The Substrate For Antibiotic Production Biology Essay

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Acremonium chrysogenum MTCC 431 were procured from Chandigarh and maintained on potato dextrose agar medium slant. Sub culturing was done at subsequent intervals.

Substrate for Antibiotic Production

Commercial quality of wheat rawa, Bombay rawa and rice bran, were procured from a local market. They were used as the solid substrate and were assessed to study their effect on the production of cephalosporin c. The best solid substrate achieved by this step was fixed for subsequent experiments. In solid state fermentation different substrate used for the production of cephalosporin c, such as wheat rawa (100g), Bombay rawa (100g), barley (100g) and Rice bran (100g).

Salt solution

Salt solution is used as an additional nutrient in the production medium. [K2HPO4-0.5g/l, MgSO4 7H2O-0.5g/l, FeSO4 7H20-0.5g/l, Nacl-0.5g/l]

Methods

Solid State Fermentation

Static experiments were in each 500 ml completely dried conical flask containing 100g of each substrate. 10ml of salt solution was added in each flasks containing substrate and distilled H2O was added and the moisture content was made to 80%. The flasks were autoclaved at 121ËšC at 15psi for 20 minutes.

Estimation of Moisture Content

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The moisture content of the solid substrate (wheat rawa, Bombay rawa, Barley and rice bran) was estimated by drying 10 g of solids to constant weight at 105°C and the dry weight was recorded. To fix the initial moisture content of the solid medium, wheat rawa was soaked with the desired quantity of water. After soaking, the sample was again dried and percent moisture content was calculated as follows,

After sterilization by autoclaving flasks were cooled and inoculated with a 5% inoculum under appropriate experimental conditions.

Optimization

Factors such as initial moisture content (50, 60, 70 and 80 %), inoculum level (5, 10, 15 and 20 %), incubation temperature (25oC, 30oC, 37oC and 50oC), inoculum age (2, 4, 6 and 8 days), incubation time (6, 8, 10 and 12 days), initial pH (varies from 5.0 - 8.0 with 1N HCl or 1N NaOH), various carbon sources (maltose, fructose, sucrose, starch, rhamnose and lactose), nitrogen additives (tryptone, peptone, yeast extract and casein) and the different inorganic nitrogen sources (sodium nitrate, ammonium sulphate, potassium nitrate and dipotassium hydrogen phosphate) are influencing in the secretion of cephalosporin C antibiotic by Acremonium chrysogenum MTCC431 under solid state fermentation were optimized by varying parameters one at a time.

Extraction of cephalosporin c from fermented medium:

At the end of fermentation period, 10g of fermented wheat rawa, barley, Bombay rawa, Rice bran, was taken. To which 10ml of distilled water was added and stirred by glass rod thoroughly.

The contents were filtered by muslin cloth and centrifuged at 10,000 rpm for 15 minutes. The liquid above was decanted until 10 ml of filtrate was obtained later on the filtrate was subjected to analysis by antibiotic assay techniques and the cephalosporin c content was estimated.

Estimation of Cephalosporin C Concentration by Spectrophotometer Method

Preparation of Working Standard

An accurately measured 10mg of the drug was transferred into a beaker with 50 ml distilled water sonicated for 5 min and filtered into a 100ml volumetric flask then completed to volume with distilled water.

Procedure

The drug sample is allowed to react with 2.5ml of iron (III) nitrate in a 25ml of potassium hexaferrocyanate (III), heating in a water bath at 90°C for 60 min then completed to volume with distilled water. The absorbance at 777nm against a blank solution is treated similarly. Calibration graphs were prepared by plotting the absorbance against the drug concentration.

Antibiotic sensitivity test Assay

Melted Muller- Hinton agar medium was added to each sterilized petri dish and allowed to solidify. The test organism was swabbed on the petri plates evenly. The test organisms used are Staphylococcus aureus, E. coli, Proteus sp, Klebsiella sp, Pseudomonos sp.

The supernatant collected from fermented medium used as the antibiotic source. The sterile paper discs (6mm) integrated with supernatant and were placed aseptically. The plates were incubated at 35Ëšcfor 24hr. The zone of inhibition was measured and compared with standard drug.

Identification, Separation and Purification of Cephalosporin C

Thin Layer Chromatography method is used for identification of cephalosporin C by Nabi et al., 2004.

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Since cpc is heat labile, chromatography was performed at room temperature.. Antibiotic were quantities by using a Shimatzu liquid chromatography with data analysis system, utilizing UV detection at 254 nm. Antibiotics were separated with a Shimatzu C-18 10µm particle µ- bondapak reverse phase column (30 cm by 4 mm) including 10 µm particle C-18 precolumn packing (4 cm by 4mm) to predict the integrity of the analytical column. An aqueous / organic mobile phase consisted of 25 mM Dihydrogen potassium phosphate - 80 % / Methanol - 20 % at a flow rate of 1.5.ml/min.

RESULT AND DISCUSSION

The solid state fermentation process has been observed to be less sensitive to contamination than submerged fermentation. In solid state fermentation, the selection of a suitable solid substrate for a fermentation process is a critical factor and thus involves the screening of a number of agro-industrial materials for microbial growth and product formation (Ellaiah, 2003).

Solid State Fermentation

Culture and substrate selection

In the present study, four substrates, viz. wheat rawa, rice bran, Bombay rawa, and barley were used for the growth and cephalosporin c production by Acremonium chrysogenum MTCC431.(Plate-6.3.2)

Estimation of moisture content

Adinarayana et al., 2003 worked with different moisture content of solid state fermentation and found to be the maximum yield of 4445µg/g were obtained with 80 percentage of moisture. There study shows that 80 percentages is suitable moisture content of solid state fermentation by using the same substrates used in this study.

The critical importance of moisture level in solid state fermentation media and its influence on the biosynthesis and secretion of antibiotics can be attributed to the interference of moisture in the physical properties of the solid particles.

The amount of antibiotic yield produced in the present research was 2000µg/g in 80% of moisture content.

Cephalosporin C production by solid state fermentation

In the present investigation the different substrates supported growth and antibiotic formation by the culture, while wheat rawa was proved superior to other substrates. The maximum yield of cephalosporin C (2000µg/g) was obtained in a medium containing wheat rawa alone as the substrates followed by Barley (1900µg/g), Bombay rawa (1000 µg/g) and Rice bran (800µg/g). This result showed in (Figure 6.2.1).

Adinarayana 2003, reported that high titer of cephalosporin (2805µg/g) was obtained in a medium containing wheat rawa alone as the substrate followed by Bombay rawa, Barley and Rice bran. Hence, wheat rawa was selected and used for subsequent studies. In the present investigation, the production of Cephalosporin C was less when compared to author yield, because various factors to decrease the Cephalosporin C production, such as quality of the substrate, time of incubation period, the percentage of moisture content and inoculum level.

5.1.5. Reducing sugar estimation

The reducing sugar estimation proved that high amount of reducing sugar in Acremonium chrysogenum were inoculated on wheat rawa yield (600µg/g) on medium followed by Rice bran (400µg/g), Bombay rawa (350µg/g) and Barley (300µg/g), (Table-6.1.2)

Antibiotic Sensitivity Test Assay

Antibiotic sensitivity test Assay proved that both Gram positive and Gram negative bacteria are sensitive to Cephalosporin C. The Antibiotic Sensitivity Test Assay using both gram positive and Gram negative bacteria such as S.aureus, Proteus sp, Klebsiella sp, Pseudomonas sp, E.coli. In solid state fermentation Wheat rawa showed the maximum zone of inhibition 30mm in diameter against S.aureus. (Figure 6.2.2.a)

TLC for separation and identification of cephalosporin C

The TLC method is simple, rapid, selective, reproducible and applicable to separation and determination of CPC. In the present investigation the hRf value of cephalosporin after chromatography for solid state fermentation (hRf - 72) have similar hRf value for standard antibiotic (hRf- 72.22) and as well as sample. (Fig 2)

Purification and quantification of cephalosporin C by HPLC

HPLC analysis can be considered specific quantification is based upon absorption of UV radiation by the chromatographically resolved antibiotic. In this present investigation HPLC was performed for purified and quantitative measurement proved that the produced antibiotics was cephalexin (Fig: 1a and 1b).

Table (5) showed the retention time (8.2 mins) of both standard as well as sample has similar value. The quantification of sample was about 0.032 µg/g.

Discussion

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The solid state fermentation process has been observed to be less sensitive to contamination than submerged fermentation. In solid state fermentation, the selection of a suitable solid substrate for a fermentation process is a critical factor and thus involves the screening of a number of agro-industrial materials for microbial growth and product formation (Ellaiah, 2003).

Adinarayana et al. 2003 worked with different moisture content of solid state fermentation and found to be the maximum yield of 4445 µg/g were obtained with 80 percentage of moisture. Their study shows that 70 % is suitable moisture content of solid state fermentation by using the same substrates used in this study. The critical importance of moisture level in solid state fermentation media and its influence on the biosynthesis and secretion of antibiotics can be attributed to the interference of moisture in the physical properties of the solid particles. Naggar et al. (2008) worked with moisture content of solid fermentation and found to be the maximum yield of 450 μg/g was achieved when the initial moisture level was 60 %.

Inoculum level was also an important factor for the production of cephalosporin C. The present study concludes a lower inoculum density may give insufficient biomass causing reduced product formation, whereas, a higher inoculum may produce too much bio mass leading to the poor product formation. Similarly, Lee et al. (2001) reported that maximum concentration of CPC (1.9 g/l) obtained 6th day, which the highest level was obtained with any of the inoculum. It was observed that the strain produced the antibiotic in similar inoculum age as compared to the reported data.

As the metabolic activities of the microorganisms were very sensitive to change in pH level was higher or lower when compared to the optimum level. Adinarayana et al. (2003) reported that the maximum production of CPC (7045 μg/g) was obtained at pH 6.5. In the present investigation indicated that the maximum production of CPC (800 μg/g) obtained at pH 6.

Nigam et al. (2006) reported that ammonium sulphate was observed to be the best inorganic nitrogen for the higher production of CPC. While, when different inorganic nitrogen sources were added to the synthetic production medium. It has been found that only a few are used by mold for maximum synthesis of CPC. Others are not utilized by mold at the same extent. Moreover, high content of nitrogen was found to decrease the production. It might be due to the reason that if interferes to the process of differentiation of mycelium to swollen hyphal fragments and arthrospores during the production age.

Nigam et al. (2007) reported that highest productivity (3 g/l) was achieved at 28o C for 144 hrs on the synthetic production medium. An increase in the biomass concentration also causes an increase in phase volume. It has been observed that broth density varies with the age of cephalosporin fermentation.

Similarly, Matsumura et al. (1978) studied on the fermentation of cephalosporin C production during early fermentation density approximately 1.150 g/l and increased upto 1600 g/l at 60 hrs. Kim et al. (2005) studied the experiment by higher nutrient and the yield was estimated that 0.598 g/l were obtained at 8th day of incubation.

The present research concludes that ammonium sulphate inoculated medium gave high antibiotic production when compared to others, because ammonium uptake rate quickly increased when compared to the others. Similarly, Toll nick (2004) reported that the medium fed with ammonium sulphate as inorganic nitrogen additive gave 15.9 g l-1 of CPC antibiotic production.

Nabi et al. (2004) reported that the TLC was performed with different cephalosporin compound. Among all the antibiotics, cephalexin has (hRf - 72.22) similar hRf value were obtained in our present data conforms the produced antibiotic was cephalexin.