Submerged Culture Research Articles

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English

Advances in batch culture fermented Coriolus versicolor medicinal mushroom for the production of antibacterial compounds

Abstract Bioactive compounds obtained from Coriolus versicolor (Trametes versicolor (L.: Fr) Lloyd, 1920.) mushrooms cultivated in a stirred-tank bioreactor were tested to determine their antimicrobial potential. Extracellular polysaccharides were isolated from the fermentation broth by ethanol precipitation. A methanol extract was prepared from mycelium. The cultivation conditions applied during the fermentation process provided for significant biomass 6.63 ± 0.31 g dry weight L− 1 and yield of extracellular polysaccharides (EPS) (0.74 ± 0.12 g L− 1). Microscopic analyses revealed that the mycelium grew predominately in the form of fluffy pellets. The methanol extract demonstrated very good activity against all the tested Gram-positive bacteria. Bacillus spizizeni and Staphylococcus epidermidis were the most sensitive strains (minimum inhibitory concentration (MIC) Our results suggested that the submerged cultivation of Coriolus versicolor followed by ethanol precipitation of EPS and the methanol extraction of mycelia can be a promising process to obtain biological active compounds with significant antimicrobial activity. Industrial Relevance Mushrooms contain a large number of chemicals with potential use as antimicrobial compounds. One of the biggest challenges for providing biologically active compounds from mushrooms is short-term process standardization with a low risk for contamination. Submerged culture cultivation is the best choice for providing antimicrobial compounds from mushrooms. The submerged culture method represents an effective and energy-efficient means to produce novel antibacterial compound from mushrooms. Antibacterial activity testing revealed that methanol extract and isolated exopolysaccharides exhibited strong antibacterial activity, especially against Gram-positive bacteria.

Physicochemical characteristics and biological activities of polysaccharide fractions from Phellinus baumii cultured with different methods

Nine polysaccharide fractions were obtained from the fruiting bodies, submerged mycelia, and solid state fermented products of Phellinus baumii using different concentrations of ethanol precipitation. The chemical characteristics and in vitro immunological activities of the nine polysaccharide fractions were compared and studied. Results indicated that the fractions precipitated with 50% ethanol had higher yields of polysaccharides and submerged mycelia contributed to high extraction yields of polysaccharides and possessed higher polysaccharide contents. HPSEC–MALLS–RI analysis showed that the molecular weight (Mw) of polysaccharide fractions from these three materials decreased with the increasing of precipitated ethanol concentration. The Mw of fruiting body polysaccharide fractions ranged from 1.98×104Da to 1.89×106Da. Large-molecular-weight polysaccharides (from 2.11×106Da to 2.01×107Da) were found in submerged mycelia. Some lower-molecular-weight polysaccharide components were found in solid fermented products. Different culture methods contributed to significant differences in monosaccharide components and molar ratios. The 50% ethanol precipitated fractions exhibited more complexity on monosaccharide compositions comparing with fractions precipitated with 30% and 70% ethanol. Polysaccharide fractions derived from submerged mycelia exhibited higher macrophages stimulation activities. Submerged culture was found to be a suitable method to prepare active polysaccharides because of its short culture span and reasonable cost.

Fatty Acids of Xylotrophic Basidiomycetes of the Genus Pleurotus in Submerged Culture

Fatty Acids of Xylotrophic Basidiomycetes of the Genus Pleurotus in Submerged Culture

Antitumor and Immunomodulating Activities of Exopolysaccharide Produced by Big Cup Culinary- Medicinal Mushroom Clitocybe maxima (Higher Basidiomycetes) in Liquid Submerged Culture.

Water-soluble polysaccharides extracted from mushrooms have been found to have some physiological effects. In this study, exopolysaccharides (EPSs) were extracted by alcohol precipitation from cultivated broth of the mushroom Clitocybe maxima. EPSs with molecular weights of 10(4) and 10(5) Da were obtained by ultrafiltration; they are referred to as EPA and EPB, respectively. The major components of these EPSs were glucose, galactose, mannose, rhamnose, and arabinose. ICR mice with artificially induced metastatic pulmonary tumors were fed a daily diet containing EPA or EPB at doses of 8, 20, or 50 mg/kg. Results showed that the proliferation of pulmonary sarcoma lesions was lower in the groups fed EPS. In addition, the numbers of total T cells, CD4+ cells, CD8+ cells, and macrophages significantly increased in EPS-fed mice compared with the negative control group. The antitumor and immunomodulating effects observed in the EPB-fed groups were higher than those of EPA-fed groups. These results demonstrate the ability of EPSs of C. maxima to inhibit tumor cells while enhancing immune response.

Submerged culture process for biomass and exopolysaccharide production by Antarctic yeast: some engineering considerations

Production of biomass and extracellular polysaccharide (EPS) from psychrophilic Sporobolomyces salmonicolor AL1 in a stirred bioreactor was studied. The aspects of production technical-scale parameters, namely, bioreactor flow field, biomass and EPS production rates, oxygen mass transfer per input power, as well as important product properties, such as rheology and stability of EPS mixtures, were considered. The bioprocess was found to proceed in non-Newtonian flow with consistency coefficient rising typically to 0.03Pa.s(n) and flow index declining to 0.7. Flow modeling was carried out and showed good homogenization for substrate delivery at agitation rates exceeding 400rpm. Agitation rates lower than 400rpm were considered counterproductive due to flow field non-uniformity. The cell density reached 5g/l and EPS production yield reached 5.5g/l at production rate 0.057g EPS/l per hour (0.01g EPS/g biomass per hour). Oxygen uptake rate and oxygen transfer rate were in the range of 0.5-1.7 mmolO2/l per hour and 2-4.7 mmolO2/l per hour, respectively. The mass transfer coefficient at reaction conditions was found to be in the range [Formula: see text]. The bioprocess biological performance was higher at moderate agitation speed and revealed biomass diminution and cell inactivation by increasing impeller revolutions and shear rate. The product EPS was found to introduce shear-thinning behavior in water solutions with apparent viscosity of up to 30mPa.s and to stabilize 1-2% oil-in-water emulsions improving their lipophilic properties. The emulsion dispersion index was found to be comparable with the one of Arlacel 165, the emulsifier used in cosmetic. The long-term performance of the complex cream mixtures of the glucomannan prepared in commercial format was found promising for further application.

Inulinase Production by <i>Penicillium citrinum</i> ESS in Submerged and Solid-State Cultures

Problem statement: This work reports the Penicillium citrinum ESS strain as a new source of inulinase (2,1-β-D fructanhydrolase, E.C. 3.2.1.7). Approach: The enzyme was produced in both, Solid-State (SSC) and Submerged Culture (SmC) using a basal medium added either with inulin or sucrose (5 gL-1). Results: The culture grew faster in SmC than in SSC with both carbon sources; however, yields of inulinase activity per gram of biomass were higher in SSC (1,658,237.28 for inulin and 66598.96 for sucrose) than in SmC (441.05 for inulin and 183.91 for sucrose). Conclusion: The highest inulinase production was obtained by SSC, either with inulin or sucrose, reaching values of 6650 and 2970 U/L respectively. In contrast, in SmC were obtained higher levels of inulinase activity (2241 U/L) with sucrose as carbon source (24 h), while with inulin, the enzyme production was negatively affected and retarded.

Cellulases and Hemicellulases from Endophytic Acremonium Species and Its Application on Sugarcane Bagasse Hydrolysis

The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L -arabinose, D -xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using D -xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.

Optimization of Culture Conditions on Mycelial Grown in Submerged Culture of Cordyceps militaris

Statistical experimental design strategy (SES) was employed to optimize the culture conditions for the mycelial growth of C. militaris by submerged culture. Using Plackett-Burman design (PBD), liquid volumes in flask and fermentation time were identified as the significant variables that would highly influence mycelial growth, and these variables were subsequently optimized using response surface method (RSM). The steepest ascent method was used to access the optimal region of the culture condition. The results showed that the optimum values of the tested variables were 47.3 mL of liquid volumes in flask and 7.6 days of fermentation time. Under optimized culture conditions, the mycelial production was enhanced from 10.33 to 19.97 g/L. In comparison with that of original culture conditions, 1.9-fold increase was obtained. The validation experiment showed that the experimentally determined production values were in close agreement with the statistically predicted ones.

Laboratory Studies on Phosphorus Removal from Nigeria’s Agbaja Iron Ore by Bacillus Subtilis

The investigation into whether or not Bacillus subtilis can remove phosphorus from the Nigerian Agbaja iron ore was carried out with careful monitoring of the population of the removing agent as well as pH of the system. 1.00mm/0.50mm, 0.50mm/0.25mm, and 0.25mm/0.125mm ore fractions obtained from sieving of the crushed iron ore in Shital Test kits, were used in sub-merged culture of nutrient broth (NB) medium for 10 weeks. B. subtilis which was part of the rich microflora found on the ore surface was cultivated in nutrient-rich media and later inoculated in sterilized 100ml of NB in 250ml conical flask and 1g of each of the equally sterilized ore fractions was added. At weekly intervals, a set of samples was removed, treated through series of chemical reactions to obtain ammonium phosphomolybdate precipitate which was back-titrated with 0.1 N-HCl to determine the amount of phosphorus left in samples and consequently, the amount removed. The laboratory investigations found out that B. subtilis has the capability to remove phosphorus from the Nigerian Agbaja iron ore, recording an impressive average of 65.73% P. Also found out was the systematic reduction in bacterial cells count in colony forming unit per mililitre, the initial load 3.4x105 cfu/ml increased to 4.8x107 cfu/ml from where it declined to 1.3x106 cfu/ml, which justified the pH trend observed during the process of cumulative phosphorus removal. The reduction in microbial activity may be attributed to antimicrobial components of the ore, pyrite, and other heavy metals which may have affected the phosphorus uptake from ore.

Bioprocess Development for Large Scale Production of Anticancer Exo-Polysaccharide by Pleurotus ostreatus in Submerged Culture

In the recent years, mushroom derived active metabolites have emerged as an important class of bioactive compounds with several therapeutic applications. Most of the production methods were based on cultivation of mushroom on solid substrate in controlled temperature green house. For the production of bioactive compounds for therapeutic application, production should be carried out under sterile and well controlled condition. Thus, the interest of cultivation of mushroom in bioreactors for bioactive compound production was increased during the last few years. In the present study, mushroom cells were cultivated in submerged culture for the overproduction of anticancer exo-polysaccharides (EPS). Cell cultivation was optimized in fully controlled stirred tank bioreactor in batch and fed-batch culture to improve the process and to increase the anticancer EPS production. In the first part of this study, significant improvement in EPS production was achieved upon transferring the process from shake flask to 15 L bioreactor. Further development in the process was conducted through optimization of some process parameters in bioreactor batch culture. In fed-batch culture, among different feeding strategies, optimized glucose feeding based on using the in-line data for oxygen and carbon dioxide obtained from out-gas analyzer was the best. The maximal yield of EPS obtained was 2.1 g L-1 in optimized fed-batch culture. The obtained EPS was almost two fold higher than those obtained in batch culture.

Submerged culture medium composition for the antioxidant activity by Grifola frondosa TFRI1073

Effects of nutritional requirements for superoxide anion scavenging activity and reducing power by a newly screened Grifola frondosa TFRI1073 were studied in submerged cultures. The results clearly showed that the culture medium significantly affected superoxide anion scavenging activity and reducing power. In addition, both superoxide anion scavenging capability and reducing power were found to be greatly affected by varying the carbon source, the nitrogen source, the growth factor, and the mineral elements. The best superoxide anion scavenging activity could be obtained when the medium compositions (carbon source, nitrogen source, growth factor, and the mineral elements) were sucrose, ammonium chloride, nicotinic acid, K3PO4, and CuSO4; while a maximal reducing power could be achieved when the medium composition setting as: xylose, arginine, ascorbic acid, K2HPO4, and MgSO4. From a series experiments, the maximal superoxide anion scavenging activity and reducing power were 98.36±0.59% and 0.947±0.06 at 100 μg/mL, respectively. This study proved that modifying the composition of the medium can dramatically enhance the superoxide anion scavenging ability and reducing power by a newly screened G. frondosa TFRI1073.

Effect of Cultivation Conditions on the Production of Mycelial Biomass and Exopolysaccharide by Submerged Culture of a Rooting Shank Medicinal Mushroom, Oudemansiella radicata (Relhan) Singer (Agaricomycetideae)

Effect of Cultivation Conditions on the Production of Mycelial Biomass and Exopolysaccharide by Submerged Culture of a Rooting Shank Medicinal Mushroom, Oudemansiella radicata (Relhan) Singer (Agaricomycetideae)

A Novel Tannase from the Xerophilic Fungus Aspergillus niger GH1

Aspergillus niger GH1 previously isolated and identified by our group as a wild tannase producer was grown under solid-state (SSC) and submerged culture (SmC) conditions to select the enzyme production system. For tannase purification, extracellular tannase was produced under SSC using polyurethane foam as the inert support. Tannase was purified to apparent homogeneity by ultrafiltration, anion-exchange chromatography, and gel filtration that led to a purified enzyme with a specific activity of 238.14 IU/mg protein with a final yield of 0.3% and a purification fold of 46. Three bands were found on the SDS-PAG with molecular masses of 50, 75, and 100 kDa. PI of 3.5 and 7.1% Nglycosylation were noted. Temperature and pH optima were 60 degrees and 6.0 [methyl 3,4,5-trihydroxybenzoate (MTB) as substrate], respectively. Tannase was found with a KM value of 0.41 x 10-4 M and the value of Vmax was 11.03 micromoL/min at 60 degrees for MTB. Effects of several metal salts, solvents, surfactants, and typical enzyme inhibitors on tannase activity were evaluated to establish the novelty of the enzyme. Finally, the tannase from A. niger GH1 was significantly inhibited by PMSF (phenylmethylsulfonyl fluoride), and therefore, it is possible to consider the presence of a serine or cysteine residue in the catalytic site.

Adsorption of major endoglucanase from Thermoascus aurantiacus on cellulosic substrates

A thermostable endoglucanase (EndoI) was produced by the thermophilic fungus Thermoascus aurantiacus when grown on cellulosic materials under submerged culture (SC) and solid-state fermentation (SSF). In both cultivation techniques a considerable amount of enzyme activity remained adsorbed onto solid particles, and this was taken into consideration when modeling enzyme production. The results were compatible with the assumption that, following its synthesis, an amount of EndoI was bound on substrate and gradually released into the liquid medium. Adsorption of the enzyme on crystalline cellulose was confirmed in vitro by experiments with purified endoglucanase, which was isolated by anion exchange chromatography. The Langmuir isotherm could efficiently describe the adsorption kinetics, and the estimated Amax and Kad values compared with those obtained for cellulases bearing a binding domain. EndoI displayed high affinity for crystalline cellulose and low binding capacity, which could be beneficial in textile processing.

Mycelial Submerged Culture of New Medicinal Mushroom, Humphreya coffeata (Berk.) Stey. (Aphyllophoromycetideae) for the Production of Valuable Bioactive Metabolites with Cytotoxicity, Genotoxicity, and Antioxidant Activity

The mycelial submerged culture of the Colombian mushroom Humphreya coffeata, the production of exopolysaccharides (EPS), and the evaluation of the supernatant components in terms of bioactivity were investigated. Three carbon sources, three concentrations of carbon and nitrogen sources, and three pH values were evaluated. The optimal source was lactose (over sucrose and glucose), and the maximal biomass and EPS concentrations (15.5 g/L and 6.9 g/L, respectively) were obtained using lactose at 50 g/L, yeast extract 10 g/L, and an initial pH of 4.5. The cytotoxic, proliferative, genotoxic, and antioxidant activities were evaluated in lyophilized culture fi ltrates. Cytotoxicity was analyzed on human nontumorigenic keratinocytes (HaCaT), two human epithelial cell lines of cervical cancer (HeLa and InBl), and one lymphoma cell line (Jurkat). None of the fi ltrate concentrations that showed a cytotoxic effect on Jurkat cells (250-2500 μg/mL) were toxic for control HaCat cells and cervical cancer (HeLa and InBl). Furthermore, DNA damage was observed at a high extract concentration (2500 μg/mL), and genoprotection was observed using the lowest concentration tested (250 μg/mL). In addition, antioxidant activity was observed via the superoxide anion and the DPPH scavenging. This suggests that H. coffeata extracts can repress initiation and/or break the chain of oxidation reactions.

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

Submerged culture conditions for the production of mycelial biomass and antimicrobial metabolites by Polyporus tricholoma Mont.

Basidiomycete fungi of the Polyporus genus are a source of secondary metabolites which are of medicinal interest as antibacterial compounds. As these substances are produced in a small amount by the fungi, the study of the cultivation conditions in vitro that could possibly optimize their production seems of major importance. The effects of glucose and lactose, pH and agitation on biomass concentration and on the specific growth rate caused by the basidiomycete Polyporus tricholoma were investigated. The initial pH (4.5, 6.5 and 8.5) was autoregulated at pH 5.5, and the agitation increased the mycelial growth and the specific growth rate. The high concentration of carbon sources (4%) increased biomass production. The lactose concentration and the absence of agitation were determinant in the production of antibacterial metabolites. The characterization of the antibacterial substance by GC-MS indicated a major compound, isodrimenediol, produced by the fungus Polyporus tricholoma with activity against Staphylococcus aureus.

Submerged culture of a mycelial formulation of a bioherbicidal strain of Myrothecium verrucaria with mitigated mycotoxin production

A mycelial formulation of the fungus Myro- thecium verrucaria (IMI 361690) containing 0.20% Silwet L-77 surfactant was found to be highly efficacious in controlling the exotic invasive weed kudzu. The mycelium can be rapidly (48-72 h) produced in several media, including an inexpensive soy flour-corn meal medium. Mycelial yields were 2, 10, and 25 g dry weight l -1 in Czapek-Dox, Richard's V-8, and soy flour-corn meal media, respectively. Scale-up production in soy flour-corn meal medium using laboratory fermenters (10-25 l), resulted in a mycelial formulation that caused 90% mor- tality of naturally-occurring mature (0.9-1.0 m in height) kudzu within 48 h after application in field experiments. HPLC analyses revealed that the mycelium produced in this liquid culture contained no detectable amounts of the trichothecene mycotoxins roridin A and verrucarin A (limit of detection 2 l gm l -1 ). This has resulted in a safer, yet effective bioherbicidal product. We anticipate that these findings should improve the probability of EPA registration and subsequent commercial development of this bioherbicide.

Inhibition of Keratinocyte-Driven Contraction of Tissue-Engineered Skin In Vitro by Calcium Chelation and Early Restraint But Not Submerged Culture

Skin graft contracture remains a significant cause of patient morbidity with reduction in joint mobility and cosmetic deformity. Despite recent advances, its mechanism is largely unknown. The authors have previously demonstrated the importance of the keratinocyte in the contraction of tissue-engineered skin in vitro. In this study, they investigate the effect of reducing keratinocyte differentiation on contraction by adding 0.8 mM ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetra acetic acid (EGTA) to the culture medium and by culturing tissue-engineered skin submerged in medium rather than at air-liquid interface. They also simulate the effect of early mechanical splinting in vitro to study its effect on contraction. Here the study shows that removal of the epidermis after 16 days culture at air-liquid interface results in immediate dermal relaxation with a return to the original dermal surface area. Lowering extracellular calcium concentration with EGTA reduces keratinocyte differentiation and reduces the rate of contraction. Submerged culture does not significantly reduce differentiation of tissue-engineered skin and does not reduce contraction. However, following an initial short period of mechanical constraint, the rate of contraction of tissue-engineered skin is reduced. Reducing keratinocyte differentiation by lowering extracellular calcium with EGTA, reduces contraction. However, submerged culture of tissue-engineered skin is ineffective. A short period of splinting of meshed skin grafts during the initial phase of epithelialization and keratinocyte differentiation may be most effective in the prevention of subsequent contractures in vivo but additional studies are needed to establish this.