Novel experimental design paradigm for development of eco-friendly gradient chromatographic method for simultaneous determination of metronidazole and spiramycin.

This work describes the innovative experimental design-assisted development of a green gradient chromatographic method for concomitant analysis of metronidazole (MTR) and spiramycin (SPR). Two different designs including fractional factorial and Box-Behnken designs were implemented for screening and optimization steps, respectively. The optimum chromatographic conditions involved a mobile phase consisting of ethanol and 20 mM sodium dihydrogen phosphate solution (pH adjusted to 2.5) in the ratio 2:98 (v/v) for 2 min then the ratio changed to 30:70 (v/v). The flow rate was 1.3 mL/minute. Separation and analysis were performed on X-bridge C18 (150 mm × 4.6 mm × 3.5 µm) column with diode array detector set at 230 nm. Column oven temperature was 40°C. A linear response was acquired over the range of 5-125 µg/mL for both drugs. Detection and quantitation limits were 0.86 and 2.62 µg/mL for MTR and 0.92 and 2.83 µg/mL for SPR, respectively. The method was implemented for determination of both drugs in three tablet formulations. The method was proved to be green as evaluated by three assessment tools. The application of experimental designs assists in development of a robust green chromatographic method in gradient elution mode for determination of both drugs within reasonable time.

A liquid chromatography coupled to tandem mass spectrometry method for the quantification of spiramycin and its active metabolite neospiramycin in milk of major and minor species: Validation using the accuracy profile.

A liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous quantification of residues of spiramycin, a macrolide antibiotic, and its active metabolite neospiramycin in cow's milk as well as in minor species 'milk, goat and ewe. Spiramycin-d3 was used as internal standard for quantification of both analytes. This analytical method was validated using a global accuracy profile as a graphical decision tool built according to the trueness and the precision of the method. A unique and optimal linear model with logarithm transformation (with a determination coefficient of 0.9991) allowed the measurement of both analytes in the milks of the three animal species, in a wide range from 0.2 to 10 times the Maximal Residue Limit (MRL) (40-2000 µg.kg-1). The limits of detection and quantification were 13 µg.kg-1 and 40 µg.kg-1, respectively. The accuracy profile was established to get 80% of future measurements in routine assays that will fall within the acceptance limits. Trueness of the method, expressed as relative bias, was comprised between -1.6% and 5.7% over the whole range of concentrations. The mean relative standard deviation for repeatability and intermediate precision were comprised between 1.1% and 2.7%; 2.5 and 4.2%, respectively, in all levels of concentration for the three milks. Moreover, a two-order polynomial function was used to model the relative expanded uncertainty with a determination coefficient of 0.834. This function aimed to determine the uncertainty of the future quantifications within the validated dosing range. Overall, the global accuracy profile highlighted the reliability of the method for the routine assays of spiramycin and neospiramycin even in milk from minor species (goat, ewe) by using the most accessible milk (often from cow), while guaranteeing a very high proportion of samples within the fixed acceptance limits. The applicability of this method was tested during a depletion study of spiramycin and neospiramycin in the milk of cow, goat and ewe. The developed analytical method will be useful to assess the distribution profile of the antibiotic and its metabolite in milk of minor species where few studies are available.

Gold Immunochromatography Assay for the Rapid Detection of Spiramycin in Milk and Beef Samples Based on a Monoclonal Antibody.

Spiramycin (SP) residues in food do harm to human health. It is necessary to establish rapid detection method for SP. In this work, a monoclonal antibody (mAb)-based gold immunochromatography assay (GICA) is developed for the rapid detection of SP. Under optimum conditions, the half-maximal inhibitory concentration of SP-mAb is 0.43 ng mL-1 . The subtype of SP-mAb is IgG2b. This antibody has no cross-reactivity with other analogues and has high affinity (4.52 × 1010 L mol-1 ). Qualitative results can be visualized with the naked eye, with a visual detection limit of 1.0 ng mL-1 and cut-off value of 10 ng mL-1 . A hand-held strip scanner is used for the quantitative analysis, with LOD 0.43 ng mL-1 in assay buffer. The recoveries of SP ranged from 72.3% to 112% in milk and 98.5% to 115% in beef, with variable coefficient ranging from 9.4% to 11.7% in milk and 8.14% to 15.4% in beef. Besides, the proposed GICA method for SP is confirmed by LC-MS/MS in SP-spiked milk and beef samples. Overall, the developed GICA can be a useful tool for SP residues on-site screening in milk and beef samples.

Effectively remediating spiramycin from production wastewater through hydrolyzing its functional groups using solid superacid TiO2/SO4.

Breaking down the structural bonds and eliminating the functional groups are more efficient than destroying the whole molecule in antibiotic production wastewater (APW) pretreatment before further biotreatment. Two sulfated titania (TiO2/SO4) solid superacids, SSA1 and SSA2 were synthesized, characterized and used for hydrolytic pretreatment of spiramycin in APW. Spiramycin removal followed an order of SSA2>SSA1>TiO2≈pH = 3>control. The hydrolytic efficiencies increased at elevated temperature from 25 °C to 65 °C. The hydrolytic kinetics followed a first-order model and SSA2 performed the fastest. The performances were positively correlated with both the total acidity determined by n-butylamine titration and the strength of acid sites measured by NH3-temperature-programmed desorption (TPD). The residual solution for SSA2 presented the least antibacterial potency and anaerobic inhibition among all treatments. The hydrolyzed product was identified as the m/z 699.4321 fragment using UPLC-Q/TOF-MS, which was formed after losing a functional mycarose moiety from the parent molecular. The solid superacids were effective in selectively eliminating 433 mg/L of spiramycin and the antibacterial potencies of the spiramycin production wastewater, which contained very high concentrations of COD (33,000 mg/L). This hydrolytic method avoids using and handling hazardous and corrosive mineral acids on site. It is attractive as a selective catalytic pretreatment method to cleave antibiotics' functional groups and to reduce its inhibitory effects before sequential biotreatments.

Pretreatment of spiramycin fermentation residue using hyperthermophilic digestion: quick startup and performance.

This study aimed to evaluate the feasibility of hyperthermophilic anaerobic digestion at 70 °C in the pretreatment of spiramycin fermentation residue. By feeding municipal excess sludge under a solid retention time of 5 days, the hyperthermophilic digester was successfully started up within 3 days from mesophilic digestion by a one-step temperature increase from 35 to 70 °C. MiSeq sequencing showed the fast establishment of thermophilic fermenting bacterial communities in 3 days immediately after the temperature increase, with increases in abundance of Coprothermobacter, Spirochaetaceae_uncultured and Fervidobacterium from <0.001%, 1.06% and <0.001% to 33.77%, 11.65% and 3.42%, respectively. The feasibility of hyperthermophilic digestion for spiramycin residue was evaluated in batch experiments for 7 days. Hyperthermophilic digestion considerably reduced antibiotic concentrations, with removal efficiencies of 55.3% and 99.0% for the spiramycin residue alone and its mixture with hyperthermophilic sludge, respectively. At the same time, the abundances of four macrolide-lincosamide-streptogramin resistance genes were also reduced within 7 days, due to the decrease of their corresponding hosts. These results suggest that hyperthermophilic digestion could easily be started up from mesophilic digestion and might be a suitable pretreatment approach for spiramycin residue.

Influence of dextrins on the production of spiramycin and impurity components by Streptomyces ambofaciens.

Spiramycin is a 16-membered macrolide antibiotic produced by Streptomyces ambofaciens and used in human medicine for the treatment of various respiratory tract and genital infections. Several impurities were detected in spiramycin-fermentation broth, especially impurities D and F, which decreased the separation-extraction yield and increased production cost. Dextrins, as the main carbon source, influence the accumulation of spiramycin and impurities. In this work, two types of dextrin from vendor Y and Z were compared to study their influences on spiramycin production. Our results showed that final spiramycin production with dextrin Z was enhanced twofold as compared with dextrin Y; however, the content of impurities F and D were higher with dextrin Z relative to dextrin Y. Several parameters (adenosine triphosphate, total sugar, reducing sugar, and reducing sugar to total sugar) were analyzed to reveal differences in the fermentation process. In vitro dextrin hydrolysis by amylase revealed structural differences in the two types of dextrin, and real-time quantitative polymerase chain reaction analyses showed that the transcription of srm7 and srm21 (involved in forosaminyl methylation) was enhanced and potentially related to the reduced formation of impurity F with dextrin Y. Furthermore, the srm20/srm33 ratio, representing flux balance of forosaminyl and mycarosyl, was ~ 1, implying that forosaminyl and mycarosyl biosynthesis were well balanced, resulting in reduced production of impurity D with dextrin Y.

iTRAQ-based quantitative proteomic analysis of Microcystis aeruginosa exposed to spiramycin at different nutrient levels.

Research on the combined effects of antibiotic contaminants and environmental factors in cyanobacteria is still limited. This study focused on the action and its mechanism of spiramycin combined with changes in nitrogen and phosphorus level in Microcystis aeruginosa at environmentally relevant concentrations. Though photosynthetic activity was stimulated by spiramycin at a high nutrient level, no significant correlation (p>0.05) was found between photosynthesis-related proteins and growth-related proteins, and the growth rate was inhibited by 200ngL-1 of spiramycin. At low nitrogen and low phosphorus levels, up-regulated photosynthesis-related proteins were closely correlated with (p<0.05) stress response-related, transcription-related and cell division-related proteins, which consequently led to stimulated growth of M. aeruginosa under spiramycin exposure. Spiramycin exposure also regulated the production of microcystins (MCs) and the expression of two microcystin synthetases (mcyB and mcyC). The spiramycin-induced protein secretion process and the up-regulation of ATP binding cassette transporters might contribute to the increased MC release. Enolase, superoxide dismutase, protein GrpE, DNA-directed RNA polymerase subunit alpha and serine protease were candidate target proteins of spiramycin in M. aeruginosa under different nutrient conditions. Coexisting spiramycin mitigated the threat of cyanobacteria to aquatic environments at a high nutrient level but aggravated cyanobacterial bloom at a low nitrogen level.

Synthesis and characterization of a molecularly imprinted polymer for the determination of spiramycin in sheep milk.

A series of molecularly imprinted polymers (MIPs) comprising reactionary sites which are complementary to macrolide antibiotic spiramycin (SPI) were synthetized by noncovalent bulk polymerization technique. MIPs were synthesized under different polymerization process and their recognition efficiency was evaluated in binding studies in comparison with non-imprinted polymers. The best MIP was morphologically characterized and equilibrium assays were carried out. The MIP was evaluated as a sorbent for extraction and preconcentration of SPI from aqueous and sheep milk samples, and an off-line MISPE method followed by high-performance liquid chromatography with UV diode-array detection was established. Good linearity were obtained for SPI in a range of 24-965µgkg-1 and the average recoveries at three spiked levels in milk samples were higher than 90% (RSD<5%). Limit of quantification was 24.1µgkg-1. Cross-reactivity studies from other macrolides with similar structure were tested. The optimum imprinted polymer showed a good selectivity and affinity for SPI, demonstrating the potential of the proposed MISPE for rapid, sensitive and effective sample pretreatment for selective determination of SPI in sheep milk samples.

Development and Optimization of HPLC Analysis of Metronidazole, Diloxanide, Spiramycin and Cliquinol in Pharmaceutical Dosage Forms Using Experimental Design.

A new simple, sensitive, rapid and accurate gradient reversed-phase high-performance liquid chromatography with photodiode array detector (RP-HPLC-DAD) was developed and validated for simultaneous analysis of Metronidazole (MNZ), Spiramycin (SPY), Diloxanidefuroate (DIX) and Cliquinol (CLQ) using statistical experimental design. Initially, a resolution V fractional factorial design was used in order to screen five independent factors: the column temperature (°C), pH, phosphate buffer concentration (mM), flow rate (ml/min) and the initial fraction of mobile phase B (%). pH, flow rate and initial fraction of mobile phase B were identified as significant, using analysis of variance. The optimum conditions of separation determined with the aid of central composite design were: (1) initial mobile phase concentration: phosphate buffer/methanol (50/50, v/v), (2) phosphate buffer concentration (50 mM), (3) pH (4.72), (4) column temperature 30°C and (5) mobile phase flow rate (0.8 ml min-1). Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9999. Limits of detection for all of the analyzed compounds ranged between 0.02 and 0.11 µg ml-1; limits of quantitation ranged between 0.06 and 0.33 µg ml-1 The proposed method showed good prediction ability. The optimized method was validated according to ICH guidelines. Three commercially available tablets were analyzed showing good % recovery and %RSD.

High Concentrations of the Antibiotic Spiramycin in Wastewater Lead to High Abundance of Ammonia-Oxidizing Archaea in Nitrifying Populations.

To evaluate the potential effects of antibiotics on ammonia-oxidizing microbes, multiple tools including quantitative PCR (qPCR), 454-pyrosequencing, and a high-throughput functional gene array (GeoChip) were used to reveal the distribution of ammonia-oxidizing archaea (AOA) and archaeal amoA (Arch-amoA) genes in three wastewater treatment systems receiving spiramycin or oxytetracycline production wastewaters. The qPCR results revealed that the copy number ratios of Arch-amoA to ammonia-oxidizing bacteria (AOB) amoA genes were the highest in the spiramycin full-scale (5.30) and pilot-scale systems (1.49 × 10(-1)), followed by the oxytetracycline system (4.90 × 10(-4)), with no Arch-amoA genes detected in the control systems treating sewage or inosine production wastewater. The pyrosequencing result showed that the relative abundance of AOA affiliated with Thaumarchaeota accounted for 78.5-99.6% of total archaea in the two spiramycin systems, which was in accordance with the qPCR results. Mantel test based on GeoChip data showed that Arch-amoA gene signal intensity correlated with the presence of spiramycin (P < 0.05). Antibiotics explained 25.8% of variations in amoA functional gene structures by variance partitioning analysis. This study revealed the selection of AOA in the presence of high concentrations of spiramycin in activated sludge systems.

Hydrolysis of amphenicol and macrolide antibiotics: Chloramphenicol, florfenicol, spiramycin, and tylosin.

Antibiotics that enter the environment can present human and ecological health risks. An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence as biologically active contaminants. In this study, hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for two amphenicol (chloramphenicol and florfenicol) and two macrolide (spiramycin and tylosin) antibiotics. Antibiotic hydrolysis rates in pH 4-9 buffer solutions at 25°C, 50°C, and 60°C were quantified, and degradation products were characterized. All of the antibiotics tested remained stable and exhibited no observable hydrolysis under ambient conditions typical of aquatic ecosystems. Acid- and base-catalyzed hydrolysis occurred at elevated temperatures (50-60°C), and hydrolysis rates increased considerably below pH 5 and above pH 8. Hydrolysis rates also increased approximately 1.5- to 2.9-fold for each 10°C increase in temperature. Based on the degradation product masses found, the functional groups that underwent hydrolysis were alkyl fluoride, amide, and cyclic ester (lactone) moieties; some of the resultant degradation products may remain bioactive, but to a lesser extent than the parent compounds. The results of this research demonstrate that amphenicol and macrolide antibiotics persist in aquatic systems under ambient temperature and pH conditions typical of natural waters. Thus, these antibiotics may present a risk in aquatic ecosystems depending on the concentration present.

Monoclonal antibody production and the development of an indirect competitive enzyme-linked immunosorbent assay for screening spiramycin in milk.

To monitor spiramycin (SP) residue in milk, a monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed. This study described the preparation of three immunogens and the production of a high-affinity mAb. After optimization, the 50% inhibition concentration (IC50) for the developed icELISA was estimated as 0.97 ng/mL in the assay buffer, and the limit of detection and limit of quantitation were 2.51 and 4.40 µg/L in the milk matrix. The newly developed assay demonstrated negligible cross-reactivity with 15 other macrolide antibiotics, but not with kitasamycin (23.4%). The mean recoveries ranged from 81 to 103% for the spiked samples (5, 10, and 50 µg/L), and the coefficient of variation ranged from 5.4 to 9.6%. The icELISA was validated by LC-MS/MS method, and all results demonstrated that it was a suitable screening method for detecting SP residue in milk without requiring a cleanup process.

[Impurity profiling of macrolide antibiotics by liquid chromatography-mass spectrometry].

Macrolide antibiotics are broad-spectrum, with activity against a range of Gram-positive, Gram-negative organisms and some anaerobes. The components of macrolide antibiotics are generally complicated. Therefore, it is very important to establish impurity profiles of these antibiotics to ensure their safety and process control. Compared with classical methods, the liquid chromatography-mass spectrometry method is particularly advantageous to characterize minor components at trace levels in terms of sensitivity, efficiency and selectivity, thus more and more widely used in establishments of impurity profiles. In this study, the general approaches to characterize minor components in complex pharmaceutical matrix, fragmentation pathways of 14- and 16-membered macrolide antibiotics and the establishment of the impurity profile of acetylspiramycin were given to provide valuable enlightenments to establish the impurity profiles of pharmaceutical products.

Identification of the components of bitespiramycin by liquid chromatography-mass spectrometry.

Reversed-phase liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was used to characterize the components of bitespiramycin, a group of 4"-acylated spiramycins produced by bioengineered strains. In total 38 components were characterized in commercial samples, including 12 impurities that had never been reported before and 12 other that were partially characterized. The structures of these unknown compounds were deduced by comparison of their fragmentation patterns with those of known major components. Their ultraviolet spectra were used to confirm the presence of an α-, ß-, γ-, δ-unsaturated butadiene in the macrocyclic lactone. Compared with the classical method, LC/ESI-MS/MS is particularly advantageous in terms of sensitivity and efficiency to characterize minor components at trace levels in multi-component antibiotics.

Development and validation of a potentiometric biosensor assay for tylosin with demonstrated applicability for the detection of two other antimicrobial growth-promoter compounds in feedstuffs.

A potentiometric biosensor assay based on a commercially available polyclonal antibody was developed to detect tylosin residues in animal feed. The method can be used as a rapid (less than 45 min) laboratory-based procedure or as a portable field-test for the simultaneous measurement of up to 12 different samples. For both procedures the qualitative detection capability (CCß) for tylosin was determined as 0.2 mg kg(-1) in a range of animal feeds with a measurement repeatability at concentrations between 0.2 and 4 mg kg(-1) of ≤13% coefficient of variation (%CV). The field-test format was capable of detecting tylosin residues at operating (external air) temperatures ranging between +4 and 37°C, although some reduction in signal was observed at the lower temperatures. The laboratory-based tylosin assay was evaluated using 16 medicated and 22 non-medicated feeds and was found to give comparable data with a confirmatory method based upon liquid chromatography-tandem mass spectrometry (LC-MS/MS). The potential to develop a multi-probe format assay for the simultaneous detection of tylosin, spiramycin and virginiamycin was also demonstrated. Cross-validation in a second laboratory showed the assay to be transferable, reliable and robust.

Simultaneous determination of metronidazole and spiramycin in bulk powder and in tablets using different spectrophotometric techniques.

Metronidazole (MZ) is an anti-infective drug used in the treatment of anaerobic bacterial and protozoa infections in humans. It is also used as a veterinary antiparasitic drug. Spiramycin (SP) is a medium-spectrum antibiotic with high effectiveness against Gram-positive bacteria. Three simple, sensitive, selective and precise spectrophotometric methods were developed and validated for the simultaneous determination of MZ and SP in their pure form and in pharmaceutical formulations. In methods A and B, MZ was determined by the application of direct spectrophotometry and by measuring its zero-order (D(0)) absorption spectra at its λ(max) = 311 nm. In method A, SP was determined by the application of first derivative spectrophotometry (D(1)) and by measuring the amplitude at 218.3 nm. In method B, the first derivative of the ratio spectra (DD(1)) was applied, and SP was determined by measuring the peak amplitude at 245.6 nm. Method C entailed mean centering of the ratio spectra (MCR), which allows the determination of both MZ and SP. The methods developed were used for the determination of MZ and SP over a concentration range of 5-25 µg ml(-1). The proposed methods were used to determine both drugs in their pure, powdered forms with mean percentage recoveries of 100.16 ± 0.73 for MZ in methods A and B, 101.10 ± 0.90 in method C, 100.09 ± 0.70, 100.02 ± 0.88 and 100.49 ± 1.26 for SP in methods A, B and C, respectively. The proposed methods were proved using laboratory-prepared mixtures of the two drugs and were successfully applied to the analysis of MZ and SP in tablet formulation without any interference from each other or from the excipients. The results obtained by applying the proposed methods were compared statistically with a reported HPLC method and no significant difference was observed between these methods regarding both accuracy and precision.

Electrochemical study of spiramycin and its determination in pharmaceutical preparation.

Spiramycin (SPY) is a medium-spectrum antibiotic with high effectiveness against Gram-positive bacteria. The voltammetric behaviour of spiramycin was studied using differential pulse polarography (DPP) and square wave polarography (SWP). The drug in Britton-Robinson buffer (pH 11.5) is reduced at - 1.45 V, giving rise to a well-defined cathodic peak using hanging mercury drop electrode (HMDE) versus Ag/AgCl electrode. This peak is attributed to the reduction of the aldehyde group. The results proved that the reduction of SPY is an irreversible diffusion-controlled process. The diffusion current-concentration relationship was shown to be rectilinear over the range of 20-80 and 0.8-80 µg ml(-1) using DPP and SWP modes, respectively, with detection limit of 8.5 µg ml(-1) (1.01 × 10(-5) M) and 0.46 µg ml(-1) (5.46 × 10(-7) M) for DPP and SWP modes, respectively. A mechanism is postulated for the reduction of SPY. The proposed techniques were successfully applied to the determination of the studied compound either in pure form or in its formulation.

On-line identification of 4''-isovalerylspiramycin I in the genetic engineered strain of S. spiramyceticus F21 by liquid chromatography with electrospray ionization tandem mass spectrometry, ultraviolet absorbance detection and nuclear magnetic resonance spectrometry.

LC-hyphenated techniques were applied to the on-line identification of isovalerylspiramycin I (isp I), a spiramycin-like macrolide in the crude extract of fermentation broth from a genetically engineered strain of S. spiramyceticus F21. In the structural characterization of the large molecular secondary metabolite of isp I, LC-DAD-UV-ESI-MS(n) analysis played a crucial role, and stop-flow LC-(1)H NMR measurement, with bitespiramycin used as reference, was a valuable complement approach. This rational approach proved to be an efficient means for the rapid and accurate structural determination of known microbial secondary metabolites, by which targeted isolation of component(s) of interest can be subsequently performed for further biological and pharmacological studies in drug development.

Fate of antibacterial spiramycin in river waters.

Spiramycin, a widely used veterinary macrolide antibiotic, was found at traceable levels (nanograms per litre range) in Po River water (N-Italy). The aqueous environmental fate of this antibiotic compound was studied through drug decomposition, the identification of the main and secondary transformation products (TPs), assessment of mineralisation and the investigation of drug TPs toxicity. Initially, laboratory experiments were performed, with the aim of stimulating the antibacterial transformation processes followed in aquatic systems. The TPs were identified through the employment of the liquid chromatography (LC)-mass spectrometry technique. Under illumination, spiramycin degraded rapidly and transformed into numerous organic (intermediate) compounds, of which 11 could be identified, formed through five initial transformation routes. These laboratory simulation experiments were verified in situ to check the mechanism previously supposed. Po River water was sampled and analysed (by LC-high-resolution mass spectrometry) at eight sampling points. Among the previously identified TPs, five of them were also found in the river water. Three of them seem to be formed through a direct photolysis process, while the other two are formed through indirect photolysis processes mediated by natural photo sensitisers. The transformation occurring in the aquatic system involved hydroxylation, demethylation and the detachment of forosamine or mycarose sugars. Toxicity assays using Vibrio fischeri proved that even if spiramycin did not exhibit toxicity, its transformation proceeded through the formation of toxic products.

Effect of branched-chain amino acids, valine, isoleucine and leucine on the biosythesis of bitespiramycin 4"-O-acylspiramycins

Bitespiramycin, a group of 4"-O-acylated spiramycins with 4"-O-isovalerylspiramycins as the major components, was produced by recombinantspiramycin-producing strain Streptomyces spiramyceticus harboring a 4"-O-acyltransferase gene. The experiment was initially performed in synthetic medium with 0.5 g l-1 Valine, Isoleucine or Leucine feeding at 36 h cultivation. When valine was fed, the biological titer of bitespiramycin was 45.3 percent higher than that of the control group, but the relative content of total isovalerylspiramycin components decreased by 22.5 percent. In the case of ilecine, the biological titer of bitespiramycin and the total isovalerylspiramycins alone were 85 percent and 72.1 percent of the control group, respectively. In contrast, the relative content of other acylated spiramycins increased by 54.41 percent. However, leucine feeding increased the relative content of total isovalerylspiramycins by 41.9 percent while the biological titer of bitespiramycin was nearly equal to that of the control group. The improvement effect of leucine on the biosynthesis of isovalerylspiramycins was further confirmed by feeding of 2.0 g l-1 leucine to the culture with complex medium. After batch feeding with a total amount of 2.0 g l-1 leucine to the culture from 70 h to 90 h, the biological titer of bitespiramycin was almost unreduced, and the final relative content of total isovalerylspiramycins increased from 31.1 percent to 46.9 percent.