Guimarães, R.M.1, Alves, M.C.1*, Fusconi, R.2 Machado, A.E.H.2 1
Nanobrax
,
Soluções Tecnológicas e Prestação de Serviço, Ltda. R. Cruzeiro do Peixotos, 499 sala 1104 CEP 38400-608, Uberlândia (MG)2
Laboratório de Fotoquímica, Instituto de Química Universidade Federal de Uberlândia, CX 593, CEP38400-902 Uberlândia, Minas Gerais, Brasil
*e-mail: [email protected] Keywords: Bacterial exopolysaccharides, emulsification, hydrocarbons
INTRODUCTION: Exopolysaccharides (EPS) are produced by several bacteria and have important structural role in biofilms, the natural habitat of many microbial communities, both in natural or artificial environments where a solid subtract remains exposed to moisture. Moreover, EPS are of great industrial interest. They possess a wide variety of properties that may not be found in traditional polymers of plant origin (Guezennec, 2002). The variety of chemical structures found in EPS lead to a broad spectrum of physical properties, allowing a great diversity of applications, such as emulsifying activity. The preset work aimed at the isolation, selection of EPS-producing bacteria from Sagittaria rombifolia Cham as well as the evaluation of its emulsifying properties on different hydrocarbons.
MATERIAL AND METHODS: EPS-producing bacteria were isolated from leaves of Sagittaria rombifolia Cham (of a palm swamp stream in Uberlândia, MG) on sugarcane 4% molasses agar
and selected based on the mucoid mode of colonies and on Alcian Blue staining (Fusconi & Godinho, 2002). For emulsification assays (Iqbal et al., 1995), selected strains were cultured in sugarcane molasses medium for 48 hours at 30°C and 150 rpm. Emulsification assays were carried out using cell free 4% sugarcane molasses media with benzene, toluene and o-xylene. Triton X-100 was used as chemical surfactant and a control was prepared using the same method but replacing the sample by non-inoculated media. Results were tested by analysis of variance (Anova), followed by the Tukey test at a 0.05 level of significance.
RESULTS AND DISCUSSION: A total of 40 bacterial strains were isolated. F1CC and F1CA exhibited a high mucoid aspect and positive EPS production tested with Alcian Blue staining. Both strains were selected as EPS-producing bacteria for emulsification activity evaluation. The emulsifying index for Triton X-100 in benzene and toluene was higher than the one obtained in sugarcane molasses F1CC and F1CA culture supernatant (F= 30.226, p=0,001; F=35.762; p<0,001 respectively) suggesting a low activity of the bioemulsifiers on those hydrocarbons. However, in the culture supernatant of F1CC and F1CA with o-xylene, the emulsifying activity was similar than Triton X-100 (F= 3.523; p=0,097).
CONCLUSION: F1CC and F1CA were selected as EPS-producing bacteria. EPS produced by the strains showed significant emulsifying activity with o-xylene, suggesting their potential application in bioremediation studies in contaminated sites with this hydrocarbon.
REFERENCES:
FUSCONI, R. & GODINHO, M.J.L. Screening for exopolysaccharide-producing bacteria from sub-tropical polluted groundwater. Braz. j. biol., 62:363-369, 2002.
GUEZENNEC, J. Deep sea hydrothermal vents: a new source of innovative bacterial exopolysaccharides of biotechnological interest? J. ind. microbiol. biotechnol., 19: p.204-208, 2002.
IQBAL, S., KHALID, Z.M. & MALIK, K.A. Enhanced biodegradation and emulsification of crude oil and hyperproduction of biosurfactants by a gamma ray-induced mutant of Pseudomonas
aeruguinosa. Lett. Appl. Microbiol., 21:176-179, 1995. Financial Support: CNPq, Nanobrax e FAPEMIG
KINETICS OF GROWTH ASSOCIATED TO THE PRODUCTION OF TOXINS BY Bacillus
thuringiensis subsp. Israelensis
Angelo, E. A1*.; Vilas-Bôas, G. F. L. T2.; Castro-Gómez, R. J. H.1 1
Department of Food Science and Technology, State University of Londrina; 2Department of General Biology, State University of Londrina .
Keywords: Bacillus thuringiensis, biological control, Aedes aegypti, bioinsecticide.
INTRODUCTION: Bacillus thuringiensis is a Gram-positive bacteria spore, which produces protein crystals with insecticidal activity. Despite the widespread use of B. thuringiensis in biological control, there are few published studies on its production, since many details are trade secrets. Therefore, the objective of this work was to study the kinetics of growth of B. thuringiensis israelensis involving the toxicity of culture, in an alternative medium based on meal chrysalis of Bombyx mori and also determine the time of cultivation of fermentation for the production of toxins.
MATERIAL AND METHODS: The microorganism used in this study was B. thuringiensis subsp. israelensis HD537, which was grown in a medium of cultivation-based on meal chrysalis of B. mori,
glucose, ammonium sulphate, glucose and salts, at 30 ° C, 120 rpm for a period of 96 hours. The growth was evaluated through tests of gravity, the cell morphology by light microscopy and toxicity by bioassay against
Aedes aegypti according to the protocol of the World Health Organization (WHO, 2005).
RESULTS AND DISCUSSION: The phase lag of the growth curve had the lowest toxicity and corresponded to about 9,4% of the time of cultivation. The exponential phase, which presented the highest growth rate, was about 76% of the time of cultivation, in which at the end of the biomass was 15.13 mg / mL. The toxicity increased gradually during cultivation, being maximum during the stationary and declining phases, when it corresponded to a lethal concentration for 50% of the larvae (LC50) of 0.50 ppm (v / v). In general, sporulation occurs in the stationary phase, when the bacteria has consumed most of the sources of nutrients and it becomes a factor limiting to their growth. Furthermore, during the fermentation, the microorganism may produce metabolites, which with accumulation, ultimately inhibit its growth. Most of the toxins of B. thuringiensis synthesized during sporulation is therefore different from other species, the stationary phase is very important in the fermentation of this bacteria. (Bravo et al., 2007; SCHNEPF et al, 1998). The lower values of LC50, ie the higher toxicity, was observed after 96 hours of cultivation. However, when analyzing it slides, the occurrence of germination of some spores can be noticed at 96 hours. The major toxicity of the period of 96 hours, probably occurred because the vast majority of cells of the growth phase had already released their crystals, while in 72 hours some cells, although little, were seen at the end of the sporulation.
CONCLUSION: The major toxicities of culture of B. thuringiensis israelensis HD537 occured in the final stages of cultivation, being up to 96 hours, time considered ideal for fermentation under testing conditions. Alternatively, the culture can be kept for 72 hours and submitted to a rest period of 24 hours so that the cells can release their toxins.
REFERENCES:
BRAVO, A. et al. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon, v.49, p. 423-435, 2007.
SCHNEPF, E. et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Review, v. 62, p. 775-806, 1998.
ALTERTHUM, F.; CARVALHAL, M. L. Crescimento bacteriano, In: TRABULSI, L R. et al. (ed.) Microbiologia, São Paulo: Atheneu, 3.ed, 1999. 586p.
WORLD HEALTH ORGANIZATION (WHO). Guidelines for laboratory and field testing of mosquito larvicides. WHO/CDS/WHOPES/GCDPP/2005.13, 2005, 39p.
LACTIC ACID BACTERIA AND BACTERIOCINS IN WINE BIOTECHNOLOGY
Ruiz-Larrea, F.*1,2, Díez, L.1,2, Portugal, C.B. 1,2, Fernández, R. 1,2, Rojo- Bezares, B.3, Sáenz, Y. 3, Oliva, T. 1,2, Tenorio, C. 1, Zarazaga, M. 2 and Torres, C.2,3 1
Instituto de Ciencias de la Vid y del Vino (ICVV), 2Universidad de La Rioja, Av. Madre de Dios 51, 26006 Logroño, Spain. 3
4. Lonvaud-Funel, A. Lactic acid bacteria in the quality improvement and depreciation of wine. Antonie
Van Leeuwenhoek 76: 317-331. 1999.
5. Mills, D.A., Rawsthorne, H., Parker, C., Tamir, D. & Makarova, K.. Genomic analysis of Oenococcus
oeni PSU-1 and its relevance to winemaking. FEMS Microbiology Reviews 29(3): 465-475. 2005.
6. De Vuyst, L. & Leroy, F. Bacteriocins from lactic acid bacteria: production, purification, and food applications. Journal of Molecular Microbiology and Biotechnology 13(4): 194-199. 2007.
7. Cotter, P. D., C. Hill, & R. P. Ross. Bacteriocins: developing innate immunity for food. Nature Reviews
Microbiology 3: 777-788. 2005.
8. Papagianni, M. & Anastasiadou, S. Pediocins: the bacteriocins of Pediococci. Sources, production, Centro de Investigación Biomédica de La Rioja (CIBIR), Unidad de Microbiología Molecular, c/ Piqueras 98, 26006 Logroño, Spain.
*e.mail: [email protected]
Keywords: microbiological control, wine, bacteriocin, Oenococcus oeni, lactic acid bacteria
Abstract
Lactic acid bacteria (LAB) are responsible for the secondary fermentation of wine, and this fermentation is a requirement for quality red wines that are going to be submitted to the process of ageing in oak barrels. This secondary fermentation is named malolactic fermentation (MLF) and essentially it involves the degradation of L-malic acid into L-lactic acid and carbon dioxide, the consequence of which is a reduction in total acidity (deacidification) of the wine [1]. This biological deacidification is always accompanied by the metabolism of other molecules, which takes place on a modest scale and has as a result the increase of aroma components such as acetaldehyde, acetic acid, acetoin, diacetyl, etc. [2, 3, 4]. As a whole, wine gains in mellowness, roundness and fullness, and becomes more pleasant to the palate. The capacity of LAB to grow in wine is largely determined by wine pH, alcohol concentration, temperature, and sulphur dioxide concentration. Oenococcus oeni is the LAB species considered preferable for achieving MLF in wine because of its tolerance to low pH and high ethanol levels [5], and thus, this LAB is currently used as starter culture for MLF in wine.
Bacteriocins are peptides with antimicrobial activity that are naturally produced by some bacteria to inhibit the growth of other competing microorganisms. Currently, bacteriocins produced by LAB arouse most interest because LAB possess the status of QPS (qualified presumption of safety), i.e. they are regarded as safe microorganisms for human consumption because they and their metabolites have been consumed in fermented foods for countless generations without adverse effects in the population. Bacteriocins produced by LAB have found important applications as natural preservatives in food industry because they offer the possibility of preventing the development of food spoilage bacteria [6]. In the future this application could be useful as well in preservation of wine and it offers the additional advantage of allowing a decrease of the sulphurous anhydride levels that are currently used in wine preservation. Two of the most widely used bacteriocins in food industry are nisin [7] and pediocin [8]. A number of results will be presented showing the effect of nisin and pediocin, alone and in combination with sulphurous anhydride, to preserve wine during the ageing and storage process. Currently a number of studies are being carried out searching for bacteriocin active fermentates and wine indigenous lactic acid bacteria showing bacteriocin activity. References:
1. Boulton, R.B., Singleton, V.L., Bisson, L.F., & Kunkee, R.E. Malolactic fermentation. In: Principles and practices of winemaking, The Chapman and Hall Enology Library, New York, pp 244-728. 1996. 2. Bartowsky, E.J. & Henschke, P.A. The “buttery” attribute of wine-diacetyl-desirability, spoilage and
beyond. International Journal of Food Microbiology 96 (3): 235-252. 2004.
3. Liu, S.Q. Malolactic fermentation in wine beyond deacidification. Journal of Applied Microbiology 92: 589-601. 2002.