Optimization of Culture Conditions on the Proliferation of Aspergillus Terreus N-GL1 Strain Isolated from Curcuma Longa L. by Design-Expert 6.0.6 and BC Pharsoft Software

Curcuma longa L. is widely used as spices and medicines. Leaves, stems and rhizomes of the plant contain essential oils and provide a useful habitat for different endophytic fungi groups. Strain of Aspergillus terreus was isolated from Curcuma longa L. and was determined to be capable of producing secondary metabolites against bacteria by disc diffusion method and optimization of culture conditions by Design-Expert 6.0.6 and BC Pharsoft software. This research identified of the culture conditions for A. terreus N-GL1 strain produced secondary metabolites against Staphylococcus aureus and MRSA (Methicillin resistant Staphylococcus aureus) good: appropriate carbon source was saccharose 1% or molasses 2,2%; suitable nitrogen sources are potatoes 10% at pH 7; initial amount of yeast cells was 104 CFU/ml; incubated at room temperature for seven day. This study was found out as suitable conditions for A. terreus N-GL1 strain produced the secondary metabolites against S. aureus and MRSA.


Introduction
Endophyte that grow in the deep tissues of the plant, but usually do not cause disease the host plant. Endophytic fungi close relationship with the host, they use the nutrients in the plants to survive, bringing many benefits to plants as generating metabolic products have biological activity as the growth hormone, antibiotics have the ability to protect plants from disease-causing microorganisms. The secretions from the endophyte has antibacterial, antifungal and antioxidant, etc. [7] Some of the fungi are as Aspergillus, Penicillium, Fusarium have produced of many metabolites for biological effects such as antibacterial, antiviral, anticancer, etc. In which A. terreus for some compounds antibiotics such as terremid A, terremid B, terrein. A. terreus also produces enzymes such as invertase and β-glucosidase when grown in submersible fermentation containing barley flour black as carbon source; production of β-xylanase; cis-aconitic decarboxylase (CAD) -an important enzyme which produced of itaconic acid, beta-glucosidase,etc. [9] In this study, A. terreus N-GL1 were isolated from the leaf of Curcuma longa L. (Zingiberaceae family) has identified capable of produced secondary metabolites against S. aureus and MRSA. Research on the impact of environmental conditions on endogenous fungi capable of producing highly active metabolites is a new research direction and potential for future application.A. terreus N-GL1 were isolated from the leaf of Curcuma longa L. (Zingiberaceae family) has been identified capable of produced secondary

The effect of different pH media on the production of antibacterial substance of N-GL1
Surveying the effect of pH media on the production of antibacterial substance by N-GL1 on the PDB, the pH media is adjusted in the range of 3, 4, 5, 6, 7, 8 in static conditions, incubated at room temperature. After 7th days, the culture fluid adjusted to neutral pH, and examined the impact of resistance to S. aureus and MRSA by disc diffusion method. [3] 2.2.3. Screening for endophytic fungi antibacterial metabolites Endophytic fungi culture on PDA from 5-12 days at room temperature were tested for the antibacterial activity against Staphylococcus aureus, E. coli, Streptococcus faecalis, MRSA by disc diffusion method.
Antibacterial assay: The antibacterial activity of the secondary metabolites which were extracted from the culture of endophytes were determined by disc diffusion method. The suitable conditions for endophytes produced antibacterial substance were determined basing on diameter of inhibition zone.
Incubation time on the production of antibacterial substance of N-GL1: N-GL1 were cultured on PDB with the initial amount 10 4 CFU/L, glucose 2% (w/w), potatoes 20% (w/v), incubated at room temperature for static conditions. Monitor antimicrobial substances produced by fungi into the culture medium over time by means of diffusion through after 5, 7, 9, 12 days.

2.2.4.
The effect of carbon source, nitrogen source, aeration, oil on the production of antibacterial substances by Design-Expert 6.0.6 Software N-GL1 were cultured as designed in the same conditions, the amount of the initial spore 10 4 CFU/ml. Determine to the antibacterial substances produced by fungi in culture media from time to time by disc diffusion method. The culture conditions were determined based on diameter of inhibited zones [2], [5], [8], [11].
Vegetable oil was used to supplement the carbon source on the production of antibacterial substances. N-GL1 was determined the composition and culture conditions for high antibacterial activity with the carbon source was molasses 2.7% were added 1% sesame oil, 1% sunflower oil, 1% soybean oil, 1% corn oil, 1% olive oil. Survey antibacterial action S. aureus and MRSA of culture fluid [6], [10]. A. terreus were cultured as designed in the same conditions and processes at room temperature, static conditions. Evaluated of results in antibacterial of culture fluid and biomass obtained at day 7 by means of diffusion through wells. [8] 2.2.6. The optimal culture conditions Data input is the environment and the value parameters yi of recipes 26. Environmental optimization is done by BC Pharsoft software. Verify the optimal environment, 3 batches cultured under optimal culture conditions. Culture fluid was determined the antibacterial diameter and similar biomass obtained in environmental design stage to compare the results from the software.

Effect of pH on the the Production of Antibacterial Production of N-GL1 Strain
The pH of culture medium is one of the determinant factors for the microbial growth, pigment production and biosynthesis of secondary metabolites. N-GL1 strain was grown at different initial pH values (3.0 to 8.0) in static conditions. The pH 7.0 was suitable for the maximum production of antibacterial agent. (Table 1)

Effect of incubation time on the production of antibacterial activity of N-GL1 strain
After seven days incubated at room temperature, A. terreus N-GL1 strain can produce the substances against S. aureus and MRSA in higher level than other days. (Table 2).

Effect of carbon sources, nitrogen sources, aeration, oil on the production of Antibacterial Substances from A. terreus N-GL1
Based on the basic medium with nitrogen source was potatoes 20%, carbon source was glucose 2%, changes nitrogen and carbon source base on equivalent to the nitrogen content of potatoes 20% and glucose 2%. Concentration of carbon and nitrogen sources were presented in Table 3. Using the Design Expert 6.0.6 software to design experiments with x1 variable was source of nitrogen, x2 was carbon source, x3 was ventilation conditions (shaking 200 cycles/min or static). The amount of input is 104CFU/ml, incubation at room temperature. The culture conditions were identified based on diameter of inhibited zones of S. aureus and MRSA after 5, 7, 9, 12 days.
In the medium components, carbon sources, nitrogen sources and aeration conditions were changed by independent variables. The survey by the independent variables are presented in the following table: Saccharose 1% Peptone 1% Static 9 Rice starch 2% Yeast extract 1% 12 Molasses 2.7% Soybean 10% An empirical model was designed by the Design-Expert 6.0.6 software includes environments 23. Environmental data and diameter of inhibited zones (mm) is presented in Table 4. Twenty-three environments were cultured in the same process and experimental conditions. Peptone Impact of oil on the production of antibacterial substances of A. terreus N-GL1: Surveying the influence of oil on the production of antibacterial substances of A. terreus N-GL1, vegetable oils such as sesame oil, sunflower oil, soybean oil, corn oil, olive oil (1% concentration) was added culture media with ingredients including potatoes 20%, molasses 2.7%, in static conditions from culture fluids after seventh days by disc diffusion method. Implemented in parallel with culture fluid of the same composition and culture conditions without additional vegetable oil. (Table 5). Oil-supplemented culture medium have not impact on the production of antibacterial substances by A. terreus N-GL1.

Design and optimization of culture conditions
Experimental models were designed in software Design -Expert 6.0.6 with the following variables: x1: Potatoes concentration 10%, 20%, 30%. x2: Glucose concentration of carbon sources: molasses 2%, 3%, 4%; saccharose 0.5%, 1%, 2%. x3: Carbon source is molasses, saccharose x4: The amount included in the initial yeast with OD: 0.05; 0.1; 0.2. The survey by the independent xi variable was presented in Table 6. Empirical model designed by Design-Expert 6.0.6. software includes 26 environmental. Environmental data, the antibacterial diameter and biomass were presented in Table 7. Twenty-six environments were cultured in the same process and the experimental conditions. Results of R 2 training and R 2 test and try to find an association between components causal formula with properties parameters were reviewed. Results optimized by intelligent software includes BC Pharsoft optimal parameters of culture conditions and the predicted value of the product properties are as follows: Optimal composition formula: y1= 21.344; y2=2.608; Optimal environmental elements include potatoes predicted 10%; molasses 2.2%, OD530nm= 0.14 (1 ml fungal to 100 ml cultures); Verify the optimal recipe empirically, compared with predicted results 3 batch cultures conducted with optimal conditions in the same conditions and determine the parameters yi. (Table 9); Process repetitive culture and nature products between 3 different batches without statistical significance (p = 0.87> 0.05). The result is the average of 3 lots of culture and anticipated results of the different software without statistical significance (p> 0.05). Therefore, the BC Pharsoft software has correctly predicted optimal culture conditions.

Conclusions
This study have surveyed the factors for A. terreus N-GL1 produce secondary metabolites against S. aureus and MRSA (pH media, vegetable oil, carbon source, nitrogen source, ventilation conditions and incubation time). The results show that in 23 test culture medium with different sources of nitrogen and carbon, A. terreus N-GL1 for antibacterial substances highest in culture media as nitrogen source was potatoes, carbon source was saccharose or molasses. Using software components Design Expert 6.0.6 and BC pharsoft predicted optimal culture for A. terreus N-GL1 produce antibacterial substances highest: nitrogen source was potatoes 10%; source of carbon was molasses 2.2%; initial amount of yeast cells was 10 4 CFU/ml; incubated in static conditions for seventh days. Identifying antibacterial efficacy by diffusion method showed that the experimental results fit predictions of software.