Enzyme-Triggered Polyelectrolyte Complex for Responsive Delivery of α-Helical Polypeptides to Optimize Antibacterial Therapy.

Responsive nanomaterials hold significant promise in the treatment of bacterial infections by recognizing internal or external stimuli to achieve stimuli-responsive behavior. In this study, we present an enzyme-responsive polyelectrolyte complex micelles (PTPMN) with α-helical cationic polypeptide as a coacervate-core for the treatment of Escherichia coli (E. coli) infection. The complex was constructed through electrostatic interaction between cationic poly(glutamic acid) derivatives and phosphorylation-modified poly(ethylene glycol)-b-poly(tyrosine) (PEG-b-PPTyr) by directly dissolving them in aqueous solution. The cationic polypeptide adopted α-helical structure and demonstrated excellent broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria, with a minimum inhibitory concentration (MIC) as low as 12.5 µg mL-1 against E. coli. By complexing with anionic PEG-b-PPTyr, the obtained complex formed ß-sheet structures and exhibited good biocompatibility and low hemolysis. When incubated in a bacterial environment, the complex cleaved its phosphate groups triggered by phosphatases secreted by bacteria, exposing the highly α-helical conformation and restoring its effective bactericidal ability. In vivo experiments confirmed accelerated healing in E. coli-infected wounds.

Toward Synthetic Intrinsically Disordered Polypeptides (IDPs): Controlled Incorporation of Glycine in the Ring-Opening Polymerization of N-Carboxyanhydrides.

Intrinsically disordered proteins (IDPs) do not have a well-defined folded structure but instead behave as extended polymer chains in solution. Many IDPs are rich in glycine residues, which create steric barriers to secondary structuring and protein folding. Inspired by this feature, we have studied how the introduction of glycine residues influences the secondary structure of a model polypeptide, poly(l-glutamic acid), a helical polymer. For this purpose, we carried out ring-opening copolymerization with γ-benzyl-l-glutamate and glycine N-carboxyanhydride (NCA) monomers. We aimed to control the glycine distribution within PBLG by adjusting the reactivity ratios of the two NCAs using different reaction conditions (temperature, solvent). The relationship between those conditions, the monomer distributions, and the secondary structure enabled the design of intrinsically disordered polypeptides when a highly gradient microstructure was achieved in DMSO.

Layer-by-layer assembly of quercetin-loaded zein/γPGA/low-molecular-weight chitosan/fucoidan nanosystem for targeting inflamed blood vessels.

Chitosan acts as a versatile carrier in polymeric nanoparticle (NP) for diverse drug administration routes. Delivery of antioxidants, such as quercetin (Qu) showcases potent antioxidant and anti-inflammatory properties for reduction of various cardiovascular diseases, but low water solubility limits uptake. To address this, we developed a novel layer-by-layer zein/gamma-polyglutamic acid (γPGA)/low-molecular-weight chitosan (LC)/fucoidan NP for encapsulating Qu and targeting inflamed vessel endothelial cells. We used zein (Z) and γPGA (r) to encapsulate Qu (Qu-Zr NP) exhibited notably higher encapsulation efficiency compared to zein alone. Qu-Zr NP coated with LC (Qu-ZrLC2 NP) shows a lower particle size (193.2 ± 2.9 nm), and a higher zeta potential value (35.2 ± 0.4 mV) by zeta potential and transmission electron microscopy analysis. After coating Qu-ZrLC2 NP with fucoidan, Qu-ZrLC2Fa NP presented particle size (225.16 ± 0.92 nm), zeta potential (-25.66 ± 0.51 mV) and maintained antioxidant activity. Further analysis revealed that Qu-ZrLC2Fa NP were targeted and taken up by HUVEC cells and EA.hy926 endothelial cells. Notably, we observed Qu-ZrLC2Fa NP targeting zebrafish vessels and isoproterenol-induced inflamed vessels of rat. Our layer-by-layer formulated zein/γPGA/LC/fucoidan NP show promise as a targeted delivery system for water-insoluble drugs. Qu-ZrLC2Fa NP exhibit potential as an anti-inflammatory therapeutic for blood vessels.

Balancing mechanical property and swelling behavior of bacterial cellulose film by in-situ adding chitosan oligosaccharide and covalent crosslinking with γ-PGA.

Bacterial cellulose (BC) is an ideal candidate material for drug delivery, but the disbalance between the swelling behavior and mechanical properties limits its application. In this work, covalent crosslinking of γ-polyglutamic acid (γ-PGA) with the chitosan oligosaccharide (COS) embedded in BC was designed to remove the limitation. As a result, the dosage, time, and batch of COS addition significantly affected the mechanical properties and the yield of bacterial cellulose complex film (BCCF). The addition of 2.25 % COS at the incubation time of 0.5, 1.5, and 2 d increased the Young's modulus and the yield by 5.65 and 1.42 times, respectively, but decreased the swelling behavior to 1774 %, 46 % of that of native BC. Covalent γ-PGA transformed the dendritic structure of BCCF into a spider network, decreasing the porosity and increasing the swelling behavior by 3.46 times. The strategy balanced the swelling behavior and mechanical properties through tunning hydrogen bond, electrostatic interaction, and amido bond. The modified BCCF exhibited a desired behavior of benzalkonium chlorides transport, competent for drug delivery. Thereby, the strategy will be a competent candidate to modify BC for such potential applications as wound dressing, artificial skin, scar-inhibiting patch, and so on.

Genomic characterization and related functional genes of γ- poly glutamic acid producing Bacillus subtilis.

γ- poly glutamic acid (γ-PGA), a high molecular weight polymer, is synthesized by microorganisms and secreted into the extracellular space. Due to its excellent performance, γ-PGA has been widely used in various fields, including food, biomedical and environmental fields. In this study, we screened natto samples for two strains of Bacillus subtilis N3378-2at and N3378-3At that produce γ-PGA. We then identified the γ-PGA synthetase gene cluster (PgsB, PgsC, PgsA, YwtC and PgdS), glutamate racemase RacE, phage-derived γ-PGA hydrolase (PghB and PghC) and exo-γ-glutamyl peptidase (GGT) from the genome of these strains. Based on these γ-PGA-related protein sequences from isolated Bacillus subtilis and 181 B. subtilis obtained from GenBank, we carried out genotyping analysis and classified them into types 1-5. Since we found B. amyloliquefaciens LL3 can produce γ-PGA, we obtained the B. velezensis and B. amyloliquefaciens strains from GenBank and classified them into types 6 and 7 based on LL3. Finally, we constructed evolutionary trees for these protein sequences. This study analyzed the distribution of γ-PGA-related protein sequences in the genomes of B. subtilis, B. velezensis and B. amyloliquefaciens strains, then the evolutionary diversity of these protein sequences was analyzed, which provided novel information for the development and utilization of γ-PGA-producing strains.

Enhancing Poly-γ-glutamic Acid Production in Bacillus tequilensis BL01 through a Multienzyme Assembly Strategy and Expression Features of Glutamate Synthesis from Corynebacterium glutamicum.

The enhancement of intracellular glutamate synthesis in glutamate-independent poly-γ-glutamic acid (γ-PGA)-producing strains is an essential strategy for improving γ-PGA production. Bacillus tequilensis BL01ΔpgdSΔggtΔsucAΔgudB:P43-ppc-pyk-gdhA for the efficient synthesis of γ-PGA was constructed through expression of glutamate synthesis features of Corynebacterium glutamicum, which increased the titer of γ-PGA by 2.18-fold (3.24 ± 0.22 g/L) compared to that of B. tequilensis BL01ΔpgdSΔggtΔsucAΔgudB (1.02 ± 0.11 g/L). To further improve the titer of γ-PGA and decrease the production of byproducts, three enzymes (Ppc, Pyk, and AceE) were assembled to a complex using SpyTag/Catcher pairs. The results showed that the γ-PGA titer of the assembled strain was 31.31% higher than that of the unassembled strain. To further reduce the production cost, 25.73 ± 0.69 g/L γ-PGA with a productivity of 0.48 g/L/h was obtained from cheap molasses. This work provides new metabolic engineering strategies to improve the production of γ-PGA in B. tequilensis BL01. Furthermore, the engineered strain has great potential for the industrial production of γ-PGA from molasses.

Production of ultra-high-molecular-weight poly-γ-glutamic acid by a newly isolated Bacillus subtilis strain and genomic and transcriptomic analyses.

Poly-γ-glutamic acid (γ-PGA) is a microbial-derived polymer with molecular weight (Mw) from 104 to 107 Da, and the high-Mw (> 7.0 × 105 Da) or ultra-high-Mw (> 5.0 × 106 Da) γ-PGA has important application value as a tissue engineering material, as a flocculant, and as a heavy metal remover. Therefore, how to produce these high-Mw γ-PGAs with low cost and high efficiency has attracted wide attention. In this study, a γ-PGA producer was isolated from the natural environment, and identified and named Bacillus subtilis GXD-20. Then, the ultra-high-Mw (> 6.0 × 106 Da) γ-PGA produced by GXD-20 was characterized. Interestingly, GXD-20 could produce γ-PGA at 42°C, and exhibited a γ-PGA titer of up to 22.29 ± 0.59 g L-1 in a 5-L fermenter after optimization of the fermentation process. Comparative genomic analysis indicated that the specific protein sequence and subcellular localization of PgdS (a γ-PGA-degrading enzyme) were closely related to the ultra-high-Mw of γ-PGA. Transcriptomic analysis revealed that the high γ-PGA titer at 42°C was mainly related to the high expression of genes encoding enzymes for sucrose transportation and utilization, nitrogen transportation, endogenous glutamate synthesis, and γ-PGA synthesis. These results provide new insights into the production of ultra-high-Mw γ-PGA by Bacillus at high temperatures.

[Effects of different fertilization patterns on soil improvement and vegetation restoration of desertified grassland in northwest Liaoning Province, China]. / 不同培肥模式对辽西北沙化草地土壤改良与植被恢复的影响.

Improving soil fertility is one of the key approaches for ecological restoration of the wind-sand area in northwest Liaoning Province. Taking wind-sand area in northwest Liaoning Province as test object, we conducted a fertilization experiment with treatments of inorganic fertilizer (nitrogen, phosphorus and potassium fertilizers), organic fertilizer, combined application of organic and inorganic fertilizers, and organic fertilizer combined with a biologically organic matrix (γ-polyglutamic acid), and no fertilizer as control. We measured soil organic matter content and extractable cations concentrations, vegetation coverage, and biomass under different fertilization treatments and determine the suitable fertilization mode. The results showed that compared to the control, inorganic fertilizer rapidly increased vegetation coverage and biomass, but high levels of inorganic fertilizer (150 kg N·hm-2) led to soil acidification and Ca2+ leaching. Organic fertilizer increased soil organic matter content, exchangeable K+, Ca2+, and Mg2+ contents, as well as coverage and biomass vegetation, especially combined with γ-polyglutamic acid. Overall, the combination of low levels of inorganic fertilizer (50 kg N·hm-2) and moderate levels of organic fertilizer (30000 kg·hm-2) was the best fertilization practice for the rapid and stable restoration of grassland in wind-sand area. Moreover, the extra addition of γ-polyglutamic acid (60 kg·hm-2)could effectively improve soil fertility.

Effects of γ-polyglutamic acid supplementation on alfalfa growth and rhizosphere soil microorganisms in sandy soil.

This study aimed at exploring the effects of γ-polyglutamic acid on the growth of desert alfalfa and the soil microorganisms in the rhizosphere. The study examined the effects of varying concentrations of γ-polyglutamic acid (0%-CK, 2%-G1, 4%-G2, 6%-G3) on sandy soil, the research investigated its impact on the growth characteristics of alfalfa, nutrient content in the rhizosphere soil, and the composition of bacterial communities. The results indicated that there were no significant differences in soil organic matter, total nitrogen, total phosphorus, total potassium, and available phosphorus content among the G1, G2, and G3 treatments. Compared to CK, the soil nutrient content in the G2 treatment increased by 14.81-186.67%, showing the highest enhancement. In terms of alfalfa growth, the G2 treatment demonstrated the best performance, significantly increasing plant height, chlorophyll content, above-ground biomass, and underground biomass by 54.91-154.84%. Compared to the CK treatment, the number of OTUs (operational taxonomic units) in the G1, G2, and G3 treatments increased by 14.54%, 8.27%, and 6.84%, respectively. The application of γ-polyglutamic acid altered the composition and structure of the bacterial community, with Actinobacteriota, Proteobacteria, Chloroflexi, Acidobacteriota, and Gemmatimonadota accounting for 84.14-87.89% of the total bacterial community. The G2 treatment significantly enhanced the diversity and evenness of soil bacteria in the rhizosphere. Redundancy analysis revealed that organic matter, total nitrogen, total potassium, moisture content, and pH were the primary factors influencing the structure of bacterial phyla. At the genus level, moisture content emerged as the most influential factor on the bacterial community. Notably, moisture content exhibited a strong positive correlation with Acidobacteriota, which in turn was positively associated with indicators of alfalfa growth. In summary, the application of γ-polyglutamic acid at a 4% ratio has the potential for improving sandy soil quality, promoting plant growth, and regulating the rhizosphere microbial community.

Electrofermentation increases concentration of poly γ-glutamic acid in Bacillus subtilis biofilms.

Fluctuations in redox conditions in bioprocesses can alter the end-products, reduce their concentration, and lengthen the process time. Electrofermentation enables rapid metabolic modulation of biosynthesis and allows control of redox imbalances in biofilm-based fermentation processes. In this study, electrofermentation is used to boost the production of the bacterial biopolymer poly-γ-glutamic acid (γ-PGA) from Bacillus subtilis ATCC 6051. When compared to control experiments (3.3 ± 0.99 g L-1 ), the application of an electrode potential E = 0.4 V versus Ag/AgCl results in a more than two-fold increase in the production of γ-PGA (9.13 ± 1.4 g L-1 ). Using an engineered B. subtilis strain, in which γ-PGA production is driven by isopropyl ß-d-1-thiogalactopyranoside, electrofermentation improves polymer concentrations from 15.4 ± 1.5 to 23.1 ± 1.6 versus g L-1 . These results confirm that electrofermentation conditions can be adopted to increase the concentration of γ-PGA and perhaps other extracellular biopolymers in industrial strains.

Enhancing the tumor penetration of multiarm polymers by collagenase modification.

Tumor penetration is a critical determinant of the therapy efficacy of nanomedicines. However, the dense extracellular matrix (ECM) in tumors significantly hampers the deep penetration of nanomedicines, resulting in large drug-untouchable areas and unsatisfactory therapy efficacy. Herein, we synthesized a third-generation PAMAM-cored multiarm copolymer and modified the polymer with collagenase to enhance its tumor penetration. Each arm of the copolymer was a diblock copolymer of poly(glutamic acid)-b-poly(carboxybetaine), in which the polyglutamic acid block with abundant side groups was used to link the anticancer agent doxorubicin through the pH-sensitive acylhydrazone linkage, and the zwitterionic poly(carboxybetaine) block provided desired water solubility and anti-biofouling capability. The collagenase was conjugated to the ends of the arms via the thiol-maleimide reaction. We demonstrated that the polymer-bound collagenase could effectively catalyze the degradation of the collagen in the tumor ECM, and consequently augmented the tumor penetration and antitumor efficacy of the drug-loaded polymers.

Effective Removal of Different Heavy Metals Ion (Cu, Pb, and Cd) from Aqueous Solutions by Various Molecular Weight and Salt Types of Poly-γ-Glutamic Acid.

In light of industrial developments, water pollution by heavy metals as hazardous chemicals has garnered attention. Addressing the urgent need for efficient heavy metal removal from aqueous environments, this study delves into using poly-γ-glutamic acid (γ-PGA) for the bioflocculation of heavy metals. Utilizing γ-PGA variants from Bacillus subtilis with different molecular weights and salt forms (Na-bonded and Ca-bonded), the research evaluates their adsorption capacities for copper (Cu), lead (Pb), and cadmium (Cd) ions. It was found that Na-bonded γ-PGA with a high molecular weight showed the highest heavy metal adsorption (92.2-98.3%), particularly at a 0.5% concentration which exhibited the highest adsorption efficiency. Additionally, the study investigated the interaction of γ-PGA in mixed heavy metal environments, and it was discovered that Na-γ-PGA-HM at a 0.5% concentration showed a superior adsorption efficiency for Pb ions (85.4%), highlighting its selectivity as a potential effective biosorbent for wastewater treatment. This research not only enlightens the understanding of γ-PGA's role in heavy metal remediation but also underscores its potential as a biodegradable and non-toxic alternative for environmental cleanup. The findings pave the way for further exploration into the mechanisms and kinetics of γ-PGA's adsorption properties.

Examining the wound healing potential of curcumin-infused electrospun nanofibers from polyglutamic acid and gum arabic.

Advancements in medicine have led to continuous enhancements and innovations in wound dressing materials, making them pivotal in medical care. We used natural biological macromolecules, γ-polyglutamic acid and gum arabic as primary raw materials to create nanofibers laden with curcumin by blending electrostatic spinning technology in the current investigation. These nanofibers were meticulously characterized using fluorescence microscopy, scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction (XRD). Our comprehensive analyses confirmed the successful encapsulation of curcumin within the nanofiber carrier and it has uniform diameter, good water absorption and mechanical properties. Subsequently, we evaluated the antimicrobial effects of these curcumin-loaded nanofibers against Staphylococcus aureus through an oscillating flask method. We created a mouse model with acute full-thickness skin defects to further investigate the wound healing potential. We conducted various biochemical assays to elucidate the mechanism of action. The results revealed that curcumin nanofibers profoundly impacted wound healing. They bolstered the expression of TGF-ß1 and VEGF and reduced the expression of inflammatory factors, leading to an accelerated re-epithelialization process, enhanced wound contraction, and increased regeneration of new blood vessels and hair follicles. Furthermore, these nanofibers positively influenced the proportion of three different collagen types. This comprehensive study underscores the remarkable potential of curcumin-loaded nanofibers to facilitate wound healing and lays a robust experimental foundation for developing innovative, natural product-based wound dressings.

Fabrication of yeast ß-glucan/sodium alginate/γ-polyglutamic acid composite particles for hemostasis and wound healing.

Particles with a porous structure can lead to quick hemostasis and provide a good matrix for cell proliferation during wound healing. Recently, many particle-based wound healing materials have been clinically applied. However, these products show good hemostatic ability but with poor wound healing ability. To solve this problem, this study fabricated APGG composite particles using yeast ß-glucan (obtained from Saccharomyces cerevisiae), sodium alginate, and γ-polyglutamic acid as the starting materials. The structure of yeast ß-glucan was modified with many carboxymethyl groups to obtain carboxymethylated ß-glucan, which could coordinate with Ca2+ ions to form a crosslinked structure. A morphology study indicated that the APGG particles showed an irregular spheroidal structure with a low density (<0.1 g cm-3) and high porosity (>40%). An in vitro study revealed that the particles exhibited a low BCI value, low hemolysis ratio, and good cytocompatibility against L929 cells. The APGG particles could quickly stop bleeding in a mouse liver injury model and exhibited better hemostatic ability than the commercially available product Celox. Furthermore, the APGG particles could accelerate the healing of non-infected wounds, and the expression levels of CD31, α-SMA, and VEGF related to angiogenesis were significantly enhanced.

Poly(Glutamic Acid-Lysine) Hydrogels with Alternating Sequence Resist the Foreign Body Response in Rodents and Non-Human Primates.

The foreign body response (FBR) to implanted biomaterials and biomedical devices can severely impede their functionality and even lead to failure. The discovery of effective anti-FBR materials remains a formidable challenge. Inspire by the enrichment of glutamic acid (E) and lysine (K) residues on human protein surfaces, a class of zwitterionic polypeptide (ZIP) hydrogels with alternating E and K sequences to mitigate the FBR is prepared. When subcutaneously implanted, the ZIP hydrogels caused minimal inflammation after 2 weeks and no obvious collagen capsulation after 6 months in mice. Importantly, these hydrogels effectively resisted the FBR in non-human primate models for at least 2 months. In addition, the enzymatic degradability of the gel can be controlled by adjusting the crosslinking degree or the optical isomerism of amino acid monomers. The long-term FBR resistance and controlled degradability of ZIP hydrogels open up new possibilities for a broad range of biomedical applications.

Cucumber (Cucumis sativus L.) with heterologous poly-γ-glutamic acid has skin moisturizing, whitening and anti-wrinkle effects.

Three genes involved in poly-γ-glutamic acid(γ-PGA)synthesis cloned from Bacillus licheniformis were transformed into cucumber for the first time. Compared with control, its water content increased by 6-14 % and water loss rate decreased by 11-12 %. In zebrafish and human skin experiments, the moisturizing effect of transgenic cucumber was significantly higher than that of CK, γ-PGA and hyaluronic acid group. Transgenic cucumber reduced facial wrinkles and roughness by 19.58 % and 24.97 %, reduced skin melanin content by 5.27 %, increased skin topological angle and L-value by 5.89 % and 2.49 %, and increased the R2 and Q1 values of facial elasticity by 7.67 % and 5.64 %, respectively. The expressions of aqp3, Tyr, silv and OCA2 were down-regulated, eln1, eln2, col1a1a and col1a1b were up-regulated in zebrafish after treated with transgenic cucumber. This study provides an important reference for the endogenous synthesis of important skin care functional molecules in plants.

Incorporation of poly(γ-glutamic acid) in lipid nanoparticles for enhanced mRNA delivery efficiency in vitro and in vivo.

Messenger RNA (mRNA)-based therapy shows immense potential for broad biomedical applications. However, the development of safe and efficacious mRNA delivery vectors remains challenging due to delivery barriers and inefficient intracellular payload release. Herein, we presented a simple strategy to boost the mRNA intracellular release by incorporation of anionic poly(γ-glutamic acid) (PGA) into an ionizable lipid-based LNP/mRNA. We systematically investigated the impact of PGA incorporation on mRNA transfection both in vitro and in vivo. The molecular weights and formulation ratios of PGA greatly affected the transfection efficacy of LNP/mRNA. From in vitro study, the optimized LNP/mRNA/PGA was formulated by incorporation of PGA with the molecular weight of 80 kDa or 200 kDa and the charge ratio (N/P/C) of 25/1/1. The optimized formulation achieved around 3-fold mRNA expression in HeLa cells compared to the bare LNP/mRNA. The intracellular releasing study using specific DNA probe revealed that this enhancement of transfection efficacy was attributed to the elevated mRNA release into cytoplasm. Moreover, the optimized LNP/mRNA/PGA achieved up to 5-fold or 3-fold increase of luciferase mRNA expression in vivo after being injected into mice systematically or intramuscularly, respectively. In addition, the incorporation of PGA did not significantly alter the biodistribution profile of the complexes on both organ and cellular levels. Therefore, our work provides a simple strategy to boost mRNA delivery, which holds great promise to improve the efficacy of mRNA therapeutics for various biomedical applications. STATEMENT OF SIGNIFICANCE: The process of designing and screening potent mRNA carriers is complicated and time-consuming, while the efficacy is not always satisfying due to the delivery barriers and inefficient mRNA release. This work presented an alternative strategy to boost the mRNA delivery efficacy by incorporating an anionic natural polymer poly(γ-glutamic acid) (PGA) into LNP/mRNA complexes. The optimized LNP/mRNA/PGA achieved up to 3-fold and 5-fold increase in transfection efficacy in vitro and in vivo, respectively. Intracellular releasing analysis revealed that the enhancement of transfection efficacy was mainly attributed to the elevated intracellular release of mRNA. In addition, the incorporation of PGA did not alter the biodistribution or the biosafety profile of the complexes. These findings indicate that PGA incorporation is a promising strategy to improve the efficacy of mRNA therapeutics.

Effects of γ-polyglutamic acid on grassland sandy soil properties and plant functional traits exposed to drought stress.

The current study provides field experimental data that support the use of γ-polyglutamic acid (γ-PGA) in drought stress and proposes its application in grassland management. We hypothesized that water treatment combined with PGA application to sandy soil would reduce drought stress in grasslands more effectively than watering alone. A randomized block design was used, with three replicate watering blocks (no watering, weekly watering, and monthly watering) and PGA treatments at four different concentrations (0%, 0.3%, 1%, and 2% PGA). The results showed that PGA acts as a biostimulant, alleviating the effects of stress in plants by: (1) increasing the availability of ions, especially K+, Zn2+, Mn2+, Fe2+/3+, Ca2+, and Mg2+, as well as N-NH4+, and N-NO3-, (2) elongating plant roots, (3) increasing the aboveground biomass, (4) improving the resprouting capacity of the dominant grass Nardus stricta, and (5) improving the regeneration of dicotyledons. In the case of meadows on sandy soils, the use of low PGA concentrations (0.3% or 1%) was the most beneficial for the availability of macro- and microelements and improving the functional traits of plants. Irrigation had a greater effect than using PGA only for the dicotyledon to monocotyledon ratio.

Culture-dependent and -independent analyses of bacterial compositions and its contributions to formation of γ-aminobutyric acid and poly-γ-glutamic acid in Cheonggukjang.

This study was performed to unveil bacterial compositions and their contributions to the formation of γ-aminobutyric acid (GABA) and poly-γ-glutamic acid (γ-PGA) in Cheonggukjang. To predict possible key factors contributing to the content of the bioactive compounds in Cheonggukjang, commercial products were analyzed for various parameters. The content of GABA and γ-PGA showed a negative (R2 = 0.61 - 0.73) and positive correlation (R2 = 0.53 - 0.96) with antioxidative activity. Consistently, GABA content showed a moderate negative correlation with γ-PGA content (R2 = 0.58). Among the physicochemical and microbial parameters, only salinity showed a moderate negative correlation with γ-PGA content (R2 = 0.75), which might be due to the inhibition of bacterial growth. It was also suggested that multiple factors (including bacterial species) were involved in the formation of GABA and γ-PGA in Cheonggukjang. To reveal dominant bacterial species and further presume their contributions to the bioactive compound formation in Cheonggukjang, both culture-independent (metagenomic) and -dependent (culturomic) methods were used. Culture-independent method showed that Bacillus piscis was dominant (23.37 - 94.89 %), followed by B. hisashii (0.00 - 62.45 %) and B. coagulans (0.00 - 13.82 %). Considering the quantitative speciation data on the bioactive compound content in Cheonggukjang (and bacterial production capability) together, it was further elucidated that B. piscis contributed primarily to the bioactive compound formation. Unlike this, culture-dependent analysis revealed that B. licheniformis and B. subtilis were dominant (30.0 - 47.6 and 17.5 - 39.5 %, respectively). Based on the quantitative speciation data on the bacterial production capability of GABA and γ-PGA, B. subtilis was the primarily contributing bacterial species to the bioactive compound formation. Consequently, it was observed that the bacterial compositions and their contributions to the bioactive compound formation determined by the two methods differed considerably, i.e., B. piscis and B. subtilis were identified to be prominent bacterial contributors, respectively, depending on the method used.

Antidiabetic effects and mechanism of γ-polyglutamic acid on type II diabetes mice.

Diabetes is one of the foremost chronic non-communicable diseases worldwide, which significantly impacts people's quality of life. This study aimed to investigate the hypoglycemic effects of γ-polyglutamic acid (γ-PGA) on STZ-induced type II diabetes mice and its potential mechanisms. The results indicated that γ-PGA intervention contributed to reducing fasting blood glucose levels in diabetic mice, regulating lipid metabolism in type II diabetes mice, and improving insulin resistance. Additionally, γ-PGA could alleviate liver inflammation, enhancing the activity of hepatic antioxidant enzymes. Investigation into the insulin signaling pathway revealed that γ-PGA significantly increased the expression of INSR, IRS-1, Akt, PI3K in diabetic mice, thereby enhancing insulin sensitivity and improving insulin resistance to regulate glucose metabolism. High-throughput sequencing of mouse gut microbiota using 16S rRNA showed that γ-PGA increased the abundance and evenness of beneficial bacteria in the intestines of type II diabetic mice, inhibited the growth of harmful bacteria, and may exerted hypoglycemic effects by modulating and improving relevant metabolic pathways associated with diabetes symptoms. This study provides new insights into the treatment of type II diabetes and highlights the significant potential of γ-PGA in treating type II diabetes.