Hydrolysis of wheat bran,rice bran and jute powder by immobilized enzymes from Macrophomina phaseolina

The stability of cellulolytic and hemicellulolytic enzymes from Macrophomina phaseolina improved on immobilization and was 1.5 to 2-fold more active against pre-treated wheat bran, rice bran or jute powder. The hydrolysis efficiency of the catalyst increased with a decrease in its particle size. About 80% (w/v) of the sugar obtained from wheat bran was assimilated by Saccharomyces sp., whereas the corresponding values for rice bran and jute powder were about 70 and 50% (w/v), respectively.     

Effect of Medium Components on Bacteriocin Production by Lactobacillus Pentosus ST151BR,a Strain Isolated from Beer Produced by the Fermentation of Maize,Barley and Soy Flour

Lactobacillus pentosus ST151BR, isolated from home-brewed beer, produces a 3.0 kDa antibacterial peptide (bacteriocin ST151BR) active against Lactobacillus casei, Lactobacillus sakei, Pseudomonas aeruginosa, Enterococcus faecalis and Escherichia coli. Treatment with Proteinase K or Pronase resulted in loss of activity. Bacteriocin levels of 6400 AU/ml were recorded in MRSbb (De Man-Rogosa-Sharpe broth without Tween 80) at pH 5.5, 6.0 and 6.5. The same growth conditions at pH 4.5 yielded only 1600 AU/ml bacteriocin. Inclusion of Tween 80 in the growth medium reduced bacteriocin production by more than 50%. Growth in the presence of tryptone or tryptone plus meat extract stimulated bacteriocin production, whereas much lower activity was recorded when the bacteria were grown in the presence of meat extract, yeast extract, tryptone plus yeast extract, meat extract plus yeast extract, or a combination of tryptone, meat extract and yeast extract. MRSbb supplemented with maltose, lactose or mannose (2.0%, w/v) yielded bacteriocin levels of 6400 AU/ml. Sucrose or fructose at these concentrations reduced the activity by 50 and 75%, respectively. Growth in the presence of 4.0%(w/v) glucose resulted in 50% activity loss. Glycerol levels as low as 0.1%(w/v) repressed bacteriocin production. Addition of cyanocobalamin, ascorbic acid, thiamine and thioctic acid (1.0 mg/l) to the growth medium did not lead to an increase in bacteriocin production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Formulation of Bacillus amyloliquefaciens B190 for Control of Lily Grey Mould (Botrytis elliptica)

Calcium hydroxide (0.1%) significantly increased the growth of Bacillus amyloliquefaciens B190, inhibited completely the germination of Botrytis elliptica, and decreased the disease severity caused by B. elliptica on lily. Spraying B. amyloliquefaciens B190 mixed with either 0.025% calcium hydroxide, 0.05% sodium carbonate or 0.025% ammonium nitrate decreased the grey mould disease on lily leaves. B. amyloliquefaciens B190 mixed with 0.025% calcium hydroxide and 0.05% sodium carbonate, or mixed with 0.025% calcium hydroxide and 0.025% ammonium nitrate controlled lily grey mould completely. When the concentration of tested adjuvants was below 0.1% (v/v), adhesive adjuvant, i.e. carboxymethyl cellulose and spreader, i.e. Tween 80 were equally effective to assist B. amyloliquefaciens B190 to control lily grey mould. Calcium hydroxide (0.025%) and 0.05% sodium carbonate mixed with 0.1% Tween 80 significantly controlled lily grey mould. B. amyloliquefaciens B190 mixed with 0.025% calcium hydroxide and 0.05% sodium carbonate, and these two chemicals plus or without 0.1% Tween 80 and 0.05% mineral oil (i.e. emulsion and wettable powder, respectively) was consistently able to control grey mould on lily as well as 100 p.p.m. flusilazole in greenhouse and field trials, respectively.     

Lipase Activity from Strains of Pasteurella multocida

Thirteen clinical isolates of Pasteurella multocida from a variety of different animals and humans were examined for their ability to produce lipase. Lipase substrates used included Tween 20, Tween 40, Tween 80, and Tween 85. Lipase activity was detected in the filtrates of organisms grown to the exponential phase in Roswell Park Memorial Institute-1640 defined media (RPMI-1640), but activity increased in the filtrates when the cultures were allowed to proceed to the stationary phase. All strains examined (except for serotype 2) showed lipase activity against at least one of the Tweens. Tween 40 was the best substrate to demonstrate lipase activity. Pasteurella multocida serotype 8 produced the most active lipase against Tween 40 (3,561.7 units of activity/μg of protein). This activity continued to increase after P. multocida entered a stationary growth phase. P. multocida lipase activity was optimal at pH 8.0. Lipase activity of P. multocida serotype 8 was eluted from a Sepharose 2B column at several points, indicating that several lipases may be produced in vitro by this organism. These data demonstrate that clinical isolates of P. multocida produce lipase; therefore, this enzyme should be considered a potential virulence factors for this organism. Received: 16 September 1999 / Accepted: 22 November 1999     

Surfactant-enhanced anaerobic acidogenesis of Canna indica L. by rumen cultures

Polyoxyethylene sorbitan monoolate (Tween 80) was used to enhance the anaerobic acidogenesis of Canna indica L. (canna) by rumen culture in this study. Dose of Tween 80 at 1 ml/l enhanced the volatile fatty acids (VFA) production from the acidogenesis of canna compared to the control. However, Tween 80 at higher dosages than 5 ml/l inhibited the rumen microbial activity and reduced the VFA yield. Response surface methodology was successfully used to optimize the VFA yield. A maximum of VFA yield of 0.147 g/g total solids (TS) added was obtained at canna and Tween 80 concentrations of 6.3g TS/l and 2.0 ml/l, respectively. Dosage of Tween 80 at 1-3.75 ml/l reduced the unproductive adsorption of microbes or enzymes on the lignin part in canna and increased microbial activity. A high VFA production was achieved from canna presoaked with Tween 80, suggesting that the structure of canna was disrupted by Tween 80.     

Studies on Pathogenic Leptospirae

Effect of Tween 80 on the growth of Leptospira canicola strain Utrecht and L. icterohaemorrhagiae strain Mikawajima was examined. The suspension of washed leptospira was inoculated into modified Korthof's basal medium containing varied amounts of Tween 80 and cultured at 30 C. Cell numbers were counted by using Petroff-Hausser counting chamber every other day. Optimum Tween 80 concentrations for L. canicola were 0.0125 and 0.025%. Cell counts in the second sub-cultures reached 10⁸ per ml the same as the primary cultures. Generation time of L. canicola in 0.025% Tween 80 medium was about 13 hours. Growth of L. icterohaemorrhagiae was inhibited at concentrations greater than 0.0125 per cent. Cell numbers increased about 4 times at concentration of 0.0000125% Tween 80. L. canicola utilizes Tween 80 as a nutrient while L. icterohaemorrhagiae appears sensitive to it. A difference of more than 1,000 times in maximal growth-supporting concentration between L. canicola and L. icterohaemorrhagiae exists. This difference appears to be caused by difference in surface structure and metabolic requirements.     

Entomotoxicity,protease and chitinase activity of Bacillus thuringiensis fermented wastewater sludge with a high solids content

This study investigated the production of biopesticides, protease and chitinase activity by Bacillus thuringiensis grown in raw wastewater sludge at high solids concentration (30 g/L). The rheology of wastewater sludge was modified with addition of Tween-80 (0.2% v/v). This addition resulted in 1.6 and 1.3-fold increase in cell and spore count, respectively. The maximum specific growth rate (μ_max) augmented from 0.17 to 0.22 h⁻¹ and entomotoxicity (Tx) increased by 29.7%. Meanwhile, volumetric mass transfer coefficient (k_La) showed marked variations during fermentation, and oxygen uptake rate (OUR) increased 2-fold. The proteolytic activity increased while chitinase decreased for Tween amended wastewater sludge, but the entomotoxicity increased. The specific entomotoxicity followed power law when plotted against spore concentration and the relation between Tx and protease activity was linear. The viscosity varied and volume percent of particles increased in Tween-80 amended wastewater sludge and particle size (D₅₀) decreased at the end of fermentation. Thus, there was an increase in entomotoxicity at higher suspended solids (30 g/L) as Tween addition improved rheology (viscosity, particle size, surface tension); enhanced maximum growth rate and OUR.     

Medium optimization of a hydrophilic solvent‐stable protease from Serratia sp. SYBC H

Two statistical methods were used for medium optimization for a hydrophilic solvent‐stable protease production by Serratia sp. SYBC H with duckweed as the nitrogen source. Orthogonal design was applied to find the significant variables, then response surface methodology (RSM), including Box–Behnken central composite experiments, was used to determine the optimal concentrations and interaction of the significant variables. Results demonstrated that duckweed powder, wheat flour, Tween 80, sodium chloride had significant effects on the solvent‐stable protease production. The interaction between duckweed and wheat flour was significant. The optimal level of the variables for the maximum protease production was duckweed 43.9 g/L, wheat flour 20 g/L, sodium chloride 0.08 M, Tween 80 1% v/v, initial pH 11.0, and inoculum size 7% v/v. The maximum protease activity reached 1922.8 U/mL in the optimized medium, with about 18.3‐fold higher than that in the unoptimized medium. Most importantly, the protease from Serratia sp. SYBC H has successfully catalyzed the specific acylation of sucrose in a two‐solvent medium consisting of pyridine and n‐hexane (1:1, v/v), and non‐specific acylation of sucrose in anhydrous DMSO. These results demonstrated that the protease from Serratia sp. SYBC H is a solvent‐stable protease and it could be an ideal biocatalyst for sugar esters syntheses in non‐aqueous media.     

Permeabilization of elicited suspension culture of madder (Rubia akane Nakai) cells for release of anthraquinones

To enhance the productivity of anthraquinone colorants during madder (Rubia akane Nakai) cell cultures, the effects of permeabilizing agents on the production of anthraquinone colorants were investigated. Tween 80 was the best among the permeabilizing agents tested. Addition of 1% Tween 80 increased the total and released concentrations of anthraquinones about 1.6 times (159 mg l–1) and 14 times (71 mg l–1), respectively. In addition, anthraquinone production was increased to 220 mg l–1, 2.2 times as the level of control culture by simultaneous use of 1% Tween 80, 5 mg chitosan/l and 2% (w/v) XAD-7. Also, 47% (105 mg l–1) of total anthraquinones was released to medium or adsorbed on XAD-7.     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

Transport of histone intoEscherichia coli

Histone appears to be transported intoEscherichia coli cells in two ways: by an adsorption-like process and by a system which is temperature-dependent and is inhibited by 2,4-dinitrophenol. The adsorbed component cannot be washed out with a salt solution but can be partly exchanged for labelled histone and can be displaced with Tween 80. The actively transported histone can be replaced with unlabelled histone and with polylysine, and released by a high concentration of Mg²⁺ or by Tween 80. The active transport is observed only at concentrations greater than 100 μg histone/ml; the adsorption-like process occurring at lower concentrations.     

Production of cellulolytic and xylanolytic enzymes by an Arthrographis species

A fungal isolate, Arthrographis sp. strain F4, when grown in shake-flask culture, produced cellulolytic and xylanolytic enzymes optimally at 30°C with an initial pH of 5.0 to 6.0. Coarsely-ground filter paper was the most suitable carbon substrate for production of the enzymes. Inorganic nitrogen sources gave higher activities of the enzymes than organic nitrogen sources: NH₄NO₃ and yeast extract was the most effective combination. Significant stimulation (P<0.05) of enzyme production was achieved with 0.1% (v/v) Tween 80.B.C. Okeke was and S.K.C. Obi is with the Department of Microbiology, University of Nigeria, Nsukka, Nigeria. B.C. Okeke is now with the Department of Bioscience and Biotechnology, Royal College Building, University of Strathclyde, Glasgow G1 1XW, UK     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Effects of Tween 80 and rhamnolipid on the extracellular enzymes of Penicillium simplicissimum isolated from compost

Extracellular enzymes of microorganisms play an important role in the decomposition of macromolecules in the composting process. In this study, the effects of Tween 80 and rhamnolipid on the extracellular amylase, carboxymethyl cellulose enzyme (CMCase), xylanase and protease of Penicillium simplicissimum isolated from compost were investigated during solid-state fermentation. The results showed that the enzyme activities of amylase, CMCase and xylanase were increased by Tween 80 and rhamnolipid, which, however, had a negative effect on the protease production. The stimulative effects on the three enzymes were quite different during the whole fermentation process. Tween 80 and rhamnolipid also increased the fungal biomass slightly. As a result of the enhanced enzyme activities, the organic matter were also improved to different extents by both surfactants, and the decomposition rates of hemicellulose and cellulose were increased about 8.0% and 11.6% by Tween 80 at best, respectively, as well as 5% and 5.5% by rhamnolipid.     

The products from lipase-catalysed hydrolysis of bovine milkfat kill Helicobacter pylori in vitro

Free fatty acids and monoglycerides released from milkfat by partial pregastric lipase-catalysed hydrolysis are bactericidal towards Helicobacter pylori. Two milkfat preparations were investigated: a normal bovine milkfat, and a fractionated milkfat preparation, termed ModFat, enriched in triglycerides containing short- and medium-chain fatty acids. The released products were tested for bactericidal potency against H. pylori. The potencies of the respective preparations were consistent with expected potencies calculated from individual free fatty acid and monoglyceride concentrations and their lauric acid equivalence factors (Ki). ModFat products were more bactericidal, in accordance with release of free fatty acid types of high potency, and addition of the surfactant Tween 80 to the hydrolysed lipid increased potency eight times more than did addition of lecithin. Tween 80 micelles have smaller aggregation numbers, and the mixed micelles of Tween 80/free fatty acids would be more likely to expose the bacteria to higher apparent free fatty acid concentrations.     

Biocides formulation of essential oils having antimicrobial activity

Essential oils of fennel, peppermint, caraway, eucalyptus, geranium and lemon were tested for their antimicrobial activities against some plant pathogenic micro-organisms (Fusarium oxysporum, Alternaria alternate, Penicilium italicum Penicilium digitatum and Botyritus cinerea). Essential oils of fennel, peppermint, caraway were selected as an active ingredient for the formulation of biocides due to their efficiency in controlling the tested micro-organisms. Successful emulsifiable concentrates (biocides) were prepared from these oils using different emulsifiers (Emulgator B.L.M. Tween20 and Tween80) and different fixed oils (sesame, olive, cotton and soybean oils). Physico-chemical properties of the formulated biocide (spontaneous emulsification, emulsion stability test, cold stability and heat stability tests as well as viscosity, surface tension and pH) were measured. The prepared biocides were ready to be tested for application in a future work as a safe pesticide against different pathogens.     

Direct ethanol fermentation from lignocellulosic biomass by Antarctic basidiomycetous yeast Mrakia blollopis under a low temperature condition

Antarctic basidiomycetous yeast Mrakia blollopis SK-4 has unique fermentability for various sugars under a low temperature condition. Hence, this yeast was used for ethanol fermentation from glucose and also for direct ethanol fermentation (DEF) from cellulosic biomass without/with Tween 80 at 10 °C. Maximally, 48.2 g/l ethanol was formed from 12% (w/v) glucose. DEF converted filter paper, Japanese cedar and Eucalyptus to 12.2 g/l, 12.5 g/l and 7.2 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80, ethanol concentration increased by about 1.1–1.6-fold compared to that without Tween 80. This is the first report on DEF using cryophilic fungi under a low temperature condition. We consider that M. blollopis SK-4 has a good potential for ethanol fermentation in cold environments.     

Methotrexate-Loaded Chitosan- and Glycolchitosan-Based Nanoparticles: A Promising Strategy for the Administration of the Anticancer Drug to Brain Tumors

Brain tumor treatment employing methotrexate (MTX) is limited by the efflux mechanism of Pg-p on the blood–brain barrier. We aimed to investigate MTX-loaded chitosan or glycol chitosan (GCS) nanoparticles (NPs) in the presence and in the absence of a coating layer of Tween 80 for brain delivery of MTX. The effect of a low Tween 80 concentration was evaluated. MTX NPs were formulated following the ionic gelation technique and size and zeta potential measurements were acquired. Transport across MDCKII-MDR1 monolayer and cytotoxicity studies against C6 glioma cell line were also performed. Cell/particles interaction was visualized by confocal microscopy. The particles were shown to be cytotoxic against C6 cells line and able to overcome MDCKII-MDR1 cell barrier. GCS-based NPs were the most cytotoxic NPs. Confocal observations highlighted the internalization of Tween 80-coated fluorescent NPs more than Tween 80-uncoated NPs. The results suggest that even a low concentration of Tween 80 is sufficient for enhancing the transport of MTX from the NPs across MDCKII-MDR1 cells. The nanocarriers represent a promising strategy for the administration of MTX to brain tumors which merits further investigations under in vivo conditions.     

Effect of Tween 80 on the production of curdlan by Alcaligenes faecalis ATCC 31749

This study aims to investigate the effects of Tween 80 on curdlan production, cell growth, and glucosyltransferase activity. The addition of Tween 80 to the culture medium increased curdlan production. However, curdlan production did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. The addition of Tween 80 to the culture medium did not affect cell growth. The glucosyltransferase activity involved in the curdlan synthesis increased with the increase of Tween 80 concentration. The glucosyltransferase activity did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. Maximum curdlan was observed at day 5 and then levelled off. The biomass continued to increase until the end of the experimental period (6 d). Maximum glucosyltransferase activity was also observed at day 5 and decreased thereafter. The results indicate that the enhanced curdlan production by Tween 80 is highly correlated with glucosyltransferase activity.     

Improvement of direct ethanol fermentation from woody biomasses by the Antarctic basidiomycetous yeast, Mrakia blollopis, under a low temperature condition

The Antarctic basidiomycetous yeast Mrakia blollopis SK-4 can quite uniquely ferment various sugars under low temperature conditions. When strain SK-4 fermented lignocellulosic biomass using the direct ethanol fermentation (DEF) technique, approximately 30% to 65% of the theoretical ethanol yield was obtained without and with the addition of the non-ionic surfactant Tween 80, respectively. Therefore, DEF from lignocellulosic biomass with M. blollopis SK-4 requires the addition of a non-ionic surfactant to improve fermentation efficiency. DEF with lipase converted Eucalyptus and Japanese cedar to 12.6 g/l, and 14.6 g/l ethanol, respectively. In the presence of 1% (v/v) Tween 80 and 5 U/g-dry substrate lipase, ethanol concentration increased about 1.4- to 2.4-fold compared to that without Tween 80 and lipase. We therefore consider that the combination of M. blollopis SK-4 and DEF with Tween 80 and lipase has good potential for ethanol fermentation in cold environments.