Water content and water activity for the production of cyclodepsipeptides in solid-state fermentation by Metarhizium anisopliae

Metarhizium anisopliae was grown by solid-state fermentation on a series of compositions of rice, rice bran, or rice husk media for the production of cyclodepsipeptides, destruxins A and B. The best yield for destruxin A and destruxin B were 2.9 mg kg^–1 substrate and 227 mg kg^–1 substrate, respectively, after 14 days cultivation with rice/bran/husk medium at 71% water content together with water activity of 0.921.     

In vitro effect of fungal cyclodepsipeptides on leukemic cells: study of destruxins A, B and E.

The activities of three mycotoxins isolated from the hyphomycete Metarhizium anisopliae: destruxin A, B, and E (DA, DB and DE) are described and compared in vitro on leukemic cells. Their antitumor effect was investigated by flow cytometry on growth, cell viability and cell cycle perturbation 48 h after destruxin exposure. Against P388 leukemic cells, DE displayed greater antiproliferative activity than DA and DB. The minimum concentration required to inhibit 50% of cell proliferation is 0.33 microgram/ml for DE, 11.7 micrograms/ml for DA and 9.4 micrograms/ml for DB. Cell cycle modifications were only observed with DE at 50 and 10 micrograms/ml and consisted in an accumulation of the cells in G0/1 phase. DA and DB did not modify the number of cells in G0/1 of the cell cycle. Nevertheless a decrease in the number of cells in G2+M phase was induced by the three destruxins.     

绿僵菌素的分离制备及其对蛴螬的毒力

以金龟子绿僵菌金龟子变种Metarhizium anisopliae var. anisopliae菌株MaQ10发酵液为材料,采用萃取、浓缩、制备色谱及重结晶技术,分离纯化出5种绿僵菌素的晶体,经与标准样品以及参考文献相对照,此5种晶体与绿僵菌素A、A2、B、C和E相吻合。进而,采用浸渍法测定了绿僵菌素A和B对大等鳃金龟Exolontha serrulata (Gyllenhall) 和卵圆齿爪鳃金龟Holotrichia ovata Chang 1龄幼虫的触杀毒力。结果表明： 绿僵菌素A和B对大等鳃金龟的触杀活性,在处理后96～120 h内最高,LC₅₀分别为78.1571 mg/L和88.7562 mg/L; 处理浓度为300 mg/L时,LT₅₀分别为13.4159 h和10.5331 h。绿僵菌素A和B对卵圆齿爪鳃金龟的触杀活性,在处理后96 h时最高,LC₅₀分别为66.5308 mg/L和79.4309 mg/L;处理浓度为300 mg/L时,LT₅₀分别是13.6399 h和9.9451 h。     

Medium optimization of carbon and nitrogen sources for the production of spores from Bacillus amyloliquefaciens B128 using response surface methodology

The production of spores from Bacillus amyloliquefaciens B128 was investigated in shaker-flask cultivation. Optimization of the culture medium was carried out by using a two-step approach. A quick identification of a suitable source of carbon and nitrogen was obtained by a simple screening experiment, which was followed by an application of response surface methodology (RSM) for further optimization design. A five-level four-factor central composite design was employed to determine the maximum spore yield at optimum levels for lactose, tapioca, ammonium sulfate and peptone. Tapioca and peptone showed a significant linear main effect, while lactose and ammonium sulfate had no significant linear effect. The spore production was also significantly affected by lactose–ammonium sulfate and lactose–peptone. Optimum cultivation parameters were (g/L): 12.7 of lactose, 16.7 of tapioca, 1.8 of ammonium sulfate and 8.0 of peptone. The prediction spore yield was 5.93 × 10⁸ (no/mL). The actual experimental results were in agreement with the prediction.     

Sporulation of several biocontrol fungi as affected by carbon and nitrogen sources in a two-stage cultivation system

The development of fungal biopesticides requires the efficient production of large numbers spores or other propagules. The current study used published information concerning carbon concentrations and C:N ratios to evaluate the effects of carbon and nitrogen sources on sporulation of Paecilomyces lilacinus (IPC-P and M-14) and Metarhizium anisopliae (SQZ-1-21 and RS-4-1) in a two-stage cultivation system. For P. lilacinus IPCP, the optimal sporulation medium contained urea as the nitrogen source, dextrin as the carbon source at 1 g/L, a C:N ratio of 5:1, with ZnSO(4)·7H(2)O at 10 mg/L and CaCl(2) at 3 g/L. The optimal sporulation medium for P. lilacinus M-14 contained soy peptone as the nitrogen source and maltose as the carbon source at 2 g/L, a C:N ratio of 10:1, with ZnSO(4)·7H(2)O at 250 mg/L, CuSO(4)·5H(2)O at 10 mg/L, H(3)BO(4) at 5 mg/L, and Na(2)MoO(4)·2H(2)O at 5 mg/L. The optimum sporulation medium for M. anisopliae SQZ-1-21 contained urea as the nitrogen source, sucrose as the carbon source at 16 g/ L, a C:N ratio of 80:1, with ZnSO(4)·7H(2)O at 50 mg/L, CuSO(4)·5H(2)O at 50 mg/L, H(3)BO(4) at 5 mg/L, and MnSO(4)·H(2)O at 10 mg/L. The optimum sporulation medium for M. anisopliae RS-4-1 contained soy peptone as the nitrogen source, sucrose as the carbon source at 4 g/L, a C:N ratio of 5:1, with ZnSO(4)·7H(2)O at 50 mg/L and H(3)BO(4) at 50 mg/L. All sporulation media contained 17 g/L agar. While these results were empirically derived, they provide a first step toward low-cost mass production of these biocontrol agents.     

Kinetics of α-amylase production in a continuous culture ofBacillus licheniformis

As found during continuous cultivation ofBacillus licheniformis on a semisynthetic medium (glucose or maltose as C source), the specific rate of α-amylase production is proportional to growth rate but is repressed by higher substrate concentrations. Besides glucose or maltose, peptone was also used as an alternative carbon source during cultivation. The specific rate of production of the enzyme on maltose is half that found with glucose.     

Optimization of medium composition for actinomycin X2 production by <Emphasis Type="Italic">Streptomyces </Emphasis><Emphasis Type="Italic">spp</Emphasis> JAU4234 using response surface methodology

The effects of cultivation medium compositions including soybean meal, peptone, soybean oil and cornstarch for actinomycin X2 production by Streptomyces spp JAU4234 were accessed by using response surface methodology. The 2(4) full factorial designs and the paths of steepest ascent were effective in searching for the major factors of actinomycin X2 production. In this study, cornstarch and soybean oil showed negative effect on actinomycin X2 production based on the first-order regression coefficients derived from MINITAB software. Subsequently, a central composite design for optimization was further investigated. Preliminary studies showed that soybean meal and peptone were believed to be the major factors for actinomycin X2 production. Estimated optimum compositions for the production of actionmycin X2 were as follows (g/l): soybean meal 21.65 and peptone 9.41, and result in a maximum actionmycin X2 production of 617.4 mg/l. This value was closed to the 612 mg/l actionmycin X2 production from actual experimental observations. The yield of actionmycin X2 was increased by 36.9% by culturing the strain Streptomyces spp JAU4234 in the nutritionally optimized fermentation medium.     

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomyceteMonascus ruber in mixed solid-liquid (or submerged) cultures at 25°C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate, the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8, , glucose 129, KNO₃ 8.0, MgSO₄·7H₂O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

Optimization of a liquid medium for beauvericin production in <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial culture

Beauvericin (BEA) is a proven and potent antibiotic compound useful for bio-control and a potential antifungal and anticancer agent for human. This study was to evaluate and optimize the nutrient medium for BEA production in mycelial liquid culture of a high BEA-producing fungus Fusarium redolens Dzf2 isolated from a medicinal plant. Among various organic and inorganic carbon and nitrogen sources, glucose and peptone were found the most favorable for the F. redolens Dzf2 mycelial growth and BEA production. Through a Plackett-Burman screening test on a basal medium, glucose, peptone, and medium pH were identified as the significant factors for mycelial growth and BEA production. These factors were optimized through central composite design of experiments and response surface methodology, as 49.0 g/L glucose, 13.0 g/L peptone and pH 6.6, yielding 198 mg/L BEA (versus 156 mg/L in the basal medium). The BEA yield was further increased to 234 mg/L by feeding 10 g/L glucose to the culture during exponential phase. The results show that F. redolens Dzf2 mycelial fermentation is a feasible and promising process for production of BEA.     

响应面法优化多杀菌素发酵培养基的研究

采用响应面分析方法,对刺糖多孢茵（Saccharopolyspora spinosa）H-2产多杀菌素的发酵培养基进行优化研究。运用单因子试验筛选出葡萄糖和棉籽粉为最适碳源和氮源,通过Plack—ett—Burman设计试验,对影响发酵培养基的8个相关因子进行评估并筛选出具有显著效应的4个因子：葡萄糖、棉籽粉、黄豆饼粉及玉米浆。通过最陡爬坡实验逼近以上4个因子的最大响应区域后,采用Box-Behnken响应面分析法,确定发酵产多杀菌素最佳培养基为葡萄糖64．5g,麦芽糖20g,玉米浆2g,大豆油40g,棉籽粉25g,黄豆饼粉2．4g,蛋白胨25g,CaCO35g,定容至1L,pH7．0。培养基优化后多杀菌素产量由278．1mg／L提高到508．7mg／L,比初始多杀茵素产量提高了1．83倍。     

乳牛肝菌液态发酵生长条件的优化

采用响应曲面法对乳牛肝菌发酵培养基及发酵条件进行优化,以麦芽糖、蛋白胨和装液量3个因素为变量,分析乳牛肝菌液态发酵产菌丝体的最佳条件。实验结果表明,乳牛肝菌发酵的关键因子值分别为麦芽糖33．03g/L,蛋白胨6．95g/L,装液量为102．21mL／250mL时,可得到最大菌丝体生物量23．44g/L（干重）。     

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.     

Culture condition improvement for whole-cell lipase production in submerged fermentation by Rhizopus chinensis using statistical method

Rhizopus chinensis CCTCC M201021 was a versatile strain capable of producing whole-cell lipase with synthetic activity in submerged fermentation. In order to improve the production of whole-cell lipase and study the culture conditions systematically, the combination of taguchi method and response surface methodology was performed. Taguchi method was used for the initial optimization, and eight factors viz., maltose, olive oil, peptone, K2HPO4, agitation, inoculum size, fermentation volume and pH were selected for this study. The whole-cell lipase activity yield was two times higher than the control experiment under initial optimal conditions, and four significant factors (inoculum, olive oil, fermentation volume and peptone) were selected to test the effect on the lipase production using response surface methodology. The optimal fermentation parameters for enhanced whole-cell lipase yield were found to be: inoculum 4.25 x 10(8) spores/L, olive oil 2.367% (w/v), fermentation volume 18 mL/250 mL flask, peptone 4.06% (w/v). Subsequent experimental trails confirmed the validity of the model. These optimal culture conditions in the shake flask led to a lipase yield of 13875 U/L, which 120% increased compare with the non-optimized conditions.     

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。     

Regulation of Amylase Synthesis in Bacillus circulans F-2

In the usual batch cultivation, Bacillus circulans F-2 produced amylase only when granular carbon sources such as raw starch or crosslinked starches (CLP) were added. In the dialysis cultivation, where CLP and partially purified amylase were incubated inside the dialysis tubing, the bacterium inoculated outside of the tubing grew and produced the amylase. Amylase production of this bacterium was further investigated in feeding cultivation, in which maltose was fed to the cultivation medium at various rates. The bacterial growth increased with the increase of the feeding rate of maltose, but maximum amylase production was observed at a feeding rate of 45 mg/hr/1. No amylase was produced on the media containing monosaccharides, sucrose, lactose, or isomaltose in the feeding cultivation although bacterial growth was observed. The amylase of this bacterium was found to be inducible. Replacement of 20% of the maltose with glucose resulted in a great decrease (70%) in the amylase production. This shows that the amylase synthesis of B. circulans F-2 is severely repressed by glucose.     

Design and optimization of fermentation medium for enhanced bacteriocin production by probiotic bacterium Enterococcus faecium MC13

Statistics-based experimental designs were used to develop a cost-effective medium for enhanced production of viable cells and bacteriocin by probiotic Enterococcus faecium MC13. Carbon, nitrogen, and mineral sources were first screened by one-variable-at-a-time (OVAT) methods. In order to increase yield production, the selected variables were further statistically optimized using response-surface methodology (RSM) with central composite design (CCD). The maximum and minimum levels of the selected variables were determined and a set of 34 experimental runs was performed. The optimum concentrations of the tested variables for production of viable cells (12.24 log CFU mL(-1)) and bacteriocin activity (25,600 AU mL(-1)) were tryptone (10.0 g/L), peptone (6.0 g/L), maltose (3.0 g/L), glucose (9.0 g/L), NaCl (15.0 g/L), sodium citrate (2.5 g/L), sodium acetate (1.0 g/L), and dipotassium PO(4) (0.1 g/L). Threefold increased yield of bacteriocin was achieved in optimized medium compared to the unoptimized counterpart, and this was two times less cost than commercial MRS medium.     

Study of mycelial growth and bioactive polysaccharide production in batch and fed-batch culture of Grifola frondosa

The fermentation of Grifola frondosa was investigated in the shake flasks and a 5-L jar fermenter in batch and fed-batch modes. In the shake-flask experiments, the preferable mycelial growth and exopolysaccharide (EPS) production was observed at relatively low pH; maltose and glucose were preferred carbon sources for high mycelial production. The EPS was doubled after 13 d of cultivation when glucose was increased from 2% to 4%. Yeast extract (YE) (0.4%) in combination with corn steep powder (CSP) (0.6%) and YE (0.8%) in combination with CSP (1.2%) were preferred nitrogen sources for high mycelial production and EPS production, respectively. All plant oils tested significantly stimulate cell growth of G. frondosa but they failed to enhance EPS production. The EPS products usually consisted of two fractions of different molecular sizes varied by the plant oils used. The fed-batch fermentation by glucose feeding was performed when the glucose concentration in the medium was lower than 0.5% (5g/L), which greatly enhanced the accumulation of mycelial biomass and EPS; the mycelial biomass and EPS were 3.97g/L and 1.04g/L before glucose feeding, which reached 8.23g/L and 3.88g/L at 13 d of cultivation. In contrast, the mycelial biomass and EPS in the batch fermentation were 6.7g/L and 3.3g/L at 13 d of cultivation.     

Effects of nutrients on germination of Verticillium lecanii (=Lecanicillium sp.) conidia and infection of greenhouse whitefly, (Trialeurodes vaporariorum)

Laboratory experiments were conducted to assess effects of nutrients on germination of Verticillium lecanii (=Lecanicillium sp.) conidia and infection of the greenhouse whitefly, Trialeurodes vaporariorum. Suspensions of V. lecanii conidia were prepared in four nutrient solutions: 2% glucose, 2% sucrose, 2% maltose, and 2% peptone. Suspensions in de-mineralized water served as the control. At 23°C the germination rate was highest in the 2% glucose solution, followed by sucrose, maltose, demineralized water, and peptone, respectively. Germ tube growth was greatest at 23°C in the 2% glucose solution after 10 h incubation. Results of the bioassays indicated that the nutrients influenced whitefly infection. Infection levels were highest for conidial suspensions (1×10⁶ conidia/mL) prepared in 2% glucose, and were significantly greater than for peptone, demineralized water and maltose. Infection levels at 1×10⁸ conidia/mL were not significantly different from each other for all materials tested. The potential use of nutrients in a spray formulation as a means of enhancing field efficacy are discussed.     

Effect of medium composition on the production of tetanus toxin by Clostridium tetani

The tetanus toxin is a neurotoxin synthesized by the bacillus Clostridium tetani that, after detoxification with formaldehyde, still exhibits antigenic and immunologic properties, hence its denomination of tetanus toxoid. Such a neurotoxin is produced by cultivation of the microorganism in vegetative form on a relatively complex specific medium containing glucose and peptone. The simultaneous effects of the starting levels of glucose (G0) and N-Z Case TT (NZ0) as carbon and nitrogen sources, respectively, on the production of tetanus toxin have been investigated in this work in static cultivations by means of a five-level star-shaped experimental design and evaluated by response surface methodology (RSM) for optimization purposes. The highest final average yield of tetanus toxin (72 Lf/mL), achieved at G0= 9.7 g/L and NZ0= 43.5 g/L, was 80% higher than that obtained with standard cultivations (G0= 8.0 g/L and NZ0= 25.0 g/L).