Jingxin LIU, Yunhong HUANG, Leng DAI, Zhonger LONG

College of Life Science, Jiangxi Provincial Key Lab of Protection and Utilization of Subtropical Plant Resources, Jiangxi Normal University, Nanchang 330022, China

Actinomycetes are widely distributed in nature,and are typically useful in the pharmaceutical industry for their seemingly unlimited capacity to produce antibiotics with biological activities[1-2]. The approximately two-thirds of naturally occurring antibiotics have been isolated from actinomycetes. Among microorganisms, actinomycetes, particularly members of the genus Streptomyces,are one of the most investigated groups,from which a large number of antibiotics were obtained.However,with the different types of antibiotics prevailing in the market, the drug and multidrugresistant bacteria has emerged in the world[3-4].Therefore, there is an urgent need to find new, safer and broadspectrum antibiotics from potential actinomycetes to control the spread of antibiotic resistant bacteria.

According to the taxonomy, Micromonospora carbonacea belongs to genus Micromonospora, Micromonosporaceae, Actinomycetal, Actinobacteria[5-6].The M.carbonacea could produce more than one component of the sugar derivatives of antibiotics- Everninomicin complexes. The Daidzein and Genistein, which showed the immunosuppressive and anti-tumor activity rather than antibacterial activity,were produced by a marine source of M.carbonacea[7-8].According to the literature survey,it was not reported that the other active products were fermented by M. carbonacea yet[9-10]. In our laboratory, a strain of rare actinomycetes, with broad-spectrum antimicrobial activity, isolated from the soil samples from the farmland in the area of Yaohu in Nanchang,was identified as M.carbonacea JXNU-1[11-12].

The aim of the present study was to investigate the feasibility of resins to extract antibacterial substances in fermentation broth of M. carbonaceaJXNU-1.Various parameters including the type of resins, the time of adsorption and the initial pH of sample on the extraction efficiency were investigated and optimized.

Materials and Methods

Strains

M. carbonacea JXNU-1 was isolated from soil samples and was stored as previously described[13].

Staphylococcus aureus was used as test microorganism, obtained from Microbiology Laboratory of the College of Life Sciences, Jiangxi Normal University, and stored in nutrient agar slant at 4 ℃.

Media

The beef extract peptone medium(pH 7.0-7.2)consisted of 3 g beef extract,10 g peptone,5 g NaCl,15-20 g agar, in 1 000 ml distilled water. The microorganism was cultivated by this medium.

The Gause’s No.1 medium (pH 7.2-7.4)contained 20 g soluble starch,1 g KNO3, 0.5 g NaCl, 0.5 g K2HPO4,0.5 g MgSO4,0.01 g FeSO4,20 g agar in 1 000 ml distilled water.The M.carbonacea JXNU-1 was stored by this medium. Full detail procedures have been previously described[14].

The seed culture medium(pH 7.2)was composed of 22.5 g sucrose,12.5 g soy flour, 0.2 g K2HPO4, 1 g NaCl,0.1 g Na2SO4,0.01 g FeSO4·7H2O,2 g CaCO3in 1 000 ml distilled water.

The fermentation medium (pH 7.7) contained 20 g starch, 13 g sucrose, 35 g peanut meal, 0.1 g K2HPO4, 1 g NaCl, 0.1 g Na2SO4, 0.01 g FeSO4·7H2O,3 g CaCO3in 1 000 ml distilled water.

Methodology

Fermentation cultivation The procedure of fermentation was described by Long et al[13]. The preparation process of fermentation was briefly described as follows.The M.carbonacea JXNU-1 was first spread onto the Gause’s No.1 medium slopes and incubated at 28 ℃for 7 d. Then the slopes were washed with sterile water,and about 1×106spores were inoculated into 50 ml of seed culture medium in a 250 ml triangle flask.After incubation at 28 ℃and 200 r/min for 72 h,the seed cultures were collected for further fermentation. The seed cultures were inoculated into 20 ml of fermentation medium in a 250 ml triangle flask when the inoculation ratio was 6%(v/v). For the production of antibiotics, the fermentation was also carried out at 28 ℃and 200 r/min for 108 h. The fermentation broth was collected for further study.

Preparation of fermentation broth

The process for the preparation of fermentation broth was briefly described as follows. The supernatant was firstly collected by the centrifugation at 4 000 r/min for 20 min.Then the small hypha and floating oil in the upper layer were removed when the supernatant was filtered through Xinhua filter paper. The filtrate was added by twice the volume of anhydrous ethanol and was precipitated at 4 ℃for 4 h. The supernatant adjusted to pH 7.0 was secondly collected by the centrifugation at 4 000 r/min for 15 min. The alcohol in the supernatant was removed under vacuum at 45 ℃. The supernatant was added to the original volume of broth by distilled water and was sterilized for 20 min at 121 ℃for further work.

Preparation of bacterium suspension The S.aureus were first spread onto the beef extract peptone medium slopes and incubated at 37 ℃. Then the cultures were inoculated into 50 ml of MH medium in a 250 ml triangle flask.After incubation at 37 ℃and 200 r/min, the cultures were collected for further antibacterial activity when the growth of S.aureus was at logarithmic phase.The bacterium suspension was counted by a complete blood counting plate method and was diluted to be about 106CFU/ml.

Antibacterial activity of antibiotics The antibacterial activity of antibiotics was determined by agar diffusion method and was previously described[14]. The 200 μl of fermentation broth prepared was transferred into the different Oxford cups in the same plate with S. aureus. After incubation at 37 ℃for 24 h, the zone of inhibition was measured and recorded. Each experiment was performed in triplicate.

Adsorption experiments The adsorption experiments were conducted by using a batch method. 10 g of the resins were immersed in a 250 ml triangle flask containing preparation of fermentation broth. The mixture in flask was incubated at 25 ℃on a rotary shaker at 200 r/min. Following centrifugation, the antibacterial activity was analyzed by agar diffusion method in the residue. Various parameters including the type of resins, the initial pH and the time of adsorption on the extraction efficiency were investigated and optimized.Each experiment was performed in triplicate.

Adsorption isotherm of resins To investigate the adsorption isotherm of the resins, 10 g of the resins, and 30 ml of fermentation broth with different concentration antibiotics ranging from 60 to 160 μg/ml were respectively placed into 250 ml triangle flask[15].The mixture in flask was incubated at 25 ℃on a rotary shaker at 200 r/min for 2.5 h. Following centrifugation, the antibacterial activity was analyzed by agar diffusion method in the residue.Each experiment was performed in triplicate.

The equilibrium adsorption capacity Qe(μg/g),was calculated based on the difference of antibiotics concentration before and after equilibrium adsorption,the volume of aqueous solution and the mass of the resins according to:

Where C0is the initial antibiotics concentration (μg/ml), Ceis the equilibrium concentration of antibiotics(μg/ml), V is the volume of fermentation broth (ml), and m is the mass of resins(g).

Statistical analysis

The collected and recorded data were performed by using SPSS 19.0 Version software.Means and standard errors for each treatment were reported.The data obtained were subjected to analysis of variance(ANOVA).

Results and Analysis

The established standard curve of penicillin

Many researches and practices showed that, by the same way for measurement of the inhibition zone,there is a linear relationship between the logarithm of antibiotic concentration and the square of the inhibition zone radius in a certain range of antibiotic concentration,namely:

Where r is the inhibition zone ra-dius, C is the antibiotic concentration,a and b are constant.

As the antagonistic activity was expressed as the titer of penicillin which could form the same inhibition zone.

The linear relationship between the logarithm of penicillin concentration and the square of the inhibition zone radius in Fig.1 is y=5.765 8x-14.060. The regression constant is calculated as 0.992 3. The titer of antibiotics in fermentation broth of M. carbonacea JXNU-1 can be calculated on radius of inhibition zone.

Effect of the type of resins on adsorption

Resins have been used in large bio-molecules and metabolites separation as well as in isolation of chemical entities from traditional Chinese medicine[16]. Four kinds of resins 001x7, 7732, 201x7 and 717 were applied for separating antibiotics in fermentation broth of M.carbonacea JXNU-1. 10 g of the resins were immersed in a 250 ml triangle flask containing 20 ml preparation of fermentation broth.The mixture in flask was incubated at 25 ℃on a rotary shaker at 200 r/min for 3 h. The effect of resins on adsorption capacities and adsorption ratio were shown in Table 1.

The results showed that the adsorption amount of 001x7, 7732,201x7 and 717 resins was 0, 0, 515 and 713 U/g, respectively. It was shown that the anion resins well adsorbed the antibiotics in fermentation broth of M. carbonacea JXNU-1. The adsorption ratio of 201x7 and 717 anion resins was 72.2% and 100%, respectively. It was indicated that the 717 anion resin was the optimal for separating antibiotics in fermentation broth of M.carbonacea JXNU-1.

Effect of pH on adsorption

The effect of pH on the adsorption of antibiotics in the fermentation broth was studied by adding 10 g of the 717 anion resin to 30 ml of fermentation broth with equal titer at different pH values in a 250 ml triangle flask. The pH of fermentation broth was adjusted by acetic acid or sodium hydroxide. The mixture in flask was incubated at 25 ℃on a rotary shaker at 200 r/min for 3 h.The solutions were separated from the resins and the antibacterial activity of supernatant was analyzed by agar diffusion method.

From the Fig.2, it could be seen that adsorption amount of the resins for antibiotics reached 1 221 U/g at the acidic range of pH (pH 5). Therefore,pH 5 was selected as the optimum pH for adsorption experiments.

Adsorption kinetics of resin

Adsorption kinetics experiments were carried out for the resins by using a batch method[17-18]. 10 g of the 717 anion resin was immersed in a 250 ml triangle flask containing 30 ml preparation of fermentation broth. The mixture in flask was incubated at 25 ℃on a rotary shaker at 200 r/min for 4 h.

Fig.3 presents the adsorption kinetics of the resins for antibiotics. The adsorption amount of the resin for antibiotics increased within 2.5 h. It was noted that the adsorption amount of the resins for antibiotics reached equilibrium after 2.5 h and was 1 221 U/g,which further suggested 717 anion resin quickly reached the equilibrium and had good adsorption capacity for antibiotics from M. carbonacea JXNU-1.Therefore,2.5 h was selected as adsorption time for further studied.

Table 1 Results of adsorption capacities and adsorption ratio

Adsorption isotherm of resins

The adsorption activity of resin is often investigated by description of the adsorption isotherm.There are Langmuir model, Freundlich model and Langmuir-Freundlich model in the analysis of adsorption isotherm[19-20].The Freundlich model is widely applied to describe adsorption isotherm curves of the resin under condition that the concentration of adsorbed substance is lower. The Freundlich equation is described as follows:

Where Qeis the equilibrium adsorption capacity(μg/g),Ceis the equilibrium concentration of antibiotics(μg/ml), K and n are the adsorption equilibrium constant.

The adsorption isotherm of the 717 anion resin for antibiotics was generally estimated by Freundlich equation. The adsorption capacity of the resin for antibiotics was increasing under condition that the concentration of antibiotics was increasing. The adsorption isotherm curves of the resin for antibiotics were shown in Fig.4.

The linear regression equation for the experimental dots in Fig.4 was y=2.090 1x -2.475 8. The regression constant was calculated as 0.994 3.The results indicated that the Freundlich model was fit for the adsorption model of the 717 anion resin for antibiotics. The adsorption model of the 717 anion resin for antibiotics was multilayer adsorption.

Conclusions

In this study, a highly efficient method for separating and purifying antibiotics from M. carbonacea JXNU-1 was established. We successfully optimized related parameters and treated fermentation broth from M.carbonacea JXNU-1 with resins. The 717 anion resin was chosen from four alternative resins, and its adsorption capacity reached 1 221 U/g within 150 min. The Freundlich model was fit for the adsorption model of the 717 anion resin for antibiotics.

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