Solid-state fermentation of Apocynum venetum L. by Aspergillus niger: Effect on phenolic compounds, antioxidant activities and metabolic syndrome-associated enzymes.

This study aimed to evaluate the effect of solid-state fermentation (SSF) with Aspergillus niger on the total phenolic content (TPC), the total flavonoid content (TFC), individual phenolic contents, and antioxidant and inhibitory activities against metabolic syndrome-associated enzymes in an ethanol extract from Apocynum venetum L. (AVL). TPC, TFC, and the contents of quercetin and kaempferol during SSF were 1.52-, 1.33-, 3.64-, and 2.22-fold higher than those of native AVL in the ethyl acetate (EA) subfraction of the ethanol extract. The ABTS·+, DPPH· scavenging, and inhibitory activities against α-glucosidase and pancreatic lipase were found to be highest in the EA subfraction. Fermentation significantly increased the ABTS radical cation, DPPH radical scavenging, and pancreatic lipase inhibitory activities by 1.33, 1.39, and 1.28 times, respectively. TPC showed a significantly positive correlation with antioxidant activities or inhibition against metabolic syndrome-associated enzymes. This study provides a theoretical basis for producing tea products with enhanced antioxidant, antidiabetic, and antihyperlipidemic activities.

Liquid hot water pretreatment combined with high-solids enzymatic hydrolysis and fed-batch fermentation for succinic acid sustainable processed from sugarcane bagasse.

In order to sustainable process of bio-succinic acid (SA), response surface methodology (RSM) was applied to optimize liquid hot water pretreatment pretreatment of sugarcane bagasse (SCB), followed by high-solids enzymatic hydrolysis of pretreated residual that without washing, then the hydrolysates and partial pretreatment liquid were used as carbon sources for SA fermentation. Results showed that the highest sugars yield could be achieved at pretreatment conditions of temperature 186 °C, time 25 min and solid-to-liquid ratio 0.08; enzymatic digestion the pretreated residuals at 20 % (w/v) solid content via enzymes reconstruction and fed-batch strategy, the obtained sugars reached to 121 g/L; by controlling the nutrition and conditions of the fermentation process, most of the C5 and C6 sugars in the hydrolysate and pretreatment liquid were converted into SA with a conversion rate high to 280 mg/g SCB. This study can provide a novel clue for clean and efficient biorefining of chemicals.

Probiotic assessment and antioxidant characterization of Lactobacillus plantarum GXL94 isolated from fermented chili.

Oxidative stress is caused by an imbalance between prooxidants and antioxidants, which is the cause of various chronic human diseases. Lactic acid bacteria (LAB) have been considered as an effective antioxidant to alleviate oxidative stress in the host. To obtain bacterium resources with good antioxidant properties, in the present study, 113 LAB strains were isolated from 24 spontaneously fermented chili samples and screened by tolerance to hydrogen peroxide (H2O2). Among them, Lactobacillus plantarum GXL94 showed the best antioxidant characteristics and the in vitro antioxidant activities of this strain was evaluated extensively. The results showed that L. plantarum GXL94 can tolerate hydrogen peroxide up to 22 mM, and it could normally grow in MRS with 5 mM H2O2. Its fermentate (fermented supernatant, intact cell and cell-free extract) also had strong reducing capacities and various free radical scavenging capacities. Meanwhile, eight antioxidant-related genes were found to up-regulate with varying degrees under H2O2 challenge. Furthermore, we evaluated the probiotic properties by using in vitro assessment. It was showed that GXL94 could maintain a high survival rate at pH 2.5% or 2% bile salt or 8.0% NaCl, live through simulated gastrointestinal tract (GIT) to colonizing the GIT of host, and also show higher abilities of auto-aggregation and hydrophobicity. Additionally, the usual antibiotic susceptible profile and non-hemolytic activity indicated the safety of the strain. In conclusion, this study demonstrated that L. plantarum GXL94 could be a potential probiotic candidate for producing functional foods with antioxidant properties.

Improving saccharification of ramie stalks by synergistic effect of in-house cellulolytic enzymes consortium.

The high cost of cellulase is one of the main obstacles hindering the large-scale biorefining of lignocellulosic biomass. Therefore, developing efficient method for preparation of cellulase is promising. In the present study, the production of cellulase by Trichoderma reesei, Trichoderma harzianum, and Aspergillus niger was optimized, and the synergistic effect of these cellulase on enzymatic hydrolysis of pretreated ramie stalks was also evaluated. The maximum CMCase (Carboxymethyl Cellulase) and filter paper activity (FPA) produced by T. reesei reached to 3.12 IU/mL and 0.13 IU/mL, respectively. The maximum activities of CMCase (3.68 IU/mL), FPA (0.04 IU/mL) and ß-glucosidase (8.44 IU/mL) were obtained from A. niger. The results also showed that under the premise of the same FPA activity, the contribution of ß-glucosidase activity to yield of reducing sugar was greater than that of CMCase. Besides, cellulase produced by T. reesei and A. niger had the best synergistic effect on enzymatic hydrolysis of pretreated ramie stalks. The highest reducing sugars yield (417 mg/g dry substrate) was achieved when enzyme cocktail was prepared at the ratio of 1:1, which was 1.36-3.35 folds higher than that of different single enzymes. The present research has provided a novel method for efficient preparation of enzymes consortium for enzymatic hydrolysis of ramie stalks.

Landscape of meiotic crossovers in Hericium erinaceus.

Meiotic crossover shows marked interspecific and intraspecific variation, and knowledge about the molecular mechanism of crossover variation remains limited. Herein, we described the genome-wide scanning of crossover in one mushroom-forming fungus Hericium erinaceus. Utilizing the whole-genome single-nucleotide polymorphism (SNP) data-sets of a 127 F1 haploid progeny, we localized a total of 1316 crossover events and found that they were more likely to occur in the genic than intergenic regions. More than 30 % of the crossovers were concentrated in 59 crossover hotspots that were preferentially located close to chromosome ends. We then examined the genomic features around crossover hotspots. Results showed that the crossover hotspots were associated with increased gene density and guanine-cytosine (GC) content. An 8-bp GC-rich motif (GCGTCAGC) was found to be significantly enriched in these hotspots. The presence of mating-type loci affected the crossover at local scale rather than the overall crossover number. In order to dissect the genetic mechanisms shaping crossover variation, we then conducted quantitative trait locus (QTL) mapping for the total crossovers (TCO) and the crossover events that solely occurred within hotspots (HCO). Genome-wide QTL scanning identified four TCO-QTLs and two HCO-QTLs, which all located within or next to the crossover-hotspots. Crossover variations were shaped by multiple small-effect loci, with individual QTL contributing 6.9 %-11.7 % of variation. A few recombination pathway genes, including Spo11, Msh5, and Smc5 were found to be co-localized with the mapped crossover QTLs. Taken together, findings of this study offer insights into the crossover distribution and genetic factors conferring crossover variation in H. erinaceus, and advance our understandings for meiotic recombination in mushroom-forming fungi.

Comparative secretome of white-rot fungi reveals co-regulated carbohydrate-active enzymes associated with selective ligninolysis of ramie stalks.

In the present research, Phanerochaete chrysosporium and Irpex Lacteus simultaneously degraded lignin and cellulose in ramie stalks, whereas Pleurotus ostreatus and Pleurotus eryngii could depolymerize lignin but little cellulose. Comparative proteomic analysis of these four white-rot fungi was used to investigate the molecular mechanism of this selective ligninolysis. 292 proteins, including CAZymes, sugar transporters, cytochrome P450, proteases, phosphatases and proteins with other function, were successfully identified. A total of 58 CAZyme proteins were differentially expressed, and at the same time, oxidoreductases participated in lignin degradation were expressed at higher levels in P. eryngii and P. ostreatus. Enzyme activity results indicated that cellulase activities were higher in P. chrysosporium and I. lacteus, while the activities of lignin-degrading enzymes were higher in P. eryngii and P. ostreatus. In addition to the lignocellulosic degrading enzymes, several proteins including sugar transporters, cytochrome P450 monooxygenases, peptidases, proteinases, phosphatases and kinases were also found to be differentially expressed among these four species of white-rot fungi. In summary, the protein expression patterns of P. eryngii and P. ostreatus exhibit co-upregulated oxidoreductase potential and co-downregulated cellulolytic capability relative to those of P. chrysosporium and I. lacteus, providing a mechanism consistent with selective ligninolysis by P. eryngii and P. ostreatus.

Lactiplantibacillus plantarum AR113 Exhibit Accelerated Liver Regeneration by Regulating Gut Microbiota and Plasma Glycerophospholipid.

Emerging evidence indicates that probiotics have been proved to influence liver injury and regeneration. In the present study, the effects of Lactiplantibacillus plantarum AR113 on the liver regeneration were investigated in 70% partial hepatectomy (PHx) rats. Sprague-Dawley (SD) rats were gavaged with L. plantarum AR113 suspensions (1 × 1010 CFU/mL) both before and after partial hepatectomy. The results showed that L. plantarum AR113 administration 2 weeks before partial hepatectomy can accelerate liver regeneration by increased hepatocyte proliferation and tumor necrosis factor-α (TNF-α), hepatocyte growth factor (HGF), and transforming growth factor-ß (TGF-ß) expression. Probiotic administration enriched Lactobacillus and Bacteroides and depleted Flavonifractor and Acetatifactor in the gut microbiome. Meanwhile, L. plantarum AR113 showed decline of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidyl serine (PS), and lysophosphatidyl choline (LysoPC) levels in the serum of the rats after the L. plantarum AR113 administration. Moreover, L. plantarum AR113 treated rats exhibited higher concentrations of L-leucine, L-isoleucine, mevalonic acid, and lower 7-oxo-8-amino-nonanoic acid in plasma than that in PHx. Spearman correlation analysis revealed a significant correlation between changes in gut microbiota composition and glycerophospholipid. These results indicate that L. plantarum AR113 is promising for accelerating liver regeneration and provide new insights regarding the correlations among the microbiome, the metabolome, and liver regeneration.

Comparative Study on Bioactivities from Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), Gives an Insight into the Fermentation Broth Showing Greater Antioxidative Activities.

Ganoderma lucidum is one of the most famous mushrooms in traditional Chinese medicine. At present, the fully utilized parts of G. lucidum are mainly fruiting body and spore powder. The wild and artificially cultivated G. lucidum fruiting body is costly and rare. Therefore, how to improve the utilization of G. lucidum by means of fermentation is worth investigating. The present study was to perform submerged fermentation of G. lucidum and compare the bioactivities of G. lucidum submerged fermentation broth and fruiting body extract. After the extraction and submerged fermentation methods were optimized, the optimum conditions for extraction were determined as ethanol extraction at 80°C with a solid-to-liquid ratio of 1:30, and those for submerged fermentation were cultivation on malt extract medium for 6 days at 30°C. Under the optimum conditions, the antioxidative activity and tyrosinase inhibition rate of the fermentation broth were 1.2-4.1 fold higher than those of the ethanol extract. Cytotoxicity analysis showed that the ethanol and water extracts and the fermentation broth effectively inhibited pancreatic cancer cells and prostate cancer cells, with much smaller effect on nontumor human embryonic kidney (HEK293T). These results demonstrate that the submerged fermentation could improve the utilization value of G. lucidum and the fermentation broth can be used as an antioxidant additive applied in food, drugs, and cosmetics.

Whole genome sequence of an edible and medicinal mushroom, Hericium erinaceus (Basidiomycota, Fungi).

Hericium erinaceus is a well-known culinary and medicinal mushroom in China. The biological and genetic studies on this mushroom is rare, thereby hindering the breeding of elite cultivars. Herein, we performed de novo sequencing and assembly of H. erinaceus monokaryon CS-4 genome using the Illumina and PacBio platform. The generated genome was 41.2 Mb in size with a N50 scaffold size of 3.2 Mb, and encoded 10,620 putative predicted genes. A wide spectrum of carbohydrate-active enzymes, with the total number of 341 CAZymes, involved in lignocellulose degradation were identified in H. erinaceus. A total of 447 transcription factors were identified. This present study also characterized genome-wide microsatellites and developed markers in H. erinaceus. A comprehensive microsatellite markers database (HeSSRDb) containing the information of 904 markers was generated. These genomic resources and newly-designed molecular markers would enrich the toolbox for biological and genetic studies in H. erinaceus.

Mapping the Secretome and Its N-Linked Glycosylation of Pleurotus eryngii and Pleurotus ostreatus Grown on Hemp Stalks.

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.

A Resequencing-Based Ultradense Genetic Map of Hericium erinaceus for Anchoring Genome Sequences and Identifying Genetic Loci Associated With Monokaryon Growth.

Hericium erinaceus has attracted tremendous interest owing to its compelling health-promoting properties. However, breeding of elite cultivars of H. erinaceus is hindered by the lack of a genetic and molecular toolbox. Here, we performed resequencing analysis of 127 F1 single-spore isolates and constructed the first high-resolution genetic map of H. erinaceus. With the use of recombination bins as markers, an ultradense genetic map consisting of 1,174 bins (including 37,082 single-nucleotide polymorphisms) was generated. This newly developed genetic map covered 1,096.5 cM, with an average bin spacing of 0.95 cM. High collinearity between genetic map and H. erinaceus genome assembly was revealed by aligning scaffolds to this genetic map using bin markers as anchors. The application of this newly developed genetic map in quantitative trait locus (QTL) mapping was also elucidated, and four QTLs for monokaryon growth were recovered. One QTL, mgr1, which contributes 12.1% of growth variations, was located near the mating type A (MAT-A) loci. Overall, this newly constructed high-resolution genetic map (or bin map) could be used as reference in future genetic, genomic, and breeding studies on H. erinaceus.

A novel mechanism of Gamma-aminobutyric acid (GABA) protecting human umbilical vein endothelial cells (HUVECs) against H2O2-induced oxidative injury.

Vascular endothelial cell damage is related to many vascular diseases, including cardiovascular disease (CVD). Reactive oxygen species (ROS) play a vital role in the pathogenesis of many cardiovascular diseases. Herein, H2O2-induced human umbilical vein endothelial cell (HUVEC) injury model was used to explore the mechanisms involved in the pathogenesis of ROS-induced oxidative stress and cell dysfunction. Gamma-aminobutyric acid (GABA), a naturally occurring four-carbon non-protein amino acid, has antioxidant activity and anti-inflammatory action. In the present study, we demonstrated that GABA could scavenge free radicals including DPPH and ABTS, reverse H2O2-induced suppression on HUVEC proliferation, HUVEC apoptosis and ROS formation via p65 signaling. Interestingly, GABA treatment alone did not cause significant changes in p65 phosphorylation, suggesting that GABA will not cause imbalance in NF-κB signaling and ROS formation without oxidative stress. Moreover, GABA also modulated Keap1-Nrf2 and Notch signaling pathways upon H2O2 stimulation, suggesting that GABA may exert its effect via multi mechanisms. In conclusion, the present study demonstrated that GABA inhibits H2O2-induced oxidative stress in HUVECs via inhibiting ROS-induced NF-κB and Caspase 3 pathway activation. GABA may, therefore, have potential as a pharmacological agent in the prevention or treatment of oxidative injury-related cardiovascular disease.

Comparative transcriptomics of Pleurotus eryngii reveals blue-light regulation of carbohydrate-active enzymes (CAZymes) expression at primordium differentiated into fruiting body stage.

Blue light is an important environmental factor which could induce mushroom primordium differentiation and fruiting body development. However, the mechanisms of Pleurotus eryngii primordium differentiation and development induced by blue light are still unclear. The CAZymes (carbohydrate-active enzymes) play important roles in degradation of renewable lignocelluloses to provide carbohydrates for fungal growth, development and reproduction. In the present research, the expression profiles of genes were measured by comparison between the Pleurotus eryngii at primordium differentiated into fruiting body stage after blue light stimulation and dark using high-throughput sequencing approach. After assembly and compared to the Pleurotus eryngii reference genome, 11,343 unigenes were identified. 539 differentially expressed genes including white collar 2 type of transcription factor gene, A mating type protein gene, MAP kinase gene, oxidative phosphorylation associated genes, CAZymes genes and other metabolism related genes were identified during primordium differentiated into fruiting body stage after blue light stimulation. KEGG results showed that carbon metabolism, glycolysis/gluconeogenesis and biosynthesis of amino acids pathways were affected during blue light inducing primordia formation. Most importantly, 319 differentially expressed CAZymes participated in carbon metabolism were identified. The expression patterns of six representative CAZymes and laccase genes were further confirmed by qRT-PCR. Enzyme activity results indicated that the activities of CAZymes and laccase were affected in primordium differentiated into fruiting body under blue light stimulation. In conclusion, the comprehensive transcriptome and CAZymes of Pleurotus eryngii at primordium differentiated into fruiting body stage after blue light stimulation were obtained. The biological insights gained from this integrative system represent a valuable resource for future genomic studies on this commercially important mushroom.

Biodegradation of ramie stalk by Flammulina velutipes: mushroom production and substrate utilization.

In the textile industry, ramie stalk is byproducts with a low economic value. The potential use of this leftover as a substrate ingredient for Flammulina velutipes (F. velutipe) cultivation was evaluated. The degradation and utilization of ramie stalk by F. velutipes was evaluated through mushroom production, lignocelluloses degradation and lignocellulolytic enzymes activity. The best substrate mixture for F. velutipes cultivation comprised 50% ramie stalk, 20% cottonseed hulls, 25% wheat bran, 4% cornstarch and 2% CaCO3. The highest biological efficiency of fruiting bodies was reached 119.7%. F. velutipes appears to degrade 12.7-32.0% lignin, 14.4-30.2% cellulose and 9.3-25.7% hemicellulose during cultivation on the different substrates. The results of enzymes activities showed that laccase and peroxidase were higher before fruiting; while cellulase and hemicellulase showed higher activities after fruiting. The biological efficiency of fruiting bodies was positively correlated with the activities of cellulase, hemicellulase and ligninolytic enzyme. The results of this study demonstrate that ramie stalk can be used as an effective supplement for increasing mushroom yield in F. velutipes.

White-rot fungi pretreatment combined with alkaline/oxidative pretreatment to improve enzymatic saccharification of industrial hemp.

White-rot fungi combined with alkaline/oxidative (A/O) pretreatments of industrial hemp woody core were proposed to improve enzymatic saccharification. In this study, hemp woody core were treated with only white rot fungi, only A/O and combined with the two methods. The results showed that Pleurotus eryngii (P. eryngii) was the most effective fungus for pretreatment. Reducing sugars yield was 329mg/g with 30 Filter Paper Unit (FPU)/g cellulase loading when treated 21day. In the A/O groups, the results showed that when treated with 3% NaOH and 3% H2O2, the yield of reducing sugars was 288mg/g with 30FPU/g cellulase loading. After combination pretreatment with P. eryngii and A/O pretreatment, the reducing sugar yield from enzymatic hydrolysis of combined sample increased 1.10-1.29-fold than that of bio-treated or A/O pretreatment sample at the same conditions, suggesting that P. eryngii combined with A/O pretreatment was an effective method to improve enzyme hydrolysis.

Effects of Different Substrates on Lignocellulosic Enzyme Expression, Enzyme Activity, Substrate Utilization and Biological Efficiency of Pleurotus Eryngii.

BACKGROUND/AIMS: Pleurotus eryngii is one of the most valued and delicious mushrooms which are commercially cultivated on various agro-wastes. How different substrates affect lignocellulosic biomass degradation, lignocellulosic enzyme production and biological efficiency in Pleurotus eryngii was unclear. METHODS AND RESULTS: In this report, Pleurotus eryngii was cultivated in substrates including ramie stalks, kenaf stalks, cottonseed hulls and bulrush stalks. The results showed that ramie stalks and kenaf stalks were found to best suitable to cultivate Pleurotus eryngii with the biological efficiency achieved at 55% and 57%, respectively. In order to establish correlations between different substrates and lignocellulosic enzymes expression, the extracellular proteins from four substrates were profiled with high throughput TMT-based quantitative proteomic approach. 241 non-redundant proteins were identified and 74 high confidence lignocellulosic enzymes were quantified. Most of the cellulases, hemicellulases and lignin depolymerization enzymes were highly up-regulated when ramie stalks and kenaf stalks were used as carbon sources. The enzyme activities results suggested cellulases, hemicellulases and lignin depolymerization enzymes were significantly induced by ramie stalks and kenaf stalks. CONCLUSION: The lignocelluloses degradation, most of the lignocellulosic enzymes expressions and activities of Pleurotus eryngii had positive correlation with the biological efficiency, which depend on the nature of lignocellulosic substrates. In addition, the lignocellulosic enzymes expression profiles during Pleurotus eryngii growth in different substrates were obtained. The present study suggested that most of the lignocellulosic enzymes expressions and activities can be used as tools for selecting better performing substrates for commercial mushroom cultivation.

Secretome analysis of Pleurotus eryngii reveals enzymatic composition for ramie stalk degradation.

Pleurotus eryngii (P. eryngii) can secrete large amount of hydrolytic and oxidative enzymes to degrade lignocellulosic biomass. In spite of several researches on the individual lignolytic enzymes, a direct deconstruction of lignocellulose by enzyme mixture is not yet possible. Identifying more high-performance enzymes or enzyme complexes will lead to efficient in vitro lignocelluloses degradation. In this report, secretomic analysis was used to search for the new or interesting enzymes for lignocellulose degradation. Besides, the utilization ability of P. eryngii to ramie stalk substrate was evaluated from the degradation of cellulose, hemicellulose, and lignin in medium and six extracellular enzymes activities during different growth stages were discussed. The results showed that a high biological efficiency of 71% was obtained; cellulose, hemicelluloses, and lignin decomposition rates of P. eryngii were 29.2, 26.0, and 51.2%, respectively. Enzyme activity showed that carboxymethyl cellulase, xylanase, laccase, and peroxidase activity peaks appeared at the primordial initiation stage. In addition, we profiled a global view of the secretome of P. eryngii cultivated in ramie stalk media to understand the mechanism behind lignocellulosic biomass hydrolysis. Eighty-seven nonredundant proteins were identified and a diverse group of enzymes, including cellulases, hemicellulases, pectinase, ligninase, protease, peptidases, and phosphatase implicated in lignocellulose degradation were found. In conclusion, the information in this report will be helpful to better understand the lignocelluloses degradation mechanisms of P. eryngii.