Production and molecular characteristics of four groups of exopolysaccharides from submerged culture of Phellinus gilvus

AIMS: The objective of the present study was to determine the optimal culture conditions for the production of four groups of exopolysaccharides (EPSs) in Phellinus gilvus by submerged culture and to investigate their molecular properties by multi-angle laser-light scattering (MALLS) analysis. METHODS AND RESULTS: The optimal temperature and initial pH for the production of both mycelial biomass and EPSs in P. gilvus by submerged flask cultures were found to be 30 degrees C and pH 9.0, respectively. Glucose and corn steep powder were the most suitable carbon and nitrogen source for both mycelial biomass and EPS production. Optimal medium composition was determined to be glucose 30 g l-1, corn steep powder 5 g l-1, MgSO4 1.23 g l-1, KH2PO4 0.68 g l-1, and K2HPO4 0.87 g l-1. Four groups of EPSs (Fr-I, II, III, and IV) were obtained from the culture filtrates by gel filtration chromatography on Sepharose CL-4B and characterized by size exclusion chromatography (SEC) coupled with MALLS. The weight average molar mass (Mw) of Fr-I, Fr-II, Fr-III and Fr-IV were determined to be 8.628 x 106 (+/-129 420), 1.045 x 106 (+/-19 855), 61.09 x 104 (+/-1244), and 33.55 x 104 (+/-134) g mol-1, respectively. CONCLUSIONS: Under optimal culture conditions, the maximum EPS production in a 5-l stirred fermenter indicated 5.3 g l-1 after 11 days of fermentation. The SEC/MALLS analysis revealed that Fr-I, which has extremely high molecular weight, was presumably an aggregate of complex polysaccharides forming a compact globular shape; whereas Fr-II was nearly spherical, Fr-III and Fr-IV were rod-like chains in an aqueous solution. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the production of high amounts of EPSs from liquid-culture of the basidiomycete, P. gilvus. The SEC/MALLS approach used in this study could be useful in providing greater insight into the characterization of the mushroom polysaccharides without carrying out elaborate fractionation procedures prior to analysis.     

Production and characterization of exopolysaccharides and antioxidant from Paenibacillus sp. TKU023

Using squid pen powder (SPP) as the sole C/N source, Paenibacillus sp. TKU023 produced exopolysaccharides (EPS) and antioxidant. With medium containing 1.5% SPP, 0.1% K(2)HPO(4), and 0.05% MgSO(4)·7H(2)O, pH 7.23, the culture was incubated at 37°C in liquid (50 mL/250 mL) for five days. The resultant culture supernatant had higher EPS productivity (4.55 g/L). The crude EPS were isolated by centrifugation, methanol precipitation and deproteinization. The characterization of the EPS demonstrated that it was mainly composed of glucose and maltose. In addition, the culture supernatant incubated for four days by using baffled base flask showed the strongest antioxidant activities and the highest total phenolic content, but maximum EPS production was found at the fifth day by using flat base flask. The production of two invaluable environmental-friendly biomaterials (EPS and antioxidant) is unprecedented. Besides, the use of SPP (waste) is green that made the whole process more valuable and attractive.     

Production of exopolysaccharides by a submerged culture of an entomopathogenic fungus, Paecilomyces sp

Exopolysaccharide basic was obtained from a submerged culture of a native Paecilomyces sp. strain isolated from Chilean soil.     

Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Fractionation,characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus

Exopolysaccharide (EPS) fractions P_1/5, P_2/5, P₁, P₂ and P₅ were isolated from the fermentation medium of a medicinal fungus Cordyceps sinensis by gradient precipitation with ethanol at 1/5, 2/5, 1, 2, and 5 volume ratios to the liquid medium. P_1/5 and P_2/5 were mainly composed of polysaccharides with negligible protein content and a large molecular or particle size (intrinsic viscosity [η] 2025 mL/g; hydrodynamic radius R_h 905 nm). The fraction attained at a higher ethanol volume ratio had a much higher protein and lower carbohydrate content, and a smaller molecular size. In particular, P₅ was composed of mainly protein with an average molecular weight 16 kDa, [η] 4.3 mL/g and R_h 23.5 nm. The antioxidant activities of EPS fractions showed a significant dependence on the protein content, being negligible of P_1/5 and P_2/5, low to moderate of P₁ and P₂, and very strong of P₅. Gradient ethanol precipitation was proven a simple and workable method for initial fractionation of polysaccharides, proteins and their complexes with different molecular sizes and for further identification of the bioactive components.     

季也蒙假丝酵母胞外多糖的分离纯化及抗氧化活性研究

对一株源于海参肠道的季也蒙假丝酵母产胞外多糖进行分离纯化并对其抗氧化活性进行研究.采用乙醇沉淀、Sevag除蛋白等方法得到胞外粗多糖EPS.EPS经Sepharose强阴离子交换层析分离后,分别得到3个组分EPS1、EPS2和EPS3,对抗氧化活性较高的EPS2采用Sephacryal凝胶过滤层析进行纯化,得到1个单一组分EPS2-1,采用气相色谱法分析其单糖组成,并验证其抗氧化活性.结果表明:EPS2-1是由木糖、甘露糖、葡萄糖和半乳糖组成的;它具有较强的抗氧化活性,当其浓度为0.36 mg/mL时,对羟基自由基的清除率可达100％,明显高于同浓度下Vc对羟基自由基的清除率14.42％;当EPS2-1浓度为0.60 mg/mL时,对超氧阴离子自由基的清除率可达53.22％;同时EPS2-1表现出一定的还原力.研究证明该活性多糖具有很好的应用潜力,值得进一步研究开发.     

Characterization and antioxidant activity for exopolysaccharide from submerged culture of Boletus aereus

Three polysaccharide fractions (designated as Fr-I, Fr-II and Fr-III) were successfully purified from the crude exopolysaccharide (EPS) produced from submerged culture of Boletus aereus by gel filtration chromatography on Sepharose CL-6B. The size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS) system showed that the average molecular weights (Mws) of these three fractions were 1.365 × 10⁶, 1.048 × 10⁵ and 2.471 × 10⁴ g/mol, respectively. The SEC/MALLS also revealed that the molecular conformation of the Fr-I was a random coil, with Fr-II being a rigid rod in aqueous solution. Moreover, monosaccharide composition analysis indicated that Fr-I was mainly composed of glucose, while both of Fr-II and Fr-III were mainly composed of mannose and glucose. Then, FT-IR spectral analysis of the purified EPS revealed prominent characteristic groups. Furthermore, thermo gravimetric analysis (TGA) indicated the degradation temperature of the Fr-I (170 °C) was higher than those of Fr-II (156 °C) and Fr-III (155 °C). Finally, on the basis of the antioxidant activity test in vitro, Fr-I exhibited the highest antioxidant ability among these samples, which might be attributed to the monosaccharide composition and molecular weight in the EPS fraction.     

Production,characterization and antioxidant activities in vitro of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

The production, characterization and antioxidant activities in vitro of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3 were investigated. For EPS production, the preferable culture conditions were 24 °C and pH 8 for 60 h with sucrose and yeast extract as the carbon and nitrogen sources, respectively. Notably, sucrose concentration was the prominent factor, and the maximum yield of EPS (22.82 g/L) was obtained at a sucrose concentration of 160 g/L. The crude EPS was purified by chromatography of DEAE-52 and Sephadex G-100, affording EPS-1 and EPS-2 with molecular weights of 1.22 × 10⁶ and 8.69 × 10⁵ Da, respectively. They were composed of mannose, fructose and glucose in a molar ratio of 2.59:29.83:1 and 4.23:36.59:1, respectively. In addition, both crude and purified EPS showed strong scavenging activities on superoxide and hydroxyl radicals, and their antioxidant activities decreased in the order of crude EPS > EPS-2 > EPS-1.     

Immunomodulating activities of water-soluble exopolysaccharides obtained from submerged culture of Lentinus lepideus

Immunomodulating activities of water-soluble exopolysaccharides (LL-EX) obtained from submerged mycelial culture of Lentinus lepideus were studied and their effectiveness was compared with lipopolysaccharide (LPS). The influence of the LL-EX on macrophage cellular lysosomal enzyme activity was to stimulate up to 267%, 392%, and 464% at the level of 10, 50, and 100 mug/ml, respectively. When the LL-EX was further fractionated into LL-Fr.I and Fr.II by Sepharose CL-6B gel chromatography, the cellular lysosomal enzyme activity of LL-Fr.II (2.1- fold) was higher than Fr.I (1.2-fold). Moreover, both LL-Fr.I and Fr.II stimulated the cytokines IL-1beta, TNF-alpha, and IL-6 in macrophages. In mixed lymphocyte reaction, LL-Fr.I and Fr.II enhanced the splenocyte proliferation up to 1.2-fold and 1.4-fold (50 mg/ml), respectively, stimulating only T lymphocytes. The fractions of LL-EX not show any direct toxicity against human gastric adenocarcinoma cell (AGS). The molecular masses of LL-Fr.I and Fr.II were estimated to be about 1,986 kDa and 21 kDa, respectively. The total sugar and protein contents of the two fractions were 84.97% and 69.88% and 15.03% and 30.12%, respectively. The sugar and amino acid compositions of the LL-Fr.I and Fr.II were also analyzed in detail.     

六妹羊肚菌多糖的提取工艺优化及结构表征和抗氧化活性

六妹羊肚菌Morchella sextelata是一种珍贵的食药用真菌,具有重要的经济价值。本研究在单因素试验基础上,采用响应面分析法优化其多糖提取工艺,利用Sevage法及透析法对粗多糖进行初步纯化,获得六妹羊肚菌多糖（Morchella sextelata polysaccharide,MSP）组分。采用凝胶色谱-示差-多角度激光光散射（gel permeation chromatography- refractive index-multi angle laser light scattering,GPC-RI-MALLS）、高效阴离子交换色谱（high performance anion exchange chromatography,HPAEC）、傅里叶变换红外光谱仪（fourier transform infrared spectrometer,FTIR）和气相色谱质谱联用仪（gas chromatography-mass spectrometry,GC-MS）等对其进行结构分析,并检测了该多糖清除自由基活性的能力。结果表明,六妹羊肚菌多糖最优提取工艺为：提取温度89.94℃、液料比31.07mL/g、提取时间162.86min。在此工艺条件下,提取率为23.98%。该多糖主要由分子量为4.655×10 ⁶Da和6.571×10 ⁴Da的两个组分组成,其单糖组成为葡萄糖、甘露糖、半乳糖,摩尔百分比分别为71.60%、23.70%和4.70%。该多糖的糖苷键主要有T-Glc、1,2-Man、1,4-Glc、1,6-Man、1,3,4-Man、1,4,6-Gal。此外,该多糖具有较强的清除2,2‐联氮基双‐3‐乙基苯并噻唑啉‐6‐磺酸[2,2‐azino bis (3‐ethylbenzothiazoline‐6‐sulfonic acid),ABTS]、1,1‐二苯基‐2‐苦基肼（1,1‐diphenyl‐2‐picrylhydrazyl,DPPH）和羟自由基活性的能力。本研究结果为六妹羊肚菌多糖功能食品的开发和利用提供研究基础。     

Production of exopolysaccharides by submerged culture of an enthomopathogenic fungus, Paecilomyces tenuipes C240 in stirred-tank and airlift reactors

The objective of this study was to investigate the effect of shearing effect on the production of exopolysaccharides (EPS) from an enthomopathogenic fungus, Paecilomyces tenuipes C240 in a stirred-tank reactor (STR) and in an airlift reactor (AR). The optimal agitation rate for the production of EPS in the STR was 150 rpm with the mycelial morphology of hairy pellets, where the final concentration and the specific production rate of EPS were 2.33 g l(-1) and 0.312 gg(-1) h(-1), respectively. However, the maximum concentration of biomass (21.06 g l(-1)) in the STR was obtained at a high agitation speed of 300 rpm. The specific production rate of EPS (0.456 gg(-1) h(-1)) in the AR was significantly higher than that achieved in the STR, in which the typical morphological form of mycelium was a loose clump. The three EPS groups in the STR (designated as STR-I, -II, and -III) and two groups of EPS in the AR (designated as AR-I and -II) were obtained from the culture filtrates by a gel filtration chromatography on Sepharose CL-6B. The molecular weights of STR-I, STR-II, STR-III, AR-I, and AR-II were determined to be 1,820, 25, 1.8, 1,160, and 6.7 kDa, respectively. An agitation rate of 150 rpm in the STR was selected as the optimal culture condition for maximum EPS production (2.33 g l(-1)), which was similar to the level achieved in the AR (2.30 g l(-1)). The carbohydrate composition in each EPS was quite different from each other: the major component was glucose (in STR-I, -III, and AR-I), mannose (in STR-II), and arabinose (in AR-II). In contrast, no significant difference in amino acid composition was observed.     

Optimization of medium composition for the production of exopolysaccharides from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of exopolysaccharides

Optimization of medium composition for the production of exopolysaccharides (EPS) from Phellinus baumii Pilát in submerged culture and the immuno-stimulating activity of EPS were carried out. Firstly, the medium components having significant effect on EPS production were screened out to be glucose, yeast extract and diammonium oxalate monohydrate by using a 2⁽⁷⁻³⁾ fractional factorial design. Secondly, the concentrations of the three factors were optimized using central composite design in response surface methodology. As results, a quadratic model was found to fit for EPS production, and the optimal medium composition was determined as following (g/l): 34.12 glucose, 4 peptone, 5.01 yeast extract, 0.88 diammonium oxalate monohydrate, 0.75 MgSO₄ and 1 KH₂PO₄ and 0.0075 thiamine (VB₁). A yield of 2.363 ± 0.04 g/l for EPS was observed in verification experiment. Finally, EPS from P. baumii Pilát was found to have direct immuno-stimulating activity in vitro on splenocyte proliferative response and acid phosphatase activity in peritoneal macrophages in a dose-dependent manner.     

Production of metabolites with antioxidant activity by Botryosphaeria dothidea in submerged fermentation

Bioprocess and Biosystems Engineering - Antioxidants are able of inhibiting free radicals and play an important role in human diet, protection of foods, anti-aging cosmetics, among others. Fungi...     

Production and characterization of exopolysaccharides from an enthomopathogenic fungus Cordyceps militaris NG3

Optimization of the submerged culture conditions for the mycelial growth and production of exopolysaccharide (EPS) from a newly isolated Cordyceps species (C. militaris NG3) was studied in flask cultures. The optimal temperature and initial pH for EPS production were 30 degrees C and 8.0, respectively. Sucrose (30 g/L) and corn steep powder (10 g/L) were the most suitable carbon and nitrogen source for both mycelial growth and EPS production. There was a distinguishable morphological changes in mycelium grown between organic and inorganic nitrogen sources. A smooth pellet growth with heavy hyphal thickness was observed in the medium containing organic nitrogen sources, whereas irregular pellets with less hairy region were formed in the medium containing inorganic nitrogen sources. More highly branched cells appearing in the medium of organic nitrogen sources seemed a favorable morphological form for both EPS production and mycelial growth. Under optimal culture conditions, the maximum concentrations of mycelial growth and EPS were 17.6 and 3.4 g/L in a 5-L stirred-tank fermenter. Four groups of EPSs (designated as Fr-I, Fr-II, Fr-III, and Fr-IV) were obtained from the culture filtrates by size exclusion chromatography (SEC), and their molecular characteristics were examined by a multiangle laser-light scattering (MALLS) and refractive index (RI) detector system. The weight-average molar masses of the Fr-I, Fr-II, Fr-III, and Fr-IV of EPS were determined to be 2.262 x 10(6), 3.348 x 10(5), 1.049 x 10(5), and 5.059 x 10(4) g/mol, respectively. All four EPSs showed very low polydispersity indices ranging from 1.00 to 1.18. The SEC/MALLS analysis revealed that the molecular shape of the Fr-I was a rigid sphere suspected to be an aggregate of complex polysaccharides, the Fr-II and Fr-III were nearly globular in shape, and the Fr-IV was an almost rodlike structure.     

粗柄羊肚菌菌丝体多糖及胞外多糖的提取工艺

为了研究粗柄羊肚菌菌丝体多糖及胞外多糖的最佳提取工艺条件,采用超声波提取菌丝体多糖、菌丝体发酵液浓缩提取胞外多糖的方法。通过单因素试验和L9(34)正交试验设计,探讨二者的最佳提取工艺条件。研究结果表明菌丝体多糖的最佳提取工艺条件:料液比1∶20(g∶m L),超声波处理温度70℃,超声时间20 min,超声提取次数2次;胞外多糖的最佳提取工艺条件:浓缩倍数1∶5,浓缩温度50℃,醇沉浓度95%,p H为6。该项工艺研究得到菌丝体粗多糖平均含量56.761 2 mg/g,胞外多糖平均含量1.275 4 mg/m L,多糖产量稳定,且此试验方法稳定可行。     

Production of exopolysaccharides by submerged mycelial culture of <Emphasis Type="Italic">Grifola frondosa</Emphasis> TFRI1073 and their antioxidant and antiproliferative activities

The exopolysaccharide (EPS) extract of a newly screened Grifola frondosa TFRI1073 was evaluated antioxidant activity, including superoxide anion scavenging activity and reducing power, and the antiproliferative activities by using lung (A549 cells) and breast (MDA-MD-231 cells) cancer cell line. The exopolysaccharide of G. frondosa plays the important role on antioxidant and antiproliferative activities. Nutritional requirements for mycelial biomass and EPS production by G. frondosa were studied. Flushing the culture medium with carbon sources has a stimulating effect on both mycelial biomass and EPS production. In addition, nitrogen sources appeared to be an important and significant component for mycelial biomass. Vitamins were probably not essential for mycelial biomass and EPS production. Inorganic salts appeared to have a significant effect on both mycelial biomass and EPS productions. The effect of phosphate ion on mycelial biomass was better than on EPS production. The information generated in this study will facilitate physiological research of a newly screened G. frondosa TFRI1073, helping to develop the exopolysaccharide of a new dietary supplement and functional food from G. frondosa.     

Production and characterization of laccase from Pleurotus ferulae in submerged fermentation

Pleurotus ferulae is a mushroom typically found in arid steppe that is distributed widely in the Junggar Basin of Xinjiang, China. In this work, laccase production by P. ferulae JM30X was optimized in terms of medium composition and culture conditions. After optimization, the highest laccase activity obtained was 6,832.86 U/L. A single isozyme with a molecular weight of 66 kDa was observed by SDS-PAGE and native-PAGE. Optimum pH and temperature were 3.0 and 50–70 °C, respectively. The best laccase substrate was ABTS, for which the Michaelis-Menten constant (K _m) and catalytic efficiency (K _cat/K _m) value for P. ferulae laccase were 0.193 mM and 2.73?×?10⁶ (mM s)^?1, respectively. The activity of purified laccase was increased by more than four-fold by Cu²⁺, Mn²⁺ and Mg²⁺, while it was completely inhibited by Fe²⁺ and Fe³⁺. The production of laccase was influenced by the initial pH and K⁺ concentration, and the activity of purified laccase was enhanced by Cu²⁺, Mn²⁺ and Mg²⁺. This Pleurotus genus laccase from P. ferulae JM30X was analyzed by MS spectrum and the results are conducive to furthering our understanding of Pleurotus genus laccases.     

Production ofPaecilomyces Fumosoroseus conidia in submerged culture

Mexican isolates ofPaecilomyces fumosoroseus (Wize) Brown & Smith virulent to nymphs and adults ofBemisia tabaci Gennadius (Homoptera: Aleyrodidae) were screened in terms of spore production in submerged culture. Effects of light, temperature stress and yeast extract on sporulation were studied. Cycles of 12 hours light/12 hours dark increased spore production as well as an incubation for 24 hours at 37°C prior to incubation at 30°C. In absence of organic nitrogen both fungal growth and sporulation were very low. Spore production in fermentors with a culture media of a C:N ratio of 25 was doubled as compared to a media with a C:N ratio of 11. Both conidia and blastospores were produced. Production of conidia directly from blastospores through microcyclic sporulation was observed. The proportion of conidia obtained under optimal conditions was 88.8%. Submerged culture ofP. fumosoroseus seemed advantageous compared to ricefilled plastic bags production method because of shorter fermentation times, higher spore yields and substantially higher volumetric spore productivity. Results indicated that careful manipulation of nutritional and environmental conditions allowed for production of conidia during submerged growth ofP. fumosoroseus, microcyclic sporulation being induced under a set of environmental conditions including temperature stress and nutrients limitation.     

Optimization of submerged culture condition for the production of mycelial biomass and exopolysaccharides by Agrocybe cylindracea

The optimization of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) from Agrocybe cylindracea ASI-9002 using the statistically based experimental design in a shake flask culture. Both maximum mycelial biomass and EPS were observed at 25 degrees C. The optimal initial pH for the production of mycelial biomass and EPS were found to be pH 4.0 and pH 6.0, respectively. Subsequently, optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth was as follows: maltose 80 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 1.4 g/l, and CaCl2 1.1 g/l; for EPS production: maltose 60 g/l, Martone A-1 6 g/l, MgSO4 x 7H2O 0.9 g/l, and CaCl2 1.1 g/l. Under the optimal culture condition, the maximum EPS concentration achieved in a 5-l stirred-tank bioreactor indicated 3.0 g/l, which is about three times higher than that at the basal medium.