Antimicrobial activity of Anonna mucosa (Jacq.) grown in vivo and obtained by in vitroculture

Brazilian flora includes numerous species of medicinal importance that can be used to develop new drugs. Plant tissue culture offers strategies for conservation and use of these species allowing continuous production of plants and bioactive substances. Annona mucosa has produced substances such as acetogenins and alkaloids that exhibit antimicrobial activities. The widespread use of antibiotics has led to an increase in multidrug-resistant bacteria, which represents a serious risk of infection. In view of this problem, the aim of this work was to evaluate the antibacterial potential of extracts of A. mucosa obtained by in vitro techniques and also cultured under in vivo conditions. Segments from seedlings were inoculated onto different culture media containing the auxin picloram and the cytokinin kinetin at different concentrations. The calluses obtained were used to produce cell suspension cultures. The materials were subjected to methanol extraction and subsequent fractionation in hexane and dichloromethane. The antimicrobial activity against 20 strains of clinical relevance was evaluated by the macrodilution method at minimum inhibitory and minimum bactericidal concentrations. The extracts showed selective antimicrobial activity, inhibiting the growth of Streptococcus pyogenes and Bacillus thuringiensis at different concentrations. The plant tissue culture methods produced plant materials with antibacterial properties, as well as in vivo grown plants. The antibacterial activity of material obtained through biotechnological procedures of A. mucosa is reported here for the first time.

Application of statistical experimental design for optimisation of bioinsecticides production by sporeless Bacillus thuringiensis strain on cheap medium

In order to overproduce bioinsecticides production by a sporeless Bacillus thuringiensis strain, an optimal composition of a cheap medium was defined using a response surface methodology. In a first step, a Plackett-Burman design used to evaluate the effects of eight medium components on delta-endotoxin production showed that starch, soya bean and sodium chloride exhibited significant effects on bioinsecticides production. In a second step, these parameters were selected for further optimisation by central composite design. The obtained results revealed that the optimum culture medium for delta-endotoxin production consists of 30 g L-1 starch, 30 g L-1 soya bean and 9g L-1 sodium chloride. When compared to the basal production medium, an improvement in delta-endotoxin production up to 50% was noted. Moreover, relative toxin yield of sporeless Bacillus thuringiensis S22 was improved markedly by using optimised cheap medium (148.5 mg delta-endotoxins per g starch) when compared to the yield obtained in the basal medium (94.46 mg delta-endotoxins per g starch). Therefore, the use of optimised culture cheap medium appeared to be a good alternative for a low cost production of sporeless Bacillus thuringiensis bioinsecticides at industrial scale which is of great importance in practical point of view.

Use of by-products rich in carbon and nitrogen as a nutrient source to produce Bacillus thuringiensis (Berliner)-based biopesticide / Produção de biopesticida à base de Bacillus thuringiensis (Berliner) usando meios alternativos ricos em carbono e nitrogênio como fontes de nutrientes

The amount and sources of carbon and nitrogen used to produce Bacillus thuringiensis (Berliner)-based biopesticide may infl uence the quality of the fi nal product. The objective of this research was to test different levels of carbon and nitrogen: medium 1 - 1.5% maize glucose + 0.5% soy fl our,medium 2 - 3.0% maize glucose + 1.0% soy fl our, medium 3 - 1.0% maize glucose + 3.0% soy fl our and medium 4 - Luria Bertani (LB) + salts (FeSO4, ZnSO4, MnSO4, MgSO4). The seed culture was produced in LB medium plus salt, under agitation (200 rpm) for 18h at 30oC. The strain 344 of Bt wasused (B. thuringiensis var tolworthi – belonging to the Embrapa´s Bt Bank). The pH was measured at regular intervals. and After culturing for 96h, the pH of the four tested media was basifi ed (6.91 and 8.15), the number of spores yielded 4.39 x 109 spores/ml in medium 3, where the amount of proteinis high. The dry biomass weight accumulated in media 3 was 39.3 g/l. Mortality of 2-day-old larvae Spodoptera frugiperda (J.E. Smith) was 100% when using Bt produced in media 3 and 4. CL50 for medium 3 was 8.4 x 106 spores/ml. All tested media were satisfactory to Bt growth, and medium 3wass the most promising to be used on a large scale Bt-based biopesticide production.

A quantidade de carbono e nitrogênio usados para produzir biopesticidas à base de Bacillus thuringiensis (Berliner) pode infl uenciar a qualidade fi nal do produto. O objetivo deste trabalho foi testar meios com diferentes níveis de carbono e nitrogênio: meio 1 - glicose de milho a 1.5% + farinha de soja a 0,5%, meio 2 - glicose de milho a 3,0% + farinha de soja a 1,0%, meio 3 - glicose de milho a 1,0% + farinha de soja a 3,0% e meio 4 - Luria Bertani (LB) + sais (FeSO4, ZnSO4, MnSO4 e MgSO4). O inóculo semente foi produzido usando 150 ml de meio LB mais sais incubados por 18h a 30oC, sob agitação de 200 rpm. A cepa utilizada foi 344 (B. thuringiensis var tolworthi – pertencente ao Banco de Microorganismos da Embrapa). O pH foi medido a intervalos regulares. Após 96h de cultivo, o pH de todos os meios testados tenderam ao básico (entre 6,91 e 8,15), o maior número de esporos foi de 4,39 x 109 sporos/ml no meio 3, onde o teor de proteína usado foi o mais alto. A produção de massa celular foi maior no meio 3, com total acumulado de 39,3 g/l. A mortalidade de larvas de Spodoptera frugiperda (J.E. Smith) de dois dias de idade nos meios 3 e 4 foi de 100%. A CL50 para o meio 3 foi de 8,4 x 106 esporos/ml. Os meios alternativos usados promoveram crescimento satisfatório de Bt, sendo o meio 3 o mais promissor para ser usadona produção de biopesticida à base de Bt.

Impact of larviciding with a Bacillus thuringiensis israelensis formulation, VectoBac WG, on dengue mosquito vectors in a dengue endemic site in Selangor State, Malaysia.

The field bioefficacy of a wettable granule (WG) formulation of Bacillus thuringiensis israelensis (Bti), VectoBac WG (Bti strain AM65-52) against dengue vectors, Aedes aegypti and Ae albopictus; was evaluated in a suburban residential area (TST) and in a temporary settlement site (KB) in the state of Selangor, Malaysia. Pre-control ovitrap surveillance of the trial sites indicated a high population of both types of Aedes mosquitoes. The populations were monitored continuously by weekly ovitrapping. Bti was sprayed biweekly at a dosage of 500 g/ha by using a mist-blower. The spray application was targeted into outdoor larval habitats. If required, Bti formulation was also applied directly into indoor water-holding containers at 8 g/1,000 l. Based on ovitrap surveillance, a significant reduction in Aedes populations was evident 4 weeks after initiating the first Bti treatment. The ovitrap index (OI) and the larvae density decreased drastically in both trial sites. In TST, the indoor OI was significantly reduced from 57.50 +/- 7.50% to 19.13 +/- 5.49% (p<0.05), while the outdoor OI decreased from 38.89 +/- 11.11% to 15.36 +/- 5.93%. In KB, similarly, the OI was significantly reduced by more than half, from 66.66 +/- 6.67% to 30.26 +/- 2.99% (p< 0.05). In all cases, the reduction in OI was paralleled by reduction in larval density.

Estudo da influência do suprimento de oxigênio e da concentraçäo de glicose sobre o cultivo de Bacillus thuringiensis var. israelensis IPS82 em regime descontínuo / Influence study of the oxygen supply and concentration of glucose on the cultivation of Bacillus thuringiensis var. israelensis IPS82 in discontinuous regime

Neste trabalho foi avaliada a influência do suprimento de oxigênio e da concentraçäo inicial de substrato (S `IND. 0') sobre o cultivo de Bacillus thuringiensis var. israelensis IPS82, em meio semi-sintético (Meio GYS), em regime descontínuo. Foram realizados, ainda, ensaios com meio composto de resíduos agroindustriais (Meio MA), eficientes para a produçäo de bioinseticidas com esta bactéria. Em cultivos sem limitaçÝo de oxigênio, o ciclo celular apresentou quatro fases com características morfológicas e fisiológicas bem definidas: Fase I, crescimento vegetativo; Fase II, transiçÝo para a esporulaçÝo; Fase III, esporulaçäo; Fase IV, maturaçäo dos esporos e lise celular. Durante as Fases III e IV, verificou-se a formaçäo dos cristais protéicos responsáveis pela atividade larvicida...

A carbon-limited medium for growth and sporulation of Bacillus thuringiensis var. kurstaki.

A culture medium for batch production of d-endotoxin by Bacillus thuringiensis (B., t.) has been modified. Through batch and continuous cultivation studies, the original medium was diagnosed to be limited in organic nitrogen. Corn steep liquor was found to be an excellent source for the organic nitrogen and its addition resulted in a carbon limited medium and in a significant increase in the amount of spore-toxin complex formed in shake flasks. Results of bioassay, conducted on Trichoplusia ni, suggest enhancement of larvicidal efficacy under carbon-limited growth conditions.

Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.

Production & formulation of Bacillus thuringiensis var. israelensis & B. sphaericus 1593.

Three fermentation media each for bulk growth of B. thuringiensis var. israelensis and B. sphaericus 1593 were formulated using defatted groundnut cake (Arachis hypogea) as the first nitrogen source and gram flour (Cicer arientinum), soy bean (Glycine max) and defatted milk powder as the second nitrogen source. Medium containing gram flour showed highest toxicity (14.45 micrograms/l) in case of B. thuringiensis var. israelensis whereas medium containing milk powder was found to be highly toxic with B. sphaericus 1593 (51.39 micrograms/l). Sustained release floating pellet formulations of B. thuringiensis var. israelensis and B. sphaericus 1593 exhibited toxicity of 77 per cent and above for 42 days at a dose of 500 micrograms/l for 4th instar larvae of Culex pipiens quinquefasciatus Say.

Effect of corn-steep liquor on growth and mosquito larvicidal activity of Bacillus thuringiensis var israelensis de Barjac 1978 and B. sphaericus Neide 1904.

Influence of corn steep liquor on the cell yield and toxicity of three strains of B. thuringiensis var israelensis and two strains of B. sphaericus was studied and compared with peptone-yeast extract using a laboratory fermentor. Large increase in the cell yield of all the three strains of B. thuringiensis var israelensis was observed when cornsteep liquor was used as the sole nitrogen source. Significant increase in toxicity was also observed in B. thuringiensis var israelensis strains B17 and B113. Among the two B. sphaericus strains tested, the strain 1593 showed no significant change in cell yield and toxicity, whereas the strain VCRC B42 showed increased cell yield and toxicity in this medium. The results indicate that cornsteep liquor can effectively replace both peptone and yeast extract in the media presently used for large scale multiplication of the two larvicidal bacilli.

Growth & toxicity of Bacillus thuringiensis var israelensis.

Chemically defined media containing glutamic acid, glutamine and aspartic acid at a 20 mM concentration individually supported abundant growth and sporulation of Bacillus thuringiensis var israelensis. The parasporal crystals produced in these media were toxic to fourth instar larvae of Culex pipiens quinquefasciatus Say. The medium containing 20 mM of glutamine induced early sporulation of B. thuringiensis var israelensis. An LC50 value of 3.92 micrograms/l was obtained. Calcium alginate and agar agar immobilized formulations of Bacillus thuringiensis var israelensis exhibited toxicity of 90 per cent and above to C. pipiens quinquefasciatus Say larvae, over a period of 90 days.