Production of bacterial cellulose tubes for biomedical applications: Analysis of the effect of fermentation time on selected properties.

Biosynthesis of bacterial cellulose (BC) in cylindrical oxygen permeable molds allows the production of hollow tubular structures of increasing interest for biomedical applications (artificial blood vessels, ureters, urethra, trachea, esophagus, etc.). In the current contribution a simple set-up is used to obtain BC tubes of predefined dimensions; and the effects of fermentation time on the water holding capacity, nanofibrils network architecture, specific surface area, chemical purity, thermal stability, mechanical properties, and cell adhesion, proliferation and migration of BC tubes are systematically analysed for the first time. The results reported highlight the role of culture time on key properties of the BC tubes produced, with significant differences arising from the denser and more compact fibril arrangements generated at longer fermentation intervals.

Facile synthesis of cobalt ferrite nanotubes using bacterial nanocellulose as template.

A facile method for the preparation of cobalt ferrite nanotubes by use of bacterial cellulose nanoribbons as a template is described. The proposed method relays on a simple coprecipitation operation, which is a technique extensively used for the synthesis of nanoparticles (either isolated or as aggregates) but not for the synthesis of nanotubes. The precursors employed in the synthesis are chlorides, and the procedure is carried out at low temperature (90 °C). By the method proposed a homogeneous distribution of cobalt ferrite nanotubes with an average diameter of 217 nm in the bacterial nanocellulose (BC) aerogel (3%) was obtained. The obtained nanotubes are formed by 26-102 nm cobalt ferrite clusters of cobalt ferrite nanoparticles with diameters in the 9-13 nm interval. The nanoparticles that form the nanotubes showed to have a certain crystalline disorder, which could be attributed in a greater extent to the small crystallite size, and, in a lesser extent, to microstrains existing in the crystalline lattice. The BC-templated-CoFe2O4 nanotubes exhibited magnetic behavior at room temperature. The magnetic properties showed to be influenced by a fraction of nanoparticles in superparamagnetic state.

Statistical optimization of culture conditions for biomass production of probiotic gut-borne Saccharomyces cerevisiae strain able to reduce fumonisin B1.

AIM: To evaluate the ability of probiotic Saccharomyces cerevisiae RC016 strain to reduce fumonisin B(1) (FB(1)) in vitro and to optimize the culture conditions for the growth of the yeast employing surface response methodology. METHODS AND RESULTS: Using Plackett-Burman screening designs (PBSD) and central composite designs (CCD), an optimized culture medium containing (g l(-1)) fermentable sugars provided by sugar cane molasses (CMs), yeast extract (YE) and (NH(4))(2) HPO(4) (DAP) was formulated. The S. cerevisiae RC016 strain showed the greatest binding at all assayed FB1 concentration. The CMs, YE, DAP concentrations and incubation time influenced significantly the biomass of S. cerevisiae RC016. CONCLUSION: A combination of CMs 17%; YE 4·61 g l(-1) and incubation time 60 h was optimum for maximum biomass of S. cerevisiae RC016. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of this work lies in the search for live strains with both probiotic and fumonisin B1 decontamination properties that could be sustainably produced in a medium just containing cheap carbon, nitrogen and phosphorus sources and would be included in a novel product to animal feed.

Statistical optimization of simple culture conditions to produce biomass of an ochratoxigenic mould biocontrol yeast strain.

AIM: To maximize biomass production of an ochratoxigenic mould-controlling strain of Lachancea thermotolerans employing response surface methodology (RSM). METHODS AND RESULTS: Using Plackett-Burman screening designs (PBSD) and central composite designs (CCD), an optimized culture medium containing (g l(-1) ): fermentable sugars (FS), 139·2, provided by sugar cane molasses (CMz), (NH(4) )(2) HPO(4) (DAP), 9·0, and yeast extract (YE), 2·5, was formulated. Maximal cell concentration obtained after 24 h at 28°C was 24·2 g l(-1) cell dry weight (CDW). The mathematical model obtained was validated in experiments performed in shaken-flask cultures and also in aerated bioreactors. Maximum yield and productivity values achieved were, respectively, of 0·23 g CDW/g FS in a medium containing (g l(-1) ): FS, 87·0; DAP, 7·0; YE, 1·0; and of 0·96 g CDW l(-1) h(-1) in a medium containing (g l(-1) ): FS, 150·8 plus DAP, 6·9. CONCLUSIONS: Optimized culture conditions for maximizing yeast biomass production determined in flask cultures were applicable at a larger scale. The highest yield values were attained in media containing relatively low-CMz concentrations supplemented with DAP and YE. Yeast extract would not be necessary if higher productivity is the aim. SIGNIFICANCE AND IMPACT OF THE STUDY: Cells of L. thermotolerans produced aerobically could be sustainably produced in a medium just containing cheap carbon, nitrogen and phosphorus sources. Response surface methodology allowed the fine-tuning of cultural conditions.

Modelling the freezing response of baker's yeast prestressed cells: a statistical approach.

AIMS: To study the effect of prestress conditions on the freezing and thawing (FT) response of two baker's yeast strains and the use of statistical analysis to optimize resistance to freezing. METHODS AND RESULTS: Tolerance to FT of industrial strains of Saccharomyces cerevisiae was associated to their osmosensitivity and growth phase. Pretreatments with sublethal stresses [40 degrees C, 0.5 mol l(-1) NaCl, 1.0 mol l(-1) sorbitol or 5% (v/v) ethanol] increased freeze tolerance. Temperature or hyperosmotic prestresses increased trehalose contents, nevertheless no clear correlation was found with improved FT tolerance. Plackett-Burman design and response surface methodology were applied to improve freeze tolerance of the more osmotolerant strain. Optimal prestress conditions found were: 0.779 mol l(-1) NaCl, 0.693% (v/v) ethanol and 32.15 degrees C. CONCLUSIONS: Ethanol, saline, osmotic or heat prestresses increased freezing tolerance of two phenotypically distinct baker's yeast strains. A relationship among prestresses, survival and trehalose content was not clear. It was possible to statistically find optimal combined prestress conditions to increase FT tolerance of the osmotolerant strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Statistically designed combination of prestress conditions that can be applied during the production of baker's yeast could represent a useful tool to increase baker's yeast FT resistance.

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process.

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 degrees C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1 mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Exploring the use of natural antimicrobial agents and pulsed electric fields to control spoilage bacteria during a beer production process / Exploración del uso de agentes antimicrobianos naturales y de campos eléctricos pulsantes para el control de bacterias contaminantes durante el proceso de elaboración de cerveza

Different natural antimicrobials affected viability of bacterial contaminants isolated at critical steps during a beer production process. In the presence of 1 mg/ml chitosan and 0.3 mg/ml hops, the viability of Escherichia coli in an all malt barley extract wort could be reduced to 0.7 and 0.1% respectively after 2 hour- incubation at 4 °C. The addition of 0.0002 mg/ml nisin, 0.1 mg/ml chitosan or 0.3 mg/ml hops, selectively inhibited growth of Pediococcus sp. in more than 10,000 times with respect to brewing yeast in a mixed culture. In the presence of 0.1mg ml chitosan in beer, no viable cells of the thermoresistant strain Bacillus megaterium were detected. Nisin, chitosan and hops increased microbiological stability during storage of a local commercial beer inoculated with Lactobacillus plantarum or Pediococcus sp. isolated from wort. Pulsed Electric Field (PEF) (8 kV/cm, 3 pulses) application enhanced antibacterial activity of nisin and hops but not that of chitosan. The results herein obtained suggest that the use of these antimicrobial compounds in isolation or in combination with PEF would be effective to control bacterial contamination during beer production and storage.

Diferentes antimicrobianos naturales disminuyeron la viabilidad de bacterias contaminantes aisladas en etapas críticas del proceso de producción de cerveza. En un extracto de malta, el agregado de 1 mg/ml de quitosano y de 0,3 mg ml de lúpulo permitió reducir la viabilidad de Escherichia coli a 0,7 y 0,1%, respectivamente, al cabo de 2 horas de incubación a 4 °C. El agregado de 0,0002 mg/ml de nisina, 0,1 mg/ml de quitosano o de 0,3 mg/ml de lúpulo inhibió selectivamente (10.000 veces más) el crecimiento de Pediococcus sp. respecto de la levadura de cerveza en un cultivo mixto. El agregado de 0,1 mg/ml de quitosano permitió disminuir la viabilidad de una cepa bacteriana termorresistente, Bacillus megaterium, hasta niveles no detectables. Por otra parte, el agregado de nisina, quitosano y lúpulo aumentó la estabilidad microbiológica durante el almacenamiento de cervezas inoculadas con Lactobacillus plantarum y Pediococcus sp. aislados de mosto de cerveza. La aplicación de campos eléctricos pulsantes (CEP) (3 pulsos de 8kV/cm) aumentó el efecto antimicrobiano de la nisina y del lúpulo, pero no el del quitosano. Los resultados obtenidos indicarían que el uso de antimicrobianos naturales en forma individual o en combinación con CEP puede constituir un procedimiento efectivo para el control de la contaminación bacteriana durante el proceso de elaboración y almacenamiento de la cerveza.

[Increase of rising activity of commercial yeasts by application of stress conditions during their propagation]. / Aumento de la actividad panificante de levaduras comerciales por aplicación de condiciones de estrés durante su propagación.

Rising activity determined as CO2 production of two commercial strains of Saccharomyces cerevisiae could be increased mainly in sweet bread doughs by introducing a "starvation/pulse feeding" schedule of sugar cane molasses during a fed-batch propagation. Such increase was strain dependent. Except for the trehalose intracellular level, other traits related to the yeast industrial performance were unaffected. Applicability of method for baker's yeast industrial production is discussed.

Aumento de la actividad panificante de levaduras comerciales por aplicación de condiciones de estrés durante su propagación / Increase of baking activity of commercial yeasts by application of stress conditions during their propagation

La actividad panificante valorada como producción de CO2 de dos cepas comerciales de Saccharomyces cerevisiae pudo ser incrementada, principalmente en amasijos azucarados, por la aplicación de un esquema de “hambreado/ pulso¼ de melaza de caña de azúcar durante su propagación bajo la forma de lote alimentado. Dicho incremento fue dependiente de la cepa utilizada. Otras características relacionadas con el comportamiento industrial de las levaduras no se vieron afectadas, con excepción de la concentración intracelular de trehalosa.Se discute la aplicabilidad del método para la producción industrial de levaduras de panificación.

Baking activity determined as CO2 production of two commercial strains of Saccharomyces cerevisiae could be increased mainly in sweet bread doughs by introducing a “starvation/ pulse feeding” schedule of sugar cane molasses during a fed-batch propagation . Such increase was strain dependent. Except for the trehalose intracellular level, other traits related to the yeast industrial performance were unaffected. Applicability of method for baker‘s yeast industrial production is discussed.

Aumento de la actividad panificante de levaduras comerciales por aplicación de condiciones de estrés durante su propagación. / [Increase of rising activity of commercial yeasts by application of stress conditions during their propagation]

Rising activity determined as CO2 production of two commercial strains of Saccharomyces cerevisiae could be increased mainly in sweet bread doughs by introducing a [quot ]starvation/pulse feeding[quot ] schedule of sugar cane molasses during a fed-batch propagation. Such increase was strain dependent. Except for the trehalose intracellular level, other traits related to the yeast industrial performance were unaffected. Applicability of method for bakers yeast industrial production is discussed.

[Optimization of the conditions for electroporation and the addition nisin for Pseudomonas aeruginosa inhibition]. / Optimización de las condiciones de electroporación y agregado de nisina en la inhibición de Pseudomonas aeruginosa.

A mathematical approach was applied in order to optimize the effect of electroporation by application of pulsed electric fields (PEF) and nisin addition on the inhibition of a strain of Pseudomonas aeruginosa isolated from river sediments. This strain showed to be highly resistant to nisin as only two log cycles reduction of viable cells were obtained in the presence of 84,000 IU/ml nisin. But when a combination of bacteriocin and selected PEF treatment conditions were applied, 4.4-decimal log cycle reduction could be achieved. PEF and nisin interaction seems to be complex, as at lower electric field intensities (i.e., 5 kV/cm) an increment in the number of pulses applied clearly induced a lower inhibitory effect of nisin. At higher PEF intensities (i.e., 11 kV/cm), the inhibitory effect of nisin increased with the number of pulses applied. Results overall, the obtained indicate the possibility of combining PEF and nisin treatments in order to improve the inhibition of resistant microorganisms. The Doehlert experimental design and surface response methodology was an interesting tool to obtain or predict the optimal combination of the stress factors applied.

Optimización de las condiciones de electroporación y agregado de nisina en la inhibición de Pseudomonas aeruginosa. / [Optimization of the conditions for electroporation and the addition nisin for Pseudomonas aeruginosa inhibition]

A mathematical approach was applied in order to optimize the effect of electroporation by application of pulsed electric fields (PEF) and nisin addition on the inhibition of a strain of Pseudomonas aeruginosa isolated from river sediments. This strain showed to be highly resistant to nisin as only two log cycles reduction of viable cells were obtained in the presence of 84,000 IU/ml nisin. But when a combination of bacteriocin and selected PEF treatment conditions were applied, 4.4-decimal log cycle reduction could be achieved. PEF and nisin interaction seems to be complex, as at lower electric field intensities (i.e., 5 kV/cm) an increment in the number of pulses applied clearly induced a lower inhibitory effect of nisin. At higher PEF intensities (i.e., 11 kV/cm), the inhibitory effect of nisin increased with the number of pulses applied. Results overall, the obtained indicate the possibility of combining PEF and nisin treatments in order to improve the inhibition of resistant microorganisms. The Doehlert experimental design and surface response methodology was an interesting tool to obtain or predict the optimal combination of the stress factors applied.

Optimización de las condiciones de electroporación y agregado de nisina en la inhibición de Pseudomonas aeruginosa / [Optimization of the conditions for electroporation and the addition nisin for Pseudomonas aeruginosa inhibition]

A mathematical approach was applied in order to optimize the effect of electroporation by application of pulsed electric fields (PEF) and nisin addition on the inhibition of a strain of Pseudomonas aeruginosa isolated from river sediments. This strain showed to be highly resistant to nisin as only two log cycles reduction of viable cells were obtained in the presence of 84,000 IU/ml nisin. But when a combination of bacteriocin and selected PEF treatment conditions were applied, 4.4-decimal log cycle reduction could be achieved. PEF and nisin interaction seems to be complex, as at lower electric field intensities (i.e., 5 kV/cm) an increment in the number of pulses applied clearly induced a lower inhibitory effect of nisin. At higher PEF intensities (i.e., 11 kV/cm), the inhibitory effect of nisin increased with the number of pulses applied. Results overall, the obtained indicate the possibility of combining PEF and nisin treatments in order to improve the inhibition of resistant microorganisms. The Doehlert experimental design and surface response methodology was an interesting tool to obtain or predict the optimal combination of the stress factors applied.

Inactivation of Escherichia coli by a combination of nisin, pulsed electric fields, and water activity reduction by sodium chloride.

The effect of nisin combined with pulsed electric fields (PEF) and water activity reduction by sodium chloride (NaCl) on the inactivation of E. coli in simulated milk ultrafiltrate media was studied with a Doehlert design and a response surface method. The reduction of water activity from 0.99 to 0.95 by the addition of NaCl (without any other hurdle) did not affect E. coli viability of approximately 10(8) CFU/ml. A reduction in PEF effectiveness occurred when the NaCl concentration was increased because of an increase in conductance, which reduced the pulse decay time. In cells submitted to PEF nisin activity was decreased, probably as a consequence of the nonspecific binding of nisin to cellular debris or the emergence of new binding sites in or from cells. However, the lethal effect due to nisin was reestablished and further improved when water activity was reduced to 0.95. A synergistic effect was evidenced when low-intensity PEF were applied. Decreasing water activity to 0.95 and applying PEF at 5 kV/cm (a nonlethal intensity when no other hurdle is used) with the further addition of nisin (1,200 IU/ml) resulted in a 5-log cycle reduction of the bacterial population.

Combined effect of water activity and pH on the inhibition of Escherichia coli by nisin.

The Doehlert design and surface response methodology were used to study the influence of pH and water activity (aw) on Escherichia coli inhibition by nisin. Combining stress factors at levels where they are not inhibitory by themselves, a reduction of E. coli survival fraction can be achieved with lower nisin doses than in a single nisin treatment. For all the pH values assayed, a synergistic effect of aw and nisin concentration was detected, and the isoresponse lines showed the existence of an area of maximum inhibition. Factors that reduced viable cell counts by 4 to 5 log cycles were 1,000 to 1,400 IU of nisin per ml at pH 5.5 to 6.5 and a water activity of 0.97 and 0.98. The addition of different ionic and nonionic solutes to control aw suggested that the effect of aw in the inhibitory action of nisin on E. coli cells was not solute-specific. The use of the Doehlert experimental design was effective to determine the optimal combination of stress factors, as well as to point out the most important variables that affected E. coli inhibition.

Commercial baker's yeast stability as affected by intracellular content of trehalose, dehydration procedure and the physical properties of external matrices.

The effects of vacuum-drying and freeze-drying on the cell viability of a commercial baker's yeast, Saccharomyces cerevisiae, strain with different endogenous contents of trehalose were analyzed. An osmotolerant Zygosaccharomyces rouxii strain was used for comparative purposes. Higher viability values were observed in cells after vacuum-drying than after freeze-drying. Internal concentrations of trehalose in the range 10-20% protected cells in both dehydration processes. Endogenous trehalose concentrations did not affect the water sorption isotherm nor the Tg values. The effect of external matrices of trehalose and maltodextrin was also studied. The addition of external trehalose improved the survival of S. cerevisiae cells containing 5% internal trehalose during dehydration. Maltodextrin (1.8 kDa) failed to protect vacuum-dried samples at 40 degrees C. The major reduction in the viability during the freeze-drying process of the sensitive yeast cells studied was attributed to the freezing step. The suggested protective mechanisms for each particular system are vitrification and the specific interactions of trehalose with membranes and/or proteins. The failure of maltodextrins to protect cells was attributed to the fact that none of the suggested mechanisms of protection could operate in these systems.

[Inhibition of Bacillus coagulans growth in laboratory media and in fruit purees]. / Inhibición del desarrollo de Bacillus coagulans en medios de laboratorio y en puré de frutas.

The growth of two strains of B. coagulans was inhibited in laboratory media at pH < or = 4.5, and at water activity (aw) levels of 0.96 for B. coagulans NRS 609 and 0.95 for B. coagulans ATCC 803. The growth of both strains was also inhibited in apple and strawberry purees (pH = 3.5) stored at 37 degrees C for over two months. B. coagulans was able to grow in banana puree (pH approximately equal to 5.0) but acidification of the puree at pH = 3.5 was enough to prevent growth. The addition of up to 3,000 ppm vainillin ("natural" preservative) or 1,000 ppm potassium sorbate (traditional preservative) at pH higher than the inhibitory level previously determined could not prevent growth of B. coagulans in laboratory or in fruits, but 100 ppm lysozyme retarded growth in laboratory media at different pH levels (from 4.5 to 6.7) and in banana puree. As lysozyme showed to be effective at pH < or = 6.7, it might be used to prevent growth of B. coagulans at an eventual increment of pH during storage.

Combined effect of nisin and pulsed electric fields on the inactivation of Escherichia coli.

The Doehlert design was applied in order to investigate the combined effect of nisin and high voltage pulsed electric fields (PEF) on the inactivation of Escherichia coli in simulated milk ultrafiltrate media. Nisin alone was totally inactivated by PEF, but in the presence of bacterial cells a protective effect was observed. However, the effectiveness of nisin was still decreased when bacterial cells were subjected to the combined treatment. In spite of this phenomenon, an almost additive response emerged as a consequence of the combined treatment. A 4-log cycle reduction may be accomplished with around 1,000 IU/ml (7.15 microM) of nisin and three pulses of 11.25 kV/cm or 500 IU/ml for five pulses of the same intensity. The observed efficacy arising from the combination of both treatments suggests the possibility of using PEF for improving the action spectrum of natural antimicrobials.

Inhibición del desarrollo de Bacillus coagulans en medios de laboratorio y en puré de frutas / Inhibition of Bacillus coagulans growth in laboratory media and in fruit purees

The growth of two strains of B. coagulans was inhibited in laboratory media at pH < or = 4.5, and at water activity (aw) levels of 0.96 for B. coagulans NRS 609 and 0.95 for B. coagulans ATCC 803. The growth of both strains was also inhibited in apple and strawberry purees (pH = 3.5) stored at 37 degrees C for over two months. B. coagulans was able to grow in banana puree (pH approximately equal to 5.0) but acidification of the puree at pH = 3.5 was enough to prevent growth. The addition of up to 3,000 ppm vainillin ("natural" preservative) or 1,000 ppm potassium sorbate (traditional preservative) at pH higher than the inhibitory level previously determined could not prevent growth of B. coagulans in laboratory or in fruits, but 100 ppm lysozyme retarded growth in laboratory media at different pH levels (from 4.5 to 6.7) and in banana puree. As lysozyme showed to be effective at pH < or = 6.7, it might be used to prevent growth of B. coagulans at an eventual increment of pH during storage.(AU)

Inhibición del desarrollo de Bacillus coagulans en medios de laboratorio y en puré de frutas / Inhibition of Bacillus coagulans growth in laboratory media and in fruit purees

The growth of two strains of B. coagulans was inhibited in laboratory media at pH < or = 4.5, and at water activity (aw) levels of 0.96 for B. coagulans NRS 609 and 0.95 for B. coagulans ATCC 803. The growth of both strains was also inhibited in apple and strawberry purees (pH = 3.5) stored at 37 degrees C for over two months. B. coagulans was able to grow in banana puree (pH approximately equal to 5.0) but acidification of the puree at pH = 3.5 was enough to prevent growth. The addition of up to 3,000 ppm vainillin ("natural" preservative) or 1,000 ppm potassium sorbate (traditional preservative) at pH higher than the inhibitory level previously determined could not prevent growth of B. coagulans in laboratory or in fruits, but 100 ppm lysozyme retarded growth in laboratory media at different pH levels (from 4.5 to 6.7) and in banana puree. As lysozyme showed to be effective at pH < or = 6.7, it might be used to prevent growth of B. coagulans at an eventual increment of pH during storage.