Calibration Graph

Different volumes (5 µl, 10 µl, 15 µl, 20 µl, and 25 µl) of the three extracts were loaded onto 6x6 mm filter paper disks and allowed to dry and antibiotic activity determined as before.

Two bacteria were used to create the calibration curve (S. aureus ATCC 25923 strain and E. coli ATCC 10536).

2.17: Purification and determination of the structure of the antibiotics

2.17.1High Performance Liquid Chromatography: Purification of the antibiotics for Es1 extract

In this study, reverse phase, analytical and semi-preparative, high performance liquid chromatography (HPLC Agilent Technologies 1220 Infinity LC) was used for

fractionation and purification of the extracts. Reverse-phase (RP) was chosen as this method works best for low molecular mass molecules as well as neutral compounds (Cseke et al., 2016). The success of HPLC separations in this study depends on the selection of mobile phases (solvent) and column type. Isocratic elution was used in this study to choose the best column and solvent system to use for fractionation and purification of the extracts, according to the study conducted by (Friesen et al., 2015), this method is easy to perform, reliable, and sufficient for separations, gradient elution was then used with semi-preparative column (Hichrom Ltd, UK) for collecting

fractions with antimicrobial activity.

Solvent systems

HPLC (Agilent Technologies 1220 Infinity LC), analytical column, (HP Hewlett Packard Spherisorb ODS-2 5µm 250 mm x 4.6 mm I.D) was used. The separations were conducted by injecting 20 µl of the extract (from isolates Es1 and M1 11) onto the analytical column. Six solvent systems were used in this study for optimising the analytical RP-HPLC parameters. All systems used isocratic elution with different mobile phases (water: methanol 70:30 (v/v), water: methanol 50:50 (v/v), water: methanol 40:60 (v/v), water: methanol 30:70 (v/v), 30:70 (v/v) of water: methanol containing 0.1 % trifluoroacetic acid
 (TFA) and a ratio 10:90 (v/v) of water: methanol).

Chromatograms were obtained using a UV-VIS HPLC detector (Agilent 1200 series, G1311B), the solvent system that gave best separation was used and the peaks of the chromatogram collected separately (called fractions). One mililitre of each fraction was placed on to two 6 mm filter paper disks in a petri dish in a fume cupboard for 24 h to evaporate the solvent. The filter paper disks were placed on MHA plates seeded with E.coli ATCC 10536 and S.aureus ATCC 25923 and incubated at 37°C to identify the fractions containing compounds with antimicrobial properties.

Columns

Three other analytical columns, ACE Excel 5 C-18 amide, ACE Excel 5 SuperC18 and ACE Excel 5 CN-ES (Hichrom Ltd, UK) were tried, the system was an isocratic elution using a mobile phase of water: methanol 30:70 (v/v), the separations were conducted by injecting an equal amount of sample (20µl) onto the analytical columns. The peaks of the chromatogram of the column that gave the best separation were collected separately and again 1 ml of each fractions was assayed for antimicrobial activity as before.

Comparison of fractionation from Es1 extracts from solid medium (AIA), broth medium (AIB) and mycelium extraction obtained from dense cultures of Es1 in AIB.

HPLC Agilent Technologies 1220 Infinity LC, analytical ACE Excel 5 C-18 amide column was used. The chromatogram was conducted by injecting an equal amount of the samples and the peaks of the chromatogram were collected separately and the fractions tested for antibacterial activity.

Table 2.7: Summary of amounts of extract loaded on 6mm filter paper disc for different samples

Extract Amount

After extraction of antibiotics from filtered broth medium 2.12.2 20µl After two different methods were used to extract the antibiotic 2.12.4.1 300µl After three different inoculation methods used 2.12.4.2 300µl After scale up number of plates extracted 2.12.4.3 300µl

After 7 and 21d of incubation 2.11.4.4 300µl

After different solvents were used for antibiotic extraction 2.12.4.5 300µl After extraction of antibiotics from different isolates 2.13 300µl

2.17.2 Collection of the fractions with antimicrobial properties

Based on the analytical HPLC separation results obtained from the first stage, a semi- preparative ACE Excel 5 C-18 amide column, Gradient HPLC system with a mobile phase of water: methanol 30:70 (v/v) and a flow rate of 5 ml/min was used. The separations were conducted by automated injecting 500 µl of the sample (Es1extract) each time onto the semi-preparative column, automated collector collected each fraction with antibacterial activity. All fractions were evaporated by rotary

evaporator, after that the crud of each fraction eluted on Methanol-d4, than analyzed by NMR spectroscopy to check purity of the antibiotics.

The system used was an isocratic elution using a mobile phase of water: acetonitrile (Fisher, UK) 33:67 (v/v) for each fraction to remove impurities from the antibiotics. Two hundred and fifty microlitres of the semi pure antibiotic was injected into a semi- preparative ACE Excel 5 C-18 amide column; the peaks of the chromatogram were collected separately, and antibiotic activity determined as before. Fractions from each run were pooled and the solvent evaporated by rotary evaporator, the dried extracts were dissolved in methanol-d4.

2.17.3High Performance Liquid Chromatography: Purification of the antibiotics from isolate M1 11 extracts

The same procedure as above was used for purification the antibiotics produced by M1 11 isolate, except that the solvent system was different and to get pure antibiotic three different solvent system were used, Gradient HPLC system, mobile phase of water: acetonitrile 33:67(v/v) with a flow rate of 5 ml/min for collecting the fractions with antimicrobial properties, than an isocratic system was used with a mobile phase of water: methanol: acetonitrile 30:35:35 (v/v) to remove impurities from the

antibiotics. When analyzed with NMR the proton spectrum showed that the

antibiotics were not yet pure and a further purification step using an isocratic system with a mobile phase of water: methanol 30:70 (v/v) was used to further purify the antibiotics.

2.17.4 Identification of Chemical Structures

NMR spectroscopy

After purified fractions were obtained by HPLC all the samples were analyzed by NMR spectroscopy, all NMR measurements were performed on a Bruker Ultra-shield Avance AMX 400 MHz spectrometer, using 5-mm NMR tubes (Wilmad NMR tubes, 5 mm diam., precision 500 MHz Sigma-Aldrich, UK). Samples (5 mg of the

compound for some fractions and the available amount of the compounds for rest of the fractions) were dissolved in a 600 μl deuterated methanol-D4 (CD3OD) or deuterated Dimethyl sulfoxide-D_{6 (DMSO-D6).}

FTIR

FTIR analysis of the each compound was carried out with a Thermo Nicolet iS10 IR spectrometer following the manufacturer’s instructions. For each sample, 16 scans were collected.

Mass Spectroscopy (MS) of the active compounds

MS for pure compounds (Es1 fractions) were obtained first by LC-MS (Agilent Technologies 6120 Quadrupole LC/MS) at the University of Salford by injecting 10µl of each compound into instrument in positive and negative ion mode. Then, accurate MS were obtained by sending samples to the Cambridge University Chemical Laboratory to confirm the final structures of the compounds.