Formation mechanism, environmental sensitivity and functional characteristics of succinylated ovalbumin/ε-polylysine electrostatic complexes: The roles of succinylation modification and ε-polylysine combination.

Biocomplex materials formed by oppositely charged biopolymers (proteins) tend to be sensitive to environmental conditions and may lose part functional properties of original proteins, and one of the approaches to address these weaknesses is protein modification. This study established an electrostatic composite system using succinylated ovalbumin (SOVA) and ε-polylysine (ε-PL) and investigated the impact of varying degrees of succinylation and ε-PL addition on microstructure, environmental responsiveness and functional properties. Molecular docking illustrated that the most favorable binding conformation was that ε-PL binds to OVA groove, which was contributed by the multi­hydrogen bonding and hydrophobic interactions. Transmission electron microscopy observed that SOVA/ε-PL had a compact spherical structure with 100 nm. High-degree succinylation reduced complex sensitivity to heat, ionic strength, and pH changes. ε-PL improved the gel strength and antibacterial properties of SOVA. The study suggests possible uses of SOVA/ε-PL complex as multifunctional protein complex systems in the field of food additives.

MoO3/MIL-125-NH2 with boosted peroxidase-like activity for electrochemical staphylococcus aureus sensing via specific recognition of bacteriophages.

Herein, novel nanozyme mimics MoO3/MIL-125-NH2 were reported and conjugated with bacteriophages as a new electrochemical probe for high sensitivity and specific electrochemical detection of staphylococcus aureus. The excellent peroxidase-like activity of MoO3/MIL-125-NH2 composites was attributed to the integration of MIL-125-NH2 with MoO3, which can boost the generation of superoxide radicals (O• 2-) and thus promote the oxidation of TMB in the presence of H2O2. In this work, two bacteriophages named SapYZU04 and SapYZU10 were isolated from sewage samples by using staphylococcus aureus YZUsa12 as the host. In comparison, MoO3/MIL-125-NH2@SapYZU04 was selected as a recognition agent. The DPV current declined linearly with staphylococcus aureus YZUsa12 concentration in the range of 101-108 CFU mL-1, with a low detection limit of 16 CFU mL-1 (S/N = 3). 20 strains including 13 host strains and 7 non-host strains were used to evaluate the selectivity of the proposed sensor. Regardless of the differences in the degrees of lytic performance for phage SapYZU04, all selected host strains can be screened with merely the same DPV current. Host spectrum-oriented bacteriophage sensing is of great importance for the practical application of bacteriophage-based biosensors in the future.

Calcium-mediated modulation of Pseudomonas fluorescens biofilm formation.

Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca2+) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca2+ involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca2+ significantly reduced the swarming motility of P. fluorescens strains (P.F2, P.F4, and P.F17), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca2+. By the supplementation of Ca2+, complex structures with more cell clusters glued together in P. fluorescens P.F4 biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F4 biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F4 showed a transcriptional response to the supplementation of 10 mM Ca2+, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca2+ might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.

Comparative Transcriptomic Analyses Propose the Molecular Regulatory Mechanisms Underlying 1,8-Cineole from Cinnamomum kanehirae Hay and Promote the Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation.

Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea.

WGS analysis of two Staphylococcus aureus bacteriophages from sewage in China provides insights into the genetic feature of highly efficient lytic phages.

The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of specific genes associated with DNA replication, DNA packaging and the lytic cycle.

Low Weight Polysaccharide of Hericium erinaceus Ameliorates Colitis via Inhibiting the NLRP3 Inflammasome Activation in Association with Gut Microbiota Modulation.

Ulcerative colitis (UC), one of the typical inflammatory bowel diseases caused by dysregulated immunity, still requires novel therapeutic medicine with high efficacy and low toxicity. Hericium erinaceus has been widely used to treat different health problems especially gastrointestinal sickness in China for thousands of years. Here, we isolated, purified, and characterized a novel low weight polysaccharide (HEP10, Mw: 9.9 kDa) from the mycelia of H. erinaceus in submerged culture. We explored the therapeutic effect of HEP10 on UC and explored its underlying mechanisms. On one hand, HEP10 suppressed the production of TNF-α, IL-1ß, IL-6, inducible iNOS, and COX-2 in LPS challenged murine macrophage RAW264.7 cells, as well as in colons from DSS-induced colitis mice. On the other hand, HEP10 treatment markedly suppressed the activation of NLRP3 inflammasome, NF-κB, AKT, and MAPK pathways. Moreover, HEP10 reversed DSS-induced alternation of the gut community composition and structure by significantly increasing Akkermansia muciniphila and also promoting functional shifts in gut microbiota. Structural equation modeling also highlighted that HEP10 can change widely through gut microbiota. In conclusion, HEP10 has a better prebiotic effect than the crude polysaccharides of H. erinaceus, which can be used as a novel dietary supplement and prebiotic to ameliorate colitis.

Molecular Regulatory Mechanism of the Iron-Ion-Promoted Asexual Sporulation of Antrodia cinnamomea in Submerged Fermentation Revealed by Comparative Transcriptomics.

Antrodia cinnamomea is a precious edible and medicinal fungus with activities of antitumor, antivirus, and immunoregulation. Fe2+ was found to promote the asexual sporulation of A. cinnamomea markedly, but the molecular regulatory mechanism of the effect is unclear. In the present study, comparative transcriptomics analysis using RNA sequencing (RNA-seq) and real time quantitative PCR (RT-qPCR) were conducted on A. cinnamomea mycelia cultured in the presence or absence of Fe2+ to reveal the molecular regulatory mechanisms underlying iron-ion-promoted asexual sporulation. The obtained mechanism is as follows: A. cinnamomea acquires iron ions through reductive iron assimilation (RIA) and siderophore-mediated iron assimilation (SIA). In RIA, ferrous iron ions are directly transported into cells by the high-affinity protein complex formed by a ferroxidase (FetC) and an Fe transporter permease (FtrA). In SIA, siderophores are secreted externally to chelate the iron in the extracellular environment. Then, the chelates are transported into cells through the siderophore channels (Sit1/MirB) on the cell membrane and hydrolyzed by a hydrolase (EstB) in the cell to release iron ions. The O-methyltransferase TpcA and the regulatory protein URBS1 promote the synthesis of siderophores. HapX and SreA respond to and maintain the balance of the intercellular concentration of iron ions. Furthermore, HapX and SreA promote the expression of flbD and abaA, respectively. In addition, iron ions promote the expression of relevant genes in the cell wall integrity signaling pathway, thereby accelerating the cell wall synthesis and maturation of spores. This study contributes to the rational adjustment and control of the sporulation of A. cinnamomea and thereby improves the efficiency of the preparation of inoculum for submerged fermentation.

Enhanced Antibacterial Efficiency and Anti-Hygroscopicity of Gum Arabic-ε-Polylysine Electrostatic Complexes: Effects of Thermal Induction.

The aim of this investigation was to scrutinize the effects of a thermal treatment on the electrostatic complex formed between gum arabic (GA) and ε-polylysine (ε-PL), with the goal of improving the antibacterial properties and reducing the hygroscopicity of ε-PL. The heated complex with a ratio of 1:4 exhibited an encapsulation efficiency of 93.3%. Additionally, it had an average particle size of 350.3 nm, a polydispersity index of 0.255, and a zeta potential of 18.9 mV. The formation of the electrostatic complex between GA and ε-PL was confirmed through multispectral analysis, which demonstrated the participation of hydrogen bonding and hydrophobic and electrostatic interactions, as well as the enhanced effect of heat treatment on these forces within the complex. The complex displayed a core-shell structure, with a regular distribution and a shape that was approximately spherical, as observed in the transmission electron microscopy images. Additionally, the heated GA-ε-PL electrostatic composite exhibited favorable antibacterial effects on Salmonella enterica and Listeria monocytogenes, with reduced minimum inhibitory concentrations (15.6 µg/mL and 62.5 µg/mL, respectively) and minimum bactericidal concentrations (31.3 µg/mL and 156.3 µg/mL, respectively) compared to free ε-PL or the unheated electrostatic composite. Moreover, the moisture absorption of ε-PL reduced from 92.6% to 15.0% in just 48 h after being incorporated with GA and subsequently subjected to heat. This research showed a way to improve the antibacterial efficiency and antihygroscopicity of ε-PL, reducing its application limitations as an antimicrobial substance to some extent.

Review of Bioactivity, Isolation, and Identification of Active Compounds from Antrodia cinnamomea.

Antrodia cinnamomea is a precious and popular edible and medicinal mushroom. It has attracted increasing attention due to its various and excellent bioactivities, such as hepatoprotection, hypoglycemic, antioxidant, antitumor, anticancer, anti-inflammatory, immunomodulation, and gut microbiota regulation properties. To elucidate its bioactivities and develop novel functional foods or medicines, numerous studies have focused on the isolation and identification of the bioactive compounds of A. cinnamomea. In this review, the recent advances in bioactivity, isolation, purification, and identification methods of active compounds from A. cinnamomea were summarized. The present work is beneficial to the further isolation and discovery of new active compounds from A. cinnamomea.

Comparative Transcriptomic Analyses Reveal the Regulatory Mechanism of Nutrient Limitation-Induced Sporulation of Antrodia cinnamomea in Submerged Fermentation.

Antrodia cinnamomea is a precious edible and medicinal mushroom with various biological activities, such as hepatoprotection, antitumor, antivirus, immunoregulation, and intestinal flora regulation. However, the wild fruiting bodies of A. cinnamomea are scarce and expensive. Submerged fermentation based on spore inoculation has become the most efficient and popular artificial culture method for A. cinnamomea. In order to complement the mechanism of asexual sporulation of A. cinnamomea in submerged fermentation, and provide a theoretical basis to further improve the sporulation, comparative transcriptomics analysis using RNA-seq and RT-qPCR were conducted on A. cinnamomea mycelia cultured under different nutritional conditions to reveal the regulatory mechanism underlying the asexual sporulation induced by nutrient limitation. The obtained mechanism is as follows: under nitrogen starvation, the corresponding sensors transmit signals to genes, such as areA and tmpA, and promote their expression. Among these genes, AreA has a direct or indirect effect on flbD and promotes its expression, further enhancing the expression of brlA. Meanwhile, TmpA has a direct or indirect effect on brlA and promotes its expression; under carbon starvation, transport protein Rco-3, as a glucose sensor, directly or indirectly transmits signals to brlA and promotes its expression. BrlA promotes the expression of abaA gene, which further enhances the expression of wetA gene, and wetA then directly leads to asexual sporulation and promotes spore maturation; meanwhile, gulC can also promote cell autolysis, which provides energy and raw materials for sporulation.

Isolation and Characterization of a Lytic Vibriophage OY1 and Its Biocontrol Effects Against Vibrio spp.

Vibrio species are important pathogens of marine animals and aquaculture populations and some of them can cause serious infections in humans through consumption of contaminated seafood and aquaculture products. Lytic bacteriophages can potentially alleviate Vibrio contamination in the aquaculture organisms and in the processing of aquatic products and have gained significant scientific attention in recent years. In the present study, bacteriophages were isolated from sewage of local aquatic products markets and grown using Vibrio mimicus CICC 21613 as host cells. The lytic vibriophage OY1 belonging to the newly proposed family Autographiviridae and the genus Maculvirus was identified by observation under electron microscope and comparative genomic analysis. The phage OY1 showed lytic activity against 24 among 32 tested strains belonging to eight Vibrio species. The complete phage OY1 genome consists of a single circular double-stranded DNA of 43,479 bp with a total GC content of 49.27% and was predicted to encode 40 open reading frames (ORFs). To evaluate its potential against vibrios, the one-step growth curve, thermal and pH stability, host range, and lytic activity of the OY1 phage against Vibrio species were evaluated. The results showed that phage OY1 had a range of thermal and pH tolerance, and exhibited a significant inhibitory effect on the growth of tested Vibrio species. Bacterial growth in the fish muscle extract juice (FMEJ) inoculated with Vibrio mimicus CICC 21613, Vibrio parahaemolyticus CICC 21617, Vibrio alginolyticus VJ14, and the mixed bacterial culture was reduced by 2.65 log CFU/ml, 2.42 log CFU/ml, 1.93 log CFU/ml, and 2.01 log CFU/ml, respectively, by incubation with phage OY1 at 25°C for 36 h. Phage OY1 also showed a strong ability to prevent biofilm formation and destroy formed Vibrio species biofilms. These results indicate that phage OY1 is a potential biocontrol agent against Vibrio species in the aquaculture industry and in food safety control.

N, O-codoped hierarchical porous graphitic carbon for electrochemical immunosensing of Lactobacillus rhamnosus GG.

An ultrasensitive label-free electrochemical immunosensor was fabricated for quantitative detection of Lactobacillus rhamnosus GG (LGG). The N/O co-doped three-dimensional hierarchical porous graphitic (THPG) carbon was synthesized by a one-step synthesis of polyaniline hydrogel, and followed by simple carbonization and chemical activation procedures. Because of the unique structure design, the obtained THPG carbon networks possess an ultra-large specific surface area of 4859 m2 g-1 along with a class of highly graphitic carbons. The results offer an enormous surface area and excellent electrical conductivity for label-free electrochemical immunosensing of probiotic L. rhamnosus strain. Under optimal conditions, the immunosensor showed a good linear relationship between peak current and concentration of LGG (R2 = 0.9976), with a detection limit of 2 CFU mL-1. Furthermore, this label-free immunosensor also shows good specificity, long-term stability, and reliability, and could be applied to detect probiotic LGG in dairy products and drinks with satisfactory results. The present protocol was shown to be quite promising for practical screening and functional evaluation of probiotic products containing LGG. A ultrasensitive label-free electrochemical immunosensor based on THPG carbon was fabricated for detection of Lactobacillus rhamnosus GG.

Prevention and detoxification of patulin in apple and its products: A review.

Patulin-producing fungi pose an unavoidable problem for apple and its product quality, thereby threatening human and/or animal health. Studies on controlling the patulin-producing fungal growth and patulin contamination in apple and its products by physical methods, chemical fungicides, and biological methods have been performed for decades, but patulin contamination has not been addressed. Here, the important of studying regulation mechanism of patulin production in apple at the protein expression and metabolism levels is proposed, which will facilitate the development of controlling patulin production by using physical, chemical, and biological methods. Furthermore, the advantages or disadvantages and effects or mechanisms of using physical, chemical, biological methods to control the decay caused by Penicillium expansum and to remove patulin in food was discussed. The development of physical methods to remove patulin depends on the development of special equipment. Chemical methods are economical and efficient, if we have ensured that there are no unknown reactions or toxic by-products by using these chemicals. The biological method not only effectively controls the decay caused by Penicillium espansum, but also removes the toxins that already exist in the food. Degradation of patulin by microorganisms or biodegradation enzymes is an efficient and promising method to remove patulin in food if the microorganisms used and the degradation products are completely non-toxic.

Enhanced human lysozyme production by Pichia pastoris via periodic glycerol and dissolved oxygen concentrations control.

In human lysozyme (hLYZ) production by Pichia pastoris, the glycerol fed-batch phase was generally implemented under the environment of "oxygen sufficient-glycerol limited" to achieve high cell-density cultivation during the cell growth phase. However, the structural and functional components in P. pastoris cells were irreversible damaged with more and more reactive oxygen species (ROS) accumulation when cells were exposed to the oxygen sufficient environments for long time, leading to a failure of hLYZ expression. In this study, a novel periodic glycerol and dissolved oxygen concentration (DO) control strategy was proposed to solve these problems. This strategy periodically switched the cultivation environments from "oxygen sufficient-glycerol limited" to "oxygen limited-glycerol sufficient" for 5 cycles. When using this strategy: (1) the highest dry cell weight (DCW) of 143.02 g-DCW/L and the lowest distribution of glycerol towards to cell maintenance (0.0400 1/h) were achieved during the glycerol feeding phase by maintaining ROS levels below 48.39 Fluorescence intensity/g-DCW; (2) the adaption time of P. pastoris cells to methanol induction environments was shortened for about 50%; (3) P. pastoris cell metabolic activities reflected by the activities of alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and methanol consumption rate, etc., in the successive induction phase were largely enhanced; (4) hLYZ activity reached the highest level of 2.45 × 105 IU/mL, which was about 2-fold than that obtained with the strategy of "oxygen sufficient-glycerol limited," when the same methanol induction strategy was adopted. KEY POINTS: • A novel periodic glycerol feeding strategy proposed/used for P. pastoris cell growth. • Higher cell density was obtained by controlling ROS at low level via this strategy. • The highest hLYZ activity was achieved when initiating induction at higher cell density.

Protective Effect of Spore Powder of Antrodia camphorata ATCC 200183 on CCl4-Induced Liver Fibrosis in Mice.

Liver fibrosis is a pathological process with intrahepatic diffused deposition of the excess extracellular matrix, which leads to various chronic liver diseases. Drugs with high efficacy and low toxicity for liver fibrosis are still unavailable. Antrodia camphorata has antioxidant, antivirus, antitumor and anti-inflammation roles, and has been used to treat liver diseases in the population. However, the hepatoprotective effects of A. camphorata spores and the mechanisms behind it have not been investigated. In this study, we evaluate the hepatoprotective effect of spore powder of A. camphorata (SP, 100 mg/kg/day or 200 mg/kg/day) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. SP groups reduced serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities compared with the CCl4 group. SP also showed a decrease in hydroxyproline (Hyp) content in liver tissues. SP improved cell damage and reduced collagen deposition by H&E, Sirius red and Masson staining. Furthermore, SP down-regulated the mRNA levels of α-SMA and Col 1, and the protein expression of α-smooth muscle actin (α-SMA), collagen I (Col 1), tumor necrosis factor alpha (TNF-α), toll like receptor 4 (TLR4) and nuclear factor-Κb (NF-κB) p65. In summary, SP has an ameliorative effect on hepatic fibrosis, probably by inhibiting the activation of hepatic stellate cells, reducing the synthesis of extracellular matrix.

Vanillin Promotes the Germination of Antrodia camphorata Arthroconidia through PKA and MAPK Signaling Pathways.

Wild fruiting bodies of medicinal mushroom Antrodia camphorata are only found on the endemic species bull camphor tree, Cinnamomum kanehirae, in Taiwan. Despite the evident importance of the host components in promoting the growth of A. camphorata, insights into the underlying mechanisms are still lacking. Here, we first evaluated effects of the compounds from C. kanehirai, C. camphora, and A. camphorata, and their structural analogs on the germination rate of A. camphorata arthroconidia. Among the 54 tested compounds, vanillin (4-hydroxy-3-methoxybenzaldehyde) was determined as the optimum germination promoter, while o-vanillin and 1-octen-3-ol as major negative regulators of arthroconidia germination. Second, the protein patterns of arthroconidia after 24 h of incubation in the presence or absence of vanillin were compared via isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics. Via bioinformatic analysis, it was found that 61 proteins might relate to the germination of arthroconidia, in which 16 proteins might involve in two potential protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signaling pathways in the vanillin-promoted germination of A. camphorata arthroconidia. Last, the mRNA expression levels of the 16 germination-related genes in the potential PKA and MAPK signaling pathways were analyzed by quantitative real time PCR. Together, our results are beneficial for the elucidation of molecular mechanisms underlying the germination of A. camphorata arthroconidia.

[Effect of calcium on sporulation of Taiwanofungus camphoratus in submerged fermentation].

Taiwanofungus camphoratus is a valuable and rare medicinal mushroom with various bioactivities, such as liver protection and anti-cancer. T. camphoratus can produce many arthroconidia at the end of submerged fermentation, but molecular mechanism underlying this submerged conidiation remains unknown. In this study, we found that Ca²âº concentration in culture medium significantly affected the arthroconidium production of T. camphoratus. Then, we identified two proteins (CaM and HSP90) involved in Ca²âº/calmodulin signaling pathway and one protein (AbaA) involved in FluG-mediated conidiation pathway by two-dimensional electrophoresis analyses. Furthermore, we proposed a Ca²âº/calmodulin- and FluG-mediated signaling pathway by bioinformatics analysis. By real-time quantitative PCR analyses of 23 key genes in the Ca²âº/calmodulin- and FluG-mediated conidiation pathway, we found that expression levels of 7 genes (crz1, hsp90, flbB, brlA, abaA, wetA and fadA) showed significant responses to Ca²âº concentration in fermentation medium. Our research is beneficial for elucidating the underlying mechanism of submerged fermentation conidiation for T. camphoratus.

Comparative Transcriptomic and Proteomic Analyses Reveal a FluG-Mediated Signaling Pathway Relating to Asexual Sporulation of Antrodia camphorata.

Medicinal mushroom Antrodia camphorata sporulate large numbers of arthroconidia in submerged fermentation, which is rarely reported in basidiomycetous fungi. Nevertheless, the molecular mechanisms underlying this asexual sporulation (conidiation) remain unclear. Here, we used comparative transcriptomic and proteomic approaches to elucidate possible signaling pathway relating to the asexual sporulation of A. camphorata. First, 104 differentially expressed proteins and 2586 differential cDNA sequences during the culture process of A. camphorata were identified by 2DE and RNA-seq, respectively. By applying bioinformatics analysis, a total of 67 genes which might play roles in the sporulation were obtained, and 18 of these genes, including fluG, sfgA, SfaD, flbA, flbB, flbC, flbD, nsdD, brlA, abaA, wetA, ganB, fadA, PkaA, veA, velB, vosA, and stuA might be involved in a potential FluG-mediated signaling pathway. Furthermore, the mRNA expression levels of the 18 genes in the proposed FluG-mediated signaling pathway were analyzed by quantitative real-time PCR. In summary, our study helps elucidate the molecular mechanisms underlying the asexual sporulation of A. camphorata, and provides also useful transcripts and proteome for further bioinformatics study of this valuable medicinal mushroom.

Efficient production of bioactive metabolites from Antrodia camphorata ATCC 200183 by asexual reproduction-based repeated batch fermentation.

Large-scale submerged fermentation (SmF) of Antrodia camphorata (A. camphorata) usually encounters challenges including tedious preparation of mycelial inoculum, long fermentation period (10-14 d), and poor repeatability. Here we developed an asexual reproduction-based repeated batch fermentation (RBF) process for bioactive metabolites production by A. camphorata ATCC 200183. Compared with traditional batch fermentation, production time was shortened to 58 d from 80 d (overall time for eight cycles) using the RBF process established in this study, and accordingly, the productivities of bioactive metabolites (including antrodins) were improved by 40-60%. Kinetic parameters (α is 2.1-18.7 times as ß) indicated that the cell growth was the major contribution for bioactive metabolites production. The RBF shows excellent batch-repeatability (Pearson correlation coefficient of 0.998±0.001), together with advantages of energy-efficient, low cost, and labor-saving, RBF process can be implemented to SmF by other filamentous fungi.

Alpha-terpineol promotes triterpenoid production of Antrodia cinnamomea in submerged culture.

Antrodia cinnamomea is a medicinal mushroom producing potent bioactive triterpenoids. However, triterpenoids of A. cinnamomea in submerged culture are much less than those in fruiting bodies. Here we evaluated effects of different extracts from a host-related species, Cinnamomum camphora, on the mycelial growth and triterpenoid production of A. cinnamomea in submerged culture. The hot water extract of the stem showed the strongest promotion of the mycelial growth. The petroleum ether extract of the stem (PES) (0.05 g L(-1)) showed the greatest stimulatory effect on content and production of triterpenoids. A total of 39 compounds including terpenoids, phenolic and aromatic compounds were identified in the PES by GC-MS analysis. Furthermore, the effects of seven compounds contained in the PES on the mycelial growth and triterpenoid production of A. cinnamomea were evaluated. Among them, α-terpineol (0.5 mg L(-1)) showed the greatest stimulatory effect on the triterpenoid content (23.31 mg g(-1)) and triterpenoid production (91.33 mg L(-1)) of A. cinnamomea. Results of LC-MS analysis showed that α-terpineol (0.5 mg L(-1)) stimulated the syntheses of six triterpenoids in the mycelia of A. cinnamomea. This indicates that α-terpineol can act as an elicitor for triterpenoid biosynthesis in A. cinnamomea.