Factors affecting mycelial biomass and exopolysaccharide production in submerged cultivation of Antrodia cinnamomea using complex media

Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).     

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.     

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.     

桑木层孔菌液体培养条件的研究

对桑木层孔菌(Phellinus mori)液体发酵条件进行了研究,以生物量和胞外多糖为指标,通过L16(45)和L9(34)正交表进行了两次正交试验,筛选出桑木层孔菌最适液体培养条件为:麦芽糖30 g/L,酵母浸粉和蛋白胨15 g/L(质量比2 1),KH2PO4和CaCl25.5 g/L(质量比1 1),初始pH6.0;通过单因素试验筛选出最适装液量为120 mL/250 mL,最适接种量为10%。在此条件下液体发酵培养7 d后,桑木层孔菌生物量达到23.375 g/L,胞外多糖产量达到3.993 g/L。     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

Methanogens in the human intestinal tract and oral cavity

Curvularia lunata var.aeria was grown in YPD (yeast extract, peptone, and dextrose) medium (pH 6.5) at 28°C with varying concentrations (10–40 g/L) of glucose for the production of rifamycin oxidase. Enzyme activity and glucose concentration were found to be indirectly related to the production of black intracellular pigment by the organism. Depletion of glucose level and rise of culture pH initiate the synthesis of pigment. Carboxymethylcellulose (CMC) was used as a carbon source to improve the enzyme yield, but utilization of the substrate in the reactor was much less. Compared with 10 g/L of CMC in the medium, low or high concentrations of CMC did not yield any better result. Addition of glucose in YPC (yeast extract, peptone, and CMC) medium did not increase the enzyme activity, and glucose was rapidly utilized byC. lunata, forming pellets rather than mycelia.     

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.     

Nutritional requirements of mycelial growth of Cordyceps sinensis in submerged culture

AIMS: The nutritional requirements for mycelial growth of Cordyceps sinensis in semi-synthetic liquid media were investigated. The results provide a basis for further physiological study and industrial fermentation of the fungus. METHODS AND RESULTS: Nutritional requirements, including 17 carbohydrates, 16 nitrogen compounds, nine vitamins, four macro-elements, four trace-elements and eight ratios of carbon to nitrogen, were studied for their effects on the mycelial growth in submerged cultures of C. sinensis by using one-factor-at-a-time and orthogonal matrix methods. Among these variables, sucrose, peptone, folic acid, calcium, zinc and a carbon to nitrogen ratio 12 : 1 were identified as the requirements for the optimum mycelial growth. The concentrations of sucrose, peptone and yeast extract were optimized and the effects of medium composition on mycelial growth were found to be in the order sucrose > yeast extract > peptone. The optimal concentration for mycelial growth was determined as 50 g l(-1) sucrose, 10 g l(-1) peptone and 3 g l(-1) yeast extract. CONCLUSIONS: Under optimal culture conditions, over 22 g l(-1) of mycelial biomass could be obtained after 40 days in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Cordyceps sinensis, one of the most valued medicinal fungi, is shown to grow in axenic culture. This is the first report on nutritional requirements and design of a simplified semi-synthetic medium for mycelial growth of this psychrophilic species, which grows slowly below 20 degrees C. The results of this study will facilitate research on mass production of the fungus under defined culture conditions.     

Morphological and rheological properties of the three different species of basidiomycetes Phellinus in submerged cultures

AIMS: The objective of this work was to investigate the morphological and rheological properties in submerged culture of the three different basidiomycetes Phellinus (P. baumii, P. gilvus and P. linteus) that produce pharmacologically important exopolysaccharides (EPS). METHODS AND RESULTS: In flask cultures, pH proved to be a critical factor affecting mycelial growth, morphological change and EPS production. The macroscopic morphologies observed under different pHs in flask cultures were also comparable: i.e. starfish-like pellets with a lesser extent of free mycelium appeared in P. baumii, whereas smooth pellets with higher amounts of free mycelium were observed in P. gilvus and P. linteus. The pelleted fermentations were further characterized in a 5-l stirred-tank fermenter by image analysis with respect to mean diameter, core area and pellet circularity. Phellinus baumii showed the largest pellet size (5.2 mm in diameter), whereas P. linteus had extremely small and spherical pellets. The culture broth of P. baumii and P. gilvus yielded extremely high apparent viscosities, ranging from 5 to 7 Pa s. CONCLUSIONS: Three important species of Phellinus showed significantly different morphological and rheological properties. The morphological variation of the three Phellinus species was closely linked to EPS productivity and the apparent viscosity of the whole broth. SIGNIFICANCE AND IMPACT OF THE STUDY: The morphological change in the three species of Phellinus was a good indicator for identifying cell activity for EPS production. Our finding may be beneficial for further optimization of other fungal fermentation processes for large-scale production of EPS.     

Solid-state fermentation of cornmeal with the basidiomycete Ganoderma lucidum for degrading starch and upgrading nutritional value

AIMS: The objective of this research was to study the ability of the basidiomycete Ganoderma lucidum to degrade starch and upgrade nutritional value of cornmeal during solid-state fermentation (SSF). METHODS AND RESULTS: On the basal medium that consisted of cornmeal and salt solution, alpha-amylase activity of G. lucidum reached its maximum value of 267 U g(-1) of culture on day 20 after inoculation. Prolongation of fermentation time from 10 to 25 days increased significantly the degradation rate of starch and ergosterol yield (a kind of physiologically active substances of G. lucidum, also as an indicator of mycelial biomass) (P < 0.01). Supplementation of glucose, sucrose or maltose to the basal medium also caused a significant increase in either the degradation rate of starch or the ergosterol yield as compared with control (P < 0.01). Among five kinds of nitrogen sources supplemented, yeast extract, casamino acid and peptone were more effective than (NH4)2SO4 and NH4NO3, and yeast extract gave the highest degradation rate of starch and ergosterol yield, followed by peptone. Through orthogonal experiments, the theoretical optimum culture medium for SSF of this fungus was the following: 100 g cornmeal, ground to 30-mesh powder, moistened with 67 ml of nutrient salt solution supplemented with 3 g yeast extract and 7.5 g glucose per litre. CONCLUSIONS: Under the optimum culture condition, the degradation rate of starch reached its maximum values of 70.4%; the starch content of the fermented product decreased from 64.5 to 25.3%, while the reducing sugar content increased from 4.2 to 20.6%. SSF also produced a significant increase (P < 0.01) from 11.0 to 16.5% in protein content. SIGNIFICANCE AND IMPACT OF THE STUDY: After SSF by G. lucidum, the digesting and absorbing ratio of cornmeal was strikingly increased and some active substances originated from G. lucidum remained in the fermented product. This implied that cornmeal could be processed into many kinds of special functional foods by SSF of G. lucidum.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju

Microbial exopolysaccharides (EPSs) are very important because they are used in biotechnological applications in different industrial areas. The aim of the study was to determine the best EPS producer Pleurotus sp., to optimize EPS production and to perform partial purification and characterization of the produced EPS. After the production conditions were optimized, the EPS was isolated and partially purified. EPS was characterized by HP-TLC, 1H-NMR, FT-IR, and TGA. Hydroxyl, superoxide, and DPPH radical scavenging activities of the EPS were also investigated spectrophotometrically. The best EPS producer and its incubation period in submerged fermentation were determined as Pleurotus sajor caju and on 5 days, respectively. Culture conditions to increase EPS production were optimized as follows (in per liter): 90 g of glucose, 10 g of yeast extract, 10 g of peptone, and 100 mM of Mg2+. The optimal initial pH, temperature, and an agitation rate of culture were determined as 5.0, 25 °C, and 150 rate min−1, respectively. The highest EPS production was determined as 33.32 ± 1.6 g L−1. After isolation of EPS, one active fraction was obtained by gel filtration chromatography. EPS is composed mainly of glucose according to HP-TLC analysis. To the results, EPS had a complex structure by having carbohydrate and protein contents. The produced EPS had high degradation temperature as well as high antioxidant activity.     

Characterization of optimal growth conditions and carotenoid production of strain Rhodotorula mucilaginosa HP isolated from larvae of Pieris rapae

Yeasts have been studied because of their production of a pigment known as carotenoid with potential application in food and feed supplements. A carotenoid‐producing yeast was isolated from the larvae of Pieris rapae, named HP. The strain HP was identified as Rhodotorula mucilaginosa classified by its carbohydrate fermentation pattern and physiological tests. Rhodotorula mucilaginosa HP produces several exogenous enzymes: alkaline phosphatase, esterase, leucine arylamidase, valine arylamidase, acid phosphatase and β‐glucosidase. Using response surface methodology, selected medium components (yeast extract, malt extract, peptone, glucose) were tested to find the optimum conditions for carotenoid production and the growth of R. mucilaginosa HP. Central composite design was used to control the concentrations of medium components. Peptone and glucose had the largest effects on carotenoid production and cell growth of R. mucilaginosa HP, respectively. The estimated optimal growth conditions of R. mucilaginosa HP were: yeast extract 3.23%, malt extract 2.84%, peptone 6.99% and glucose 12.86%. The estimated optimal conditions for carotenoid production were: yeast extract 2.17%, malt extract 2.11%, peptone 5.79% and glucose 12.46%. These results will assist in the formulation of an appropriate culture medium for optimal carotenoid production of R. mucilaginosa HP for commercial use.     

Effects of Tween 80 and pH on mycelial pellets and exopolysaccharide production in liquid culture of a medicinal fungus

This study investigated the effects of surfactant additives and medium pH on mycelial morphology and exopolysaccharide (EPS) production in liquid culture of a valuable medicinal fungus Cordyceps sinensis Cs-HK1. In the medium containing 20 g l⁻¹ glucose and 6 g l⁻¹ peptone as the sole nitrogen source, the Cs-HK1 fungal mycelia formed smooth and spherical pellets about 1.8-mm mean diameter. The mycelial pellets became less uniform at pH (4.0–5.0) lower than the optimum (pH 6.0) or turned to filamentous form at higher pH (8–9). Surfactants added to the medium inhibited pellet formation, resulting in smaller and looser pellets. Tween 80 exhibited a remarkable promoting effect on EPS production, increasing the EPS yield more than twofold at 1.5% (w/v), which was most probably attributed to the stimulation of EPS biosynthesis and release from the fungal cells by Tween 80.     

氮源对灵芝菌丝体液态深层发酵合成灵芝三萜的影响

以沪农灵芝1号为供试菌株,葡萄糖作为碳源,用硫酸铵、氯化铵、鱼粉蛋白胨、胰蛋白胨和酵母粉作为氮源,研究不同种类氮源对灵芝菌丝体液态深层发酵过程的影响。首先,确定了N-10酵母自溶粉作为发酵的氮源,降低了发酵的复杂性和不确定性;其次,考察N-10酵母自溶粉不同浓度对灵芝菌丝体发酵合成灵芝三萜过程中菌丝体的生物量、葡萄糖消耗、灵芝三萜产量等方面的影响,确定了N-10酵母自溶粉的适宜添加浓度。在此基础上,采用响应面中心组合设计,对4因素最佳水平范围进行研究,结果表明,葡萄糖、N-10酵母自溶粉、磷酸二氢钾和七水硫酸镁的含量分别为31.06g/L、2.76g/L、1.77g/L和1.99g/L时,灵芝三萜的理论产量为21.166g/kg干菌丝体,实际发酵产量提高到21.153g/kg干菌丝体。与原工艺相比,新工艺的灵芝三萜产量提高了6.22%。     

Growth and biosynthesis of rugulovasines in Penicillium variabile Sopp 1912

Production of clavine alkaloids rugulovasines by P. variabile did not depend on the habitat of the producers. During submerged cultivation on a simple synthetic medium in early growth stages, microcyclic conidiation was observed in the tested fungi; its presence or absence, as well as the activity of the cultures as to biosynthesis of rugulovasines, depended on the composition of the culture medium. On a complex medium supplemented with peptone, conidiation occurred but was considerably suppressed. Conidia were completely absent in the medium supplemented with yeast extract. In both cases, no appreciable amounts of rugulovasines were detected.     

出芽短梗霉在三种不同氮源的培养基中多糖积累以及形态变化

本研究旨在阐明出芽短梗霉在不同氮源培养基中形态和胞外多糖的积累及化学成分变化。采用摇瓶法培养出芽短梗霉。三种培养基的氮源分别为硝酸钠(培养基1,M1)、硫酸氨、酵母膏(培养基2,M2)和硫酸氨、蛋白胨和酵母膏(培养基3,M3)。M1培养基中,菌丝体和单细胞的生物量积累均比M2、M3低,但胞外多糖的产量则等于甚至略超过M2和M3。在指数生长的前期,白色菌丝体和酵母状细胞状态占优势。指数生长的后期,以厚垣孢子、肿大细胞和黑色菌丝体占优势。胞外多糖都能为茁霉多糖酶水解为麦芽糖和麦芽三糖,说明这些多糖的化学组成都具有(1→4,1→6)-α结构的茁霉多糖。但M1中产生的茁霉多糖结构单元为麦芽糖和麦芽三糖,且二者比例相当。M2中茁霉多糖的麦芽糖结构单元明显减少,而M3中144h后麦芽糖结构单元完全消失。这似乎表明氧化性的氮源和低溶解氧水平可能是造成茁霉多糖结构单元同时具有麦芽糖和麦芽三糖的原因。     

Optimization of submerged culture conditions for exopolysaccharide production in <Emphasis Type="Italic">Sarcodon aspratus</Emphasis> (Berk) S.lto TG-3

The effect of medium components (carbon, nitrogen, and mineral sources) and environmental factors (initial pH and temperature) for mycelial growth and exopolysaccharide (EPS) production in Sarcodon aspratus(Berk) S.lto TG-3 was investigated. The optimal temperature (25°C) and initial pH (5.0) for the EPS production in shake flask cultures of S. aspratus were determined using the two-dimensional contour plot. The most suitable carbon, nitrogen, and mineral sources for EPS production were glucose, yeast extract, CaCl₂ and KH₂PO₄, respectively. Notably, the EPS production was significantly enhanced by supplementation of calcium ion. Subsequently, the optimum concentration of glucose (30gl^–1), yeast extract (15gl^–1), CaCl₂ (1.1gl^–1), and KH₂PO₄ (1.2gl^–1) were determined using the orthogonal matrix method. The effects of nutritional requirement on the mycelial growth of S.aspratuswere in regular sequence of glucose>KH₂PO₄>yeast extract>CaCl₂, and those on EPS production were in the order of glucose>yeast extract>CaCl₂>KH₂PO₄. Under the optimal culture conditions, the maximum EPS concentration in a 5-l stirred-tank reactor was 2.68gl^–1 after 4days of fermentation, which was 6-fold higher than that at a basal medium. The two-dimensional contour plot and orthogonal matrix method allowed us to find the relationship between environmental factors and nutritional requirement by determining optimal operating conditions for maximum EPS production in S.asparatus. The statistical experiments used in this work can be useful strategies for optimization of submerged culture processes for other mushrooms.     

Optimization of fumaric acid production by Rhizopus delemar based on the morphology formation

The effects of temperature, agitation rate and medium composition, including concentrations of glucose, soybean peptone, and inorganic ions, on pellet formation and pellet diameter of Rhizopus delemar (Rhizopus oryzae) NRRL1526 during pre-culture were studied. Inorganic ions and soybean peptone had negative and positive effects on pellet formation, respectively. The initial glucose and soybean peptone concentrations directly affected pellet diameter. Within a certain range, pellet diameter decreased with increased initial substrate concentrations; however, above this range there was an opposite trend. Thus, optimal concentrations of substrate during pre-culture were beneficial for producing small pellets of R. delemar. Furthermore, dry cell mass and yield of fumaric acid tended to increase with decreased pellet diameter. Based on the pellet morphology optimization, the final fumaric acid concentration was improved by 46.13% when fermented in a flask and 31.82% in stirred bioreactor tank fermentation.     

Optimisation of submerged culture conditions for the production of mycelial biomass and exopolysaccharide byPleurotus nebrodensis

The optimisation of submerged culture conditions and nutritional requirements was studied for the production of exopolysaccharide (EPS) fromPleurotus nebrodensis. The optimal temperature and initial pH for both mycelial growth and EPS production in shake flask cultures were 25 °C and 8.0, respectively. Maltose was found the most suitable carbon source for both mycelial biomass and EPS production. Yeast extract was favourable nitrogen source for both mycelial biomass and EPS production. Optimum concentration of each medium component was determined using the orthogonal matrix method. The optimal combination of the media constituents for mycelial growth and EPS production was as follows: 200 g l^?1 bran, 25 g l^?1 maltose, 3 g l^?1 yeast extract, 1 g l^?1 KH₂PO₄, 1 g l^?1 MgSO₄ 7H₂O. Under the optimal conditions, the mycelial biomass (4.13 g l^?1) and EPS content (2.40 g l^?1) ofPleurotus nebrodensis was 2.3 and 3.6 times compared to the control with basal medium respectively.