sweat assays using mice, sweating is quantitated by manually counting the number of sweating spots, which can contribute to various errors owing to arbitrary judgment. In this study, we developed a mouse sweat-assay model and a method for quantitating the amount of sweating to remove possible errors. The use of the iodine-starch test in the castor oil-covered hind footpad skin of anesthetized mice resulted in the sweating area being stained blue-black. After the anesthesia and treatment with drugs (pilocarpine, glycopyrrolate, botulinum neurotoxin, myricetin, and myricetin-loaded lipid nanoparticles), the remaining area of the footpad skin was eliminated from the acquired footpad images using ImageJ. Blue pixels extracted from the footpad image are automatically adjusted using the Phansalkar method, where the percentage of the blue area was determined based on the whole hind footpad skin area, finally indicating the percentage of the sweating area. Using this mouse model and analysis for sweat assays, a clear difference between the control group and antiperspirant-administered group was observed with respect to the sweating area % with no error. In conclusion, this assay can be used as a preclinical tool to screen potential antiperspirant drugs. Graphic abstract: Overview of the mouse-model sweat assay and objective quantitation of the focal sweating area.

Development of End-Spliced Dimeric Nanodiscs for the Improved Virucidal Activity of a Nanoperforator.

Lipid-bilayer nanodiscs (NDs) wrapped in membrane scaffold proteins (MSPs) have primarily been used to study membrane proteins of interest in a physiological environment. Recently, NDs have been employed in broader applications including drug delivery, cancer immunotherapy, bio-imaging, and therapeutic virucides. Here, we developed a method to synthesize a dimeric nanodisc, whose MSPs are circularly end-spliced, with long-term thermal stability and resistance to aggregation. The end-spliced nanodiscs (esNDs) were assembled using MSPs that were self-circularized inside the cytoplasm ofEscherichia colivia highly efficient protein trans-splicing. The esNDs demonstrated a consistent size and 4-5-fold higher stability against heat and aggregation than conventional NDs. Moreover, cysteine residues on trans-spliced circularized MSPs allowed us to modulate the formation of either monomeric nanodiscs (essNDs) or dimeric nanodiscs (esdNDs) by controlling the oxidation/reduction conditions and lipid-to-protein ratios. When the esdNDs were used to prepare an antiviral nanoperforator that induced the disruption of the viral membrane upon contact, antiviral activity was dramatically increased, suggesting that the dimerization of nanodiscs led to cooperativity between linked nanodiscs. We expect that controllable structures, long-term stability, and aggregation resistance of esNDs will aid the development of novel versatile membrane-mimetic nanomaterials with flexible designs and improved therapeutic efficacy.

Rheology, Microstructure, and Storage Stability of Emulsion-Filled Gels Stabilized Solely by Maize Starch Modified with Octenyl Succinylation and Pregelatinization.

We prepared emulsion-filled gels stabilized using octenyl succinic anhydride-modified and pregelatinized maize starch (OSA-PGS). The effect of the oil volume fraction (Φ, 0.05-0.20) and OSA-PGS concentration (3-10% w/v) on the rheological and microstructural properties of the emulsion-filled gels was evaluated. Confocal fluorescence images showed that OSA-PGS stabilized the emulsion, indicated by the formation of a thick layer surrounding the oil droplets, and simultaneously gelled the aqueous phase. All of the emulsions exhibited shear-thinning flow behavior, but only those with 10% w/v OSA-PGS were categorized as Herschel-Bulkley fluids. The rheological behavior of the emulsion-filled gels was significantly affected by both the OSA-PGS concentration and Φ. The mean diameters (D1,0, D3,2, and D4,3) of oil droplets with 10% w/v OSA-PGS were stable during 30 days of storage under ambient conditions, indicating good stability. These results provide a basis for the design of systems with potential applications within the food industry.

Visible on-site detection of Ara h 1 by the switchable-linker-mediated precipitation of gold nanoparticles.

Biosensors have been widely applied in tests for allergens, but on-site detection remains a challenge. Herein, we proposed a detection procedure for peanut Ara h 1 as a representative allergen, which was extracted from a cookie, thereby minimising the need for any complex pretreatment that was difficult to perform, and enabling the visual detection of the target without the use of analytical equipment. The extraction procedure was performed in less than 30 min using a syringe and filter (0.45 µm). The detection method for Ara h 1 was based on the aggregation of switchable linkers (SL) and gold nanoparticles (AuNP), and the presence of 0.19 mg peanut protein per 30 g of cookie could be confirmed within 30 min based on the AuNP/SL concentration ratio by the precipitation. This proposed procedure could be successfully applied to the detection of a wide range of food allergens.

Reduction of focal sweating by lipid nanoparticle-delivered myricetin.

Myricetin-a flavonoid capable of inhibiting the SNARE complex formation in neurons-reduces focal sweating after skin-application when delivers as encapsulated in lipid nanoparticles (M-LNPs). The stability of M-LNP enables efficient delivery of myricetin to sudomotor nerves located underneath sweat glands through transappendageal pathways while free myricetin just remained on the skin. Furthermore, release of myricetin from M-LNP is accelerated through lipase-/esterase-induced lipolysis in the skin-appendages, enabling uptake of myricetin by the surrounding cells. The amount of sweat is reduced by 55% after application of M-LNP (0.8 mg kg-1) on the mouse footpad. This is comparable to that of subcutaneously injected anticholinergic agents [0.25 mg kg-1 glycopyrrolate; 0.8 U kg-1 botulinum neurotoxin-A-type (BoNT/A)]. M-LNP neither shows a distal effect after skin-application nor induced cellular/ocular toxicity. In conclusion, M-LNP is an efficient skin-applicable antiperspirant. SNARE-inhibitory small molecules with suitable delivery systems have the potential to replace many BoNT/A interventions for which self-applications are preferred.

Development of fluorescent Escherichia coli for a whole-cell sensor of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL), a major component of fucosylated human milk oligosaccharides, is beneficial to human health in various ways like prebiotic effect, protection from pathogens, anti-inflammatory activity and reduction of the risk of neurodegeneration. Here, a whole-cell fluorescence biosensor for 2'-FL was developed. Escherichia coli (E. coli) was engineered to catalyse the cleavage of 2'-FL into L-fucose and lactose by constitutively expressing α-L-fucosidase. Escherichia coli ∆L YA, in which lacZ is deleted and lacY is retained, was employed to disable lactose consumption. E. coli ∆L YA constitutively co-expressing α-L-fucosidase and a red fluorescence protein (RFP) exhibited increased fluorescence intensity in media containing 2'-FL. However, the presence of 50 g/L lactose reduced the RFP intensity due to lactose-induced cytotoxicity. Preadaptation of bacterial strains to fucose alleviated growth hindrance by lactose and partially recovered the fluorescence intensity. The fluorescence intensity of the cell was linearly proportional to 1-5 g/L 2'-FL. The whole-cell sensor will be versatile in developing a 2'-FL detection system.

Characterization of Ginkgo biloba Leaf Flavonoids as Neuroexocytosis Regulators.

Ginkgo biloba leaf (GBL) is known as a potential source of bioactive flavonoids, such as quercetin, arresting the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-zippering. Here, the GBL flavonoids were isolated in two different manners and then examined for their bioactivity, physicochemical stability, and biocompatibility. The majority of flavonoids in the non-hydrolyzed and acidolyzed isolates, termed non-hydrolyzed isolate (NI) and acidolyzed isolate (AI) hereafter, were rich in flavonol glycosides and aglycones, respectively. Glycosidic/aglyconic quercetin and kaempferol were abundant in both NI and AI, whereas a little of apigenin, luteolin, and isorhamnetin were found in AI. NI was more thermostable in all pH ranges than quercetin, kaempferol, and AI. NI and AI both inhibited neurotransmitter release from differentiated neuronal PC-12 cells. NI and AI showed 1/2-1/3 lower EC50/CC50 values than quercetin and kaempferol. The NI and AI exhibited no toxicity assessed by the tests on chorioallantoic membranes of hen's eggs, removing toxicological concerns of irritation potential. Moreover, GBL isolates, particularly AI, showed antioxidant and anti-inflammatory activities in the use below the CC50 levels. Taken together, these results suggest that GBL isolates that are rich in antioxidant flavonoids are effective anti-neuroexocytotic agents with high stability and low toxicity.

Enhancing the oral bioavailability of curcumin using solid lipid nanoparticles.

To control the oral bioavailability of curcumin, we fabricated solid lipid nanoparticles (SLNs) using tristearin and polyethylene glycol (PEG)ylated emulsifiers. Lipolysis of prepared SLNs via simulated gastrointestinal digestion was modulated by altering the types and concentrations of emulsifiers. After digestion, the size/surface charge of micelles formed from SLN digesta were predictable and >91% of curcumin was bioaccessible in all of the SLNs. The curcumin permeation rate through mucus-covered gut epithelium in vitro was dependent on the size/surface charge of the micelles. Curcumin loaded in long-PEGylated SLNs rapidly permeated the epithelium due to the neutral surface charge of the micelles, resulting in a >12.0-fold increase in bioavailability compared to curcumin solution in a rat model. These results suggest that the bioavailability of curcumin can be controlled by modulating the interfacial properties of SLNs, which will facilitate the development of curcumin formulations for use in functional foods and pharmaceuticals.

Interfacial and colloidal characterization of oil-in-water emulsions stabilized by interface-tunable solid lipid nanoparticles.

We evaluated the correlation between the interfacial characteristics of solid lipid nanoparticles (SLNs) and the interfacial/colloidal stability of SLN-stabilized emulsions. Herein, the interfacial properties of SLNs, particularly the surface load (Γs) of emulsifiers, were tuned by controlling the type/concentration of emulsifier used to prepare the SLNs. Increasing the Γs decreased the contact angle at the oil-water interface, which enhanced the displacement free energy of the SLNs at the interface. Moreover, the Γs of emulsifiers bound to the surface of SLNs covering oil droplets was linearly correlated with the SLN-own Γs. The size/ζ-potential of emulsions stabilized by SLNs covered by the highest concentration of emulsifiers was unchanged for 1 month, indicating good emulsion stability. The interfacial/colloidal stability of SLN-stabilized emulsions was thus enhanced by increasing the emulsifier concentration used to produce the SLNs. This study provides baseline data for developing SLN-stabilized emulsions for the food, cosmetic, and pharmaceutical industries.

Envelope-deforming antiviral peptide derived from influenza virus M2 protein.

Molecules interfering with lipid bilayer function exhibit strong antiviral activity against a broad range of enveloped viruses, with a lower risk of resistance development than that for viral protein-targeting drugs. Amphipathic peptides are rich sources of such membrane-interacting antivirals. Here, we report that influenza viruses were effectively inactivated by M2 AH, an amphipathic peptide derived from the M2 protein of the influenza virus. Although overall hydrophobicity () of M2 AH was not related to antiviral activity, modification of the hydrophobic moment (<µH>) of M2 AH dramatically altered the antiviral activity of this peptide. M2 MH, a derivative of M2 AH with a <µH> of 0.874, showed a half maximal inhibitory concentration (IC50) of 53.3 nM against the A/PR/8/34 strain (H1N1), which is 16-times lower than that of M2 AH. The selectivity index (IC50/CC50), where CC50 is the half maximal cytotoxic concentration, was 360 for M2 MH and 81 for M2 AH. Dynamic light scattering spectroscopy and electron microscopy revealed that M2 AH-derived peptides did not disrupt liposomes but altered the shape of viruses. This result suggests that the shape of virus envelope was closely related to its activity. Thus, we propose that deforming without rupturing the membranes may achieve a high selectivity index for peptide antivirals.

Development of a quantitative assay for 2´-fucosyllactose via one-pot reaction with α1,2-fucosidase and l-fucose dehydrogenase.

2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.

Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids.

Whole cell biocatalysts can be used to convert fatty acids into various value-added products. However, fatty acid transport across cellular membranes into the cytosol of microbial cells limits substrate availability and impairs membrane integrity, which in turn decreases cell viability and bioconversion activity. Because these problems are associated with the mechanism of fatty acid transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was generated to promote substrate endocytosis. Caveolin-1 ( CAV1) expression in Escherichia coli increased both the fatty acid transport rate and intracellular fatty acid concentrations via endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-hydroxyoctadec-9-enoic acid) to ( Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of hydrophobic substrates.