Carnocin KZ213 produced by<Emphasis Type="Italic"> Carnobacterium piscicola</Emphasis> 213 is adsorbed onto cells during growth. Its biosynthesis is regulated by temperature,pH and medium composition

Carnocin KZ213 is an antilisterial bacteriocin produced by Carnobacterium piscicola 213. The effects of pH and temperature were studied during batch fermentation in MRS* medium (modified MRS without ammonium citrate or sodium acetate). The optimal pH for growth is between 6 and 7. The maximum bacteriocin productivity in the supernatant occurs at pH 7. Operating at controlled pH increases the volumetric activity of the free bacteriocin by 8- to 16-fold, compared with uncontrolled pH. No bacteriocin production is observed below pH 6.5. Temperature has a dramatic effect on carnocin KZ213 production. Growth is optimal at 25 °C and 30 °C, although no bacteriocin production is detected at 30 °C. Also, bacteriocin production is observed at 25 °C in MRS*, but not in complex APT broth, where growth is optimal. The presence of glucose as a carbon and/or energy source is important for carnocin KZ213 synthesis. Hence, bacteriocin synthesis is regulated by temperature, carbon source and medium composition. Quantification studies of bacteriocin adsorbed onto producer cells show that the majority of the carnocin KZ213 secreted is adsorbed onto the producer cells during growth. Only 15% of the total bacteriocin produced is detected in the cell-free supernatant at the end of growth.     

Evaluation of environmental conditions for production of bacteriocin-like substance by <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain P34

The influence of temperature, pH and media on bacteriocin production by Bacillus sp. P34 was investigated. The effect of temperature and initial pH was evaluated by factorial design and response surface methodology (RSM). Statistical analysis of results showed that, in the range studied, the two variables have a significant effect on bacteriocin production. Response-surface data showed maximum antimicrobial activity production at initial pH values between 6.0 and 8.0 and temperatures between 25 and 37 °C. No relationship between bacterial growth and bacteriocin production was observed. RSM proved to be a powerful tool in optimizing the production of antimicrobial activity by Bacillus sp. P34. When different media were tested, maximum bacteriocin production was observed in soybean protein-based medium, but antimicrobial activity was not achieved by cultivation on fish meal, feather meal, whey and grape waste.     

Temperature significantly affects retroviral vector production and deactivation rates, and thereby determines retroviral titers

The retroviral titer obtained from the pMFG/ψCRIP producer cell line is determined by a dynamic interplay of vector production and deactivation rates. Both these rates are influenced by temperature. It was determined that; (i) the retroviral half-lives are strongly influenced by temperature and the temperature dependency can be described by the Arrhenius equation with an activation energy of 39 kcal/gmol; (ii) the actual retroviral vector productivity per cell is highest at 37?°C with retroviral production rate of 24.4(±7.0; ±?standard deviation) colony forming unit (CFU)/cell/day; (iii) the dynamic interplay of these two factors produces an optimal temperature of 34?°C for pMFG/ψCRIP cells under the culture conditions used; and (iv) the cellular growth rate is highest at 37?°C at 26.8 hr doubling time. Taken together, these parameters can be used to optimize a two-step retroviral production protocol, where the cells are first grown under optimal growth conditions (37?°C) and second, the virus is produced at 34?°C to yield the highest titer. These results have significant implications for optimal retroviral production protocols.     

Influence of pH,temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

AIMS: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. METHODS AND RESULTS: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. CONCLUSIONS: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.     

Effects of culture temperature shift and light-dark time cycle on the cellular sugar accumulation of Chlorella pyrenoidosa

We investigated the effect of culture temperature on the maximum specific growth rate and the cellular sugar accumulation, and the effect of a temperature shift on the sugar accumulation of Chlorella pyrenoidosa cells in a batch culture system. Increase in temperature below 30?°C appeared to correlate with increase in the maximum specific growth rate, on the contrary the cellular sugar content showed a reverse tendency against temperature. We attempted to utilize this tendency for improving sugar productivity in Chlorella. First, we cultured Chlorella at 28?°C during the logarithmic growth phase to obtain a high specific growth rate. The culture temperature was then shifted from 28?°C to 22?°C at the late logarithmic growth phase in order to reduce the specific growth rate and enhance the cellular sugar accumulation. As a result, we obtained a 15% increase in sugar production over that obtained by cultivation at 28?°C throughout the culture. We also investigated the effect of light-dark time cycle on the sugar productivity and found that this operating variable did not affect the cellular sugar content but influenced the final cell concentration. Among the examined light-dark time cycles, maximum sugar productivity was obtained in the case of 12?h light and 12?h dark period.     

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.     

Effect of physical factors on the production of bacteriocin from Pediococcus acidilactici ITV 26

The effect of pH, temperature and agitation on growth and bacteriocin production by Pediococcus acidilactici ITV 126 was investigated. Experiments were made in flasks containing MRS medium at 30 to 40°C, pH 5 to 7 and agitation 0 to 200 rpm. Factor levels were arranged in a 2³ factorial design with central and axial points. Anova and Tukey paired comparison tests showed that a temperature of 35°C favored bacteriocin production, whereas 40°C was best for cell growth. A statistical interaction of temperature and agitation was observed affecting microbial growth. pH 5 favored both cell growth and bacteriocin production. Journal of Industrial Microbiology & Biotechnology (2001) 26, 191–195. Received 30 October 1999/ Accepted in revised form 31 January 2001     

Influence of complex nutrients, temperature and pH on bacteriocin production by Lactobacillus sakei CCUG 42687

The effects of process conditions and growth kinetics on the production of the bacteriocin sakacin P by Lactobacillus sakei CCUG 42687 have been studied in pH-controlled fermentations. The fermentations could be divided into phases based on the growth kinetics, phase one being a short period of exponential growth, and three subsequent ones being phases of with decreasing specific growth rate. Sakacin P production was maximal at 20 °C. At higher temperatures (25–30 °C) the production ceased at lower cell masses, when less glucose was consumed, resulting in much lower sakacin P concentrations. With similar media and pH, the maximum sakacin P concentration at 20 °C was seven times higher than that at 30 °C. The growth rate increased with increasing concentrations of yeast extract, and the maximum concentration and specific production rate of sakacin P increased concomitantly. Increasing tryptone concentrations also had a positive influence upon sakacin P production, though the effect was significantly lower than that of yeast extract. The maximum sakacin P concentration obtained in this study was 20.5 mg l⁻¹. On the basis of the growth and production kinetics, possible metabolic regulation of bacteriocin synthesis is discussed, e.g. the effects of availability of essential amino acids, other nutrients, and energy. Received: 7 June 1999 / Received revision: 15 September 1999 / Accepted: 17 September 1999     

Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi

The influence of growth parameters on the fermentative production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi was studied. The bacteriocin production was greatly affected by carbon and nitrogen sources. Strain A164 produced at least 4-fold greater bacteriocin in M17 broth supplemented with lactose than other carbon sources. The amount of 3% yeast extract was found to be the optimal organic nitrogen source. While the maximum biomass was obtained at 37 degrees C, the optimal temperature for the bacteriocin production was 30 degrees C. The bacteriocin production was also affected by pH of the culture broth. The optimal pH for growth and bacteriocin production was 6.0. Although the cell growth at pH 6.0 was nearly the same level at pH 5.5 and 6.5, the greater bacteriocin activity was observed at pH 6.0. Exponential growth took place only during an initial period of the cultivation, and then linear growth was observed. Linear growth rates increased from 0.160 g(DCW) x l(-1) x h(-1) to 0.245 g(DCW) x l(-1) x h(-1) with increases in lactose concentrations from 0.5 to 3.0%. Maximum biomass was also increased from 1.88 g(DCW) x l(-1) to 4.29 g(DCW) x l(-1). However, increase in lactose concentration did not prolong the active growth phase. After 20 h cultivation, cell growth stopped regardless of lactose concentration. Production of the bacteriocin showed primary metabolic kinetics. However, bacteriocin yield based on cell mass increased greatly during the late growth phase. A maximum activity of 131x10(3) AU x ml(-1) was obtained at early stationary growth phase (20 h) during the batch fermentation in M17L broth (3.0% lactose) at 30 degrees C and pH 6.0.     

Industrial Whey Utilization as a Medium Supplement for Biphasic Growth and Bacteriocin Production by Probiotic Lactobacillus casei LA-1

The ability of probiotic Lactobacillus casei LA-1 for bacteriocin production using industrial by-products, such as whey, as supplement in growth medium has been demonstrated for the first time. Whey was investigated as a sole carbon source in cooperation with other components to substitute expensive nutrients as MRS for economical bacteriocin production. Industrial whey-supplemented MRS medium was then selected as to determine the effect of four variables (temperature, initial pH, incubation time, and whey concentration) by response surface methodology on bacteriocin production. Statistical analysis of results showed that two variables have a significant effect on bacteriocin production. Response surface data showed maximum bacteriocin production of 6,132.33?AU/mL at an initial pH of 7.12, temperature 34.29?°C, and whey concentration 13.74?g/L. The production of bacteriocin started during the exponential growth phase, reaching maximum values at stationary phase, and a biphasic growth and production pattern was observed. Our current work demonstrates that this approach of utilization of whey as substitution in costly medium as MRS has great promise for cost reduction in industry for the production of novel biological metabolic product that can be utilized as a food preservative.     

Studies on bacteriocin (thermophilin T) production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ACA-DC 0040 in batch and fed-batch fermentation modes

Growth conditions that support bacteriocin (thermophilin T) production by Streptococcus thermophilus ACA-DC 0040 were identified. Synthesis of thermophilin T occurred during primary metabolic growth, while its specific rate of synthesis seemed to be optimal at T = 30°C. Thermophilin T activity rapidly decreased in the stationary phase, especially at high growth temperature (i.e. T = 42°C). In media with high content of complex nitrogen sources, high amounts of bacteriocin were detected in the growth environment, while about an 8-fold increase of thermophilin T titer and a 2-fold increase of specific synthesis rate was achieved when a fed-batch fermentation mode was applied.     

Production of L-malate from fumarate by the yeast Dipodascus magnusii

An alternative microbiological method for the production of malate from fumarate is presented. The yeast Dipodascus magnusii was used for this bioconversion. The optimum cell growth temperature was 28°C and the working volume 120 ml. The highest level of fumarase activity during bioconversion was achieved at a pH of 7.5 and a temperature of 37°C. These conditions were determined as optimal. Using sodium fumarate (1M), the maximum specific productivity of malic acid obtained was 1.72 g/(g_DCW × h) for intact cells. In the case of ammonium fumarate, it was 2.25 g/(g_DCW × h).     

Effect of culture pH on recombinant antibody production by a new human cell line, F2N78, grown in suspension at 33.0 °C and 37.0 °C

The human host cell line, F2N78, is a new somatic hybrid cell line designed for therapeutic antibody production. To verify its potential as a human host cell line, recombinant F2N78 cells that produce antibody against rabies virus (rF2N78) were cultivated at different culture pH (6.8, 7.0, 7.2, 7.4, and 7.6) and temperatures (33.0 °C and 37.0 °C). Regardless of the culture temperature, the highest specific growth rate was obtained at a pH of 7.0–7.4. Lowering the culture temperature from 37.0 °C to 33.0 °C suppressed cell growth while allowing maintenance of high cell viability for a longer period. However, it did not enhance antibody production because specific antibody productivity did not increase at 33.0 °C. The highest maximum antibody concentration was obtained at 37.0 °C and pH 6.8. The N-linked glycosylation of the antibody was affected by the culture pH rather than the temperature. Nevertheless, G1F was dominant and G2F occupied a larger portion than G0F in all culture conditions. Compared to the same antibody produced from recombinant CHO cells, the antibody produced from rF2N78 cells has more galactose capping and was more similar to human plasma IgG. Taken together, the results obtained here demonstrate the potential of F2N78 as an alternative human host cell line for therapeutic antibody production.     

Characterisation of an Antilisterial Bacteriocin Produced by Lactobacillus sakei CWBI-B1365 Isolated from Raw Poultry Meat and Determination of Factors Controlling its Production

Amongst 101 lactic acid bacteria isolated from meat and fish samples, strain CWBI-B1365, identified as Lactobacillus sakei, was found to produce the subclass IIa bacteriocin sakacin G. Partial sequencing of the gene involved in the biosynthetic pathways revealed an unusual gene organisation in that the accessory gene associated with bacteriocin transport did not occur immediately downstream of the gene encoding an ABC transporter, but upstream of the putative immunity gene and encoded on the opposite DNA strand. Sakacin G production was strongly regulated by pH, temperature and the carbon sources used in the growth medium, as well as the concentration of carbon and nitrogen sources. The condition of pH 5.5 and the temperature of 25°C appeared to be optimal for bacteriocin production. The use of sucrose during culturing and the fed batch addition of sucrose and meat extract greatly enhanced bacteriocin production.     

Statistical optimization of physical parameters for enhanced bacteriocin production by L. casei

Antimicrobial proteinaceous compounds such as bacteriocins produced from Lactobacillus sp. are widely known. They have potential antimicrobial activities towards closely related bacteria and several pathogens associated with food spoilage and hence can be a potential food bio-preservative agent. Bacteriocin production requires optimized process, complex media and well-controlled physical conditions including pH and temperature. A probiotic strain of L. casei LA-1 isolated from mango pickle was used in the present study. The influence of physical parameters viz. temperature (15 ?? 45°C), pH (4.0 ?? 7.0), incubation time (up to 48 h) and inoculum size (0.7 ?? 2.0 O.D) on bacteriocin production was analyzed. The effect of all the parameters was first investigated using the one-factor-at-a-time method (OFAT) to see the significance of these parameters on bacteriocin production and then further optimized by response surface methodology (RSM). Following OFAT analysis, all factors were found to have a significant effect on bacteriocin production. Bacteriocin production of 2,844 AU/mL was obtained at temperature 37°C, pH 6.7 and inoculum size 1.8 O.D at an incubation time of 20 h and it was produced during the stationary phase of growth. Statistical analysis showed that three variables-pH, temperature and incubation time have significant effects on bacteriocin production. RSM proved to be a powerful tool in the optimization of bacteriocin production by L. casei LA-1 with a two-fold increase, giving a production of 4652.15 AU/mL at pH 7.19, temperature 33.3°C and incubation time of 22.2 h.     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K

Sakacin K is an antilisterial bacteriocin produced by Lactobacillus sake CTC 494, a strain isolated from Spanish dry fermented sausages. The biokinetics of cell growth and bacteriocin production of L. sake CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth. The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior. The model was validated successfully for all components. However, the specific bacteriocin production rate seemed to have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity. Furthermore, cells grew well at 35 degrees C but no bacteriocin production could be detected at this temperature. L. sake CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K.     

Optimization of laboratory cultivation conditions for the synthesis of antifungal metabolites by bacillus subtilis strains

In order to achieve the optimal number of colony forming units and a high level of antifungal metabolites synthesis, we carried out the periodic cultivation of the Bacillus subtilis BZR 336 g and Bacillus subtilis BZR 517 strains at various pH and temperature levels. In the experiment for determining the optimal temperature, the maximum titer of B. subtilis BZR 336 g bacterium (1.6–1.7 × 10⁹ CFU/ml) was recorded at a cultivation temperature of 20–25 °C. For B. subtilis BZR 517 strain, the temperature turned out to be optimal at 30 °C: the titer was 8.9 × 10⁸ CFU/ml. The maximum antifungal activity of B. subtilis BZR 336 g strain against the test culture of Fusarium oxysporum var. orthoceras was observed at a cultivation temperature of 20–25 °C; for B. subtilis BZR 517 strain, 25–30 °C. When determining the optimal pH level, it was found that a high titer of B. subtilis BZR 336 g strain cells was determined at pH 8.0 (2.7 × 10⁹ CFU/ml), for B. subtilis BZR 517 strain it was at pH 6.0–8.0 (1.0 × 10⁹ CFU/ml). The maximum antifungal activity was noted with the same indicators. Chromatographic and bioautographic analyses suggest that the synthesized antifungal metabolites belong to surfactin and iturin A. The data obtained in this research can be used in the development of the technology for the production of effective biofungicides to protect crops against Fusarium pathogens.     

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.