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γ-polyglutamic acid (γ-PGA), as an environmentally sustainable material, is extensive applied in agriculture for enhancing water and fertilizer utilization efficiency, augmenting crop yield, and ameliorating soil conditions. However, the effect of γ-PGA in conjunction with sesame cake fertilizer on the soil environment remains uncertain.The aim of this study is to investigate the effect of γ-PGA on soil nutrients, water use efficiency (WUE) and nitrogen use efficiency (NUE), and maize yield across various levels of sesame cake fertilizer. Additionally, the study seeks to identify the optimal ratio to establish a theoretical and practical foundation for sustainable agricultural development and the promotion of ecological agriculture. Through field experiments, nine treatments were established, comprising three levels of sesame cake fertilizer application rates (B1 = 900 kg/hm2 for low fertility, B2 = 1100 kg/hm2 for medium fertility, and B3 = 1300 kg/hm2 for high fertility) and three levels of γ-PGA application rates (R1 = 200 kg/hm2, R2 = 400 kg/hm2, and R3 = 600 kg/hm2). The results can be outlined as follows: (1) When γ-PGA application rate increased, total nitrogen (TN) exhibited a synergistic effect under B1 treatment, but an antagonistic effect under B2 and B3 treatments. At the 6-leaf stage (V6), 12-leaf stage (V12), and tasseling stage (VT), available phosphorus (AP) exhibited antagonistic effects. However, at the filling stage (R2) and maturity stage (R6), AP in B1 and B2 treatments at various depths underwent partial transformation into a synergistic effect. The levels of available potassium exhibited a notable antagonistic effect, leading to a decrease in harvest index (HI). B2 treatment demonstrated superior results compared to the B1 and B3 treatments, with the highest levels observed under B2R1 treatment; (2) TN content in the 0-40 cm soil layer increased during the filling period, and it was uniformly distributed in the 40-60 cm soil layer. When the soil AP was located in the 0-60 cm soil layer, there was an increase in AP content during the mature period. Following the tasseling period, different treatments exhibited varying patterns of increase in response to the presence of potassium within the 0-60 cm soil layer. Consequently, in cases where the sesame cake fertilizer content is low, the interaction between γ-PGA can compensate for the deficiency of fertilizer, thereby enhancing water and nitrogen utilization efficiency. The optimal fertilization strategy for enhancing soil nutrient distribution, WUE and NUE, and yield is proposed to be the application of 1100 kg/hm2 sesame cake fertilizer and 200 kg/hm2 γ-PGA.

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Poly-γ-glutamic acid (γ-PGA) is a carboxylic-acid-rich, bio-derived, water-soluble, edible, hydrating, non-immunogenic polymer produced naturally by several microorganisms. Here, we re-emphasise the ability of Bacillus subtilis natto to naturally produce γ-PGA on whole seaweed, as well as for the yields and chemical properties of the material to be affected by the presence of Mn(2+). Hyaluronic acid (HA) is an extracellular glycosaminoglycan which presents a high concentration of carboxylic acid and hydroxyl groups, being key in fulfilling numerous applications. Currently, there are strong environmental (solvent use), social (non-vegan extraction), and economic factors pushing for the biosynthesis of this material through prokaryotic microorganisms, which is not yet scalable or sustainable. Our study aimed to investigate an innovative raw material which can combine both superior hygroscopicity and UV protection to the cosmetic industry. Comparable hydration effect of commercially available γ-PGA to conventional moisturising agents (HA and glycerol) was observed; however, greater hydration capacity was observed from seaweed-derived γ-PGA. Herewith, successful incorporation of seaweed-derived γ-PGA (0.2-2 w/v%) was achieved for several model cream systems with absorbances reported at 300 and 400 nm. All γ-PGA-based creams displayed shear thinning behaviour as the viscosity decreased, following increasing shear rates. Although the use of commercial γ-PGA within creams did not suggest a significant effect in rheological behaviour, this was confirmed to be a result of the similar molecular weight. Seaweed-derived γ-PGA cream systems did not display any negative effect on model HaCaT keratinocytes by means of in vitro MTT analysis.

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Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

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Drought stress is the main factor restricting maize yield. Poly-γ-glutamic acid (γ-PGA), as a water-retaining agent and fertilizer synergist, could significantly improve the drought resistance and yield of many crops. However, its high production costs and unclear long-term impact on soil ecology limit its large-scale application. In this study, an environmentally friendly green material γ-PGA was heterologous synthesized in maize for the first time using the synthetic biology method. The genes (PgsA, PgsB, PgsC) participated in γ-PGA synthesis were cloned from Bacillus licheniformis and transformed into maize to produce γ-PGA for the first time. Under drought stress, transgenic maize significantly increased the ear length, ear weight and grain weight by 50 % compared to the control, whereas the yield characteristic of ear weight, grain number per ear, grain weight per ear and 100-grain weight increased by 1.67 %-2.33 %, 3.78 %-13.06 %, 8.41 %-22.06 %, 6.03 %-19.28 %, and 11.85 %-18.36 %, respectively under normal growth conditions. γ-PGA was mainly expressed in the mesophyll cells of maize leaf rosette structure and improved drought resistance and yield by protecting and increasing the expression of genes for the photosynthetic and carbon fixation. This study is an important exploration for maize drought stress molecular breeding and building resource-saving agriculture.

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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.

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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.

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Recently, metal-organic frameworks (MOFs) have been widely developed due to the rich porosity, excellent framework structure and multifunctional nature. Meanwhile, a series of MOFs crystals and MOF-based composites have been emerged. However, the widespread applications of MOFs are hindered by challenges such as rigidity, fragility, solution instability, and processing difficulties. In this study, we addressed these limitations by employing an in-situ green growth approach to prepare a zeolitic imidazolate frameworks-8@poly (γ-glutamic acid) hydrogel (ZIF-8@γ-PGA) with hierarchical structures. This innovative method effectively resolves the inherent issues associated with MOFs. Furthermore, the ZIF-8@γ-PGA hydrogel is utilized for dye adsorption, demonstrating an impressive maximum adsorption capacity of 1130 ± 1 mg/g for methylene blue (MB). The adsorption behavior exhibits an excellent agreement with both the kinetic model and isotherm. Meanwhile, because the adsorbent raw materials are all green non-toxic materials, multiple applications of materials can also be realized. Significantly, the results of antibacterial experiments showed that the ZIF-8@γ-PGA hydrogel after in-situ growth of ZIF-8 had better antibacterial properties. Thus, the ZIF-8@γ-PGA hydrogel has great potential for development in wound dressings, sustained drug owing to its biocompatibility and antibacterial activity.

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Water stress brought about by climate change is among the major global concerns threatening food security. Rice is an important staple food which requires high water resources. Being a semi-aquatic plant, rice is particularly susceptible to drought. The aim of this work was to develop techniques directed to promote rice resilience to water deprivation stress during germination by implementing specific seed priming treatments. Five popular Italian rice varieties were subjected to priming treatments using novel, sustainable solutions, like poly-gamma-glutamic acid (γ-PGA), denatured γ-PGA (dPGA), and iron (Fe) pulsing, alone or in combination. The effect of the developed priming methods was tested under optimal conditions as well as under water deprivation stress imposed by polyethylene glycol (PEG) treatments. The priming efficacy was phenotypically determined in terms of germination behavior by measuring a series of parameters (germinability, germination index, mean germination time, seed vigor index, root and shoot length, germination stress tolerance index). Biochemical analyses were carried out to measure the levels of iron uptake and accumulation of reactive oxygen species (ROS). Integrative data analyses revealed that the rice varieties exhibited a strong genotype- and treatment-specific germination behavior. PEG strongly inhibited germination while most of the priming treatments were able to rescue it in all varieties tested except for Unico, which can be defined as highly stress sensitive. Molecular events (DNA repair, antioxidant response, iron homeostasis) associated with the transition from seed to seedling were monitored in terms of changes in gene expression profiles in two varieties sensitive to water deprivation stress with different responses to priming. The investigated genes appeared to be differentially expressed in a genotype-, priming treatment-, stress- and stage-dependent manner. The proposed seed priming treatments can be envisioned as sustainable and versatile agricultural practices that could help in addressing the impact of climate challenges on the agri-food system.

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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.

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Microbial biopolymers have emerged as promising solutions for environmental pollution-related human health issues. Poly-γ-glutamic acid (γ-PGA), a natural anionic polymeric compound, is composed of highly viscous homo-polyamide of D and L-glutamic acid units. The extracellular water solubility of PGA biopolymer facilitates its complete biodegradation and makes it safe for humans. The unique properties have enabled its applications in healthcare, pharmaceuticals, water treatment, foods, and other domains. It is applied as a thickener, taste-masking agent, stabilizer, texture modifier, moisturizer, bitterness-reducing agent, probiotics cryoprotectant, and protein crystallization agent in food industries. γ-PGA is employed as a biological adhesive, drug carrier, and non-viral vector for safe gene delivery in tissue engineering, pharmaceuticals, and medicine. It is also used as a moisturizer to improve the quality of hair care and skincare cosmetic products. In agriculture, it serves as an ideal stabilizer, environment-friendly fertilizer synergist, plant-growth promoter, metal biosorbent in soil washing, and animal feed additive to reduce body fat and enhance egg-shell strength.

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BACKGROUND: Natto mucus is mainly composed of poly(γ-glutamic acid) (γ-PGA), which affects the sensory quality of natto and has some effective functional activities. The soybean metabolites that cause different γ-PGA contents in different fermented natto are unclear. RESULTS: In this study, we use untargeted metabolomics to analyze the metabolites of high-production γ-PGA natto and low-production γ-PGA natto and their fermented substrate soybean. A total of 257 main significantly different metabolites with the same trend among the three comparison groups were screened, of which 114 were downregulated and 143 were upregulated. Through the enrichment of metabolic pathways, the metabolic pathways with significant differences were purine metabolism, nucleotide metabolism, fructose and mannose metabolism, anthocyanin biosynthesis, isoflavonoid biosynthesis and the pentose phosphate pathway. CONCLUSION: For 114 downregulated main significantly different metabolites with the same trend among the three comparison groups, Bacillus subtilis (natto) may directly decompose them to synthesize γ-PGA. Adding downregulated substances before fermentation or cultivating soybean varieties with the goal of high production of such substances has a great effect on the production of γ-PGA by natto fermentation. The enrichment analysis results showed the main pathways affecting the production of γ-PGA by Bacillus subtilis (natto) using soybean metabolites, which provides a theoretical basis for the production of γ-PGA by soybean and promotes the diversification of natto products. © 2023 Society of Chemical Industry.

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Brewer's spent grain (BSG) is a main byproduct of the beer industry. BSG is rich in a variety of nutrients, and the search for its effective, high-value utilization is ongoing. Environmental probiotic factor γ-PGA was produced by fermenting Bacillus subtilis with BSG substrate and the fermenting grain components were analyzed. The γ-PGA yield reached 31.58 ± 0.21 g/kg of BSG. Gas chromatography-mass spectrometry and non-targeted metabolomics analyses revealed 73 new volatile substances in the fermenting grains. Furthermore, 2,376 metabolites were upregulated after fermentation and several components were beneficial for plant growth and development (such as ectoine, acetyl eugenol, L-phenylalanine, niacin, isoprene, pantothenic acid, dopamine, glycine, proline, jasmonic acid, etc). These results show that it is possible to synthesize adequate amounts of γ-PGA for use as a functional fertilizer.

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Previously, a novel Corynebacterium glutamicum strain for the de novo biosynthesis of tailored poly-γ-glutamic acid (γ-PGA) has been constructed by our group. The strain was based on the γ-PGA synthetase complex, PgsBCA, which is the only polyprotein complex responsible for γ-PGA synthesis in Bacillus spp. In the present study, PgsBCA was reconstituted and overexpressed in C. glutamicum to further enhance γ-PGA synthesis. First, we confirmed that all the components (PgsB, PgsC, and PgsA) of γ-PGA synthetase derived from B. licheniformis are necessary for γ-PGA synthesis, and γ-PGA was detected only when PgsB, PgsC, and PgsA were expressed in combination in C. glutamicum. Next, the expression level of each pgsB, pgsC, and pgsA was tuned in order to explore the effect of expression of each of the γ-PGA synthetase subunits on γ-PGA production. Results showed that increasing the transcription levels of pgsB or pgsC and maintaining a medium-level transcription level of pgsA led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. Notably, the expression level of pgsC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis, followed by pgsB. Next, genes encoding for PgsC from four different sources (Bacillus subtilis, Bacillus anthracis, Bacillus methylotrophicus, and Bacillus amyloliquefaciens) were tested in order to identify the influence of PgsC-encoding orthologues on γ-PGA production, but results showed that in all cases the synthesis of γ-PGA was significantly inhibited. Similarly, we also explored the influence of gene orthologues encoding for PgsB on γ-PGA production, and found that the titer increased to 17.14 ± 0.62 g/L from 8.24 ± 0.10 g/L when PgsB derived from B. methylotrophicus replaced PgsB alone in PgsBCA from B. licheniformis. The resulting strain was chosen for further optimization, and we achieved a γ-PGA titer of 38.26 g/L in a 5 L fermentor by optimizing dissolved oxygen level. Subsequently, by supplementing glucose, γ-PGA titer increased to 50.2 g/L at 48 h. To the best of our knowledge, this study achieved the highest titer for de novo production of γ-PGA from glucose, without addition of L-glutamic acid, resulting in a novel strategy for enhancing γ-PGA production.

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Oral ecto-mesenchymal stem cells-conditional medium (EMSCs-CM) is a promising strategy for treating ulcerative colitis (UC). However, this therapy is currently limited by the harsh gastrointestinal environment and poor colonic targeting ability. Herein, a glutamine transaminase 2 (TG2) crosslinked EMSCs-CM hydrogel (EMSCs-CM-Gel) was fabricated by combining EMSCs-CM with negatively chargedγ-polyglutamic acid (γ-PGA) hydrogel. Intestinal epithelial cell 6 (IEC-6) was applied to construct a cell model with lipopolysaccharide to evaluate the anti-inflammatory potential of EMSCs-CMin vitro. The crosslinked gel was orally administered to mice in liquid form to access the effects of EMSCs-CM-Gelin vivo. This study was based on the fact that the hydrogel containing EMSCs-CM has negative charges, which ensure it remains at the positively charged inflamed colon tissue. The EMSCs-CM could continuously be released in the damaged colon mucosa along with the degradation of theγ-PGA hydrogel. Immunofluorescence and western blot were performed to assess the effects of EMSCs-CM-Gel on mice. The resultsin vivoshowed that EMSCs-CM-Gel could significantly suppress the expression of inflammatory cytokines, prevent the shortening of the length of the intestine and repair the intestinal barrier. Collectively, our findings provided a novel colon-targeted strategy, hoping to benefit UC patients a lot.

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Vaccination is an innovative strategy for cancer treatment by leveraging various components of the patients' immunity to boost an anti-tumor immune response. Rationally designed nanoparticles are well suited to maximize cancer vaccination by the inclusion of immune stimulatory adjuvants. Also, nanoparticles might control the pharmacokinetics and destination of the immune potentiating compounds. Poly-γ-glutamic acid (γ-PGA) based nanoparticles (NPs), which have a natural origin, can be easily taken up by dendritic cells (DCs), which leads to the secretion of cytokines which ameliorates the stimulation capacity of T cells. The intrinsic adjuvant properties and antigen carrier properties of γ-PGA NPs have been the focus of recent investigations as they can modulate the tumor microenvironment, can contribute to systemic anti-tumor immunity and subsequently inhibit tumor growth. This review provides a comprehensive overview on the potential of γ-PGA NPs as antigen carriers and/or adjuvants for anti-cancer vaccination.

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Biological methods are currently the most commonly used methods for removing hazardous substances from land. This research work focuses on the remediation of oil-contaminated land. The biodegradation of aliphatic hydrocarbons and PAHs as a result of inoculation with biopreparations B1 and B2 was investigated. Biopreparation B1 was developed on the basis of autochthonous bacteria, consisting of strains Dietzia sp. IN118, Gordonia sp. IN101, Mycolicibacterium frederiksbergense IN53, Rhodococcus erythropolis IN119, Rhodococcus globerulus IN113 and Raoultella sp. IN109, whereas biopreparation B2 was enriched with fungi, such as Aspergillus sydowii, Aspergillus versicolor, Candida sp., Cladosporium halotolerans, Penicillium chrysogenum. As a result of biodegradation tests conducted under ex situ conditions for soil inoculated with biopreparation B1, the concentrations of TPH and PAH were reduced by 31.85% and 27.41%, respectively. Soil inoculation with biopreparation B2 turned out to be more effective, as a result of which the concentration of TPH was reduced by 41.67% and PAH by 34.73%. Another issue was the phytoremediation of the pre-treated G6-3B2 soil with the use of Zea mays. The tests were carried out in three systems (system 1-soil G6-3B2 + Zea mays; system 2-soil G6-3B2 + biopreparation B2 + Zea mays; system 3-soil G6-3B2 + biopreparation B2 with γ-PGA + Zea mays) for 6 months. The highest degree of TPH and PAH reduction was obtained in system 3, amounting to 65.35% and 60.80%, respectively. The lowest phytoremediation efficiency was recorded in the non-inoculated system 1, where the concentration of TPH was reduced by 22.80% and PAH by 18.48%. Toxicological tests carried out using PhytotoxkitTM, OstracodtoxkitTM and Microtox® Solid Phase tests confirmed the effectiveness of remediation procedures and showed a correlation between the concentration of petroleum hydrocarbons in the soil and its toxicity. The results obtained during the research indicate the great potential of bioremediation practices with the use of microbial biopreparations and Zea mays in the treatment of soils contaminated with petroleum hydrocarbons.

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(-)-Epigallocatechin gallate (EGCG) has gained considerable attention owing to its beneficial properties. However, its application as a functional food is restricted due to its instability and low bioavailability. In the present study, a food-derived nanoparticle system based on type A gelatin/γ-PGA was developed to preserve and deliver EGCG. The EGCG/gelatin/γ-PGA nanoparticles had a particle size of 155.1 ± 7.3 nm with a zeta potential of -23.9 ± 0.9 mV. Moreover, the EGCG/gelatin/γ-PGA nanoparticles enhanced the long-term storage stability and sustained antioxidant activity of EGCG compared to EGCG/gelatin nanoparticles. The nanoparticles protected EGCG in simulated gastric fluid containing pepsin while releasing it in simulated intestinal fluid. Additionally, the amount of EGCG transported in the Caco-2 monolayers treated with EGCG/gelatin/γ-PGA nanoparticles was three times higher than that of free EGCG, which might be related to the paracellular pathway and endocytosis. These results suggest that EGCG/gelatin/γ-PGA nanoparticles might be an effective delivery vehicle for EGCG, enhancing its potential applications in the functional food field.

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BACKGROUND: Poly-γ-glutamic acid (γ-PGA) is biodegradable, water-soluble, environment-friendly, and edible. Consequently, it has a variety of industrial applications. It is crucial to control production cost and increase output for industrial production γ-PGA. RESULTS: Here γ-PGA production from sugarcane molasses by Bacillus licheniformis CGMCC NO. 23967 was studied in shake-flasks and bioreactors, the results indicate that the yield of γ-PGA could reach 40.668 g/L in a 5L stirred tank fermenter. Further study found that γ-PGA production reached 70.436 g/L, γ-PGA production and cell growth increased by 73.20% and 55.44%, respectively, after FeSO4·7H2O was added. Therefore, we investigated the metabolomic and transcriptomic changes following FeSO4·7H2O addition. This addition resulted in increased abundance of intracellular metabolites, including amino acids, organic acids, and key TCA cycle intermediates, as well as upregulation of the glycolysis pathway and TCA cycle. CONCLUSIONS: These results compare favorably with those obtained from glucose and other forms of biomass feedstock, confirming that sugarcane molasses can be used as an economical substrate without any pretreatment. The addition of FeSO4·7H2O to sugarcane molasses may increase the efficiency of γ-PGA production in intracellular.

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Poly-γ-glutamic acid (γ-PGA) is a biopolymer with a wide range of applications, mainly produced using Bacillus strains. The formation and concomitant secretion of γ-PGA increases the culture broth viscosity, while enzymatic depolymerisation and degradation of γ-PGA decreases the culture broth viscosity. In this study, the recently published ViMOS (Viscosity Monitoring Online System) is applied for optical online measurements of broth viscosity in eight parallel shake flasks. It is shown that the ViMOS is suitable to monitor γ-PGA production and degradation online in shake flasks. This online monitoring enables the detailed analysis of the Ppst promoter and γ-PGA depolymerase knockout mutants in genetically modified Bacillus subtilis 168. The Ppst promoter becomes active under phosphate starvation. The different single depolymerase knockout mutants are ∆ggt, ∆pgdS, ∆cwlO and a triple knockout mutant. An increase in γ-PGA yield in gγ-PGA /gglucose of 190% could be achieved with the triple knockout mutant compared to the Ppst reference strain. The single cwlO knockout also increased γ-PGA production, while the other single knockouts of ggt and pgdS showed no impact. Partial depolymerisation of γ-PGA occurred despite the triple knockout. The online measured data are confirmed with offline measurements. The online viscosity system directly reflects γ-PGA synthesis, γ-PGA depolymerisation, and changes in the molecular weight. Thus, the ViMOS has great potential to rapidly gain detailed and reliable information about new strains and cultivation conditions. The broadened knowledge will facilitate the further optimization of γ-PGA production.

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Poly-γ-glutamic acid (γ-PGA) is a natural polymer composed of glutamic acid monomer and it has garnered substantial attention in both the fields of material science and biomedicine. Its remarkable cell compatibility, degradability, and other advantageous characteristics have made it a vital component in the medical field. In this comprehensive review, we delve into the production methods, primary application forms, and medical applications of γ-PGA, drawing from numerous prior studies. Among the four production methods for PGA, microbial fermentation currently stands as the most widely employed. This method has seen various optimization strategies, which we summarize here. From drug delivery systems to tissue engineering and wound healing, γ-PGA's versatility and unique properties have facilitated its successful integration into diverse medical applications, underlining its potential to enhance healthcare outcomes. The objective of this review is to establish a foundational knowledge base for further research in this field.