Abstract: Co-immobilization of cells and enzymes is often essential for the cascade biocatalytic processes of industrial-scale feasibility but remains a vast challenge. Herein, we create a facile co-immobilization platform integrating enzymes and cells in covalent organic frameworks (COFs) to realize the highly efficient cascade of inulinase and E. coli for bioconversion of natural products. Enzymes can be uniformly immobilized in the COF armor, which coats on the cell surface to produce cascade biocatalysts with high efficiency, stability and recyclability. Furthermore, this one-pot in situ synthesis process facilitates a gram-scale fabrication of enzyme-cell biocatalysts, which can generate a continuous-flow device conversing inulin to D-allulose, achieving space-time yield of 161.28 g L

Abstract: This research systematically assessed the changes in carbon, nitrogen and microbial profiling during pig and chicken manure transformation by black soldier fly larvae (BSFL) and subsequent composting process. BSFL had higher conversion efficiency for chicken manure. The pH, phosphorus and potassium contents in fresh BSFL frass increased than raw manure, but conductivity, total-/nitrate-/ammonium-nitrogen decreased. After BSFL conversion, pig manure had a larger nitrogen loss (25 %) while chicken manure had a larger carbon loss (32 %). During subsequent composting, the indicator changes (e.g. humus, ammonium nitrogen) in frass composts basically remained stable after 20-30 days. Compared to natural composts, frass composts had higher humification degree, cellulase activities, and more cellulose-degrading bacteria. Subsequent composting further reduced potential pathogens (reduced by 98.9 %-99.7 % than raw manure), and elevated the aromaticity and humification of frass. The findings gave an insight into the maturation management of manure-sourced insect frass.

Abstract: Bound extracellular polymeric substances (EPS) are complex, high-molecular-weight polymer mixtures that play a critical role in pore clogging, foulants adhesion, and fouling layer formation during membrane filtration, owing to their adhesive properties and gelation tendencies. In this study, a novel electrochemical anaerobic membrane bioreactor (EC-AnMBR) was constructed to investigate the effect of sludge bound-EPS solubilization on methane bioconversion and membrane fouling mitigation. During the 150-days' operation, the EC-AnMBR demonstrated remarkable performance, characterized by an exceptionally low fouling rate (transmembrane pressure (TMP) < 4.0 kPa) and high-quality effluent (COD removal > 98.2 %, protein removal > 97.7 %, and polysaccharide removal > 98.5 %). The highest methane productivity was up to 38.0 ± 3.1 mL/L

Abstract: Innovative solutions are needed to limit environmental effect and optimise resource use as food waste generation rises worldwide. This study investigates the potential of upcycling food waste from fresh markets using Black Soldier Fly (Hermetia illucens) larvae (BSFL) as a sustainable approach. This study explored four fresh market food waste substrates for BSFL bioconversion: discarded fish waste (FI), slaughtered chicken waste (CHI), vegetable waste (VEG), and a 1:1:1 combination of all three (MIX). Soybean curd residue (SCR) was treated as the control substrate. The effects on larval growth, nutritional content, and waste bioconversion rates were examined. The larvae growth rate was strongly impacted by waste type, with BSF-fed CHI and MIX gaining 18.0 and 16.7 mg/d, respectively, followed by BSF-fed with SCR (12.2 mg/d), FI (8.9 mg/d) and VEG (7.6 mg/d). The waste type did not substantially alter BSFL length. The survival rate of the BSFL fed with the food waste studied ranges from 95 to 98.47%, with SCR being the highest. Our findings indicated that BSFL can effectively convert a variety of fresh market food waste into valuable biomass. CHI waste produced the highest larval biomass and bioconversion rate followed by MIX, SCR, FI and VEG. The different waste stream has a major influence on BSFL biomass nutrition. BSFL nutritional composition is independent of the substrate's nutritional content, indicating no direct correlation between substrate and BSFL biomass nutritional composition. SCR waste produced the highest protein content of BSFL (50.49%), followed by VEG (32.61%), MIX (32.57%), FI (31.03%) and CHI (29.06%). SCR waste also produced BSFL biomass with lowest lipid content (26.55%) compared to other waste which resulted into BSFL with lipid levels ranging from 42.92% to 53.72%. BSFL-fed with SCR is the most suitable to be used as an alternative animal's feed based on the protein and lipid levels, while defatting procedure is necessary for the other waste-fed BSFL to render it suitability as animal feed alternatives. Based on bioconversion rate, BSFL growth, and lipid content, the MIX and CHI waste might be viable substrates for future research.

Abstract: Abstract The increasing prevalence of metabolic diseases and the global drive toward achieving Sustainable Development Goals (SDGs) underscore the need for sustainable, nutrient‐dense foods. Soybeans ( Glycine max ), a critical global crop, offer promising solutions; however, their predominant use as animal feed raises concerns regarding food security and environmental sustainability. Fermented soy products—including tempeh, natto, and miso—are rich in bioactive compounds such as peptides and isoflavones, which offer potential therapeutic effects and hold cultural and nutritional significance. These fermented products provide bioactive profiles with unique health‐promoting properties. This review critically examines the bioactive compounds generated through fermentation, focusing on their bioconversion pathways in the gastrointestinal tract and their metabolic implications for human health. Recent consumer demand for novel food ingredients with additional biological benefits has fueled research into advanced extraction techniques, enhancing the functional applications of bioactive compounds from these soy‐based products. This review further explores innovations in extraction methods that improve bioactive yield and sustainability, reinforcing the applicability of these compounds in health‐promoting food interventions. The originality of this review lies in its in‐depth exploration of the gastrointestinal bioconversion of fermented soy bioactive compounds alongside the latest sustainable extraction methods designed to optimize their use. Future research should aim to refine fermentation and extraction processes, investigate synergistic microbial interactions, and develop environmentally sustainable production methods. These efforts have the potential to position fermented soy products as essential contributors to global nutritional security and sustainable food systems, addressing both public health and environmental needs.

Abstract: Bioconversion of lignin into valuable chemicals could promote both lignin valorization and biomass-based economy. However, the heterogeneity of lignin and the lack of an effective conversion route limit the efficiency of lignin utilization. In this study, the cell factory of Pseudomonas putida KT2440 was successfully designed for converting three types of lignin into cis, cis-muconic acid (CCMA). Heterologous engineering of the native O-demethylation system enabled the metabolism of S-type lignin by P. putida KT2440. The biosynthesis pathway of CCMA was designed to simultaneously funnel three types of lignin-derived aromatics, achieving a remarkable titer and yield of 13.1 mM and 99.5 %, respectively. The metabolic regulation of NADPH/NADP+ co-factor and PobA dependent co-factor further improved the bioconversion of lignin-derived aromatics and CCMA production. Implementing a co-feeding strategy substantially increased NADPH availability, resulting in enhanced CCMA production of up to 46.5 mM with a molar yield of 96.9 %. The molecular mechanism analysis demonstrated that the designed cell factory exhibited superior ligninolytic capacity in cleaving β-O-4 linkage and potentially degrading S- and G-type lignin. It showcased outstanding conversion capacity of actual lignin hydrolysates by consuming all aromatic compounds and more hydrophilic lignin molecules, producing 9.6 mM CCMA in a fed-batch fermentation mode. Therefore, the P. putida KT2440 cell factory is a promising bacterial chassis for both biological lignin valorization and production of valuable chemicals.

Abstract: Biowaste generation is increasing worldwide and inadequate disposal has strong negative impacts on food systems and ecosystems. Biodigestion of biowaste using black soldier fly (Hermetia illucens) larvae (BSFL) generates valuable by-products such as animal feed (larval biomass) and organic fertiliser (frass). However, the latter is typically unstable immediately after waste conversion and is thus unsafe for use as a fertilizer in terms of maturity. This study evaluated recirculation of frass within bioconversion of post-consumer food waste (FW) as a dietary component for BSFL to improve the quality of the subsequent frass obtained. Frass was introduced at increasing inclusion levels replacing food waste (2.5-100% on wet-weight basis) as part of the larvae's feeding substrate. Bioconversion efficiency and material reduction were significantly reduced by frass inclusion, while larval yield per experimental unit remained unchanged. When considering only the waste component in the larval diet, larval yield (dry-weight basis) ranged between 207 (0% frass inclusion) and 403 (40% frass inclusion) kg tonne FW

Abstract: Black soldier fly larvae (BSFL, Hermetia illucens (L.)) are recognized for efficient biowaste reduction while yielding valuable proteins and fats for animals. However, lignocellulosic fibers in biowastes are difficult to digest by biowaste and larval digestive tract microorganisms as well as the larvae themselves. This study investigated two biowaste physical pretreatments (thermal, mechanical) for improving BSFL processing of fibrous biowastes. Cow manure, spent grain, and grass clippings were thermally pretreated at 90 °C for three durations (0.5, 1 and 4 h). Contrary to expectations, thermal pretreatment resulted in either no improvement or decreased larval performance on all substrates, regardless of treatment duration. In contrast, mechanical pretreatment of spent grain and grass clippings, involving milling with three screen sizes (0.5, 1 and 2 mm) showed promising results. Specifically, bioconversion rates on 0.5 mm-milled spent grain and grass clippings increased by 0-53 % and 25-44 % dry mass, respectively compared to untreated. Additionally, larval protein conversion increased by 41 % and 23 % on spent grain and grass clippings, respectively. However, mechanical pretreatment did not affect fiber degradation by larval conversion, as hemicellulose decreased by 25 % and 75 % for spent grain and grass clippings, respectively, regardless of particle size. Particle size reduction influenced substrate microbial respiration (CO2 mg/min), with 0.5-mm milled grass clippings exhibiting higher respiration compared to untreated, although this effect was not observed for spent grain. This study highlights mechanical pretreatment's potential in enhancing BSFL bioconversion of fibrous biowastes and the importance of understanding substrate physical properties influencing substrate microorganisms and BSFL.

Abstract: Formaldehyde is an intermediate metabolite of methylotrophic microorganisms that can be obtained from formate and methanol through oxidation–reduction reactions. Formaldehyde is also a one-carbon (C1) compound with high uniquely reactive activity and versatility, which is more amenable to further biocatalysis. Biosynthesis of high-value-added chemicals using formaldehyde as an intermediate is theoretically feasible and promising. This review focuses on the design of the biosynthesis of high-value-added chemicals using formaldehyde as an essential intermediate. The upstream biosynthesis and downstream bioconversion pathways of formaldehyde as an intermediate metabolite are described in detail, aiming to highlight the important role of formaldehyde in the transition from inorganic to organic carbon and carbon chain elongation. In addition, challenges and future directions of formaldehyde as an intermediate for the chemicals are discussed, with the expectation of providing ideas for the utilization of C1.

Abstract: Lignocellulosic biomass is a sustainable feedstock for producing alcohol-based biofuels such as bioethanol and biobutanol that can substitute petroleum-based fuels. However, to reduce the crystallinity of cellulose fibers and eliminate lignin from biomass during the conversion process, it is essential to employ an effective pretreatment method or a series of pretreatment methods before enzymatic hydrolysis and fermentation. The primary obstacles to the profitable production of bioethanol and biobutanol are proper pretreatment methods for lignocellulosic biomass, cost-effective cellulolytic enzyme production, and efficient fermentation of sugars (glucose and xylose). Although novel xylose-fermenting strains are being developed, they frequently lack resistance to inhibitory substances found in biomass hydrolysates. This chapter presents a comprehensive overview of the biological conversion route for the production of lignocellulosic bioethanol and biobutanol, concentrating on the most popular pretreatment techniques and important effective factors for enzymatic hydrolysis and fermentation taking into account sugar and ethanol yields.

Abstract: Using molecular dynamics, machine learning, and density functional theory calculations we make predictions on engineered cytochrome P450 structures and their product distributions.

Abstract: The prevention of lignin repolymerization/condensation in lignocellulose pretreatment can enhance both the bioconversion of cellulose and the quality of the obtained lignin. 2-Naphthol is a very effective pretreatment additive for suppressing lignin repolymerization, which enables overcoming the extraordinarily high recalcitrance of softwood to bioconversion. In this work, new approaches for adding 2-naphthol to the steam explosion pretreatment of softwood were studied. It was found that spray impregnation of spruce wood chips with 2-naphthol dissolved in acetone or ethanol is equally effective as the usually used soak-impregnation. However, spray-impregnation allows for an eight-fold reduction in solvent consumption. 2-Naphthol impregnation enabled practically quantitative cellulose conversion to glucose using an enzyme dosage of 30 FPU (filter paper units) g–1 cellulose, which corresponds to a digestibility enhancement of 89.1% compared to the control without additive. It was further disclosed that spraying of water-suspended 2-naphthol onto wood chips enabled a high glucose yield of 81.9% using an enzyme dosage of 30 FPU g–1 cellulose, which corresponds to an enhancement of 54.8% compared to the control and omits the use of solvents. An economic evaluation showed that spraying dissolved or water-suspended 2-naphthol is more cost-effective than soak-impregnation or admixing 2-naphthol flakes/powder to the biomass.

Abstract: Abstract So far, the use of artificial low‐calorie sweeteners, like sucralose, saccharin, and so on, to replace the conventional‐based sugars has not succeeded due to the long‐term adverse health effects, for example, hypertension, and not well‐known safety stand. In this review, we discussed the next generation SvGl (rebaudioside M [Reb M]), their biosynthetic pathway in plant, high‐yield production via microbial fermentation and enzyme engineering, physicochemical properties, taste modification, kinetic metabolism, application in food and beverages, safety and toxicological evaluation, regulation and dosage recommendation, and health benefits. In stevia, the biosynthesis of stevia glycosides, especially Reb M, is derived from the bifurcation of the pathway leading to gibberellin, followed by subsequent enzymatic modification of rubusoside. Reb M is more economically produced via microbial fermentation of modified yeast Yarrowia lipolytica and enzymatic bioconversion of rebaudioside A (Reb A) or Reb E. Reb M can serve as a suitable alternative to the conventional‐based sugars.

Abstract: The development of chitinase tailored for the bioconversion of chitin to chitin oligosaccharides has attracted significant attention due to its potential to alleviate environmental pollution associated with chemical conversion processes. In this present investigation, we purified extracellular chitinase derived from marine Bacillus haynesii to homogeneity and subsequently characterized it. The molecular weight of BhChi was approximately 35 kDa. BhChi displayed its peak catalytic activity at pH 6.0, with an optimal temperature of 37 °C. It exhibited stability across a pH range of 6.0–9.0. In addition, BhChi showed activation in the presence of Mn2+ with the improved activity of 105 U mL−1. Ca2+ and Fe2+ metal ions did not have any significant impact on enzyme activity. Under the optimized enzymatic conditions, there was a notable enhancement in catalytic activity on colloidal chitin with Km of 0.01 mg mL−1 and Vmax of 5.75 mmol min−1. Kcat and catalytic efficiency were measured at 1.91 s−1 and 191 mL mg−1 s−1, respectively. The product profiling of BhChi using thin layer chromatography and Mass spectrometric techniques hinted an exochitinase mode of action with chitobiose and N-Acetyl glucosamine as the products. This study represents the first report on an exochitinase from Bacillus haynesii. Furthermore, the chitinase showcased promising antifungal properties against key pathogens, Fusarium oxysporum and Penicillium chrysogenum, reinforcing its potential as a potent biocontrol agent.

Abstract: ConspectusAs the main component of natural gas and renewable biogas, methane is an abundant, affordable fuel. Thus, there is interest in converting these methane reserves into liquid fuels and commodity chemicals, which would contribute toward mitigating climate change, as well as provide potentially sustainable routes to chemical production. Unfortunately, specific activation of methane for conversion into other molecules is a difficult process due to the unreactive nature of methane C-H bonds. The use of methane activating enzymes, such as methyl-coenzyme M reductase (MCR), may offer a solution. MCR catalyzes the methane-forming step of methanogenesis in methanogenic archaea (methanogens), as well as the initial methane oxidation step during the anaerobic oxidation of methane (AOM) in anaerobic methanotrophic archaea (ANME). In this Account, we highlight our contributions toward understanding MCR catalysis and structure, focusing on features that may tune the catalytic activity. Additionally, we discuss some key considerations for biomanufacturing approaches to MCR-based production of useful compounds.MCR is a complex enzyme consisting of a dimer of heterotrimers with several post-translational modifications, as well as the nickel-hydrocorphin prosthetic group, known as coenzyme F

Abstract: Nowadays, enhanced health consciousness will boost the demand for natural products. Fermentation holds global attention because of its immense advantages and applicability. Natural flavors (e.g., vanillin, lactones, etc.) and sweeteners are produced through fermentation by the bioconversion of the appropriate precursor molecules. Through fermentation, we can reduce the constraints of farming and the demand for livestock, land, water, and animal feed. Recently, precision fermentation has offered broad applicability in generating specific ingredients (sweeteners, flavors, heme protein, and collagen protein) or alternatives (dairy, meat, seafood, or egg). Creating microbial cell factories (MCFs) via synthetic biology directs the potency of microorganisms to generate vital molecules. Combining the possibilities of MCFs with precision fermentation facilitates more economical and extensive production of industrially valuable compounds for the food/nutraceutical or pharmaceutical industry. The current chapter deals with recent strategies/products in precision fermentation, for example, soy leghemoglobin and alternative proteins.

Abstract: Effective production of xylooligosaccharides (XOS) with lower proportion of xylose entails unique and robust xylanases. In this study, two novel xylanases from Trichoderma asperellum ND-1 belonging to glycoside hydrolase families 10 (XynTR10) and 11 (XynTR11) were over-expressed in Komagataella phaffii X-33 and characterized to be robust enzymes with high halotolerance and ethanol tolerant. Both enzymes displayed strict substrate specificity towards beechwood xylan and wheat arabinoxylan. (Glu153/Glu258) and (Glu161/Glu252) were key catalytic sites for XynTR10 and XynTR11. Notably, XynTR11 could rapidly degrade xylan/XOS into xylobiose without xylose via transglycosylation. Direct degradation of corncob using XynTR10 and XynTR111 displayed that while XynTR10 yielded 77% xylobiose and 25% xylose, XynTR11 yielded much less xylose (11%) and comparable amounts of xylobiose (63%). XynTR10 or XynTR111 has great potential as a catalyst for bioconversion of xylan-containing agricultural waste into high-value products (biofuel or XOS), which is of significant benefit for the economy and environment.

Abstract: Typically, citrus waste is composted on land by producers or used as livestock feed. However, the biorefinery approach offers a sustainable and economically viable solution for managing and valorizing these agricultural residues. This review examines research from the period 2014 to 2024. Citrus waste can be utilized initially by extracting the present phytochemicals and subsequently by producing value-added products using it as a raw material. The phytochemicals reported as extracted include essential oils (primarily limonene), pectin, polyphenolic components, micro- and nano-cellulose, proteins, and enzymes, among others. The components produced from the waste include bioethanol, biogas, volatile acids, biodiesel, microbial enzymes, and levulinic acid, among others. The review indicates that citrus waste has technical, economic, and environmental potential for utilization at the laboratory scale and, in some cases, at the pilot scale. However, research on refining pathways, optimization, and scalability must continue to be an active field of investigation.

Abstract: Recent developments in the catalytic conversion of syngas to biofuels are focused on the conversion of CO- and CO2-rich syngas using novel catalyst designs or innovative reacting systems (including membranes). Considering the use of chemical intermediates, new routes are studied for biofuel production (ethanol, DME, and oxymethylene ether). Apart from these developments, little advancements have been done in the last decades. For the most relevant biofuels (ethanol, higher alcohols, and Fischer-Tropsch), a preliminary insight into the implications of these advancements in the design of industrial-scale processes is presented. Besides the catalytic conversion of syngas to biofuels, its bioconversion is another suitable alternative. In this sense, acetogenic anaerobic bacteria can metabolize not only different mixtures of CO, CO2, and H2 to produce ethanol but also higher alcohols such as butanol and hexanol. The main parameters affecting such bioconversion processes are addressed. Alternatively, syngas biomethanation yields methane as another interesting biofuel.

Abstract: Oleanolic acid (OA) is a vegetable chemical that is present naturally in a number of edible and medicinal botanicals. It has been extensively studied by medicinal chemists and scientific researchers due to its biological activity against a wide range of diseases. A significant number of researchers have synthesized a variety of analogues of OA by modifying its structure with the intention of creating more potent biological agents and improving its pharmaceutical properties. In recent years, chemical and enzymatic techniques have been employed extensively to investigate and modify the chemical structure of OA. This review presents recent advancements in medical chemistry for the structural modification of OA, with a special focus on the biotransformation, semi-synthesis and relationship between the modified structures and their biopharmaceutical properties.

Abstract: The current study focuses on the idea of "Energy from Waste" that intends to address energy crises and manage waste. Fruit waste is one of the most common forms of organic waste due to its inedible portion and perishable nature. In Pakistani regions, an extensive amount of mango pulp (MP)/juice waste is produced due to excessive consumption during summers, which poses huge environmental challenges. The study aims at effective valorization of perishable waste and elimination of deteriorating waste that causes a polluting environment. Experimental work has been conducted to evaluate the sucrolytic potential of

Abstract: Black soldier fly larvae (BSFL) can use food by-products or residues as a growth substrate, and their use is considered a strategic solution to improve the sustainability and efficiency of animal production. BSFL need to be reared under controlled environmental conditions, as temperature can affect the welfare of the larvae and the efficiency of their bioconversion. The goal of this experiment was to test different diets: control, vegetable, carnivorous, omnivorous and to assess their effect on larval growth performance, chemical composition, and substrate temperature. BSFL (6-days-old) were randomly selected and allocated to one of the four diets for the entire experiment (8 days). The temperature in each container was measured twice daily using a thermal imaging camera. The vegetable diet led to significantly lower growth performance (p

Abstract: Monolignols and their derivatives exhibit various pharmaceutical and physiological characteristics, such as antioxidant and anti-inflammatory properties. However, they remain difficult to synthesize. In this study, we engineered several whole-cell bioconversion systems with carboxylate reductase (CAR)-mediated pathways for efficient synthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine in Escherichia coli BL21 (DE3). By overexpressing the l-tyrosine ammonia lyase from Flavobacterium johnsoniae (FjTAL), carboxylate reductase from Segniliparus rugosus (SruCAR), alcohol dehydrogenase YqhD and hydroxylase HpaBC from E. coli, and caffeate 3-O-methyltransferase (COMT) from Arabidopsis thaliana, three enzyme cascades FjTAL–SruCAR–YqhD, FjTAL–SruCAR–YqhD–HpaBC, and FjTAL–SruCAR–YqhD–HpaBC–COMT were constructed to produce 1028.5 mg/L p-coumaryl alcohol, 1015.3 mg/L caffeyl alcohol, and 411.4 mg/L coniferyl alcohol from 1500, 1500, and 1000 mg/L l-tyrosine, with productivities of 257.1, 203.1, and 82.3 mg/L/h, respectively. This work provides an efficient strategy for the biosynthesis of p-coumaryl, caffeyl, and coniferyl alcohols from l-tyrosine.

Abstract: This study focuses on optimizing the medium composition for cellular biomass production and bioconversion of ethylene glycol (EG) to glycolic acid (GA) using Gluconobacter oxydans CCT 0552. The improvement in cellular growth in the presence of yeast extract and peptone led to a 35.7% and 32.7% increase, respectively, compared to the medium with each of these carbon sources separately. Negligible growth was produced when (NH4)2SO4 and urea were used. Optimal bioconversion results were very similar for both the stirred tank and bubble column bioreactors, with GA concentrations reaching 49.4 g/L and 47.7 g/L, volumetric productivities of 0.35 g/L∙h and 0.33 g/L∙h, and product yield factors of 1.08 g/g and 0.94 g/g, respectively. An extended fed-batch strategy using a STR-type bioreactor achieved a concentration of glycolic acid of 94.2 g/L, corresponding to a volumetric productivity of 0.41 g/L∙h and a yield factor of 1.19 g/g. The resulting efficiency of this biological transformation process achieved a remarkable value of 97.3%, simultaneously with a significant decrease in the substrate amount by 90.5%. This study demonstrates the efficiency of G. oxydans in producing GA, offering a cost-effective and environmentally sustainable production method.

Abstract: Wild ginseng is known to have better pharmacological effects than cultivated ginseng. Additionally, recently developed bioengineering technology has made it possible to produce cultured wild ginseng with the same genetic composition. In this study, we investigated the change in characteristics and the improvement of the intestinal barrier of cultured wild ginseng roots (CWG) and fermented cultured wild ginseng roots (FCWG). First, we screened nine strains of bacteria that are capable of growing on 5-brix CWG medium, and Limosilactobacillus fermentum HY7303 (HY7303) showed the highest growth. Second, changes in the characteristics of CWG due to fermentation using HY7303 showed that pH and total carbohydrates decreased, and reducing sugars increased. The contents of minor ginsenosides (Rg3(s), Rk1, and Rg5) increased. Third, extracellular vesicles (EVs) with a single peak at 493.7 nm were isolated from CWG, and EVs with three peaks at 9.0 nm, 155.6 nm, and 459.0 nm were isolated from FCWG, respectively. Finally, when we treated Caco-2 cells with FCWG and EVs, we confirmed the improvement of intestinal barrier functions, including recovery, permeability, and expression of tight-junction protein genes. In this study, we confirmed the potential pharmacological effects of minor ginsenosides and EVs derived from FCWG. In conclusion, this study suggests that CWG fermentation with HY7303 improves the intestinal barrier by increasing minor ginsenosides and producing EVs.