4.1.1 Collection of strains
The strains for this investigation were obtained via cooperations with various microbiological groups. The terrestrial strains with code names AdM 02 were iso-lated by Prof. H. Anke from soil samples provided by Prof. A. de Meijere. Strepto-mycetes with numbers like B8722 were obtained from the strain collections of E.
Helmke at the Alfred-Wegener Institute for Polar and Marine Research in Bremer-haven, and strains with code named GW14/1869 came from the Laboratory I. Grün-Wollny, respectively. Further strains were isolated from the Mediterranean Sea and the Red Sea. The organisms were described at the beginning temporarily by colour, morphology, the presence of mucus etc. The precise taxonomy will usually be de-termined later.
4.1.2 Pre-screening
From the received strains, 30% were usually able to produce metabolites with bioactivity or other interesting properties. To select these strains, a so-called pre-screening was performed. In this method, strains are selected by a number of suitable qualitative or quantitative criteria, like biological, chemical or physical interactions of metabolites with test systems.
The strains are sub-cultured on agar plates for 3-7 days and microscopically ex-amined for contaminations. Small pieces of the agar culture are then used to inocu-late 1-L Erlenmeyer flasks containing 250 ml medium, followed by incubation on a rotary shaker at 28 °C. The culture broth is then lyophilised and the dried residue extracted with ethyl acetate. The obtained crude extract was used for biological, chemical and pharmacological screenings and also for HPLC-UV-ESI MS/MS measurements as described above.
4.1.3 Chemical screening
The search and isolation of pure bioactive compounds from bacteria is a multi-ple step procedure and an expensive task. For this reason it is important to eliminate unnecessary work like the re-isolation of known metabolites from the crude extract or from a partially purified fraction. Chemical screening is a method, which allows us to reach this aim at the earliest stages of separation, and is therefore economically very important.
TLC (thin layer chromatography) is one of the cheapest and simplest methods used for the detection of bacterial constituents in the crude extract. Compared with other methods like HPLC it is easy to perform, quick requires simple equipment and is reproducible. A spot of the crude extract is separated by TLC with a mixture of e.g. CH2Cl2/MeOH. The developed TLC plate is visualized under UV light and inter-esting zones are further localized by exposure to spray reagents. In our group, anisal-dehyde/sulphuric acid, Ehrlich’s reagent, sulphuric acid and 2N NaOH are the most widely used ones.
- Anisaldehyde/sulphuric acid gives different colour reactions with many struc-tural elements (glycosides, steroids, terpenes, macrolides and phenols).
- Ehrlich’s reagent is a specific reagent used to determine indoles and some other nitrogen containing compounds; indoles turn pink, blue or violet, or brown for pyrroles and furan. Anthranilic acid derivatives change to yellow.
- Sulphuric acid is especially used for polyenes. Short conjugated chains are showing a brown or black colour, carotenoids develop a blue or green colour.
- NaOH is used for the detection of peri-hydroxy-quinones, which turn red, blue or violet. The deep red prodigiosins are showing a colour change to yel-low with base.
- Tin(II)-chloride/hydrochloric acid/ 4-dimethylamino-benzaldehyde is used for nitro compounds and gives yellow to deep yellow or orange spots. This reaction uses the reduction of the nitro group to the amino group and the for-mation of Schiff's bases.
General techniques 27
4.1.4 Pharmacological and Biological Assays
It is evident that in order to screen a crude extract for bioactive substances, an appropriate biological test is needed. In that case, all bioassays should have high ca-pacity, sensitivity, low cost, and must give rapid answers. In our group the crude extract is screened using the agar diffusion test with a few positive and Gram-negative bacteria and fungi such as Escherichia coli, Streptomyces viridochro-mogenes (Tü 57), Bacillus subtilis, Staphylococcus aureus, Mucor miehei (Tü 284), Candida albicans. The microalgae Chlorella vulgaris, Chlorella sorokiniana, and Scenedesmus subspicatus are used as test organisms to screen for phytotoxicity. In parallel, the cytotoxic activity was evaluated against brine shrimps (Artemia salina) and nematodes (Caenorhabditis elegans). The brine shrimp toxicity has a strong cor-relation with cellular cytotoxicity and is therefore a good indicator for potential anti-cancer activity.
The bio-autography on TLC gives simultaneously more information about an unknown bioactive component in the crude extract. This is readily seen with antim-icrobial compounds. The pharmacological tests in our group were carried out at BioLeads (Heidelberg), Oncotest (Freiburg) and later at the Institute of Biotechnol-ogy and Active Agent Research (Kaiserslautern).
4.1.5 Cultivation and scale-up
The cultivation and scale-up steps are carried out only after both primary screen-ings. An optimisation of the culture conditions may sometimes be done in order to choose the best medium, improve the yield or comparison of produced secondary metabolites. The optimisation is always applied when the amount of active sub-stances obtained is very small. There were two possibilities available for the culture of bacteria: the fermentation in shaking flasks or in a fermentor. A pre-culture of 2 L is to be used for the inoculation.
After harvesting, the culture broth is mixed with Celite and filtered under pres-sure. The water phase, which contains highly polar compounds like sugars, certain polyhydroxy acids, amino acids and many peptides can be submitted to extraction with ethyl acetate. However it is highly recommended to use a solid phase extraction with special adsorber resins (mostly Amberlite XAD-16 or Mitsubishi DIAION
HP20) due to the fact that is not harmful and reduces considerably the costs for sol-vents, than the extraction with a solvent of higher polarity like water-saturated ethyl acetate or even methanol. The mycelium is extracted with ethyl acetate and acetone.
The organic phases are evaporated to dryness and the remaining crude extract used for separations.
4.1.6 Isolation methods
The separation methods depend on the amount of the crude extract and the po-larity of the compounds of interest. Generally, the crude extract is first defatted using cyclohexane, than subjected to silica gel chromatography using a gradient of increas-ing polarity with various solvent systems (CH2Cl2/MeOH or cyclohexane/ethyl ace-tate etc.). Size-exclusion chromatography (Sephadex LH-20) offers the advantage of a higher recovery rates and minimizes the destruction of compounds. It is used pref-erentially when the amount of the crude extract is < 4 mg. Further methods like PTLC and HPLC are also used for some final purification.
Strains
1L Culture Storage
Crude extract Prescreening: Chemical and Biological
Isolation and Structure elucidation Dereplication: -Scidex(Antibase) -Scifinder
-Chapmann & Hall
Pure compound
!!!!!Activity test!!!!!
Extraction
Clean up
Derivatisation and/or optimisation if necessary
HPLC-UV-ESI-MS/MS
Figure 10: General screening of the selected strains
Pseudoalteromonas Strain T 268 29
5 Some Strains from Marine and other Origins