Mycelial Biomass Production Research Articles

Enhancement of Mycelial Biomass Production and Bioactive Properties Related to Cosmeceuticals of Calocybe indica (Milky Mushroom) by Optimization of Submerged Culture Conditions

Calocybe indica or milky mushroom grown in submerged culture was investigated as a new source of antioxidants and anti-collagenase substances, and for its ability to inhibit three bacteria that trigger inflammatory acne, Cutibacterium acnes, Staphylococcus epidermidis, and S. aureus. To facilitate C. indica submerged cultivation and achieve high productivity, a three-level four-factor Box-Behnken design was utilized to find the optimal culture conditions for its mycelial biomass production. The model estimated the optimal conditions as 30 °C, a medium pH of 5.9, and incubation for 14 days under static culture, with results verifi ed by validation experiments. The optimal conditions gave mycelial biomass of 7.12 g/L, which is 5-fold higher than the original condition. This statistical method facilitated rapid identification and integration of key factors for C. indica high mycelial biomass production with significantly improved cosmeceutical properties in terms of antioxidant and antibacterial activities against acne-causing bacteria, especially C. acnes at 19-fold and 18-fold, respectively, compared to the original condition. An ethanolic extract derived from C. indica mycelia was nontoxic to human skin keratinocytes (HaCaT). These diverse functionalities indicate that the mycelial extract of C. indica may represent a promising source of bioactive substances for cosmeceutical applications.

Ectomycorrhizal necromass turnover is one‐third of biomass turnover in hemiboreal Pinus sylvestris forests

Societal Impact StatementEfficient mitigation of climate change requires predictive models of forest ecosystems as sinks for atmospheric carbon. Mycorrhizal fungi are drivers of soil carbon storage in boreal forests, yet they are typically excluded from ecosystem models, because of a lack of information about their growth and turnover. Closing this knowledge gap could help us better predict future responses to climate change and guide policy decisions for sustainable management of forest ecosystems. This study provides new estimates of the production and turnover of mycorrhizal mycelial biomass and necromass. This information can facilitate the integration of mycorrhizal fungi into new predictive models of boreal forest soils.Summary In boreal forests, turnover of biomass and necromass of ectomycorrhizal extraradical mycelia (ERM) are important for mediating long‐term carbon storage. However, ectomycorrhizal fungi are usually not considered in ecosystem models, because data for parameterization of ERM dynamics is lacking. Here, we estimated the production and turnover of ERM biomass and necromass across a hemiboreal Pinus sylvestris chronosequence aged 12 to 100 years. Biomass and necromass were quantified in sequentially harvested in‐growth bags, and incubated in the soil for 1–24 month, and Bayesian calibration of mathematical models was applied to arrive at parametric estimates of ERM production and turnover rates of biomass and necromass. Steady states were predicted to be nearly reached after 160 and 390 growing season days, respectively, for biomass and necromass. The related turnover rates varied with 95% credible intervals of 1.7–6.5 and 0.3–2.5 times yr−1, with mode values of 2.9 and 0.9 times yr−1, corresponding to mean residence times of 62 and 205 growing season days. Our results highlight that turnover of necromass is one‐third of biomass. This together with the variability in the estimates can be used to parameterize ecosystem models, to explicitly include ERM dynamics and its impact on mycorrhizal‐derived soil carbon accumulation in boreal forests.

Adding-value to Ganoderma lingzhi by producing enzymes and antioxidant compounds under submerged fermentation using different culture media.

Ganoderma lingzhi is widely reported for its medicinal properties, presenting several bioactive substances with potential pharmaceutical and industrial application. This study aimed to evaluate the production of mycelial biomass, extracellular enzymes and antioxidant compounds by G. lingzhi under submerged fermentation. G. lingzhi was cultured in Polysaccharide (POL) and Melin-Norkrans (MNM) media for 7 days. The cellulases, xylanases, pectinases, laccases, and proteases activities were quantified in the culture broth, while the antioxidant potential was evaluated for the mycelial biomass. G. lingzhi showed higher biomass production in MNM. However, it exhibited similar microstructural characteristics in both culture media. In the POL there was greater activity of CMCase (0.229 U/mL), xylanase (0.780 U/mL), pectinase (0.447 U/mL) and proteases (16.13 U/mL). FPase did not differ (0.01 U/mL), and laccase was detected only in MNM (0.122 U/mL). The biomass water extract from MNM showed high levels of phenolic compounds (951.97 mg AGE/100 g). DPPH• inhibition (90.55%) and reducing power (0.456) were higher in MNM medium, while ABTS•+ inhibition (99.95%) and chelating ability (54.86%) were higher in POL. Thus, the MNM medium was more favorable to the production of mycelial biomass and phenolic compounds, while the POL medium favored the synthesis and excretion of hydrolytic enzymes.

Zn Fortification Influential Impact on the Productivity of Calocybe indica Mycelium

Calocybe indica is an edible medicinal mushroom, preferably eaten for its culinary value. It was cultivated for its higher nutritional value, medicinal properties, and high polysaccharide content, especially glucan. Some particular minerals were enriched in food substitutes, an alternative to fighting against some targeted human ailments. Thus, mineral fortification is accessible in the submerged cultivation of mushrooms to produce bioactive compounds and fortified mushrooms. In this study, the submerged cultivation of C. indica was performed to make exopolysaccharides (EPS) using a supplemented medium of Zinc with varying concentrations. Thus, research revealed that the Zn fortification enhances the production of EPS and mycelial biomass after 21 days of incubation. The maximum mycelial biomass was 7.7133±0.30 g/L (dry weight), and the highest 0.3853±0.006EPS was produced in the 175mg/L mineral concentration, respectively. The present study revealed that the Zn supplementation gradually increased the mineral concentration and directly influenced the yield of mycelial biomass and EPS production. These EPS have various biological activities and can be helpful for fortified food or pharmaceutical product development in the medicinal and pharmaceutical sectors.

Optimization of cultural and nutritional conditions to enhance mycelial biomass of Cordyceps militaris using statistical approach.

Cordyceps militaris is a fungus with numerous therapeutic properties that has gained worldwide popularity due to its potential health benefits. The fruiting body of this mushroom is highly expensive and takes a longer time to produce, making mycelial a sustainable and cost-effective alternative. The study investigates and optimizes cultural and nutritional conditions to maximize mycelial biomass. The initial optimization was done by the conventional single-factor approach, followed by Plackett-Burman design to screen the most significant variables, with yeast extract, temperature, and glucose being the most significant, contributing 11.58%, 49.74%, and 27.98%, respectively, in mycelial biomass production. These variables were then optimized using response surface methodology (RSM) based on central composite design (CCD). The study observed that temperature and glucose had the highest impact on mycelial biomass, with p-values of 0.0128 and 0.0191, respectively. Under the optimized conditions, temperature 20°C, glucose 2.5% (w/v), and yeast extract 0.8% (w/v), the maximal yield of mycelial biomass reached 547 ± 2.09mg/100mL, which was 1.95-fold higher than the yield in the basal medium. These findings suggest that optimizing the cultural and nutritional conditions can enhance mycelial biomass production of Cordyceps militaris, offering a sustainable and cost-effective source of this valuable fungus.

Yeast Lipids from Crude Glycerol Media and Utilization of Lipid Fermentation Wastewater as Maceration Water in Cultures of Edible and Medicinal Mushrooms

Four wild “red” yeast strains (Rhodosporidium kratochvilovae FMCC Y70, R. toruloides NRRL Y-27013, R. toruloides NRRL Y-17902 and R. toruloides NRRL Y-6985) were cultured in shake flasks on industrial glycerol at an initial substrate (Gly0) concentration ≈ 50 g/L under nitrogen limitation. Strains NRRL Y-27013, NRRL Y-17902 and NRRL Y-6985 presented appreciable dry cell weight (DCW) and lipid synthesis (DCW up to 18–19 g/L containing lipids in quantities ≈ 47%, w/w). Strains NRRL Y-27013 and NRRL Y-6985 were further tested in higher Gly0 concentrations (≈90 g/L and ≈110 g/L) with the same initial nitrogen quantity as in the first (“screening”) experiment. Both strains, despite the high Gly0 concentrations and C/N ratios (up to 120 moles/moles) imposed, presented significant DCW production (up to c. 29.0–29.5 g/L). Yeast biomass contained significant lipid (42–43%, w/w) and endopolysaccharide (up to 42%, w/w) quantities. Both lipids and endopolysaccharide quantities (in % w/w) noticeably increased as a response to the imposed nitrogen limitation. Lipids containing mainly oleic and palmitic acids constituted ideal candidates for biodiesel synthesis. Thereafter, the wastewaters derived from the lipid production process (lipid fermentation wastewaters—LFWs) were used as maceration waters in cultivations of edible and medicinal fungi, where novel (non-conventional) substrates were used in the performed cultures. CW (coffee residue + wheat straw), CB (coffee residue + beech wood shavings), OW (olive crop + wheat straw), OB (olive crop + beech wood shavings), RW (rice husk + wheat straw) and RB (rice husk + beech wood shavings) were soaked/sprayed with LFWs or tap water and utilized in the cultivation of Pleurotus, Ganoderma and Lentinula mushrooms. The impact of LFWs on the mycelial growth rate (mm/d) and biomass production was evaluated. The results show that regardless of the wetting method, the highest growth rates (6.2–6.6 mm/d) were noticed on RW and RB for Pleurotus eryngii and Ganoderma resinaceum, on OW, OB and RW for Ganoderma applanatum and on RW, OW and OB for Lentinula edodes. Nevertheless, high biomass production was obtained on substrates soaked with LFWs for Pleurotus ostreatus (RW: 443 mg/g d.w.), L. edodes (RB: 238 mg/g d.w.) and Ganoderma lucidum (RW: 450 mg/g d.w.). Overall, this study demonstrates the possibility of the industrial conversion of low-value agro-waste to mycelial mass and eventually to important food products.

Valorization of spent Brewers yeast in the integrated production of the fungal exopolysaccharide (1 → 6)-β-D-glucan (lasiodiplodan) and single-cell protein

Spent Brewers yeast was studied as a low-cost alternative feedstock for sustainably producing a high value-added microbial β-D-glucan (lasiodiplodan). The potential of using the mycelial biomass (single-cell protein, SCP) generated in the production of β-glucan was also evaluated as a strategy of an integrated biorefining. Five cell lysis protocols were studied to obtain a yeast cell lysate rich in nutrients. Mineral supplementation of the fermentation medium was assessed, and the SCP produced was characterized by the nutritional composition, amino acid, and lipid profiles. Lasiodiplodia theobromae MMPI was effective in the co-production of lasiodiplodan ((1 → 6)-β-D-glucan)) and SCP in a cell lyzate-based medium. Enzymatic lysis with Alcalase promoted more significant cell rupture and release of soluble intracellular components. Highest production of lasiodiplodan (2.12 gL-1) and mycelial biomass (9.95 gL-1) resulted from Alcalase-treated cell lysate-based medium (7 gproteinL−1) supplemented with sucrose (60 gL-1). Under these conditions, volumetric productivities of lasiodiplodan and mycelial biomass: 0.029 gL−1h−1 and 0.138 gL−1h−1, respectively, were obtained. Mycelial biomass generated in this cultivation was rich in protein and contained all essential amino acids, especially high concentrations of leucine (69 mgg−1protein), lysine (62.2 mgg−1protein), valine (44.2 mgg−1protein), threonine (43.2 mgg−1protein). Co-production of lasiodiplodan and SCP could be an economically promising bioprospecting alternative.

Produksi Biomassa, Analisis Nutrisi dan Senyawa Bioaktif Jamur Grigit (Schizophyllum commune)

Schizophyllum commune, a wild edible mushroom, has gained significant attention due to its potential as a valuable source of biomass, nutrients, and bioactive compounds. This study aims to explore the production of mycelial biomass and analyze its nutrient and bioactive compound content to be applied as a raw material in food production. The Schizophyllum commune was cultivated in potato dextrose broth media with temperature (25, 30, 35, and 40oC) and pH treatments (3, 7, and 8) to determine the optimal conditions for biomass production. Mycelia biomass was harvested and extracted. Nutrient and bioactive compounds were analyzed. Results showed that 30oC and pH 7 give the highest biomass production. Proximate analysis revealed that the mushroom's mycelia biomass contained high protein, low fat, and fiber. The analysis also demonstrated a rich profile of bioactive compounds, including flavonoid and phenolic compounds, respectively1 2,32 ± 0,95 dan 415,72 ± 15,23 g/100 g mycelia dry weight. Schizophyllum commune exhibits promising prospects for mycelial biomass production.

Harvesting Mycelial Biomass of Selected Basidiomycetes for Chitosan Biopolymer Extraction.

This study investigates the mycelial biomass production and chitosan extraction potential of various Basidiomycota strains, including Heterobasidion annosum, Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor, and Lentinus lepideus. Both submerged fermentation (SF) and solid-state fermentation (SSF) methods were employed. The chitosan yield in basidiocarps of Pleurotus ostreatus, Agaricus bisporus, and Ganoderma applanatum was also evaluated as a reference material. The chitosan extracted from fungal cells was characterized using elemental analyses and FTIR spectroscopy. Among the cultivated strains, P. chrysosporium exhibited the highest mycelial biomass concentration in SF (1.03 g 100 mL-1) after 14 days, while T. versicolor achieved the highest biomass concentration in SSF (3.65 g 100 mL-1). The highest chitosan yield was obtained from the mycelium of P. chrysosporium (0.38%) and T. versicolor (0.37%) in shaken SF. Additionally, commercially cultivated A. bisporus demonstrated the highest chitosan yield in fungal fruiting bodies (1.7%). The extracted chitosan holds potential as a functional biopolymer additive for eco-friendly materials, serving as an alternative to synthetic wet and dry strength agents in packaging materials.

Utilization of spent substrates and waste products of mushroom cultivation to produce new crops of Pleurotus ostreatus, Pleurotus eryngii and Agaricus bisporus

Five agro-industrial residues, their corresponding spent mushroom substrates (SMS), commercial fresh and spent mushroom compost of Agaricus (SMC) and Pleurotus ostreatus (SMS GZ) cultivation, Pleurotus and Agaricus waste (stipes/mishappen mushrooms) were combined and re-utilized as novel substrates for the cultivation of P. ostreatus, P. eryngii and A. bisporus mushrooms. SMSs/ SMC were used up to 40% (treatment 1 and 2), while 20% of mushroom waste were added in fresh and spent substrates (treatment 3 and 4). The impact of different substrates on mycelial growth rate and biomass production was examined. Then, Pleurotus spp. were cultivated on the most promising substrates and essential cultivation aspects (earliness, total mushroom yield, biological efficiency-BE) and carposomes’ quality parameters (weight, morphological characteristics) were evaluated. Laccase and endoglucanase production by Pleurotus species were also determined at 50 and 100% of colonization stages. All species showed their fastest mycelial growth rate (up to 5 mm/day) on substrates consisted of SMC, whereas many combinations of species/substrate enhanced biomass production. The SMS GZ supplementation positively affected laccase activity; in the cultures of P. ostreatus and P. eryngii the highest values were 62,539 and 17,584 U/g d.w., respectively. On the contrary, small amounts of endoglucanase were produced (0.007 to 0.322 U/g d.w.); the greatest production was recorded for P. ostreatus at full colonization. Regarding fermentation in bags, significant amount of total mushroom yield was produced in all substrates and those with SMS GZ supported the fastest earliness period and the highest BE for both Pleurotus species. BE values ranged from 54 to 133% for P. ostreatus and from 53 to 121% for P. eryngii. Concerning morphological characteristics, mushroom waste addition seemed to affect them positively. The data included in this paper support the effective re-utilization of different types of SMS and mushroom waste for fungal mass and enzymes’ production and for new high quality Pleurotus spp. carposomes.

Assessment of the Fungistatic Properties of Calendula officinalis L. Water Extract and the Effect of Its Addition on the Quality of Wheat Bread

The potential of Calendula officinalis water extract against fungi Aspergillus niger and Penicillium sp. and the effect of extract addition on the quality of wheat bread were investigated. In vitro, the extract reduced the mycelial growth and biomass production of A. niger, but there was no inhibitory effect on Penicillium sp. Enriched bread showed significantly higher total phenolic content, by about 77% and 95% in the bread, in which 10% and 15% of the water was replaced with extract, respectively. The antioxidant potential against DPPH• was significantly higher (compared to the control) in both variants used in the experiment, and the level of antioxidant activity increased with the addition of extract. The enriched bread had good quality characteristics—lower baking losses and higher volume than the control. The moisture content and acidity of the crumb of the extract-enriched bread were also higher. The extract additive used did not affect the sensory properties of the bread.

Mycelial biomass and intracellular polysaccharides production, characterization, and activities in Auricularia auricula-judae cultured with different carbon sources

The carbon source, an essential factor for submerged culture, affects fungal polysaccharides production, structures, and activities. This study investigated the impact of carbon sources, including glucose, fructose, sucrose, and mannose, on mycelial biomass and the production, structural characterization, and bioactivities of intracellular polysaccharides (IPS) produced by submerged culture of Auricularia auricula-judae. Results showed that mycelial biomass and IPS production varied with different carbon sources, where using glucose as the carbon source produced the highest mycelial biomass (17.22 ± 0.29 g/L) and IPS (1.62 ± 0.04 g/L). Additionally, carbon sources were found to affect the molecular weight (Mw) distributions, monosaccharide compositions, structural characterization, and activities of IPSs. IPS produced with glucose as the carbon source exhibited the best in vitro antioxidant activities and had the strongest protection against alloxan-damaged islet cells. Correlation analysis revealed that Mw correlated positively with mycelial biomass (r = 0.97) and IPS yield (r = 1.00), while IPS antioxidant activities correlated positively with Mw and negatively with mannose content; the protective activity of IPS was positively related to its reducing power. These findings indicate a critical structure-function relationship for IPS and lay the foundation for utilizing liquid-fermented A. aruicula-judae mycelia and the IPS in functional food production.

High-Molecular-Weight Exopolysaccharides Production from Tuber borchii Cultivated by Submerged Fermentation

Truffles are known worldwide for their peculiar taste, aroma, and nutritious properties, which increase their economic value. However, due to the challenges associated with the natural cultivation of truffles, including cost and time, submerged fermentation has turned out to be a potential alternative. Therefore, in the current study, the cultivation of Tuber borchii in submerged fermentation was executed to enhance the production of mycelial biomass, exopolysaccharides (EPSs), and intracellular polysaccharides (IPSs). The mycelial growth and EPS and IPS production was greatly impacted by the choice and concentration of the screened carbon and nitrogen sources. The results showed that sucrose (80 g/L) and yeast extract (20 g/L) yielded maximum mycelial biomass (5.38 ± 0.01 g/L), EPS (0.70 ± 0.02 g/L), and IPS (1.76 ± 0.01 g/L). The time course analysis of truffle growth revealed that the highest growth and EPS and IPS production was observed on the 28th day of the submerged fermentation. Molecular weight analysis performed by the gel permeation chromatography method revealed a high proportion of high-molecular-weight EPS when 20 g/L yeast extract was used as media and the NaOH extraction step was carried out. Moreover, structural analysis of the EPS using Fourier-transform infrared spectroscopy (FTIR) confirmed that the EPS was β-(1-3)-glucan, which is known for its biomedical properties, including anti-cancer and anti-microbial activities. To the best of our knowledge, this study represents the first FTIR analysis for the structural characterization of β-(1-3)-glucan (EPS) produced from Tuber borchii grown in submerged fermentation.

Enhancing Biomass-Exopolysaccharides Production of Lignosus rhinocerus in a High-Scale Stirred-Tank Bioreactor and Its Potential Lipid as Bioenergy

The depletion of fossil fuels and the emission of greenhouse gases have increased the demand for new and sustainable energy sources, leading to growing interest in using fast-growing filamentous fungi as a source of bioenergy. This study aimed to optimize the production of exopolysaccharides (EPS) and mycelial biomass (MB) from the native medicinal mushroom, Lignosus rhinocerus, through submerged liquid fermentation. Using response surface methodology (RSM), it was found that the glucose concentration and speed of agitation significantly influenced the production of MB and EPS (p < 0.05), while the initial pH medium had an insignificant effect. The validated optimized parameters of 50.0 g/L glucose, initial pH 4.0, and 128 rpm for speed of agitation were tested in 500 mL shake flasks, 5 L, and 13 L stirred-tank (STR) bioreactors. The production of MB and EPS increased significantly by ~1.2-fold in the 5 L STR and further increased to ~1.7-fold (MB) and ~2.4-fold (EPS) in the 13 L STR bioreactor compared to the shake flask. The lipid content of MB was also determined, with a result of 2.07% w/w using the Soxhlet extraction method. To conclude, this study emphasizes the ability of L. rhinocerus as a new source of bioenergy through large-scale production, with optimized parameters serving as a reference for future research and practical applications.

Carposome productivity of Pleurotus ostreatus and Pleurotus eryngii growing on agro-industrial residues enriched with nitrogen, calcium salts and oils

The suitability of the abundant agro-industrial residues wheat straw (WS; control), barley and oats straw (BOS) and rice husk (RH), supplemented with various sources of oils (sunflower, corn oil), nitrogen (peptone, yeast extract) and calcium salts (CaSO4·2H2O, CaCl2), as novel substrates in solid-state fermentation of selected Pleurotus ostreatus and P. eryngii mushrooms was investigated. The possible effect of different additives on mycelial growth rate, biomass production and endoglucanase, laccase and lipase biosynthesis were evaluated. Moreover, their impact on essential cultivation aspects (earliness, total mushroom yield, biological efficiency) and carposome quality parameters (weight, morphological characteristics) was assessed. Both fungi showed their highest growth rates on BOS substrates and the most positive implementation was CaSO4·2H2O 6 % w/w (Kr = 9.58 mm/ day; P. ostreatus, Kr = 9.42 mm/ day; P. eryngii), while different additives led to enhancement of biomass production. Pleurotus species demonstrated minimal levels of endoglucanase activity, with values ranging from 0.01 to 0.42 U/g of dry weight, regardless of the substrate and the stage of colonization. On the contrary, the maximum values of laccase activity were observed at 50 % of colonization on BOS and RH, while supplementation with nitrogen and calcium sources positively affected its biosynthesis. P. ostreatus and P. eryngii cultivated on BOS supplemented with peptone at 2 and 5 % w/w, synthesized significant laccase amounts, i.e., 12,165.78 and 8,624.55 U/g d.w., respectively. Satisfactory amounts of lipase were produced, especially in substrates supplemented with sunflower 2 % w/w, in quantities up to 1.42 U/g d.w., whereas the highest lipase activity was achieved by P. eryngii on WS supplemented with corn oil at 2 % w/w, with a value of 4.25 U/g d.w. being recorded. Regarding fermentation of Pleurotus species in polypropylene bags, WS and BOS supported faster colonization and shorter earliness period than RH substrates, whereas supplementation did not seem to affect these culture parameters. Furthermore, oils supplementation had a positive effect on BE of both species, with values up to 100 % for P. ostreatus and 80 % for P. eryngii on WS and BOS, whereas on RH the lowest BE values were detected. Morphological characteristics were not significantly affected by the additives. Results indicate the positive impact that certain additives have on mushroom productivity and production of enzymes with great financial and environmental importance.

Optimization of mycelial culture condition and biomass production of selected wild Agaric mushrooms from Luzon Island, Philippines

Optimization of mycelial culture condition and biomass production of selected wild Agaric mushrooms from Luzon Island, Philippines

Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

MicroRNA-like RNA Functions Are Required for the Biosynthesis of Active Compounds in the Medicinal Fungus Sanghuangporus vaninii.

miRNA-like RNAs (milRNAs) have been recognized as sequence-specific regulators of posttranscriptional regulation of gene expression in eukaryotes. However, the functions of hundreds of fungal milRNAs in the biosynthesis of metabolic components are obscure. Sanghuangporus produces diverse bioactive compounds and is widely used in Asian countries. Here, genes encoding two Dicers, four Argonautes, and four RdRPs were identified and characterized in Sanghuangporus vanini. Due to the lack of an efficient gene manipulation system, the efficacy of spray-induced gene silencing (SIGS) was determined in S. vanini, which showed efficient double-stranded RNA (dsRNA) uptake and gene silencing efficiency. SIGS-mediated gene knockdown showed that SVRDRP-3, SVRDRP-4, SVDICER-1, and SVDICER-2 were critical for mycelial biomass, flavonoid, triterpenoid, and polysaccharide production. Illumina deep sequencing was performed to characterize the milRNAs from S. vanini mycelium and fruiting body. A total of 31 milRNAs were identified, out of which, SvmilR10, SvmilR17, and SvmilR33 were Svrdrp-4- and Svdicer-1-dependent milRNAs. Importantly, SIGS-mediated overexpression of SvmilR10 and SvmilR33 resulted in significant changes in the yields of flavonoids, triterpenoids, and polysaccharides. Further analysis showed that these milRNA target genes encoding the retrotransposon-derived protein PEG1 and histone-lysine N-methyltransferase were potentially downregulated in the milRNA overexpressing strain. Our results revealed that S. vanini has high external dsRNA and small RNA uptake efficiency and that milRNAs may play crucial regulatory roles in the biosynthesis of bioactive compounds. IMPORTANCE Fungi can take up environmental RNA that can silence fungal genes with RNA interference, which prompts the development of SIGS. Efficient dsRNA and milRNA uptake in S. vanini, successful dsRNA-targeted gene block, and the increase in intracellular miRNA abundance showed that SIGS technology is an effective and powerful tool for the functional dissection of fungal genes and millRNAs. We found that the RdRP, Dicer, and Argonaute genes are critical for mycelial biomass and bioactive compound production. Our study also demonstrated that overexpressed SVRDRP-4- and SVDICER-1-dependent milRNAs (SvmilR10 and SvmilR33) led to significant changes in the yields of the three active compounds. This study not only provides the first report on SIGS-based gene and milRNA function exploration, but also provides a theoretical platform for exploration of the functions of milRNAs involved in biosynthesis of metabolic compounds in fungi.

Lithium bioaccumulation in Lentinus crinitus mycelia grown in media with different lithium sources and pH values.

Lentinus crinitus bioaccumulates lithium in mycelia, but bioaccumulation may be affected by pH of the culture medium. Lithium is used in clinical practice as a mood stabilizer and antidepressant. This study aimed to assess the effect of culture medium pH and lithium source (LiCl or Li2CO3) on lithium bioaccumulation in vegetative mycelia of L. crinitus grown in malt extract broth. Lentinus crinitus U9-1 was cultured in malt extract broth supplemented with Li2CO3 or LiCl (50mg L-1 lithium) in the pH range of 3.0 to 6.0. The pH was adjusted using HCl solution. The results showed that medium pH affected mycelial biomass production, lithium bioaccumulation in mycelial biomass, and lithium transfer from the culture medium to mycelial biomass. The effect of lithium source on the bioaccumulation capacity of mycelial biomass varied according to pH. At pH 4.0, both lithium sources stimulated mycelial biomass production compared to the control without the addition of lithium. At pH 5.5, Li2CO3 provided the highest lithium bioaccumulation in mycelial biomass. Lithium transfer from the culture medium to mycelia was highest in Li2CO3-supplemented cultures at pH 4.5. LiCl reduced hyphal width compared with Li2CO3 and the control. However, pH and lithium sources did not affect the formation of clamp connections in hyphae. For the first time, the influence of the pH of the culture medium on lithium bioaccumulation by Lentinus crinitus is reported. Finally, we conclude that the culture medium pH affected lithium transfer and bioaccumulation in mycelial biomass differently depending on the lithium source. Additionally, we report the presence of clamp connections in the hyphae of L. crinitus as an indicator of even growth.

Endophytic fungi of Brunfelsia uniflora: isolation, cryopreservation, and determination of enzymatic and antioxidant activity.

Brunfelsia uniflora (Pohl.) D. Don (Solanaceae), commonly known as manacá-de-cheiro, is widely distributed in Brazil and used by local indigenous peoples as an antirheumatic, antisyphilitic, depurative, emetic, vermifuge, and purgative agent. Several studies have examined the biological activities and phytochemical profile of Brunfelsia; however, few have focused on the diversity of endophytic microorganisms that colonize members of the genus. This study aimed to isolate and cryopreserve endophytic fungi from B. uniflora and determine their cellulase, laccase, and antioxidant activities. Endophytic fungi were isolated from B. uniflora stems, cultured on wheat grains, immersed in a 150gL-1 aqueous sucrose solution, and cryopreserved at -80°C for 1 and 6months. Cellulase activity was determined by a qualitative test using carboxymethylcellulose medium and laccase activity by a quantitative test based on the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate). Prior to antioxidant activity assays, fungi were grown in malt extract broth for production of mycelial biomass. A methanolic extract was prepared for evaluation of DPPH· scavenging activity, FRAP activity, and total phenolic content. A total of 46 endophytic fungal isolates were obtained from B. uniflora stems and classified into 24 groups according to morphological similarities. B. uniflora was shown to harbor different genera of ascomycete fungi as endophytic organisms. Mycelial viability was observed after 1 and 6months of cryopreservation at - 80°C. Fungi exhibited cellulase and laccase activities. Isolate CE23 had the highest laccase activity after 7days of cultivation. Twelve isolates were found to have low total phenolic contents and DPPH· and FRAP activities.