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Hygromycin B is one of the aminoglycoside antibiotics which destroy bacteria, fungi and higher eukaryotes by affecting proteins synthesis. The antibiotic interferes with translocation and cause mistranslation at the 70S ribosome. Hygromycin B is produced by the Streptomyceshygroscopicus. The most unique biological activity of Hygromycin B is its antiparasitic effect in swine. However, the quantities required to prevent growth of sensitive organism in vitro are large compared with those of most therapeutically useful antibiotics.
In the present study four substrates like wheat rawa, bombay rawa, rice bran and barley were screened for the ability to produced Hygromycin B under solid state fermentation. The substrates, bombay rawa produce the highest yield when compared to other substrate in solid state fermentation. The present research showed the maximum yield was obtained in bombay rawa as 966 µg/g. Similarly the moisture 60 % showed 495µg/g, pH 7 as 451.2 µg/g, temperature 28°C produces 495 µg/g, incubation period 6 days of 552.6 µg/g, 1% w/w soluble dextrose of 930 µg/g, yeast extract of 585 µg/g and ammonium sulphate of 586.8 µg/g were obtained and optimized. Antibiotic sensitivity test assay proved that both gram positive and negative microorganisms are sensitive to Hygromycin B. Among the substrates, bombay rawa showed the maximum zone of inhibition in (35mm) diameter against Klebsiella pneumoniae. These results showed the way to the pharmaceutical industries for the development of products under solid state fermentation.
Naturally the most of the strains which produces antibiotics are generally exhibiting the resistance to the antibiotics which they produced. They bind to specific sites on the ribosome and affect the ribosomal translation cycle. The modes of action of some antibiotics are now well recognized. The development of resistance through the mechanism of modifying the target region like erythromycin, thiostrpton and also self modification of antibiotics by enzymatic has been recorded for a number of aminocyclitols.
The resistance to aminoglycoside antibiotics is mainly on the basis of enzymes that inactivated by phosphorylation, adenylation or acetylation. The three families of aminoglycosidemodifying enzymes are accountable for antibiotic resistance by ATP-dependent O-phosphotransferases (APH), ATP-dependent O-adenyltransferases (ANT) and acetyl-CoA-dependent N-acetyltransferases (AAC).
Our early studies reported that strain Streptomyces is designated as School of Chemical and Biotechnology (SCBT), capable of inhibiting the growth of a wide range of Gram-negative and Gram-positive bacteria. Hence the present study orients towards the utilization of Streptomyces for the production of Hygromycin B.
Hygromycin B is one of the aminoglycoside antibiotics which are produced by the Streptomyces hygroscopicus. It has dynamic effect on both prokaryotic and eukaryotic cells by inhibiting the polypeptide synthesis. It stabilize the tRNA ribosomal acceptor site and stops the translocation process. The aminocyclitols, N-methyl-2 deoxy streptamine, is linked by a β-glycosidic bond to the talose sugar. This latter moiety is bound by orthoester formation between the group and destomic acid. Hygromycin B is weakly basic. However, the obvious mechanism of hygromycin B to arrest the protein synthesis by the ribosome is not known even though with the years of passionate study.
When compare to submerged fermentation, the Solid state fermentation is yielding higher amount of secondary metabolites. The solid state fermentation takes place on the absence or near absence of free water, thus being close to the natural environment to which microorganism are adopted. The solid state fermentation has been used for the enzyme and secondary metabolites production.
Various solid state fermentation parameters such as pH, temperature, inoculum size, incubation time, carbon sources and nitrogen sources were analyzed. Four different substrates banana peel, garlic peel, wheat bran and Rice bran were used. The earlier study revealed that a modified solid-state fermentation was used to produce mevastatin by Penicillium citrinum NCIM 768 recycling wheat bran as carrier. Hence, this paper describes the production of hygromycin B and its partial HPLC analysis of culture filtrate and partial characterization of the bioactive compounds.
MATERIALS AND METHODS
Streptomyces hygroscopicus MTCC 1105 were procured from the Institute of Microbial Technology (IMTECH) Chandigarh, India and maintained on ISP2 agar slant. Sub culturing was done by the subsequent intervals.
Substrate for antibiotic production
Commercial quality of wheat rawa, bombay rawa, barley and rice bran were procured from a local market. They were used as the solid substrate for the production of the Hygromycin B. In solid state fermentation different substrate used for the production of Hygromycin B, such as wheat rawa (10 g), bombay rawa (10 g), rice bran (10 g) and barley (10 g).
In addition to nutrient the salt solution 1 ml add to the each substrate, the composition of salt solution K2HPO4-0.5 g/l, MgSO4.7H2O-0.5 g/l, FeSO4.7H2O-0.5 g/l, Nacl-0.5 g/l.
Optimization of fermentation process under Solid State fermentation
Factors including selection of solid substrate, initial moisture content, incubation time, incubation temperature, initial pH and various carbon and nitrogen source additives are influencing the secretion of Hygromycin B antibiotic by Streptomyces hygroscopicus MTCC 1105 under solid state fermentation were optimized by varying parameters one at a time.
Estimation of moisture content
The moisture content of the solid substrate (wheat rawa, bombay rawa, rice bran and barley) was estimated by drying 10 g of solids to constant weight at 80°C and the dry weight was recorded. To fix the initial moisture content of the solid medium the substrate was soaked with the desired quantity of water. After soaking the sample was again dried and present moisture content was calculated as follows.
Present of moisture content (initial) of solid medium = (wt. of the substrate- dry wt.) - 100/ dry wt.
Effect of moisture content
To observe the initial moisture content of the substrate (wheat rawa, bombay rawa, barley and rice bran) the fermentation was carried out under various initial moisture content (50, 60, 70 and 80%) of each substrate, which was adjusted with distilled water. The other conditions were 5% inoculum level and the fermentation was carried out for 4 days at 28°C.
Effect of incubation temperature
The fermentation process was carried out in the various temperatures such as 25°C, 28°C, 37°C and 50°C and to study their effect on antibiotic production for 4 days.
Effect of incubation period
Various incubation periods (4, 6, 8 and 10 days) were employed to study their effect on Hygromycin B production. The fermentation was carried out 28°C and pH 7.
Effect of pH
pH of the basal medium varies from 5.0 -8.0 with 1N HCl or 1N NaOH. The fermentation was carried out 28°C to study their effect on antibiotic production.
Effect of carbon source
The different carbon source were used to the optimize the effect of Hygromycin B production, such as maltose, fructose, starch, dextrose and lactose, keeping all other conditions at their optimum level.
Effect of nitrogen source
(1) Effect of supplementary nitrogen source
The different supplementary nitrogen sources were used to optimize the effect of Hygromycin B production, nitrogen sources such as tryptone, peptone, yeast extract and casein.
(2) Effect of inorganic nitrogen source
Different inorganic nitrogen sources were used to optimize the effect on Hygromycin B production. Inorganic nitrogen sources such as sodium nitrate, ammonium sulphate, potassium nitrate and dipotassium hydrogen phosphate.
Analytical methods for solid state fermentation
1. Antibiotic extraction
At the end of the fermentation period the substrate was centrifuged at 6000 rpm for 30 min and the cell free supernatant was filtered through whatman No.1 filter paper. To filtrates equal volume of solvent ethyl acetate were added and kept in separating funnel. The crude extract is obtained and tested for antimicrobial activity against pathogens.
2. Antibiotic sensitivity test assay
Muller Hinton agar medium was poured to the each sterilized petriplates and allowed to solidify. The test organisms such as Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Bacillus subtilis and Pseudomonas aeruginosa are swabbed in the petridish. The antibiotic extracted was used as the antibiotic source. The sterilized discs are impregnated into the various concentrations of antibiotic extracts and were transferred to the test plates and incubated at 24-48 hours. The zones of inhibit were measured and taken as the activity against the test pathogen.
3. Absorption maximum on Hygromycin B
The absorption maximum of (µ max) the Hygromycin B was identified as 210 nm by variable scanning mode using UV-Vis spectrophotometer. Similarly the samples were analyzed by 210 nm and it was quantify with respect to standard graph plot with different concentration of standard.
4. Purification and quantification of antibiotic from HPLC
Chromatography analysis of antibiotics introduces a powerful tool for drug monitoring as well as clinical research. Chromatography was performed at room temperature. Since Hygromycin B is heat stable. 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 10 µm particle µ- bondapak reverse phase column (30 cm by 4 mm) including 10 µm particle C-18 precolumn packing (4 cm by 4 mm) to predict the integrity of the analytical column. An aqueous / organic mobile phase constited of 25 mM Dihydrogen potassium phosphate-80% / Methanol- 20% at a flow rate of 1.5 ml/ min.
RESULT AND DISCUSSIONS
Solid state fermentation
In solid state fermentation, the selection of a suitable solid substrate for fermentation process is a critical factor and thus involves the screening of a number of agro-industrial materials for microbial growth and product formation.
Optimization of fermentation process under solid state fermentation
1. Effect of various moisture content
The critical importance of moisture level in solid state fermentation media and its influence on the secondary metabolites can be attributed to the interference of moisture in the physical properties of the solid particles.
1. a. Estimation of Hygromycin B production
In the present research concludes 60% moisture gave the maximum yield of 495 µg/g on bombay rawa inoculated medium followed wheat rawa 363 µg/g, rice bran 433.8 µg/g, barley 392.4 µg/g (Fig: 1.a).
1. b. Antibiotic sensitivity test assay for various moisture content
The antibiotic sensitivity test proved both gram positive and gram negative microorganism are sensitive to Hygromycin B into the 60% moisture content (Table:1.1), showed that maximum zone of inhibition (26 mm) in diameter against K. pneumoniae and P. aeruginosa followed by S. aureus (19 mm) , E. coli (19 mm) and B. subtilis (24 mm).
Nagger et al. (2009) reported the maximum zone of inhibition (24 mm) in diameter against the S. aureus in the 60% moisture level.
2. Effect of incubation temperature
2. a. Estimation of Hygromycin B production
In the present research concluded that maximum yield of Hygromycin B (495 µg/g) was achieved by S. hygroscopicus inoculated on bombay rawa medium at 28°C followed by wheat rawa (451.2 µg/g), rice bran (405 µg/g) and barley (432.6 µg/g) (Fig: 1.b).
2. b. Antibiotic sensitivity test assay for incubation temperature
The effect of temperature was studied such as 25°C, 28°C, 37°C and 50°C. The optimum temperature of 28°C was produce the maximum zone of inhibition (24 mm) in diameter against K. pneumoniae followed by S. aureus (14 mm), E. coli (16 mm), P. aeruginosa (17 mm), B. subtilis (12 mm) in the substrate bombay rawa ( Table:1.2 ).
Oskay (2009) found the maximum zone of inhibition (18 mm) in diameter against the S. aureus in the 30°C temperature.
3. Effect of incubation period
3. a. Estimation of Hygromycin B for various incubation periods
Fig (1.c) explained that various incubation period shows the significant effect on Hygromycin B production. All the incubation period was studied, 6 days of incubation gave high amount of Hygromycin B (552.6 µg/g) on bombay rawa inoculated medium followed by wheat rawa (510 µg/g), rice bran (486 µg/g) and barley (450 µg/g).
3. b. Antibiotic sensitivity test assay for various incubation periods
Antibiotic, among the substrate bombay rawa showed the maximum zone of inhibition in (20 mm) diameter against K. pneumoniae and P. aeruginosa followed by S. aureus and E. coli (14 mm), B. subtilis (17 mm) (Table: 1.3).
The maximum zone of inhibition (24 mm) in diameter against the B. subtilis in the 10 days of incubation period.
4. Effect of pH
4. a. Estimation of Hygromycin B production of various pH
The effect of pH on Hygromycin B production was an important factor for production of Hygromycin B. In the present research concludes pH 7 of the maximum yield of Hygromycin B (451.2 µg/g) was achieved by S. hygroscopicus on bombay rawa inoculated medium followed by wheat rawa (420.6 µg/g), rice bran (409.8 µg/g) and barley (403.2 µg/g) (Fig: 1.d ).
4. b. Antibiotic sensitivity test assay for various pH
The effect of pH for antibiotic sensitivity test assay proved that pH 7 show the maximum zone of inhibition (26 mm) in diameter against K. pneumoniae followed by S. aureus (20 mm), E. coli (17 mm), P. aeruginosa (24 mm) B. subtilis (19 mm) in the substrate bombay rawa (Table: 1.4).
The effect of pH and antimicrobial metabolite production by the strain P. aeruginosa, the optimum pH was 7.0. The zone of inhibition was 23 mm.
5. Effect of carbon source
5. a. Estimation of Hygromycin B production for various carbon sources
Addition of different sugars (1 % w/w) as additives to bombay rawa result in better Hygromycin B production with dextrose (930 µg/g) followed by maltose (769 µg/g), fructose (822 µg/g), lactose (916.8 µg/g) and starch (606 µg/g) (Fig:1.e ).
5. b. Antibiotic sensitivity test assay for various carbon sources
Among all the various substrates, bombay rawa inoculated with dextrose as additive medium showed that maximum zone of (22 mm) in diameter against K. pneumoniae followed by S. aureus (18 mm), E. coli (13 mm), P. aeruginosa (19 mm) and B. subtilis (14 mm) (Table: 1.5).
The impact of different carbon sources and antibiotic production by the strain against P. aeruginosa, the carbon sources was maltose. The zone of inhibition was 14 mm.
6. Effect of nitrogen source
6.1 Effect of supplementary nitrogen source
6. 1. a. Estimation of Hygromycin B production for various supplementary nitrogen sources
The highest Hygromycin B production (585 µg/g) was obtained on bombay rawa with yeast extract as the nitrogen additives, whereas, wheat rawa (519 µg/g), rice bran (480.6 µg/g) and barley (435 µg/g) (Fig: 1.f.).
6.1.b. Antibiotic sensitivity test assay for supplementary nitrogen source
The maximum zone of inhibition (27mm) showed on bombay rawa with yeast extract as supplementary nitrogen additives against K. pneumoniae (27 mm), S. aureus (16 mm), E .coli (17 mm), P. aeruginosa (24 mm) and B. subtilis (20 mm) (Table:1.6).
The effect of nitrogen sources on antimicrobial metabolites production by the strain against P. aeruginosa, the nitrogen source was yeast extract. The zone of inhibition was 21 mm.
6.2. Effect of inorganic nitrogen source on Hygromycin B production
6.2.a. Estimation of Hygromycin B production for inorganic nitrogen source
When a number of inorganic nitrogen sources were tested using as a various substrate, showed that highest antibiotic production (586.8 µ/g) obtained on bombay rawa inoculated with ammonium sulphate followed by potassium nitrate (516 µg/g), sodium nitrate (534 µg/g) and diammonium hydrogen phosphate (450 µg/g) (Fig: 1.g).
6.2. b. Antibiotic sensitivity test assay for various inorganic nitrogen sources
The ABST assay proved that bombay rawa added with ammonium sulphate medium gave maximum zone of inhibition (23 mm) in diameter against K. pneumoniae and S. aureus, E. coli (22 mm), P. aeruginosa and B. subtilis (21 mm) (Table: 1.7).
The effect of inorganic nitrogen sources on antimicrobial metabolites production by the strain against the P. aeruginosa, the inorganic nitrogen source was sodium nitrate. The zone of inhibition was 10 mm.
7. Purification and quantification of Hygromycin B 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 antibiotics was Hygromycin B (Fig: 2.a and Fig: 2.b)
Table (2) showed the retention time 6.14 mins of standard as well as sample has 6.12 min; the quantification of sample was estimated about 500 µg/ml (or) 0.5 mg/ml.
The production of secondary metabolites are high value products of use in different industries fields. The optimiztion studies for the production Hygromycin B was carried out by soild state fermentation. In solid state fermentation the optimum productivity of Hygromycin B (966 µg/g) was achieved by employing bombay rawa and with optimized process parameters such as moisture content of solid substrate 60%, incubation temperature 28°C, 6th day of incubation period, pH 7, soluble dextrose as additive (1% w/w) and 1% w/w yeast extract as additivites and ammonium sulphate as the inorganic nitrogen additive.
Antibiotic sensitivity test assay proved that both gram positive and negative microorganisms are sensitive to Hygromycin B. Among the substrates, bombay rawa showed the maximum zone of inhibition in (35mm) diameter against K. pneumoniae. This research concludes solid state fermentation is the cheapest and empirical technology for the pharmaceutical industries.
Moreover, Hygromycin B production is possible through some other cheapest substrate as wheat bran, sweet potato, rice and others. r- DNA technology is to be used for the S. hygroscopicus to mutate their strain and can be used for higher Hygromycin B fermentor. These substrates are also use in pharmaceutical industry for Hygromycin B production. Further, fermentators such as airlift and solid state fermentor may also be used to increase the production through optimization.