Optimization And Enhanced Production Of Hygromycin B

Published Date: 02 Nov 2017

Abstract

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 current study, four substrates such as 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.

Introduction

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 development of resistance to aminoglycoside antibiotics is principally on the origin of enzymes that inactivated by phosphorylation, adenylation or acetylation. The three groups of aminoglycoside modifying enzymes are responsible 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 Streptomyces is designated as School of Chemical and Biotechnology (SCBT), capable of suppressing the growth of Gram-negative and Gram-positive bacteria widely. 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 the dynamic effect on both prokaryotic and eukaryotic cells by affecting the polypeptide synthesis. It stabilizes 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. The final moiety is bound by orthoester formation between the group and destomic acid. 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 process is free of water, and near to the natural environment to which microbes are adopted. The solid state fermentation has been used for the enzyme and secondary metabolites production.

Various studies of solid state fermentation factor likely 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 and optimization of the bioactive compounds.

MATERIALS AND METHODS

Microorganism

Streptomyces hygroscopicus MTCC 1105 were acquired 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 purchased from a local market. Each 10g of solid substrates were used for the production of the Hygromycin B under solid state fermentation.

Salt solution

In addition to nutrient, 1 ml of the salt solution (K2HPO4-0.5 g/l, MgSO4.7H2O-0.5 g/l, FeSO4.7H2O-0.5 g/l, Nacl-0.5 g/l.) was added to the each substrate.

Optimization of fermentation process under Solid State fermentation

Factors includes 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 under solid state fermentation were optimized by varying parameters one at a time.

Estimation of moisture content

Drying 10 g of solid substrate to constant weight at 80°C and dry weight was analyzed. Fixing of the initial moisture content is performed by soaking the substrate with known quantity of water and drying it again and 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 the fermentation was adjust the initial moisture content (50, 60, 70 and 80%) of each substrate with distilled water. The 5% inoculum was added and the fermentation was carried out for 4 days at 28°C.

Effect of incubation temperature

The fermentation process was carried out at various temperatures such as 25°C, 28°C, 37°C and 50°C.

Effect of incubation period

Various incubation periods (4, 6, 8 and 10 days) were employed to study their effect of Hygromycin B production. The fermentation was carried out 28°C and pH 7.

Effect of pH

The pH of the basal medium varies from 5.0 - 8.0 with 1N HCl or 1N NaOH. The fermentation was carried out at 28°C for 4 days.

Effect of carbon source

The different carbon source like maltose, fructose, starch, dextrose and lactose were optimize the effect of Hygromycin B production keeping all other conditions on their optimum level.

Effect of nitrogen source

(1) Effect of supplementary nitrogen source

The different supplementary nitrogen sources such as tryptone, peptone, yeast extract and casein were optimized the effect of Hygromycin B production.

(2) Effect of inorganic nitrogen source

Different inorganic nitrogen sources as sodium nitrate, ammonium sulphate, potassium nitrate and dipotassium hydrogen phosphate were optimized for the production of Hygromycin B.

Analytical methods for solid state fermentation

1. Antibiotic extraction

After 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. Equal volume of filtrates was extracted with ethyl acetate using separating funnel. The crude extract is obtained and tested for its 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. The extracted antibiotic was impregnated into sterile disc at various concentrations were transferred to the test plates and incubated at 24-48 hours. The zones of inhibition were measured.

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

Antibiotic study was carried out using a Shimadzu liquid chromatography with data analysis system, utilizing UV detection at 254 nm. Antibiotics were separated with a Shimadzu C-18 reverse phase column (30 cm by 4 mm) with relevant pre column to predict the integrity of the compound. A suitable organic aqueous mobile phase at flow rate of 1.5 ml/ min used.

RESULT AND DISCUSSIONS

Solid state fermentation

The selection of solid substrate is an important and decisive factor for the solid state fermentation and involved in the screening and renewable source of agricultural waste for the growth of microbes and as well as for the formation of industrially important products.

Optimization of fermentation process under solid state fermentation

1. Effect of various moisture content

The moisture level of slid particles in solid state fermentation media attributes the significance releasing of the secondary metabolites by the microbes.

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 followed by 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 on different moisture content

The antibiotic sensitivity test proved both gram positive and gram negative microorganism are sensitive to Hygromycin B at 60% of 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 at 60% of 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 as substrate at 28ºC 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 at 25°C, 28°C, 37°C and 50°C. The optimal temperature at 28°C showed 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 at various incubation periods

Fig (1.c) explained that various incubation period showed the significant effect of Hygromycin B. Among the different incubation period, 6 days of incubation gave maximun amount of Hygromycin B (552.6 µg/g) on bombay rawa and followed by wheat rawa (510 µg/g), rice bran (486 µg/g) and barley (450 µg/g).

3. b. Antibiotic sensitivity test assay at various incubation periods

The antibiotic produced at among the different substrates, 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 was found on 10th day of incubation.

4. Effect of pH

4. a. Estimation of Hygromycin B production of various pH

The effect of pH is an important factor for production of Hygromycin B. In the present research concludes pH 7 showed the maximum yield of Hygromycin B (451.2 µg/g) was achieved by S. hygroscopicus on bombay rawa 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).

5. Effect of carbon source

5. a. Estimation of Hygromycin B production using various carbon sources

Among the different sugars (1 % w/w) as additives to bombay rawa result in better production with dextrose (930 µg/g) and the array of 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 using various carbon sources

Among the substrates, bombay rawa inoculated with dextrose as additive showed the 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).

6. Effect of nitrogen source

6.1 Effect of supplementary nitrogen source

6. 1. a. Estimation of Hygromycin B production with different 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 with different nitrogen source

The maximum zone of inhibition showed on bombay rawa with yeast extract as supplementary nitrogen additives against K. pneumoniae (27 mm), followed by S. aureus (16 mm), E .coli (17 mm), P. aeruginosa (24 mm) and B. subtilis (20 mm) (Table:1.6).

6.2. Effect of inorganic nitrogen source on Hygromycin B production

6.2.a. Estimation of Hygromycin B production using inorganic nitrogen source

The different inorganic nitrogen sources were tested and the highest antibiotic production (586.8 µ/g) was obtained on bombay rawa 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 using 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).

7. Purification and quantification of Hygromycin B by HPLC

HPLC analysis can be considered as specific quantification by the absorption of UV radiation by the chromatographically resolved antibiotic. In this present investigation HPLC was performed for purifying and quantifying the antibiotic 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.

Conclusion

The production of secondary metabolites are high value products of use in different pharamaceutical industries. 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 at 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 and ammonium sulphate as the inorganic nitrogen.

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 the strain and can be used for higher Hygromycin B through fermentor vessel. These substrates are also use in pharmaceutical industries to manufacture Hygromycin B. Further, fermentators such as airlift and solid state fermentor may also be used to increase the production through optimization.