Primary Metabolite Kinetics Research Articles

Isolation and properties of antimicrobial substance produced by Sinhorhizobium melilloti from root nodules of Medicago sativa

The symbiotic association of alfalfa (Medigaco sativa) with Sinorhizobium melilloti contributes to the production of inoculum to improve plant growth and increase yield. The competitiveness of the introduced strains is a very important factor in the performance of an inoculum which is based on the use of rhizobial strains. This competitiveness is due to the production of anti-microbial substances, among them, bacteriocines. In this study, the totals of 30 rhizobial strains were isolated from M. sativa nodules sampled from sa-line soils of Algeria. Twenty strains were nodulated the host plant M. sativa. The antimicrobial activity producing strains SW16 and SW15 were identified as Sinorhizobium melliloti based on 16S rRNA sequance analysis. Partial puri-fication of this inhibitorys substances by ammonium sulfate were precipitat-ed with 75% saturation. These antimicrobial activitys showed the maximum activity against the strain SW10 estimated at 1800 with the strain SW16 and 2500 AU/ml for the strain SW15. The physicochemical properties of these antimicrobial activitys reveals thier thermoresistant nature and sensitive to protease, but theire were resistant to a wide range of pH and also at differ-ent concentrations of Nacl. This inhibitory agent display primary metabolite kinetics as its maximal production was detected in the growth phase. Antimi-crobial substances produced by sinorhizobium mielilloti SW15 and SW16 exhibited inhibitory activity against the phytopathogen (Aspergillus sp., Fusarium sp., Cladosporium sp. and Alternaria altterna).

Bacteriocinogenic properties and safety evaluation of Enterococcus faecium YT52 isolated from boza, a traditional cereal based fermented beverage

The objectives of this study were to characterize the antibacterial substances produced by Enterococcus faecium YT52 isolated from boza, and to evaluate the safety of this strain. E. faecium YT52 inhibits various Gram-positive bacteria, including foodborne pathogens such as Listeria monocytogenes and Bacillus cereus. The manner of action of its antibacterial activity, using proteolytic enzymes, indicate that it produces a bacteriocin. The bacteriocin producing isolate E. faecium YT52 was identified using 16S rRNA gene homology and species-specific polymerase chain reaction (PCR) analysis. The bacteriocin was found to be heat stable and active under both acid and alkaline conditions. The bacteriocin showed primary metabolite kinetics and a bactericidal mode of action against L. monocytogenes. Although enterocin A, B and X genes were detected in E. faecium YT52 by PCR, only one active peptide band (~ 5.5 kDa) with a molecular weight similar to enterocin B was identified by tricine–SDS-PAGE analysis. A safety evaluation of E. faecium YT52 indicated that it is nonhemolytic, gelatinase-negative, and susceptible to clinically relevant antibiotics such as ampicillin, tetracycline and vancomycin. It contains a small number of virulence factors (efaAfm, silent gelE) and antibiotic resistance genes (ermB, tetM, tetL). In conclusion, bacteriocinogenic E. faecium YT52 was found to have a low risk profile, and it may be potentially valuable as a protective culture in the food industry.

Isolation and preliminary characterisation of bacteriocin produced by Enterococcus faecium GHB21 isolated from Algerian paste of dates “ghars”

The bacteriocin-producing strain GHB21 isolated from the Algerian traditional paste of dates “ghars” was identified as Enterococcus faecium based on 16S rRNA sequence analysis. The bacteriocin produced by Enterococcus faecium GHB21 was sensitive to proteases, heat-stable, resistant to a wide range of pH and also to several chemicals. Recorded bacteriocin activity was higher when GHB21 was fermented in a bioreactor compared with static fermentation in a flask; it was also shown to be growth-associated and to display primary metabolite kinetics as its maximal production was detected in the early logarithmic growth phase. GHB21 presented a large activity spectrum towards different genera, and proved active against either Gram positive or Gram negative bacteria, including some pathogenic and/or spoilage bacteria. Partial purification of this bacteriocin by an adsorption-desorption method led to a fraction that lost its entire activity after 1 week of storage at 4 °C, or after 15 days when supplemented with bovine serum albumin; nevertheless the lyophilised fraction could remain stable with full activity even after 1 year. The partially purified bacteriocin exhibited a bactericidal effect on Listeria ivanovii subsp. ivanovii ATCC 19119 and could lead to its total inhibition after only 6 h but without concomitant cell lysis. Tricine-SDS-PAGE analysis of the partially purified bacteriocin indicated a low molecular weight of approximately 5 kDa.

Screening, identification and characterization of bacteriocins produced by wine-isolated LAB strains.

To screen and identify wine-isolated LAB strains for bacteriocin production, and to identify and characterize bacteriocins. One hundred and fifty-five LAB strains isolated from South African red wines undergoing spontaneous malolactic fermentation were screened for bacteriocin production. Eight isolates were identified to be bacteriocin producers and were identified as Enterococcus faecium. All eight isolates had the same phenotypic and genotypic profiles. The peptides were preliminarily identified as enterocin P using mass spectrometry and further confirmed by PCR-amplifying enterocin P gene. The enterocin activity was inhibited by α-Chymotrypsin, papain and proteinase K treatments. It was heat stable at 37, 60, 80 and 100°C and showed activity over a broad pH range of 2-10. The production of the enterocin followed that of primary metabolite kinetics and, it showed bactericidal effect to some wine spoilage LAB strains. Our study identified the presence of the enterocin-producing Enterococcus in wine. The enterocin was heat stable; with broad pH range and bactericidal effects to sensitive strains. This is one of very few studies that isolated Enterococcus species from wine. It is, however, the first to report presence of bacteriocin-producing Enterococcus in wine fermentation.

PROPERTIES OF DURANCIN GL, A NEW ANTILISTERIAL BACTERIOCIN PRODUCED BY ENTEROCOCCUS DURANS 41D

ABSTRACTThe properties of the antilisterial durancin GL were characterized. The bacteriocin was the product of Enterococcus durans 41D, which was isolated from Hispanic‐style cheese samples. The antibacterial activity of durancin GL was only evident against gram‐positive bacteria, including Listeria species. Bacteriocin's activity was retained after treatment with surfactants of different groups (Tween, Brij and sodium dodecyl sulfate), and reducing (β‐mercaptoethanol) or chelating (ethylenediaminetetraacetic acid) agents failed to inactivate the bacteriocin. Durancin GL was active over the pH range of 2–11 and heat resistant for at least 30 min at 100C. However, exposure to 121C for 17 min completely inactivated the bacteriocin. Durancin GL displayed primary metabolite kinetics with the bacteriocin production rate being parallel with the growth cycle of E. durans 41D. The estimated molecular mass of durancin GL was <6 kDa, and it displayed a bactericidal effect on sensitive strains without concomitant cell lysis. The apparently new antilisterial bacteriocin from the putative probiotic strain E. durans 41D may be used to protect food products against various gram‐positive bacteria, including the food‐borne pathogen Listeria monocytogenes.PRACTICAL APPLICATIONSThe bacteriocins of food‐grade lactic acid bacteria may be useful as natural antimicrobials for food preservation. Food‐borne pathogens, including Listeria monocytogenes, may develop resistance to nisin, which is currently the only bacteriocin approved for use in foods. This study describes the initial characterization of an apparently novel bacteriocin, durancin GL, which is produced by a strain of Enterococcus durans found in artisanal Mexican cheese samples. The bacteriocin was shown to effectively inhibit the growth of several L. monocytogenes strains, including the nisin‐resistant variant, L. monocytogenes NR30. The results suggest that durancin GL may be developed to serve as a food‐grade preservative to inhibit the growth of nisin‐resistant Listeria.

Growth and metabolism of a meat isolated strain of Pediococcus pentosaceus in submerged fermentation : Purification, characterization and properties of the produced pediocin SM-1

The meat isolate Pediococcus pentosaceus, characterized as P. pentosaceus Mees 1934, produces a bacteriocin which was characterized in this study. The molecule was found to have the pediocin characteristics (with respect to N-terminal sequence and the presence of the characteristic consensus motif –YGNGV–), a molecular mass of 5.370 Da, and was designated as pediocin SM-1. The new bacteriocin is inhibitory to several food spoilage and food-born pathogens, shows a remarkable stability to heat and cold treatments, as well as to a wide pHs range, but it appears to be sensitive to proteases. Its mode of action appears to be bactericidal. Fermentation studies carried out in a stirred tank bioreactor revealed that semi-aerobic conditions (60% dissolved oxygen saturation) enhance production and fermentation rate analysis indicate primary metabolite kinetics for growth under the particular conditions. Pediocin SM-1, produced by a food-grade microorganism, exhibits the characteristics of a potential biopreservative and can be efficiently produced by fermentation.

Production and Purification of a Bioactive Substance Inhibiting Multiple Drug Resistant Bacteria and Human Leukemia Cells from a Salt-Tolerant Marine Actinobacterium sp. Isolated from the Bay of Bengal

Four marine actinobacteria tolerant to 200 g NaCl l(-1) were screened for antibacterial activity against eight patient-derived multiple drug resistant (MDR) bacteria. The active compound (MW 300.2, predicted molecular formula C(20)H(28)O(2)) from an actinobacterium, was inhibitory to three Gram-positive and three Gram-negative MDR bacteria, seven non-clinical Gram-positive, four Gram-negative bacteria and five fungi (MIC: 3.5-4.0 microg ml(-1)). Also, 54% of human leukemia (HL-60) cells were killed by the compound at 0.05 microg ml(-1). Bioreactor production demonstrated unusual primary metabolite kinetics. Molecular phylogenetic analysis showed this typical intertidal inhabitant to be a member of the Streptomyces genus and distinct from other salt-tolerant actinobacteria. As no compound was found to match the properties in several electronic databases, our screening strategy should increase the possibility of discovering bioactive molecules from rare actinobacteria.

Enterocin A production by Enterococcus faecium FAIR-E 406 is characterised by a temperature- and pH-dependent switch-off mechanism when growth is limited due to nutrient depletion

The biokinetics of cell growth and bacteriocin production of Enterococcus faecium FAIR-E 406 was studied as a function of temperature (20–45 °C) and pH (5.5–8.5) using de Man–Rogosa–Sharpe medium. Growth of E. faecium FAIR-E 406 was characterized by three successive growth phases and was modelled with the mechanistic nutrient depletion model. Bacteriocin production showed primary metabolite kinetics but was limited to the early growth phase. The critical biomass for switching off bacteriocin production was dependent on medium pH and incubation temperature, and was inversely correlated with the specific bacteriocin production. Doubling the concentration of the nitrogen source as well as a step-wise pH increase shifted the bacteriocin production towards a higher switch-off cell density.

Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced byLactobacillus plantarumTF711

The screening and initial characterization of bacteriocins produced by lactic acid bacteria (LAB) from raw Tenerife goats' cheese with possible application as biopreservatives or ripening accelerators for Tenerife cheese. One hundred and eighty LAB of the genera Lactobacillus (95), Leuconostoc (64) and Lactococcus (21) isolated from raw Tenerife goats' cheese were screened for the production of antimicrobial substances. Lactobacillus plantarum TF711, which had the broadest spectrum of antimicrobial activity, was selected for further characterization. The antimicrobial compound was determined as a proteinaceous substance, as it was sensitive to proteases. The bacteriocin-like substance, which we called plantaricin TF711, was active against the Gram-positive bacteria Bacillus cereus, Clostridium sporogenes and Staphylococcus aureus; and against the Enterobacteriaceae Shigella sonnei and Klebsiella pneumoniae. It was stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 1 and 9. Plantaricin TF711 exhibited primary metabolite kinetics, a bacteriostatic mode of action and a molecular mass of c. 2.5 kDa as determined by tricine SDS-PAGE. Lact. plantarum TF711 produces a low molecular mass bacteriocin-like compound with a wide spectrum of activity and interesting technological properties (thermostability, good pH stability and stability against surfactants and organic solvents). Plantaricin TF711 was found to have potential for use as a biopreservative in the food industry.

Modelling the production of nisin by Lactococcus lactis in fed-batch culture

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l(-1) h(-1), respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml(-1), by constant addition of sucrose at a feeding rate of 7 g l(-1) h(-1); an increase in 58% over that of the batch culture (2,658 IU ml(-1)). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.

Bacteriocin production by Enterococcus faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta cheese making

Bacteriocin production by Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in batch fermentations, under conditions simulating Feta cheese preparation. Maximum enterocin activity and growth rate was obtained in de Man–Rogosa–Sharpe (MRS) broth at 37 °C with controlled pH 6.5. The enterocin was produced throughout the growth phase of the microorganism, showing primary metabolite kinetics with a peak activity during the mid-exponential phase. The use of skimmed milk as substrate revealed low enterocin activity. When fermentations were performed in skimmed milk in the presence of rennet, CaCl 2, and a mixed starter culture, no enterocin activity was observed, although the examined strain grew well under the above conditions. Finally, when E. faecium FAIR-E 198 was applied as adjunct starter in Feta cheese making, no enterocin activity was detected throughout ripening. Results obtained underline the frequently underestimated finding that in vitro production by novel bacteriocinogenic starter or co-cultures is no guarantee for in situ efficiency. It was concluded that the complex food environment thoroughly interferes with bacteriocin production levels.

Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0-1: production kinetics, enzyme activities and EPS yields.

To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0-1 and to detect possible links between EPS yields and the activity of relevant enzymes. Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest specific EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0-1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. EPS production in Lact. sakei 0-1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. The proposed results contribute to a better understanding of the physiological factors influencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei.

Antimicrobial activity of L. plantarum, isolated from a traditional lactic acid fermentation of table olives

Lactobacillus plantarumstrain LB17.2b, isolated from traditional table olive fermentation has been shown to produce thermostable antibacterial proteins. At pH 6.5, the proteinaceous fraction obtained from culture supernatant was active against Enterococcus faecalisand against natural com- petitors of L. plantarum from olive fermentation brines. Bioactivity was quantified against Weis- sella paramesenteroidesDSM20288 using the critical dilution method by a regression procedure. This bioactivity has been found to be growth-associated indicating a primary metabolite kinetics. Ultra- filtration studies with supernatants from different fermentation times have been carried out. A two- fold increase in the activity of the retentate from the 30 kg .mol -1 cut-off membrane was observed, by comparison with the total activity applied to this membrane (culture supernatant). The addition of the filtrate to this retentate caused a marked drop in activity, most probably associated with the presence of an inhibitor. These studies also demonstrated the presence of another antibacterial proteinaceous compound showing a wider inhibitory spectrum and produced during culture stationary phase. In the presence of lactic acid (pH 3.7), these antimicrobial proteinaceous compounds have been found to inhibit Gram-negative human pathogens, probably by a synergistic effect. At the present time one bac- teriocin-like compound was isolated, showing a molecular mass of approximately 50 kg .mol -1 . The presence of another compound of different properties and with a molecular mass between 3 and 10 kg.mol -1 was also detected. antimicrobial activity / bacteriocin / lactic acid / Lactobacillus plantarum

Production kinetics of acidophilin 801, a bacteriocin produced by Lactobacillus acidophilus IBB 801.

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801. Studying the relationship between growth and bacteriocin biosynthesis revealed primary metabolite kinetics of bacteriocin production with a peak activity at the end of the exponential growth phase followed by a decrease during the stationary phase. Both microbial growth and bacteriocin production was inhibited by lactic acid. Whereas volumetric bacteriocin production (activity units (AU) ml(-1)) was favoured under pH-controlled conditions, bacteriocin titres rapidly decreased because of strong adsorption of the bacteriocin molecules to the producing cells under less acidic conditions.

Production kinetics of acidophilin 801, a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801. Studying the relationship between growth and bacteriocin biosynthesis revealed primary metabolite kinetics of bacteriocin production with a peak activity at the end of the exponential growth phase followed by a decrease during the stationary phase. Both microbial growth and bacteriocin production was inhibited by lactic acid. Whereas volumetric bacteriocin production (activity units (AU) ml −1) was favoured under pH-controlled conditions, bacteriocin titres rapidly decreased because of strong adsorption of the bacteriocin molecules to the producing cells under less acidic conditions.

The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation.

The specific conditions in the batter of raw fermented sausages may reduce the efficiency of bacteriocin-producing starter cultures. In this work, using in vitro fermentation, we found that sodium chloride and sodium nitrite interfere with the growth of Lactobacillus sakei CTC 494, an organism which produces the antilisterial bacteriocin sakacin K. Because sakacin K production follows primary metabolite kinetics, a decrease in cell formation resulted in a decrease in sakacin K production as well. Sodium chloride dramatically influenced bacteriocin production by decreasing both biomass production and specific bacteriocin production. Sodium nitrite, however, had no effect on specific bacteriocin production and decreased bacteriocin production only because of its effect on cell growth. Moreover, sodium nitrite enhanced the toxic effect of lactic acid on bacterial growth.

Process characteristics of exopolysaccharide production by streptococcus thermophilus

AbstractThe kinetics of growth and exopolysaccharide (EPS) production from Streptococcus thermophilus were studied and optimized for different physical (temperature, pH, aeration rate) and chemical (carbon/nitrogen ratio) factors in milk medium. From these experiments, it was clear that EPS production displays primary metabolite kinetics. Using the optimized conditions, EPS production could also be established in de Man Rogosa Sharpe medium. Growth‐associated EPS production, bacterial growth and other fermentation data were translated into a mathematical model.

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.

Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis.

Four Streptococcus thermophilus strains (Strep. thermophilus BTC, Strep. thermophilus LY03, Strep. thermophilus 480 and Strep. thermophilus Sfi20) have been examined for their exopolysaccharide production capacity. All strains produced a polymer composed of the neutral sugars glucose and galactose, but in different ratios. It was clearly shown that the biosynthesis of exopolysaccharides from Strep. thermophilus LY03 is growth-associated and hence displays primary metabolite kinetics. The monomer ratio of the exopolysaccharide synthesized did not vary throughout the fermentation cycle. The production kinetics and exopolysaccharide yields were strongly dependent on the fermentation conditions. Physical factors such as temperature, pH and oxygen tension as well as chemical factors (medium composition, initial lactose concentration, carbon/nitrogen levels) were of utmost importance.

Modelling the growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in batch cultivation

A model was set up to describe the production of amylovorin L471 by Lactobacillus amylovorus DCE 471, on a laboratory scale, in which the cells are grown in MRS (deMau-Rogosa-Sharpe) broth. The main features of the dynamic model are : (i) increase of the biomass according to a logistic equation ; (ii) non-growth-associated consumption of substrate (maintenance metabolism) ; and (iii) primary metabolite kinetics for the bacteriocin production. The main purpose was to set up a simple empirical model to examine growth and bacteriocin production in different conditions. Parameters estimated from a fermentation with 20 g l−1 glucose (w/v) could be used to predict the evolution of cell dry mass, glucose and lactic acid concentration of fermentations, performed with 5, 30, 40 and 60 g l−1 initial glucose. The influence of the operating temperature (30, 37 and 45 °C) on the model parameters was also investigated. The proposed model was able to describe growth and bacteriocin production in all cases. The specific bacteriocin production rate was found to vary strongly with temperature, with 30 °C as the best value. Variation of the operating temperature from 37 to 30 °C appeared to significantly enhance the specific bacteriocin production.