Identification of anti-fibrotic and pro-apoptotic bioactive compounds from Ganoderma formosanum and their possible mechanisms in modulating TGF-ß1-induced lung fibrosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The Compendium of Materia Medica and the Classic of Materia Medica, the two most prominent records of traditional Chinese medicine, documented the therapeutic benefits of Ganoderma sinense particularly in addressing pulmonary-related ailments. Ganoderma formosanum, an indigenous subspecies of G. sinense from Taiwan, has demonstrated the same therapeutic properties. AIM OF THE STUDY: The aim of this study is to identify bioactive compounds and evaluate the potential of G. formosanum extracts as a novel treatment to alleviate pulmonary fibrosis (PF). Using an in-house drug screening platform, two-stage screening was performed to determine their anti-fibrotic efficacy. METHODS AND MATERIALS: G. formosanum was fractionated into four partitions by solvents of different polarities. To determine their antifibrotic and pro-apoptotic properties, the fractions were analyzed using two TGF-ß1-induced pulmonary fibrosis cell models (NIH-3T3) and human pulmonary fibroblast cell lines, immunoblot, qRT-PCR, and annexin V assays. Subsequently, transcriptomic analysis was conducted to validate the findings and explore possible molecular pathways. The identification of potential bioactive compounds was achieved through UHPLC-MS/MS analysis, while molecular interaction study was investigated by multiple ligands docking and molecular dynamic simulations. RESULTS: The ethyl acetate fraction (EAF) extracted from G. formosanum demonstrated substantial anti-fibrotic and pro-apoptotic effects on TGF-ß1-induced fibrotic models. Moreover, the EAF exhibited no discernible cytotoxicity. Untargeted UHPLC-MS/MS analysis identified potential bioactive compounds in EAF, including stearic acid, palmitic acid, and pentadecanoic acid. Multiple ligands docking and molecular dynamic simulations further confirmed that those bioactive compounds possess the ability to inhibit TGF-ß receptor 1. CONCLUSION: Potential bioactive compounds in G. formosanum were successfully extracted and identified in the EAF, whose anti-fibrotic and pro-apoptotic properties could potentially modulate pulmonary fibrosis. This finding not only highlights the EAF's potential as a promising therapeutic candidate to treat pulmonary fibrosis, but it also elucidates how Ganoderma confers pulmonary health benefits as described in the ancient texts.

Production of bacterial cellulose (BC)/nisin composite with enhanced antibacterial and mechanical properties through co-cultivation of Komagataeibacter xylinum and Lactococcus lactis subsp. lactis.

By employing co-cultivation technique on Komagataeibacter xylinum and Lactococcus lactis subsp. lactis, bacterial cellulose (BC)/nisin films with improved antibacterial activity and mechanical properties were successfully produced. The findings demonstrated that increased nisin production is associated with an upregulation of gene expression. Furthermore, results from Scanning electronic microscopy (SEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD), and Thermogravimetric analysis (TG) confirmed the integration of nisin within BC. While being biocompatible with human cells, the BC/nisin composites exhibited antimicrobial activity. Moreover, mechanical property analyses showed a noticeable improvement in Young's modulus, tensile strength, and elongation at break by 161, 271, and 195 %, respectively. Additionally, the nisin content in fermentation broth was improved by 170 % after co-culture, accompanied by an 8 % increase in pH as well as 10 % decrease in lactate concentration. Real-time reverse transcription PCR analysis revealed an upregulation of 11 nisin-related genes after co-cultivation, with the highest increase in nisA (5.76-fold). To our knowledge, this is the first study which demonstrates that an increase in secondary metabolites after co-culturing is modulated by gene expression. This research offers a cost-effective approach for BC composite production and presents a technique to enhance metabolite concentration through the regulation of relevant genes.

Enhanced exopolysaccharide production of Cordyceps militaris via mycelial cell immobilization on plastic composite support in repeated-batch fermentation.

Repeated-batch fermentation with fungal mycelia immobilized in plastic composite support (PCS) eliminates the lag phase during fermentation and improves metabolite productivity. The strategy is implemented herein, and a novel modified PCS is developed to enhance exopolysaccharide (EPS) production from the medicinal fungus Cordyceps militaris. A modified PCS (SYE + PCS) was made by compositing polypropylene (PP) with a nutrient mixture containing soybean hull, peptone, yeast extract, and minerals (SYE+). The use of SYE + PCS has consistent cell productivity throughout the multiple fermentation cycles, which resulted in a more higher cell productivity after second batch compared to unmodified PCS. The cell grown on SYE + PCS also generates a higher yield of EPS (3.36, 6.93, and 5.72 g/L in the first, second, and third fermentation cycles, respectively) up to three-fold higher than the cell immobilized on unmodified PCS. It is also worth noting that the EPS from mycelium grown on SYE + PCS contains up to 2.3-fold higher cordycepin than those on unmodified PCS. The presence of nutrients in SYE + PCS also affects the hydrophobicity and surface roughness of the PC, improving mycelial cell adhesion. This study also provides a preliminary antioxidant activity assessment of EPS from immobilized C. militaris grown with SYE + PCS.

Current and Potential Applications of Atmospheric Cold Plasma in the Food Industry.

The cost-effectiveness and high efficiency of atmospheric cold plasma (ACP) incentivise researchers to explore its potentials within the food industry. Presently, the destructive nature of this nonthermal technology can be utilised to inactivate foodborne pathogens, enzymatic ripening, food allergens, and pesticides. However, by adjusting its parameters, ACP can also be employed in other novel applications including food modification, drying pre-treatment, nutrient extraction, active packaging, and food waste processing. Relevant studies were conducted to investigate the impacts of ACP and posit that reactive oxygen and nitrogen species (RONS) play the principal roles in achieving the set objectives. In this review article, operations of ACP to achieve desired results are discussed. Moreover, the recent progress of ACP in food processing and safety within the past decade is summarised while current challenges as well as its future outlook are proposed.

Evaluation of porous bacterial cellulose produced from foam templating with different additives and its application in 3D cell culture.

Bacterial cellulose (BC) is used in biomedical applications due to its unique material properties such as mechanical strength with a high water-absorbing capacity and biocompatibility. Nevertheless, native BC lacks porosity control which is crucial for regenerative medicine. Hence, developing a simple technique to change the pore sizes of BC has become an important issue. This study combined current foaming BC (FBC) production with incorporation of different additives (avicel, carboxymethylcellulose, and chitosan) to form novel porous additive-altered FBC. Results demonstrated that the FBC samples provided greater reswelling rates (91.57 % ~ 93.67 %) compared to BC samples (44.52 % ~ 67.5 %). Moreover, the FBC samples also showed excellent cell adhesion and proliferation abilities for NIH-3T3 cells. Lastly, FBC allowed cells to penetrate to deep layers for cell adhesion due to its porous structure, providing a competitive scaffold for 3D cell culture in tissue engineering.

Sequential culture with aroma-producing yeast strains to improve the quality of Kyoho wine.

Despite many non-Saccharomyces yeasts being considered spoilage microorganisms, they can increase aroma and flavor diversity in alcoholic beverages. The purpose of this study was to investigate nontraditional inoculation strategies using aroma-producing yeast strains for Kyoho wine fermentation, followed by an instrumental analysis and sensory evaluation. The winemaking process was carried out using Saccharomyces cerevisiae Gr112, Hanseniaspora uvarum Pi235, and Pichia kluyveri Pe114. Multiple inoculation strategies were explored. In instrumental analysis results, mixed culture could promote the formation of esters (5.9-folds) and glycerol (1.3-folds) and reduce the content of ethanol (-0.5% [v/v]) in wine. The sensory analysis results suggested that the three yeast strains sequential inoculation treatment was associated with the aroma attributes "floral," "red fruity," and "tropical fruity." Co-cultivation contributed to an increase in complexity and aromatic intensity, with the three-strain inoculation treatment presenting a more distinctive appearance. PRACTICAL APPLICATION: The inoculation of S. cerevisiae improved the accumulation of volatile acids and esters by inhibiting the growth of non-Saccharomyces yeast strains. Inoculation of H. uvarum and P. kluyveri would effectively solve the defect of excessive content of higher alcohols in wines produced by S. cerevisiae. The suitable inoculation strategy between non-Saccharomyces yeasts could improve the overall quality of Kyoho wine whose starter might be widely used in fermentation industry.

Biocompatible and biodegradable copper-protocatechuic metal-organic frameworks as rifampicin carrier.

Tuberculosis (TB) is a disease caused by the M. tuberculosis bacteria infection and is listed as one of the deadliest diseases to date. Despite the development of antituberculosis drugs, the need for long-term drug consumption and low patient commitment are obstacles to the success of TB treatment. A continuous drug delivery system that has a long-term effect is needed to reduce routine drug consumption intervals, suppress infection, and prevent the emergence of drug-resistant strains of M. tuberculosis. For this reason, biomolecule metal-organic framework (BioMOF) with good biocompatibility, nontoxicity, bioactivity, and high stability are becoming potential drug carriers. This study used a bioactive protocatechuic acid (PCA) as organic linker to prepare copper-based BioMOF Cu-PCA under base-modulated conditions. Detailed crystal analysis by the powder X-ray diffraction demonstrated that the Cu-PCA, with a chemical formula of C14H16O13Cu3, crystalizes as triclinic in space group P1. Comprehensive physicochemical characterizations were provided using FTIR, SEM, XPS, TGA, EA, and N2 sorption. As a drug carrier, Cu-PCA showed a high maximum rifampicin (RIF) drug loading of 443.01 mg/g. Upon resuspension in PBS, the RIF and linkers release profile exhibited two-stage release kinetic profiles, which are well described by the Biphasic Dose Response (BiDoseResp) model. A complete release of these compounds (RIF and PCA) was achieved after ~9 h of mixing in PBS. Cu-PCA and RIF@Cu-PCA possessed antibacterial activity against Escherichia coli, and good biocompatibility is evidenced by the high viability of MH-S mice alveolar macrophage cells upon supplementations.

Applications of atmospheric cold plasma in agricultural, medical, and bioprocessing industries.

Atmospheric cold plasma (ACP) is a nonthermal technology that is extensively used in several industries. Within the scopes of engineering and biotechnology, some notable applications of ACP include waste management, material modification, medicine, and agriculture. Notwithstanding numerous applications, ACP still encounters a number of challenges such as diverse types of plasma generators and sizes, causing standardization challenges. This review focuses on the uses of ACP in engineering and biotechnology sectors in which the innovation can positively impact the operation process, enhance safety, and reduce cost. Additionally, its limitations are examined. Since ACP is still in its nascent stage, the review will also propose potential research opportunities that can help scientists gain more insights on the technology. KEY POINTS: • ACP technology has been used in agriculture, medical, and bioprocessing industries. • Chemical study on the reactive species is crucial to produce function-specific ACP. • Different ACP devices and conditions still pose standardization problems.

Improvement in Violacein Production by Utilizing Formic Acid to Induce Quorum Sensing in Chromobacterium violaceum.

Violacein has attracted increasing attention due to its various biological activities, such as antibacterial, antifungal, antioxidative, and antitumor effects. To improve violacein production, formic acid (FA) was added to a culture medium, which resulted in a 20% increase (1.02 g/L) compared to the no-FA-addition group (0.85 g/L). The use of a stirred-tank bioreactor system also improved violacein production (by 0.56 g/L). A quorum-sensing (QS)-related gene (cviI) was induced by FA treatment, which revealed that the mechanism induced by FA utilized regulation of the cviI gene to induce the vio gene cluster for violacein production. To analyze the antioxidative properties of the violacein produced, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) scavenging tests were conducted, and results reveal that the values of the 50% inhibitory concentration (IC50) of DPPH and ABTS were 0.286 and 0.182 g/L, respectively. Violacein also showed strong inhibitory activity against Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis). In summary, this study found that the addition of formic acid can promote QS of Chromobacterium violaceum, thereby promoting the synthesis of violacein. Subsequently, the promoting effect was also evaluated in a bioreactor system. These findings will be helpful in establishing an economically beneficial production model for violacein in future work.

Evaluation of detoxified sugarcane bagasse hydrolysate by atmospheric cold plasma for bacterial cellulose production.

Cellulosic waste as a major type of agricultural waste can be acid deconstructed as a carbon source for fermentation application. However, various fermented inhibitors, such as formic acid, furfural, and 5-hydroxymethylfurfural, are also produced during processing. In this study, sugarcane bagasse (SB) was hydrolyzed with sulfuric acid, and atmospheric cold plasma (ACP) was used to remove the toxic inhibitors. The detoxified SB hydrolysate was used as alternative nutrients for bacterial cellulose (BC) production. Results showed that degradation rates of formic acid, 5-hydroxymethylfurfural, and furfural respectively reached 25.2%, 78.6%, and 100% with optimized ACP conditions (argon ACP at 200 W for 25 min). In BC production, the ACP-treated SB hydrolysate group (PT) exhibited high BC production (1.68 g/L) but was lower than that from the ACP-untreated SB hydrolysate group (PUT) (1.88 g/L), which suggests that ACP detoxification might also cause some crucial nutrients loss of the SB hydrolysate, leading to a decrease in BC production. The material properties of BC produced from detoxified based medium are also evaluated. These findings have important implications for the broader domain of ACP detoxification for cellulosic acid hydrolysates applied to BC production.

Ganoderma formosanum Exopolysaccharides Inhibit Tumor Growth via Immunomodulation.

Ganoderma formosanum (GF) is a medicinal mushroom endemic to Taiwan. Previous research established the optimal culture conditions to produce exopolysaccharide rich in ß-glucan (GF-EPS) from submerged fermentation of GF. The present study investigated the antitumor effects of GF-EPS in a Lewis lung carcinoma cell (LLC1) tumor-bearing mice model. In the preventive model, GF-EPS was orally administered to mice before LLC1 injection. In the therapeutic model, GF-EPS oral administration was initiated five days after tumor cell injection. The tumor size and body weight of the mice were recorded. After sacrifice, the lymphocyte subpopulation was analyzed using flow cytometry. Spleen tissues were used to analyze cytokine mRNA expression. The results showed that GF-EPS (80 mg/kg) effectively suppressed LLC1 tumor growth in both the preventive and therapeutic models. GF-EPS administration increased the proportion of natural killer cells in the spleen and activated gene expression of several cytokines. Our results provide evidence that GF-EPS promotes tumor inhibition through immunomodulation in tumor-bearing mice.

Atmospheric cold plasma-assisted pineapple peel waste hydrolysate detoxification for the production of bacterial cellulose.

Toxic compounds in pineapple peel waste hydrolysate (PPWH), namely formic acid, 5-hydroxymethylfurfural (HMF), and furfural, are the major predicament in its utilization as a carbon source for bacterial cellulose (BC) fermentation. A rapid detoxification procedures using atmospheric cold plasma (ACP) technique were employed to reduce the toxic compounds. ACP treatment allows the breakdown of toxic compounds without causing excessive breakdown of sugars. Herein, the performance of two available laboratory ACP reactors for PPWH detoxification was being demonstrated. ACP-reactor-1 (R1) runs on plasma power of 80-200 W with argon (Ar) plasma source, while ACP-reactor-2 (R2) runs at 500-600 W with air plasma source. Treatment in R1, at 200 W for 15 min, results in 74.06%, 51.38%, and 21.81% reduction of furfural, HMF, and formic acid. Treatment in R2 at 600 W gives 45.05%, 32.59%, and 60.41% reductions of furfural, HMF, and formic acid. The BC yield from the fermentation of Komagateibacter xylinus in the R1-treated PPWH, R2-treated PPWH, and untreated-PPWH is 2.82, 3.82, and 2.97 g/L, respectively. The results show that ACP treatment provides a novel detoxified strategy in achieving agricultural waste hydrolysate reuse in fermentation. Furthermore, the results also imply that untreated PPWH can be an inexpensive and sustainable resource for fermentation media supplementation.

Enhanced bioethanol production using atmospheric cold plasma-assisted detoxification of sugarcane bagasse hydrolysate.

The current study used acid hydrolysis of lignocellulosic materials to obtain fermentable sugar for bioethanol production. However, toxic compounds that inhibit fermentation are also produced during the process, which reduces the bioethanol productivity. In this study, atmospheric cold plasma (ACP) was adopted to degrade the toxic compounds within sulfuric acid-hydrolyzed sugarcane bagasse. After ACP treatment, significant decreases in toxic compounds (31% of the formic acid, 45% of the acetic acid, 80% of the hydroxymethylfurfural, and 100% of the furfural) were observed. The toxicity of the hydrolysate was low enough for bioethanol production using Kluyveromyces marxianus. After adopting optimal ACP conditions (200 W power for 25 min), the bioethanol productivity improved from 0.25 to 0.65 g/L/h, which means that ACP could effectively degrade toxic compounds within the hydrolysate, thereby enhancing bioethanol production. Various nitrogen substitute was coordinated with detoxified hydrolysate, and chicken meal group presented the highest bioethanol productivity (0.45 g/L/h).

TEMPO-Oxidized Bacterial Cellulose Pellicle with Silver Nanoparticles for Wound Dressing.

Biocompatible bacterial cellulose pellicle (BCP) is a candidate for biomedical material such as wound dressing. However, due to lack of antibacterial activity, to grant BCP with the property is crucial for its biomedical application. In the present study, BCP was modified by 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation using TEMPO/NaClO/NaBr system at pH 10 to form TEMPO-oxidized BCP (TOBCP) with anionic C6 carboxylate groups. The TOBCP was subsequently ion-exchanged in AgNO3 solution and silver nanoparticles (AgNP) with diameter of ∼16.5 nm were in situ synthesized on TOBC nanofiber surfaces by thermal reduction without using a reducing agent. Field emission scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectra, Fourier transform infrared spectroscopy, and thermogravimetric analysis were carried out to confirm morphology and structure of the pellicles with AgNP. The AgNP continuously released Ag+ with a rate of 12.2%/day at 37 °C in 3 days. The TOBCP/AgNP exhibited high biocompatibility according to the result of in vitro cytotoxicity test (cell viability >95% after 48 h of incubation) and showed significant antibacterial activities of 100% and 99.2% against E. coli and S. aureus, respectively. Hence, the highly biocompatible and highly antibacterial TOBCP/AgNP prepared in the present study is a promising candidate for wound dressing.

Enhanced active extracellular polysaccharide production from Ganoderma formosanum using computational modeling.

Extracellular polysaccharide (EPS) is one of the major bioactive ingredients contributing to the health benefits of Ganoderma spp. In this study, response surface methodology was applied to determine the optimal culture conditions for EPS production of Ganoderma formosanum. The optimum medium composition was found to be at initial pH 5.3, 49.2 g/L of glucose, and 4.9 g/L of yeast extract by implementing a three-factor-three-level Box-Behnken design. Under this condition, the predicted yield of EPS was up to 830.2 mg/L, which was 1.4-fold higher than the one from basic medium (604.5 mg/L). Furthermore, validating the experimental value of EPS production depicted a high correlation (100.4%) with the computational prediction response model. In addition, the percentage of ß-glucan, a well-recognized bioactive polysaccharide, in EPS was 53±5.5%, which was higher than that from Ganoderma lucidum in a previous study. Moreover, results of monosaccharide composition analysis indicated that glucose was the major component of G. formosanum EPS, supporting a high ß-glucan percentage in EPS. Taken together, this is the first study to investigate the influence of medium composition for G. formosanum EPS production as well as its ß-glucan composition.

Extract of Ganoderma formosanum Mycelium as a Highly Potent Tyrosinase Inhibitor.

In this study, the inhibitory effect of Ganoderma formosanum mycelium extracts on tyrosinase, the central regulatory enzyme being responsible for cutaneous pigmentation, was investigated in both cell-free and cellular enzymatic systems, as well as in phenotype-based zebrafish model. Bioassay-guided purification indicated that the ethyl acetate fraction of G. fromosanum mycelium ethanolic extract (GFE-EA) demonstrated the highest inhibition toward cell-free tyrosinase (IC50 = 118.26 ± 13.34 ppm). The secreted and intracellular melanin of B16-F10 cells were reduced by GFE-EA through suppression of tyrosinase activity (IC50 = 102.27 ± 9.49 ppm) and its protein expression. Moreover, GFE-EA decreased surface pigmentation level of zebrafish via down-regulation of tyrosinase activity. Most of all, there is no significant difference in morphology and mortality between control and GFE-EA treated groups. Not only does GFE-EA exhibit similar depigmenting efficacy to kojic acid with lower dosage (approximately one-seventh of dose), but show less toxicity to zebrafish. It is worth noting that GFE-EA is extracted from mycelium, which subverts the general concept that mycelium lacks certain bioactivities possessed by fruit bodies. Altogether, it would appear that GFE-EA has great potential for application in the cosmetics industry.

Isolation and identification of cellulose-producing strain Komagataeibacter intermedius from fermented fruit juice.

A bacterial cellulose (BC) producing strain isolated from fermented fruit juice was identified as Komagataeibacter intermedius (K. intermedius) FST213-1 by 16s rDNA sequencing analysis and biochemical characteristics test. K. intermedius FST213-1 can produce BC within pH 4-9 and exhibit maximum BC production (1.2g/L) at pH 8 in short-term (4-day) cultivation. Results of Fourier transform infrared spectroscopy, X-ray diffraction, water content, thermogravimetric analysis and mechanical property indicated that BC produced from K. intermedius FST213-1 exhibits higher water content ability (99.5%), lower thermostability (315°C), lower crystallinity (79.3%) and similar mechanical properties in comparison with the specimen from model BC producer, Gluconacetobacter xylinus 23769. Based on these analyses, the novel based-resistant strain K. intermedius FST213-1 can efficiently produce BC, which can be applied for industrial manufacturing with potential features.

Evaluation of kojic acid production in a repeated-batch PCS biofilm reactor.

In this study, kojic acid, a secondary metabolite as an industrially important compound, was produced by Aspergillus oryzae (A. oryzae), which was immobilized in plastic composite support (PCS) bioreactor. Nitrogen deficient medium was applied to increase the production of KA in PCS-immobilized bioreactor. The efficiency of immobilized culture for kojic acid (KA) production and the effect of morphology of A. oryzae on KA production were evaluated. After three cycles of cultivation, 83.47 g/L of KA was produced in PCS bioreactor in nitrogen deficient medium with productivity of 3.09 g/L/d, which is higher than free suspension culture in batch fermentation. The morphology of A. oryzae mycelium changed under nitrogen starvation. Feather-like mycelium was observed with increasing KA production. RNA expression (kojA and kojT) results indicated that the nitrogen deficient environment had strong influence on KA production on the transcriptional level. PCS immobilized fermentation system, which allowed a repeated-batch fermentation with higher production and productivity, is a potential tool in industrial production of KA.

Epigenetic silencing of SFRP5 is related to malignant phenotype and chemoresistance of ovarian cancer through Wnt signaling pathway.

Oncogenic activation of the Wnt signaling pathway is common in cancers, but mutation of beta-catenin in ovarian cancer is rare. In addition to genetic events, epigenetic modification of secreted frizzled-related protein (SFRP) family has been shown to be important in regulating Wnt signaling. Although high degree of homology is observed in the same family, different SFRPs may have opposing effects on the same process. We reported recently that a Wnt antagonist, SFRP5, is downregulated frequently through promoter hypermethylation and that this hypermethylation is associated with overall survival in ovarian cancer. The aim of this study was to analyze the function of SFRP5 in ovarian cancer. Functional assays including measuring cell proliferation, invasion, colony formation and xenograft were performed using ovarian cancer cell lines with overexpression of SFRP5 or a short hairpin RNA silencing. The methylation status of SFRP5 in relation to cisplatin resistance in ovarian cancer patients was analyzed. Restoration of the expression of SFRP5 attenuated Wnt signaling in ovarian cancer cells and suppressed cancer cell growth, invasion of cells and tumorigenicity in mice. These effects were independent of the canonical pathway. The expression of SFRP5 inhibited epithelial-mesenchymal transition (EMT). The restoration of SFRP5 downregulated AKT2 and sensitized ovarian cancer cells to chemotherapy. These effects are consistent with the poor response to platinum-based chemotherapy in patients with methylation of SFRP5. Our data suggested that epigenetic silencing of SFRP5 leads to oncogenic activation of the Wnt pathway and contributes to ovarian cancer progression and chemoresistance through the TWIST-mediated EMT and AKT2 signaling.